US20030162262A1 - Recombinant narbonolide polyketide synthase - Google Patents

Recombinant narbonolide polyketide synthase Download PDF

Info

Publication number
US20030162262A1
US20030162262A1 US10/160,539 US16053902A US2003162262A1 US 20030162262 A1 US20030162262 A1 US 20030162262A1 US 16053902 A US16053902 A US 16053902A US 2003162262 A1 US2003162262 A1 US 2003162262A1
Authority
US
United States
Prior art keywords
pks
gene
narbonolide
domain
recombinant
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US10/160,539
Inventor
Gary Ashley
Melanie Betlach
Mary Betlach
Robert McDaniel
Li Tang
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US09/141,908 external-priority patent/US6503741B1/en
Application filed by Individual filed Critical Individual
Priority to US10/160,539 priority Critical patent/US20030162262A1/en
Publication of US20030162262A1 publication Critical patent/US20030162262A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
    • C07H17/08—Hetero rings containing eight or more ring members, e.g. erythromycins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D323/00—Heterocyclic compounds containing more than two oxygen atoms as the only ring hetero atoms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures

Definitions

  • the present invention provides recombinant methods and materials for producing polyketides by recombinant DNA technology. More specifically, it relates to narbonolides and derivatives thereof.
  • the invention relates to the fields of agriculture, animal husbandry, chemistry, medicinal chemistry, medicine, molecular biology, pharmacology, and veterinary technology.
  • Polyketides represent a large family of diverse compounds synthesized from 2-carbon units through a series of condensations and subsequent modifications. Polyketides occur in many types of organisms, including fungi and mycelial bacteria, in particular, the actinomycetes. There is a wide variety of polyketide structures, and the class of polyketides encompasses numerous compounds with diverse activities. Tetracycline, erythromycin, FK506, FK520, narbomycin, picromycin, rapamycin, spinocyn, and tylosin, are examples of such compounds.
  • PKS polyketide synthase
  • ORFs open reading frames
  • Modular PKSs are responsible for producing a large number of 12, 14, and 16-membered macrolide antibiotics including methymycin, erythromycin, narbomycin, picromycin, and tylosin. These large multifunctional enzymes (>300,000 kDa) catalyze the biosynthesis of polyketide macrolactones through multistep pathways involving decarboxylative condensations between acyl thioesters followed by cycles of varying ⁇ -carbon processing activities (see O'Hagan, D. The polyketide metabolites; E. Horwood: New York, 1991, incorporated herein by reference).
  • the modular PKS are generally encoded in multiple ORFs. Each ORF typically comprises two or more “modules” of ketosynthase activity, each module of which consists of at least two (if a loading module) and more typically three or more enzymatic activities or “domains.”
  • the resulting technology allows one to manipulate a known PKS gene cluster either to produce the polyketide synthesized by that PKS at higher levels than occur in nature or in hosts that otherwise do not produce the polyketide.
  • the technology also allows one to produce molecules that are structurally related to, but distinct from, the polyketides produced from known PKS gene clusters. It has been possible to manipulate modular PKS genes other than the narbonolide PKS using generally known recombinant techniques to obtain altered and hybrid forms. See, e.g., U.S. Pat. Nos. 5,672,491 and 5,712,146 and PCT publication No. WO 98/49315.
  • the present invention provides methods and reagents relating to the modular PKS gene cluster for the polyketide antibiotics known as narbomycin and picromycin.
  • Narbomycin is produced in Streptomyces narbonensis, and both narbomycin and picromycin are produced in S. venezuelae.
  • These species are unique among macrolide producing organisms in that they produce, in addition to the 14-membered macrolides narbomycin and picromycin (picromycin is shown in FIG. 1, compound 1), the 12-membered macrolides neomethymycin and methymycin (methymycin is shown in FIG. 1, compound 2).
  • Narbomycin differs from picromycin only by lacking the hydroxyl at position 12. Based on the structural similarities between picromycin and methymycin, it was speculated that methymycin would result from premature cyclization of a hexaketide intermediate in the picromycin pathway.
  • FIG. 1 shows the metabolic relationships of the compounds discussed above.
  • Picromycin (FIG. 1, compound 1) is of particular interest because of its close structural relationship to ketolide compounds (e.g. HMR 3004, FIG. 1, compound 3).
  • the ketolides are a new class of semi-synthetic macrolides with activity against pathogens resistant to erythromycin (see Agouridas et al., 1998 , J. Med. Chem. 41: 4080-4100, incorporated herein by reference).
  • genetic systems that allow rapid engineering of the narbonolide PKS would be valuable for creating novel ketolide analogs for pharmaceutical applications.
  • the production of picromycin as well as novel compounds with useful activity could be accomplished if the heterologous expression of the narbonolide PKS in Streptomyces lividans and other host cells were possible.
  • the present invention meets these and other needs.
  • the present invention provides recombinant methods and materials for expressing PKSs derived in whole and in part from the narbonolide PKS and other genes involved in narbomycin and picromycin biosynthesis in recombinant host cells.
  • the invention also provides the polyketides derived from the narbonolide PKS.
  • the invention provides the complete PKS gene cluster that ultimately results, in Streptomyces venezuelae, in the production of picromycin.
  • the ketolide product of this PKS is narbonolide. Narbonolide is glycosylated to obtain narbomycin and then hydroxylated at C12 to obtain picromycin.
  • the enzymes responsible for the glycosylation and hydroxylation are also provided in recombinant form by the invention.
  • the invention is directed to recombinant materials that contain nucleotide sequences encoding at least one domain, module, or protein encoded by a narbonolide PKS gene.
  • the recombinant materials may be “isolated.”
  • the invention also provides recombinant materials useful for conversion of ketolides to antibiotics. These materials include recombinant DNA compounds that encode the C12hydroxylase (the picK gene), the desosamine biosynthesis and desosaminyl transferase enzymes, and the beta-glucosidase enzyme involved in picromycin biosynthesis in S. venezuelae and the recombinant proteins that can be produced from these nucleic acids in the recombinant host cells of the invention.
  • the invention provides a recombinant expression system that comprises a heterologous promoter positioned to drive expression of the narbonolide PKS, including a “hybrid” narbonolide PKS.
  • the promoter is derived from a PKS gene.
  • the invention provides recombinant host cells comprising the vector that produces narbonolide.
  • the host cell is Streptomyces lividans or S. coelicolor.
  • the invention provides a recombinant expression system that comprises the desosamine biosynthetic genes as well as the desosaminyl transferase gene.
  • the invention provides recombinant host cells comprising a vector that produces the desosamine biosynthetic gene products and desosaminyl transferase gene product.
  • the host cell is Streptomyces lividans or S. coelicolor.
  • the invention provides a method for desosaminylating polyketide compounds in recombinant host cells, which method comprises expressing the PKS for the polyketide and the desosaminyl transferase and desosamine biosynthetic genes in a host cell.
  • the host cell expresses a beta-glucosidase gene as well. This preferred method is especially advantageous when producing desosaminylated polyketides in Streptomyces host cells, because such host cells typically glucosylate desosamine residues of polyketides, which can decrease desired activity, such as antibiotic activity.
  • beta-glucosidase the glucose residue is removed from the polyketide.
  • the invention provides the picK hydroxylase gene in recombinant form and methods for hydroxylating polyketides with the recombinant gene product.
  • the invention also provides polyketides thus produced and the antibiotics or other useful compounds derived therefrom.
  • the invention provides a recombinant expression system that comprises a promoter positioned to drive expression of a “hybrid” PKS comprising all or part of the narbonolide PKS and at least a part of a second PKS, or comprising a narbonolide PKS modified by deletions, insertions and/or substitutions.
  • the invention provides recombinant host cells comprising the vector that produces the hybrid PKS and its corresponding polyketide.
  • the host cell is Streptomyces lividans or S. coelicolor.
  • the invention provides recombinant materials for the production of libraries of polyketides wherein the polyketide members of the library are synthesized by hybrid PKS enzymes of the invention.
  • the resulting polyketides can be further modified to convert them to other useful compounds, such as antibiotics, typically through hydroxylation and/or glycosylation.
  • Modified macrolides provided by the invention that are useful intermediates in the preparation of antibiotics are of particular benefit.
  • the invention provides a method to prepare a nucleic acid that encodes a modified PKS, which method comprises using the narbonolide PKS encoding sequence as a scaffold and modifying the portions of the nucleotide sequence that encode enzymatic activities, either by mutagenesis, inactivation, insertion, or replacement.
  • the thus modified narbonolide PKS encoding nucleotide sequence can then be expressed in a suitable host cell and the cell employed to produce a polyketide different from that produced by the narbonolide PKS.
  • portions of the narbonolide PKS coding sequence can be inserted into other PKS coding sequences to modify the products thereof.
  • the narbonolide PKS can itself be manipulated, for example, by fusing two or more of its open reading frames, particularly those for extender modules 5 and 6, to make more efficient the production of 14-membered as opposed to 12-membered macrolides.
  • the invention is directed to a multiplicity of cell colonies, constituting a library of colonies, wherein each colony of the library contains an expression vector for the production of a modular PKS derived in whole or in part from the narbonolide PKS.
  • each colony of the library contains an expression vector for the production of a modular PKS derived in whole or in part from the narbonolide PKS.
  • the modular PKS is identical to that found in the PKS that produces narbonolide and is identifiable as such.
  • the derived portion can be prepared synthetically or directly from DNA derived from organisms that produce narbonolide.
  • the invention provides methods to screen the resulting polyketide and antibiotic libraries.
  • the invention also provides novel polyketides and antibiotics or other useful compounds derived therefrom.
  • the compounds of the invention can be used in the manufacture of another compound.
  • the antibiotic compounds of the invention are formulated in a mixture or solution for administration to an animal or human.
  • FIG. 1 shows the structures of picromycin (compound 1), methymycin (compound 2), and the ketolide HMR 3004 (compound 3) and the relationship of several compounds related to picromycin.
  • FIG. 2 shows a restriction site and function map of cosmid pKOS023-27.
  • FIG. 3 shows a restriction site and function map of cosmid pKOS023-26.
  • FIG. 4 has three parts.
  • Part A the structures of picromycin (A(a)) and methymycin (A(b)) are shown, as well as the related structures of narbomycin, narbonolide, and methynolide.
  • the bolded lines indicate the two or three carbon chains produced by each module (loading and extender) of the narbonolide PKS.
  • Part B shows the organization of the narbonolide PKS genes on the chromosome of Streptomyces venezuelae, including the location of the various module encoding sequences (the loading module domains are identified as sKS*, sAT, and sACP), as well as the picB thioesterase gene and two desosamine biosynthesis genes (picCII and picCII).
  • Part C shows the engineering of the S. venezuelae host of the invention in which the picAI gene has been deleted.
  • ACP is acyl carrier protein
  • AT is acyltransferase
  • DH is dehydratase
  • ER is enoylreductase
  • KR is ketoreductase
  • KS is ketosynthase
  • TE is thioesterase.
  • FIG. 5 shows the narbonolide PKS genes encoded by plasmid pKOS039-86, the compounds synthesized by each module of that PKS and the narbonolide (compound 4) and 10-deoxymethynolide (compound 5) products produced in heterologous host cells transformed with the plasmid.
  • the Figure also shows a hybrid PKS of the invention produced by plasmid pKOS038-18, which encodes a hybrid of DEBS and the narbonolide PKS.
  • the Figure also shows the compound, 3,6-dideoxy-3-oxo-erythronolide B (compound 6), produced in heterologous host cells comprising the plasmid.
  • FIG. 6 shows a restriction site and function map of plasmid pKOS039-104, which contains the desosamine biosynthetic, beta-glucosidase, and desosaminyl transferase genes under transcriptional control of actII-4.
  • the present invention provides useful compounds and methods for producing polyketides in recombinant host cells.
  • the term recombinant refers to a compound or composition produced by human intervention.
  • the invention provides recombinant DNA compounds encoding all or a portion of the narbonolide PKS.
  • the invention also provides recombinant DNA compounds encoding the enzymes that catalyze the further modification of the ketolides produced by the narbonolide PKS.
  • the invention provides recombinant expression vectors useful in producing the narbonolide PKS and hybrid PKSs composed of a portion of the narbonolide PKS in recombinant host cells.
  • the invention also provides the narbonolide PKS, hybrid PKSs, and polyketide modification enzymes in recombinant form.
  • the invention provides the polyketides produced by the recombinant PKS and polyketide modification enzymes.
  • the invention provides methods for producing the polyketides 10-deoxymethynolide, narbonolide, YC17, narbomycin, methymycin, neomethymycin, and picromycin in recombinant host cells.
  • Section IV methods for heterologous expression of the narbonolide PKS and narbonolide modification enzymes provided by the invention are described.
  • Section V the hybrid PKS genes provided by the invention and the polyketides produced thereby are described.
  • Section VI the polyketide compounds provided by the invention and pharmaceutical compositions of those compounds are described. The detailed description is followed by a variety of working examples illustrating the invention.
  • the narbonolide synthase gene is composed of coding sequences organized in a loading module, a number of extender modules, and a thioesterase domain. As described more fully below, each of these domains and modules is a polypeptide with one or more specific functions.
  • the loading module is responsible for binding the first building block used to synthesize the polyketide and transferring it to the first extender module.
  • the building blocks used to form complex polyketides are typically acylthioesters, most commonly acetyl, propionyl, malonyl, methylmalonyl, and ethylmalonyl CoA. Other building blocks include amino acid like acylthioesters.
  • PKSs catalyze the biosynthesis of polyketides through repeated, decarboxylative Claisen condensations between the acylthioester building blocks.
  • Each module is responsible for binding a building block, performing one or more functions on that building block, and transferring the resulting compound to the next module.
  • the next module is responsible for attaching the next building block and transferring the growing compound to the next module until synthesis is complete.
  • an enzymatic thioesterase activity cleaves the polyketide from the PKS. See, generally, FIG. 5.
  • Such modular organization is characteristic of the modular class of PKS enzymes that synthesize complex polyketides and is well known in the art.
  • the polyketide known as 6-deoxyerythronolide B is a classic example of this type of complex polyketide.
  • the genes, known as eryAI, eryAII, and eryAIII also referred to herein as the DEBS genes, for the proteins, known as DEBS1, DEBS2, and DEBS3, that comprise the 6-dEB synthase), that code for the multi-subunit protein known as DEBS that synthesizes 6-dEB, the precursor polyketide to erythromycin, are described in U.S. Pat. No. 5,824,513, incorporated herein by reference.
  • the loading module of DEBS consists of two domains, an acyl-transferase (AT) domain and an acyl carrier protein (ACP) domain.
  • Each extender module of DEBS like those of other modular PKS enzymes, contains a ketosynthase (KS), AT, and ACP domains, and zero, one, two, or three domains for enzymatic activities that modify the beta-carbon of the growing polyketide chain.
  • a module can also contain domains for other enzymatic activities, such as, for example, a methyltransferase or dimethyltransferase activity.
  • the releasing domain contains a thioesterase and, often, a cyclase activity.
  • the AT domain of the loading module recognizes a particular acyl-CoA (usually acetyl or propionyl but sometimes butyryl) and transfers it as a thiol ester to the ACP of the loading module.
  • the AT on each of the extender modules recognizes a particular extender-CoA (malonyl or alpha-substituted malonyl, i.e., methylmalonyl, ethylmalonyl, and carboxylglycolyl) and transfers it to the ACP of that module to form a thioester.
  • the acyl group of the loading module migrates to form a thiol ester (trans-esterification) at the KS of the first extender module; at this stage, extender module 1 possesses an acyl-KS adjacent to a malonyl (or substituted malonyl) ACP.
  • the acyl group derived from the loading module is then covalently attached to the alpha-carbon of the malonyl group to form a carbon-carbon bond, driven by concomitant decarboxylation, and generating a new acyl-ACP that has a backbone two carbons longer than the loading unit (elongation or extension).
  • the growing polyketide chain is transferred from the ACP to the KS of the next module, and the process continues.
  • modules may contain a ketodreductase (KR) that reduces the keto group to an alcohol.
  • KR ketodreductase
  • Modules may also contain a KR plus a dehydratase (DH) that dehydrates the alcohol to a double bond. Modules may also contain a KR, a DH, and an enoylreductase (ER) that converts the double bond to a saturated single bond using the beta carbon as a methylene function. As noted above, modules may contain additional enzymatic activities as well.
  • DH dehydratase
  • ER enoylreductase
  • a polyketide chain traverses the final extender module of a PKS, it encounters the releasing domain or thioesterase found at the carboxyl end of most PKSs.
  • the polyketide is cleaved from the enzyme and cyclyzed.
  • the resulting polyketide can be modified further by tailoring enzymes; these enzymes add carbohydrate groups or methyl groups, or make other modifications, i.e., oxidation or reduction, on the polyketide core molecule.
  • PKS enzymes incorporate a building block that is derived from an amino acid.
  • PKS enzymes for such polyketides include an activity that functions as an amino acid ligase or as a non-ribosomal peptide synthetase (NRPS).
  • NRPS non-ribosomal peptide synthetase
  • KS Q This inactivated KS is in most instances called KS Q , where the superscript letter is the abbreviation for the amino acid, glutamine, that is present instead of the active site cysteine required for activity.
  • the narbonolide PKS loading module contains a KS Q .
  • modules that include a methyltransferase or dimethyltransferase activity; modules can also include an epimerase activity.
  • narbonolide related polyketides in Streptomyces venezuelae and S. narbonensis.
  • the narbonolide PKS produces two polyketide products, narbonolide and 10-deoxymethynolide.
  • Narbonolide is the polyketide product of all six extender modules of the narbonolide PKS.
  • 10-deoxymethynolide is the polyketide product of only the first five extender modules of the narbonolide PKS.
  • These two polyketides are desosaminylated to yield narbomycin and YC17, respectively.
  • glycosylated polyketides are the final products produced in S. narbonensis. In S. venezuelae, these products are hydroxylated by the picK gene product to yield picromycin and either methymycin (hydroxylation at the C10 position of YC17) or neomethymycin (hydroxylation at the C12 position of YC17). (See FIG. 1)
  • the present invention provides the genes required for the biosynthesis of all of these polyketides in recombinant form.
  • the narbonolide PKS is composed of a loading module, six extender modules, and two thioesterase domains one of which is on a separate protein.
  • FIG. 4, part B shows the organization of the narbonolide PKS genes on the Streptomyces venezuelae chromosome, as well as the location of the module encoding sequences in those genes, and the various domains within those modules.
  • the loading module is not numbered, and its domains are indicated as sKS*, sAT, and ACP. Also shown in the Figure, part A, are the structures of picromycin and methymycin.
  • the loading and six extender modules and the thioesterase domain of the narbonolide PKS reside on four proteins, designated PICAI, PICAII, PICAIII, and PICAIV.
  • PICAI includes the loading module and extender modules 1 and 2 of the PKS.
  • PICAII includes extender modules 3 and 4.
  • PICAIII includes extender module 5.
  • PICAIV includes extender module 6 and a thioesterase domain.
  • TTII second thioesterase domain
  • Cosmid pKOS023-27 contains an insert of Streptomyces venezuelae DNA of 38506 nucleotides.
  • the complete sequence of the insert from cosmid pKOS023-27 is shown below. The location of the various ORFs in the insert, as well as the boundaries of the sequences that encode the various domains of the multiple modules of the PKS, are summarized in the Table below.
  • PICB shows a restriction site and function map of pKOS023-27, which contains the complete coding sequence for the four proteins that constitute narbonolide PKS and four additional ORFs.
  • One of these additional ORFs encodes the picB gene product, the type II thioesterase mentioned above.
  • PICB shows a high degree of similarity to other type II thioesterases, with an identity of 51%, 49%, 45% and 40% as compared to those of Amycolatopsis mediterranae, S. griseus, S. fradiae and Saccharopolyspora erythraea, respectively.
  • the recombinant nucleic acids, proteins, and peptides of the invention are many and diverse. To facilitate an understanding of the invention and the diverse compounds and methods provided thereby, the following description of the various regions of the narbonolide PKS and corresponding coding sequences is provided.
  • the loading module of the narbonolide PKS contains an inactivated KS domain, an AT domain, and an ACP domain.
  • the AT domain of the loading module binds propionyl CoA.
  • Sequence analysis of the DNA encoding the KS domain indicates that this domain is enzymatically inactivated, as a critical cysteine residue in the motif TVDACSSSL, which is highly conserved among KS domains, is replaced by a glutamine so is referred to as a KS Q domain.
  • Such inactivated KS domains are also found in the PKS enzymes that synthesize the 16-membered macrolides carbomycin, spiromycin, tylosin, and niddamycin. While the KS domain is inactive for its usual function in extender modules, it is believed to serve as a decarboxylase in the loading module.
  • the present invention provides recombinant DNA compounds that encode the loading module of the narbonolide PKS and useful portions thereof. These recombinant DNA compounds are useful in the construction of PKS coding sequences that encode all or a portion of the narbonolide PKS and in the construction of hybrid PKS encoding DNA compounds of the invention, as described in the section concerning hybrid PKSs below.
  • reference to a PKS, protein, module, or domain herein can also refer to DNA compounds comprising coding sequences therefor and vice versa.
  • reference to a heterologous PKS refers to a PKS or DNA compounds comprising coding sequences therefor from an organism other than Streptomyces venezuelae.
  • reference to a PKS or its coding sequence includes reference to any portion thereof.
  • the present invention provides recombinant DNA compounds that encode one or more of the domains of each of the six extender modules (modules 1-6, inclusive) of the narbonolide PKS.
  • Modules 1 and 5 of the narbonolide PKS are functionally similar.
  • Each of these extender modules contains a KS domain, an AT domain specific for methylmalonyl CoA, a KR domain, and an ACP domain.
  • Module 2 of the narbonolide PKS contains a KS domain, an AT domain specific for malonyl CoA, a KR domain, a DH domain, and an ACP domain.
  • Module 3 differs from extender modules 1 and 5 only in that it contains an inactive ketoreductase domain.
  • Module 4 of the narbonolide PKS contains a KS domain, an AT-domain specific for methylmalonyl CoA, a KR domain, a DH domain, an ER domain, and an ACP domain.
  • Module 6 of the narbonolide PKS contains a KS domain, an AT domain specific for methylmalonyl CoA, and an ACP domain. The approximate boundaries of these “domains” is shown in Table 1.
  • the invention provides a recombinant narbonolide PKS that can be used to express only narbonolide (as opposed to the mixture of narbonolide and 10-deoxymethynolide that would otherwise be produced) in recombinant host cells.
  • This recombinant narbonolide PKS results from a fusion of the coding sequences of the picAIII and picAIV genes so that extender modules 5 and 6 are present on a single protein.
  • This recombinant PKS can be constructed on the Streptomyces venezuelae or S. narbonensis chromosome by homologous recombination.
  • the recombinant PKS can be constructed on an expression vector and introduced into a heterologous host cell.
  • This recombinant PKS is preferred for the expression of narbonolide and its glycosylated and/or hydroxylated derivatives, because a lesser amount or no 10-deoxymethynolide is produced from the recombinant PKS as compared to the native PKS.
  • the invention provides a recombinant narbonolide PKS in which the picAIV gene has been rendered inactive by an insertion, deletion, or replacement. This recombinant PKS of the invention is useful in the production of 10-deoxymethynolide and its derivatives without production of narbonolide.
  • the invention provides recombinant narbonolide PKS in which any of the domains of the native PKS have been deleted or rendered inactive to make the corresponding narbonolide or 10-deoxymethynolide derivative.
  • the invention also provides recombinant narbonolide PKS genes that differ from the narbonolide PKS gene by one or more deletions.
  • the deletions can encompass one or more modules and/or can be limited to a partial deletion within one or more modules.
  • the resulting narbonolide derivative is at least two carbons shorter than the polyketide produced from the PKS encoded by the gene from which deleted PKS gene and corresponding polyketide were derived.
  • the deletion When a deletion is within a module, the deletion typically encompasses a KR, DH, or ER domain, or both DH and ER domains, or both KR and DH domains, or all three KR, DH, and ER domains.
  • FIG. 4 shows how a vector of the invention, plasmid pKOS039-16 (not shown), was used to delete or “knock out” the picAI gene from the Streptomyces venezuelae chromosome.
  • Plasmid pKOS039-16 comprises two segments (shown as cross-hatched boxes in FIG. 4, part B) of DNA flanking the picAI gene and isolated from cosmid pKOS023-27 (shown as a linear segment in the Figure) of the invention.
  • cosmid pKOS023-27 shown as a linear segment in the Figure
  • This Streptomyces venezuelae K039-03 host cell and corresponding host cells of the invention are especially useful for the production of polyketides produced from hybrid PKS or narbonolide PKS derivatives.
  • These host cells are also preferred for desosaninylating polyketides in accordance with the method of the invention in which a polyketide is provided to an S. venezuelae cell and desosaminylated by the endogenous desosamine biosynthesis and desosaminyl transferase gene products.
  • the recombinant DNA compounds of the invention that encode each of the domains of each of the modules of the narbonolide PKS are also useful in the construction of expression vectors for the heterologous expression of the narbonolide PKS and for the construction of hybrid PKS expression vectors, as described further below.
  • Section II The Genes for Desosamine Biosynthesis and Transfer and for Beta-Glucosidase
  • Narbonolide and 10-deoxymethynolide are desosaminylated in Streptomyces venezuelae and S. narbonensis to yield narbomycin and YC-17, respectively.
  • This conversion requires the biosynthesis of desosamine and the transfer of the desosamine to the substrate polyketides by the enzyme desosaminyl transferase.
  • S. venezuelae and S. narbonensis produce glucose and a glucosyl transferase enzyme that glucosylates desosamine at the 2′ position.
  • narbonensis also produce a beta-glucosidase, which removes the glucose residue from the desosamine.
  • the present invention provides recombinant DNA compounds and expression vectors for each of the desosamine biosynthesis enzymes, desosaminyl transferase, and beta-glucosidase.
  • cosmid pKOS023-27 contains three ORFs that encode proteins involved in desosamine biosynthesis and transfer.
  • the first ORF is from the picCII gene, also known as desVIII, a homologue of eryCII, believed to encode a 4-keto-6-deoxyglucose isomerase.
  • the second ORF is from the picCIII gene, also known as desVII, a homologue of eryCIII, which encodes a desosaminyl transferase.
  • the third ORF is from the picCVI gene, also known as desVI, a homologue of eryCVI, which encodes a 3-amino dimethyltransferase.
  • FIG. 3 shows a restriction site and function map of cosmid pKOS023-26. This cosmid contains a region of overlap with cosmid pKOS023-27.representing nucleotides 14252 to nucleotides 38506 of pKOS023-27.
  • the remaining desosamine biosynthesis genes on cosmid pKOS023-26 include the following genes.
  • ORF11 also known as desR, encodes beta-glucosidase and has no ery gene homologue.
  • the picCI gene, also known as desV is a homologue of eryCI.
  • ORF14 also known as desIV, has no known ery gene homologue and encodes an NDP glucose 4,6-dehydratase.
  • ORF13, also known as desIII has no known ery gene homologue and encodes an NDP glucose synthase.
  • the picCV gene also known as desII, a homologue of eryCV is required for desosamine biosynthesis.
  • the picCIV gene also known as desI is a homologue of eryCIV, and its product is believed to be a 3,4-dehydratase.
  • ORFs on cosmid pKOS023-26 include ORF12, believed to be a regulatory gene; ORF15, which encodes an S-adenosyl methionine synthase; and ORF16, which is a homolog of the M. tuberculosis cbhK gene.
  • Cosmid pKOS023-26 also encodes the picK gene, which encodes the cytochrome P450 hydroxylase that hydroxylates the C12 of narbomycin and the C10 and C12 positions of YC-17. This gene is described in more detail in the following section.
  • amino acid sequences or partial amino acid sequences of the gene products of the desosamine biosynthesis and transfer and beta-glucosidase genes are shown. These amino acid sequences are followed by the DNA sequences that encode them.
  • ORF16 (Homologous to M. tuberculosis cbhK) (SEQ ID NO:17) 1 MRIAVTGSIA TDHLMTFPGR FAEQILPDQL AHVSLSFLVD TLDIRHGGVA ANIAYGLGLL (SEQ ID NO:17) 61 GRRPVLVGAV GKDFDGYGQL LRAAGVDTDS VRVSDRQHTA RFMCTTDEDG NQLASFYAGA 121 MAEARDIDLG ETAGRPGGID LVLVGADDPE AMVRHTRVCR ELGLRRAADP SQQLARLEGD 181 SVRELVDGAE LLFTNAYERA LLLSKTGWTE QEVLARVGTW ITTLGAKGCR
  • Contig 001 from cosmid pKOS023-26 contains 2401 nucleotides, the first 100 bases of which correspond to 100 bases of the insert sequence of cosmid pKOS023-27.
  • Nucleotides 80-2389 constitute ORF11, which encodes 1 beta Glucosidase.
  • SEQ ID NO:20 1 CGTGGCGGCC GCCGCTCCCG GCGCCGCCGA CACGGCCAAT GTTCAGTACA CGAGCCGGGC (SEQ ID NO:20) 61 GGCGGAGCTC GTCGCCCAGA TGACGCTCGA CGAGAAGATC AGCTTCGTCC ACTGGGCGCT 121 GGACCCCGAC CGGCAGAACG TCGGCTACCT TCCCGGCGTG CCGCGTCTGG GCATCCCGGA 181 GCTGCGTGCC GCCGACGGCC CGAACGGCAT CCGCCTGGTG GGGCAGACCG CCACCGCGCT 241 GCCCGCCG GTCGCCCTGG CCAGCACCTT CGACGACACC ATGGCCGACA GCTACGGCAA 301 GGTCATGGGC CGCGACGGTC GCGCTCAA CCAGCACATG GTCCTGGGCC
  • Contig 002 from cosmid pKOS023-26 contains 5970 nucleotides and the following ORFs: from nucleotide 995 to 1 is an ORF of picCIV that encodes a partial sequence of an amino transferase-dehydrase; from nucleotides 1356 to 2606 is an ORF of picK that encodes a cytochrome P450 hydroxylase; and from nucleotides 2739 to 5525 is ORF12, which encodes a transcriptional activator.
  • Contig 003 from cosmid pKOS023-26 contains 3292 nucleotides and the following ORFs: from nucleotide 104 to 982 is ORF13, which encodes dNDP glucose synthase (glucose-1-phosphate thymidyl transferase); from nucleotide 1114 to 2127 is ORF14, which encodes dNDP-glucose 4,6-dehydratase; and from nucleotide 2124 to 3263 is the picCI ORF.
  • ORF13 which encodes dNDP glucose synthase (glucose-1-phosphate thymidyl transferase)
  • ORF14 which encodes dNDP-glucose 4,6-dehydratase
  • nucleotide 2124 to 3263 is the picCI ORF.
  • Contig 004 from cosmid pKOS023-26 contains 1693 nucleotides and the following ORFs: from nucleotide 1692 to 694 is ORF15, which encodes a part of S-adenosylmethionine synthetase; and from nucleotide 692 to 1 is ORF16, which encodes a part of a protein homologous to the M. tuberculosis cbhK gene.
  • Contig 005 from cosmid pKOS023-26 contains 1565 nucleotides and contains the ORF of the picCV gene that encodes PICCV, involved in desosamine biosynthesis.
  • SEQ ID NO:24 1 CCCCGCTCGC GGCCCCCCAG ACATCCACGC CCACGATTGG ACGCTCCCGA TGACCGCCCC (SEQ ID NO:24) 61 CGCCCTCTCC GCCACCGCCC CGGCCGAACG CTGCGCAC CCCGGAGCCG ATCTGGGGGC 121 GGCGGTCCAC GCCGTCGGCC AGACCCTCGC CGCCGGCGGC CTCGTGCCGC CCGACGAGGC 181 CGGAACGACC GCCCGCCACC TCGTCCGGCT CGCCCTGCGC TACGGCAACA GCCCCTTCAC 241 CCCGCTGGAG GAGGCCCGCC ACGACCTGGG CGTCGACCGG GACGCCTTCC GGCGCCTCCT 301 CGCCCTGTTC GGGC
  • the recombinant desosamine biosynthesis and transfer and beta-glucosidase genes and proteins provided by the invention are useful in the production of glycosylated polyketides in a variety of host cells, as described in Section IV below.
  • the present invention provides the picK gene in recombinant form as well as recombinant PicK protein.
  • the availability of the hydroxylase encoded by the picK gene in recombinant form is of significant benefit in that the enzyme can convert narbomycin into picromycin and accepts in addition a variety of polyketide substrates, particularly those related to narbomycin in structure.
  • the present invention also provides methods of hydroxylating polyketides, which method comprises contacting the polyketide with the recombinant PicK enzyme under conditions such that hydroxylation occurs. This methodology is applicable to large numbers of polyketides.
  • DNA encoding the picK gene can be isolated from cosmid pKOS023-26 of the invention.
  • the DNA sequence of the picK gene is shown in the preceding section. This DNA sequence encodes one of the recombinant forms of the enzyme provided by the invention.
  • the amino acid sequence of this form of the picK gene is shown below.
  • the present invention also provides a recombinant picK gene that encodes a picK gene product in which the PicK protein is fused to a number of consecutive histidine residues, which facilitates purification from recombinant host cells.
  • the recombinant PicK enzyme of the invention hydroxylates narbomycin at the C12 position and YC-17 at either the C10 or C12 position. Hydroxylation of these compounds at the respective positions increases the antibiotic activity of the compound relative to the unhydroxylated compound. Hydroxylation can be achieved by a number of methods. First, the hydroxylation may be performed in vitro using purified hydroxylase, or the relevant hydroxylase can be produced recombinantly and utilized directly in the cell that produces it. Thus, hydroxylation may be effected by supplying the nonhydroxylated precursor to a cell that expresses the hydroxylase.
  • Section IV Heterologous Expression of the Narbonolide PKS; the Desosamine Biosynthetic and Transferase Genes; the Beta-Glucosidase Gene; and the picK Hydroxylase Gene
  • the invention provides methods for the heterologous expression of one or more of the genes involved in picromycin biosynthesis and recombinant DNA expression vectors useful in the method.
  • recombinant expression systems included within the scope of the invention in addition to isolated nucleic acids encoding domains, modules, or proteins of the narbonolide PKS, glycosylation, and/or hydroxylation enzymes, are recombinant expression systems. These systems contain the coding sequences operably linked to promoters, enhancers, and/or termination sequences that operate to effect expression of the coding sequence in compatible host cells.
  • the host cells are modified by transformation with the recombinant DNA expression vectors of the invention to contain these sequences either as extrachromosomal elements or integrated into the chromosome.
  • the invention also provides methods to produce PKS and post-PKS tailoring enzymes as well as polyketides and antibiotics using these modified host cells.
  • the term expression vector refers to a nucleic acid that can be introduced into a host cell or cell-free transcription and translation medium.
  • An expression vector can be maintained stably or transiently in a cell, whether as part of the chromosomal or other DNA in the cell or in any cellular compartment, such as a replicating vector in the cytoplasm.
  • An expression vector also comprises a gene that serves to produce RNA, which typically is translated into a polypeptide in the cell or cell extract.
  • the expression vector typically comprises one or more promoter elements.
  • expression vectors typically contain additional functional elements, such as, for example, a resistance-conferring gene that acts as a selectable marker.
  • an expression vector can vary widely, depending on the intended use of the vector. In particular, the components depend on the host cell(s) in which the vector will be introduced or in which it is intended to function. Components for expression and maintenance of vectors in E. coli are widely known and commercially available, are components for other commonly used organisms, such as yeast cells and Streptomyces cells.
  • promoter which can be referred to as, or can be included within, a control sequence or control element, which drives expression of the desired gene product in the heterologous host cell.
  • Suitable promoters include those that function in eucaryotic or procaryotic host cells.
  • a control element can include, optionally, operator sequences, and other elements, such as ribosome binding sites, depending on the nature of the host.
  • Regulatory sequences that allow for regulation of expression of the heterologous gene relative to the growth of the host cell may also be included. Examples of such regulatory sequences known to those of skill in the art are those that cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus.
  • Preferred host cells for purposes of selecting vector components include fungal host cells such as yeast and procaryotic, especially E. coli and Streptomyces, host cells, but single cell cultures of, for example, mammalian cells can also be used.
  • fungal host cells such as yeast and procaryotic, especially E. coli and Streptomyces
  • single cell cultures of, for example, mammalian cells can also be used.
  • yeasts, plants, or mammalian cells that ordinarily do not produce polyketides it may be necessary to provide, also typically by recombinant means, suitable holo-ACP synthases to convert the recombinantly produced PKS to functionality. Provision of such enzymes is described, for example, in PCT publication Nos. WO 97/13845 and WO 98/27203, each of which is incorporated herein by reference.
  • promoters such as those derived from sugar metabolizing enzymes, such as galactose, lactose (lac), and maltose, can be used. Additional examples include promoters derived from genes encoding biosynthetic enzymes, and the tryptophan (trp), the beta-lactamase (bla), bacteriophage lambda PL, and T5 promoters. In addition, synthetic promoters, such as the tac promoter (U.S. Pat. No. 4,551,433), can also be used.
  • Particularly useful promoters for Streptomyces host cells include those from PKS gene clusters that result in the production of polyketides as secondary metabolites, including promoters from aromatic (Type II) PKS gene clusters.
  • Type II PKS gene cluster promoters are act gene promoters and tcm gene promoters; an example of a Type I PKS gene cluster promoter is the spiramycin PKS gene promoter.
  • a Streptomyces or other host ordinarily produces polyketides, it may be desirable to modify the host so as to prevent the production of endogenous polyketides prior to its use to express a recombinant PKS of the invention.
  • Such hosts have been described, for example, in U.S. Pat. No. 5,672,491, incorporated herein by reference. In such hosts, it may not be necessary to provide enzymatic activities for all of the desired post-translational modifications of the enzymes that make up the recombinantly produced PKS, because the host naturally expresses such enzymes. In particular, these hosts generally contain holo-ACP synthases that provide the pantotheinyl residue needed for functionality of the PKS.
  • the vectors of the invention are used to transform Streptomyces host cells to provide the recombinant Streptomyces host cells of the invention.
  • Streptomyces is a convenient host for expressing narbonolide or 10-deoxymethynolide or derivatives of those compounds, because narbonolide and 10-deoxymethynolide are naturally produced in certain Streptomyces species, and Streptomyces generally produce the precursors needed to form the desired polyketide.
  • the present invention also provides the narbonolide PKS gene promoter in recombinant form, located upstream of the picAI gene on cosmid pKOS023-27.
  • This promoter can be used to drive expression of the narbonolide PKS or any other coding sequence of interest in host cells in which the promoter functions, particularly S. venezuelae and generally any Streptomyces species. As described below, however, promoters other than the promoter of the narbonolide PKS genes will typically be used for heterologous expression.
  • any host cell other than Streptomyces venezuelae is a heterologous host cell.
  • S. narbonensis which produces narbomycin but not picromycin is a heterologous host cell of the invention, although other host cells are generally preferred for purposes of heterologous expression.
  • the recombinant vector need drive expression of only a portion of the genes constituting the picromycin gene cluster.
  • the picromycin gene cluster includes the narbonolide PKS, the desosamine biosynthetic and transferase genes, the beta-glucosidase gene, and the picK hydroxylase gene.
  • a vector may comprise only a single ORF, with the desired remainder of the polypeptides encoded by the picromycin gene cluster provided by the genes on the host cell chromosomal DNA.
  • the present invention also provides compounds and recombinant DNA vectors useful for disrupting any gene in the picromycin gene cluster (as described above and illustrated in the examples below).
  • the invention provides a variety of modified host cells (particularly, S. narbonensis and S. venezuelae ) in which one or more of the genes in the picromycin gene cluster have been disrupted. These cells are especially useful when it is desired to replace the disrupted function with a gene product expressed by a recombinant DNA vector.
  • the invention provides such Streptomyces host cells, which are preferred host cells for expressing narbonolide derivatives of the invention.
  • Particularly preferred host-cells of this type include those in which the coding sequence for the loading module has been disrupted, those in which one or more of any of the PKS gene ORFs has been disrupted, and/or those in which the picK gene has been disrupted.
  • the expression vectors of the invention are used to construct a heterologous recombinant Streptomyces host cell that expresses a recombinant PKS of the invention.
  • a heterologous host cell for purposes of the present invention is any host cell other than S. venezuelae, and in most cases other than S. narbonensis as well.
  • Particularly preferred heterologous host cells are those which lack endogenous functional PKS genes.
  • Illustrative host cells of this type include the modified Streptomyces coelicolor CH999 and similarly modified S. lividans described in PCT publication No. WO 96/40968.
  • the invention provides a wide variety of expression vectors for use in Streptomyces.
  • the origin of replication can be, for example and without limitation, a low copy number vector, such as SCP2* (see Hopwood et al., Genetic Manipulation of Streptomyces: A Laboratory manual (The John Innes Foundation, Norwich, U.K., 1985); Lydiate et al., 1985 , Gene 35: 223-235; and Kieser and Melton, 1988 , Gene 65: 83-91, each of which is incorporated herein by reference), SLP1.2 (Thompson et al., 1982 , Gene 20: 51-62, incorporated herein by reference), and pSG5(ts) (Muth et al., 1989 , Mol.
  • SCP2* see Hopwood et al., Genetic Manipulation of Streptomyces: A Laboratory manual (The John Innes Foundation, Norwich, U.K., 1985); Lydiate et al., 1985 , Gene 35:
  • High copy number vectors are generally, however, not preferred for expression of large genes or multiple genes.
  • E. coli origin of replication such as from pUC, p1P, p1I, and pBR.
  • E. coli origin of replication such as from pUC, p1P, p1I, and pBR.
  • the phage phiC31 and its derivative KC515 can be employed (see Hopwood et al., supra).
  • plasmid pSET152, plasmid pSAM, plasmids pSE101 and pSE211 all of which integrate site-specifically in the chromosomal DNA of S. lividans, can be employed.
  • Preferred Streptomyces host cell/vector combinations of the invention include S. coelicolor CH999 and S. lividans K4-114 host cells, which do not produce actinorhodin, and expression vectors derived from the pRM1 and pRM5 vectors, as described in U.S. Pat. No. 5,830,750 and U.S. patent application Ser. No. 08/828,898, filed Mar. 31, 1997, and Ser. No. 09/181,833, filed Oct. 28, 1998, each of which is incorporated herein by reference.
  • particularly useful control sequences are those that alone or together with suitable regulatory systems activate expression during transition from growth to stationary phase in the vegetative mycelium.
  • Other useful Streptomyces promoters include without limitation those from the ermE gene and the melC1 gene, which act constitutively, and the tipA gene and the merA gene, which can be induced at any growth stage.
  • the T7 RNA polymerase system has been transferred to Streptomyces and can be employed in the vectors and host cells of the invention.
  • the coding sequence for the T7 RNA polymerase is inserted into a neutral site of the chromosome or in a vector under the control of the inducible merA promoter, and the gene of interest is placed under the control of the T7 promoter.
  • one or more activator genes can also be employed to enhance the activity of a promoter.
  • Activator genes in addition to the actII-ORF4 gene described above include dnrI, redD, and ptpA genes (see U.S. patent application Ser. No. 09/181,833, supra).
  • the expression vector will comprise one or more marker genes by which host cells containing the vector can be identified and/or selected.
  • Selectable markers are often preferred for recombinant expression vectors.
  • a variety of markers are known that are useful in selecting for transformed cell lines and generally comprise a gene that confers a selectable phenotype on transformed cells when the cells are grown in an appropriate selective medium.
  • markers include, for example, genes that confer antibiotic resistance or sensitivity to the plasmid.
  • several polyketides are naturally colored, and this characteristic can provide a built-in marker for identifying cells.
  • Preferred selectable markers include antibiotic resistance conferring genes.
  • Streptomyces host cells Preferred for use in Streptomyces host cells are the ermE (confers resistance to erythromycin and lincomycin), tsr (confers resistance to thiostrepton), aadA (confers resistance to spectinomycin and streptomycin), aacC4 (confers resistance to apramycin, kanamycin, gentamicin, geneticin (G418), and neomycin), hyg (confers resistance to hygromycin), and vph (confers resistance to viomycin) resistance conferring genes.
  • ermE confers resistance to erythromycin and lincomycin
  • tsr confers resistance to thiostrepton
  • aadA confers resistance to spectinomycin and streptomycin
  • aacC4 confers resistance to apramycin, kanamycin, gentamicin, geneticin (G418), and neomycin
  • the narbonolide PKS genes were placed on a recombinant expression vector that was transferred to the non-macrolide producing host Streptomyces lividans K4-114, as described in Example 3. Transformation of S. lividans K4-114 with this expression vector resulted in a strain which produced two compounds in similar yield (-5-10 mg/L each). Analysis of extracts by LC/MS followed by 1H-NMR spectroscopy of the purified compounds established their identity as narbonolide (FIG. 5, compound 4) and 10-deoxymethynolide (FIG.
  • High copy number vectors are generally, however, not preferred for expression of large genes or multiple genes.
  • E. coli origin of replication such as from pUC, p1P, p1I, and pBR.
  • E. coli origin of replication such as from pUC, p1P, p1I, and pBR.
  • the phage phiC31 and its derivative KC515 can be employed (see Hopwood et al., supra).
  • plasmid pSET152, plasmid pSAM, plasmids pSE101 and pSE211 all of which integrate site-specifically in the chromosomal DNA of S. lividans, can be employed.
  • Preferred Streptomyces host cell/vector combinations of the invention include S. coelicolor CH999 and S. lividans K4-114 host cells, which do not produce actinorhodin, and expression vectors derived from the pRM1 and pRM5 vectors, as described in U.S. Pat. No. 5,830,750 and U.S. patent application Ser. No. 08/828,898, filed Mar. 31, 1997, and Ser. No. 09/181,833, filed Oct. 28, 1998, each of which is incorporated herein by reference.
  • particularly useful control sequences are those that alone or together with suitable regulatory systems activate expression during transition from growth to stationary phase in the vegetative mycelium.
  • Other useful Streptomyces promoters include without limitation those from the ermE gene and the melC1 gene, which act constitutively, and the tipA gene and the merA gene, which can be induced at any growth stage.
  • the T7 RNA polymerase system has been transferred to Streptomyces and can be employed in the vectors and host cells of the invention. In this system, the coding
  • the picB gene was integrated into the chromosome to provide the host cell of the invention Streptomyces lividans K39-18.
  • the picB gene was cloned into the Streptomyces genome integrating vector pSET152 (see Bierman et al., 1992 , Gene 116: 43, incorporated herein by reference) under control of the same promoter (PactI) as the PKS on plasmid pKOS039-86.
  • the invention provides methods of coexpressing the picB gene product or any other type II thioesterase with the narbonolide PKS or any other PKS in heterologous host cells to increase polyketide production.
  • transformation of a 6dEB-producing Streptomyces lividans /pCK7 strain with an expression vector of the invention that produces PIC TEII resulted in little or no increase in 6-dEB levels, indicating that TEII enzymes may have some specificity for their cognate PKS complexes and that use of homologous TEII enzymes will provide optimal activity.
  • picromycin biosynthetic genes in addition to the genes encoding the PKS and PIC TEII can be introduced into heterologous host cells.
  • the picK gene, desosamine biosynthetic genes, and the desosaminyl transferase gene can be expressed in the recombinant host cells of the invention to produce any and all of the polyketides in the picromycin biosynthetic pathway (or derivatives thereof).
  • the present invention enables one to select whether only the 12-membered polyketides, or only the 14-membered polyketides, or both 12- and 14-membered polyketides will be produced.
  • the invention provides expression vectors in which the last module is deleted or the KS domain of that module is deleted or rendered inactive. If module 6 is deleted, then one preferably deletes only the non-TE domain portion of that module or one inserts a heterologous TE domain, as the TE domain facilitates cleavage of the polyketide from the PKS and cyclization and thus generally increases yields of the desired polyketide.
  • the invention provides expression vectors in which the coding sequences of extender modules 5 and 6 are fused to provide only a single polypeptide.
  • the invention provides methods for desosaminylating polyketides or other compounds.
  • a host cell other than Streptomyces venezuelae is transformed with one or more recombinant vectors of the invention comprising the desosamine biosynthetic and desosaminyl transferase genes and control sequences positioned to express those genes.
  • the host cells so transformed can either produce the polyketide to be desosaminylated naturally or can be transformed with expression vectors encoding the PKS that produces the desired polyketide.
  • the polyketide can be supplied to the host cell containing those genes.
  • the desired desosaminylated polyketide is produced.
  • This method is especially useful in the production of polyketides to be used as antibiotics, because the presence of the desosamine residue is known to increase, relative to their undesosaminylated counterparts, the antibiotic activity of many polyketides significantly.
  • the present invention also provides a method for desosaminylating a polyketide by transforming an S. venezuelae or S.
  • narbonensis host cell with a recombinant vector that encodes a PKS that produces the polyketide and culturing the transformed cell under conditions such that said polyketide is produced and desosaminylated.
  • a recombinant vector that encodes a PKS that produces the polyketide and culturing the transformed cell under conditions such that said polyketide is produced and desosaminylated.
  • use of an S. venezuelae or S. narbonensis host cell of the invention that does not produce a functional endogenous narbonolide PKS is preferred.
  • the invention provides a method for improving the yield of a desired desosaminylated polyketide in a host cell, which method comprises transforming the host cell with a beta-glucosidase gene.
  • This method is not limited to host cells that have been transformed with expression vectors of the invention encoding the desosamine biosynthetic and desosaminyl transferase genes of the invention but instead can be applied to any host cell that desosaminylates polyketides or other compounds.
  • the beta-glucosidase gene from Streptomyces venezuelae provided by the invention is preferred for use in the method, any beta-glucosidase gene may be employed.
  • the beta-glucosidase treatment is conducted in a cell free extract.
  • the invention provides methods not only for producing narbonolide and 10-deoxymethynolide in heterologous host cells but also for producing narbomycin and YC-17 in heterologous host cells.
  • the invention provides methods for expressing the picK gene product in heterologous host cells, thus providing a means to produce picromycin, methymycin, and neomethymycin in heterologous host cells.
  • the recombinant expression vectors provided by the invention enable the artisan to provide for desosamine biosynthesis and transfer and/or C10 or C12 hydroxylation in any host cell, the invention provides methods and reagents for producing a very wide variety of glycosylated and/or hydroxylated polyketides. This variety of polyketides provided by the invention can be better appreciated upon consideration of the following section relating to the production of polyketides from heterologous or hybrid PKS enzymes provided by the invention.
  • the present invention provides recombinant DNA compounds encoding each of the domains of each of the modules of the narbonolide PKS, the proteins involved in desosamine biosynthesis and transfer to narbonolide, and the PicK protein.
  • the availability of these compounds permits their use in recombinant procedures for production of desired portions of the narbonolide PKS fused to or expressed in conjunction with all or a portion of a heterologous PKS.
  • the resulting hybrid PKS can then be expressed in a host cell, optionally with the desosamine biosynthesis and transfer genes and/or the picK hydroxylase gene to produce a desired polyketide.
  • a portion of the narbonolide PKS coding sequence that encodes a particular activity can be isolated and manipulated, for example, to replace the corresponding region in a different modular PKS.
  • coding sequences for individual modules of the PKS can be ligated into suitable expression systems and used to produce the portion of the protein encoded.
  • the resulting protein can be isolated and purified or can may be employed in situ to effect polyketide synthesis.
  • suitable control sequences such as promoters, termination sequences, enhancers, and the like are ligated to the nucleotide sequence encoding the desired protein in the construction of the expression vector.
  • the invention thus provides a hybrid PKS and the corresponding recombinant DNA compounds that encode those hybrid PKS enzymes.
  • a hybrid PKS is a recombinant PKS that comprises all or part of one or more extender modules, loading module, and/or thioesterase/cyclase domain of a first PKS and all or part of one or more extender modules, loading module, and/or thioesterase/cyclase domain of a second PKS.
  • the first PKS is most but not all of the narbonolide PKS
  • the second PKS is only a portion or all of a non-narbonolide PKS.
  • hybrid PKS includes a narbonolide PKS in which the natural loading module has been replaced with a loading module of another PKS.
  • Another example of such a hybrid PKS is a narbonolide PKS in which the AT domain of extender module 3 is replaced with an AT domain that binds only malonyl CoA.
  • the first PKS is most but not all of a non-narbonolide PKS
  • the second PKS is only a portion or all of the narbonolide PKS.
  • An illustrative example of such a hybrid PKS includes a DEBS PKS in which an AT specific for methylmalonyl CoA is replaced with the AT from the narbonolide PKS specific for malonyl CoA.
  • the DNA compounds of the invention that encode the individual domains, modules, and proteins that comprise the narbonolide PKS.
  • the narbonolide PKS is comprised of a loading module, six extender modules composed of a KS, AT, ACP, and optional KR, DH, and ER domains, and a thioesterase domain.
  • the DNA compounds of the invention that encode these domains individually or in combination are useful in the construction of the hybrid PKS encoding DNA compounds of the invention.
  • a DNA compound comprising a sequence that encodes the narbonolide PKS loading module is inserted into a DNA compound that comprises the coding sequence for a heterologous PKS.
  • the resulting construct, in which the coding sequence for the loading module of the heterologous PKS is replaced by that for the coding sequence of the narbonolide PKS loading module provides a novel PKS.
  • narbonolide PKS examples include the 6-deoxyerythronolide B, rapamycin, FK506, FK520, rifamycin, and avermectin PKS coding sequences.
  • a DNA compound comprising a sequence that encodes the narbonolide PKS loading module is inserted into a DNA compound that comprises the coding sequence for the narbonolide PKS or a recombinant narbonolide PKS that produces a narbonolide derivative in a different location in the modular system.
  • a portion of the loading module coding sequence is utilized in conjunction with a heterologous coding sequence.
  • the invention provides, for example, replacing the propionyl CoA specific AT with an acetyl CoA, butyryl CoA, or other CoA specific AT.
  • the KS Q and/or ACP can be replaced by another inactivated KS and/or another ACP.
  • the KS Q , AT, and ACP of the loading module can be replaced by an AT and ACP of a loading module such as that of DEBS.
  • the resulting heterologous loading module coding sequence can be utilized in conjunction with a coding sequence for a PKS that synthesizes narbonolide, a narbonolide derivative, or another polyketide.
  • a DNA compound comprising a sequence that encodes the narbonolide PKS first extender module is inserted into a DNA compound that comprises the coding sequence for a heterologous PKS.
  • the resulting construct, in which the coding sequence for a module of the heterologous PKS is either replaced by that for the first extender module of the narbonolide PKS or the latter is merely added to coding sequences for modules of the heterologous PKS provides a novel PKS coding sequence.
  • a DNA compound comprising a sequence that encodes the first extender module of the narbonolide PKS is inserted into a DNA compound that comprises coding sequences for the narbonolide PKS or a recombinant narbonolide PKS that produces a narbonolide derivative or into a different location in the modular system.
  • a portion or all of the first extender module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module.
  • the invention provides, for example, replacing the methylmalonyl CoA specific AT with a malonyl CoA, ethylmalonyl CoA, or carboxyglycolyl CoA specific AT; deleting (which includes inactivating) the KR; inserting a DH or a DH and ER; and/or replacing the KR with another KR, a DH and KR, a DH, KR, and ER.
  • the KS and/or ACP can be replaced with another KS and/or ACP.
  • the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the narbonolide PKS, from a gene for a PKS that produces a polyketide other than narbonolide, or from chemical synthesis.
  • the resulting heterologous first extender module coding sequence can be utilized in conjunction with a coding sequence for a PKS that synthesizes narbonolide, a narbonolide derivative, or another polyketide.
  • the invention provides recombinant PKSs and recombinant DNA compounds and vectors that encode such PKSs in which the KS domain of the first extender module has been inactivated.
  • Such constructs are especially useful when placed in translational reading frame with the remaining modules and domains of a narbonolide PKS or narbonolide derivative PKS.
  • the utility of these constructs is that host cells expressing, or cell free extracts containing, the PKS encoded thereby can be fed or supplied with N-acetylcysteamine thioesters of novel precursor molecules to prepare narbonolide derivatives. See U.S. patent application Serial No. 60/117,384, filed Jan. 27, 1999, and PCT publication Nos. WO 99/03986 and WO 97/02358, each of which is incorporated herein by reference.
  • telomere sequences for the modules of the heterologous PKS are useful for a variety of applications.
  • a DNA compound comprising a sequence that encodes the narbonolide PKS second extender module is inserted into a DNA compound that comprises the coding sequence for a heterologous PKS.
  • the resulting construct, in which the coding sequence for a module of the heterologous PKS is either replaced by that for the second extender module of the narbonolide PKS or the latter is merely added to coding sequences for the modules of the heterologous PKS provides a novel PKS.
  • a DNA compound comprising a sequence that encodes the second extender module of the narbonolide PKS is inserted into a DNA compound that comprises the coding sequences for the narbonolide PKS or a recombinant narbonolide PKS that produces a narbonolide derivative.
  • a portion or all of the second extender module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module.
  • the invention provides, for example, replacing the malonyl CoA specific AT with a methylmalonyl CoA, ethylmalonyl CoA, or carboxyglycolyl CoA specific AT; deleting (or inactivating) the KR, the DH, or both the DH and KR; replacing the KR or the KR and DH with a KR, a KR and a DH, or a KR, DH, and ER; and/or inserting an ER.
  • the KS and/or ACP can be replaced with another KS and/or ACP.
  • the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for-another module of the narbonolide PKS, from a coding sequence for a PKS that produces a polyketide other than narbonolide, or from chemical synthesis.
  • the resulting heterologous second extender module coding sequence can be utilized in conjunction with a coding sequence from a PKS that synthesizes narbonolide, a narbonolide derivative, or another polyketide.
  • telomere sequences for the modules of the heterologous PKS are useful for a variety of applications.
  • a DNA compound comprising a sequence that encodes the narbonolide PKS third extender module is inserted into a DNA compound that comprises the coding sequence for a heterologous PKS.
  • the resulting construct, in which the coding sequence for a module of the heterologous PKS is either replaced by that for the third extender module of the narbonolide PKS or the latter is merely added to coding sequences for the modules of the heterologous PKS provides a novel PKS.
  • a DNA compound comprising a sequence that encodes the third extender module of the narbonolide PKS is inserted into a DNA compound that comprises coding sequences for the narbonolide PKS or a recombinant narbonolide PKS that produces a narbonolide derivative.
  • a portion or all of the third extender module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module.
  • the invention provides, for example, replacing the methylmalonyl CoA specific AT with a malonyl CoA, ethylmalonyl CoA, or carboxyglycolyl CoA specific AT; deleting the inactive KR; and/or inserting a KR, or a KR and DH, or a KR, DH, and ER.
  • the KS and/or ACP can be replaced with another KS and/or ACP.
  • the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the narbonolide PKS, from a gene for a PKS that produces a polyketide other than narbonolide, or from chemical synthesis.
  • the resulting heterologous third extender module coding sequence can be utilized in conjunction with a coding sequence for a PKS that synthesizes narbonolide, a narbonolide derivative, or another polyketide.
  • a DNA compound comprising a sequence that encodes the narbonolide PKS fourth extender module is inserted into a DNA compound that comprises the coding sequence for a heterologous PKS.
  • the resulting construct, in which the coding sequence for a module of the heterologous PKS is either replaced by that for the fourth extender module of the narbonolide PKS or the latter is merely added to coding sequences for the modules of the heterologous PKS provides a novel PKS.
  • a DNA compound comprising a sequence that encodes the fourth extender module of the narbonolide PKS is inserted into a DNA compound that comprises coding sequences for the narbonolide PKS or a recombinant narbonolide PKS that produces a narbonolide derivative.
  • a portion of the fourth extender module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module.
  • the invention provides, for example, replacing the methylmalonyl CoA specific AT with a malonyl CoA, ethylmalonyl CoA, or carboxyglycolyl CoA specific AT; deleting any one, two, or all three of the ER, DH, and KR; and/or replacing any one two, or all three of the ER, DH, and KR with either a KR, a DH and KR, or a KR, DH, and ER.
  • the KS and/or ACP can be replaced with another KS and/or ACP.
  • the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the narbonolide PKS, from a coding sequence for a PKS that produces a polyketide other than narbonolide, or from chemical synthesis.
  • the resulting heterologous fourth extender module coding sequence can be utilized in conjunction with a coding sequence for a PKS that synthesizes narbonolide, a narbonolide derivative, or another polyketide.
  • a DNA compound comprising a sequence that encodes the narbonolide PKS fifth extender module is inserted into a DNA compound that comprises the coding sequence for a heterologous PKS.
  • the resulting construct, in which the coding sequence for a module of the heterologous PKS is either replaced by that for the fifth extender module of the narbonolide PKS or the latter is merely added to coding sequences for the modules of the heterologous PKS provides a novel PKS.
  • a DNA compound comprising a sequence that encodes the fifth extender module of the narbonolide PKS is inserted into a DNA compound that comprises the coding sequence for the narbonolide PKS or a recombinant narbonolide PKS that produces a narbonolide derivative.
  • a portion or all of the fifth extender module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module.
  • the invention provides, for example, replacing the methylmalonyl CoA specific AT with a malonyl CoA, ethylmalonyl CoA, or carboxyglycolyl CoA specific AT; deleting (or inactivating) the KR, inserting a DH or a DH and ER; and/or replacing the KR with another KR, a DH and KR, or a DH, KR, and ER.
  • the KS and/or ACP can be replaced with another KS and/or ACP.
  • the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the narbonolide PKS, from a coding sequence for a PKS that produces a polyketide other than narbonolide, or from chemical synthesis.
  • the resulting heterologous fifth extender module coding sequence can be utilized in conjunction with a coding sequence for a PKS that synthesizes narbonolide, a narbonolide derivative, or another polyketide.
  • telomere sequences for the modules of the heterologous PKS are useful for a variety of applications.
  • a DNA compound comprising a sequence that encodes the narbonolide PKS sixth extender module is inserted into a DNA compound that comprises the coding sequence for a heterologous PKS.
  • the resulting construct, in which the coding sequence for a module of the heterologous PKS is either replaced by that for the sixth extender module of the narbonolide PKS or the latter is merely added to coding sequences for the modules of the heterologous PKS provides a novel PKS.
  • a DNA compound comprising a sequence that encodes the sixth extender module of the narbonolide PKS is inserted into a DNA compound that comprises the coding sequences for the narbonolide PKS or a recombinant narbonolide PKS that produces a narbonolide derivative.
  • a portion or all of the sixth extender module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module.
  • the invention provides, for example, replacing the methylmalonyl CoA specific AT with a malonyl CoA, ethylmalonyl CoA, or carboxyglycolyl CoA specific AT; and/or inserting a KR, a KR and DH, or a KR, DH, and an ER.
  • the KS and/or ACP can be replaced with another KS and/or ACP.
  • the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the narbonolide PKS, from a coding sequence for a PKS that produces a polyketide other than narbonolide, or from chemical synthesis.
  • the resulting heterologous sixth extender module coding sequence can be utilized in conjunction with a coding sequence for a PKS that synthesizes narbonolide, a narbonolide derivative, or another polyketide.
  • the sixth extender module of the narbonolide PKS is followed by a thioesterase domain. This domain is important in the cyclization of the polyketide and its cleavage from the PKS.
  • the present invention provides recombinant DNA compounds that encode hybrid PKS enzymes in which the narbonolide PKS is fused to a heterologous thioesterase or a heterologous PKS is fused to the narbonolide synthase thioesterase.
  • a thioesterase domain coding sequence from another PKS gene can be inserted at the end of the sixth extender module coding sequence in recombinant DNA compounds of the invention.
  • Recombinant DNA compounds encoding this thioesterase domain are therefore useful in constructing DNA compounds that encode the narbonolide PKS, a PKS that produces a narbonolide derivative, and a PKS that produces a polyketide other than narbonolide or a narbonolide derivative.
  • hybrid PKSs of the invention certain general methods may be helpful. For example, it is often beneficial to retain the framework of the module to be altered to make the hybrid PKS. Thus, if one desires to add DH and ER functionalities to a module, it is often preferred to replace the KR domain of the original module with a KR, DH, and ER domain-containing segment from another module, instead of merely inserting DH and ER domains.
  • the stereochemistry of the resulting polyketide is a function of three aspects of the synthase.
  • the first aspect is related to the AT/KS specificity associated with substituted malonyls as extender units, which affects stereochemistry only when the reductive cycle is missing or when it contains only a ketoreductase, as the dehydratase would abolish chirality.
  • the specificity of the ketoreductase may determine the chirality of any beta-OH.
  • the enoylreductase specificity for substituted malonyls as extender units may influence the result when there is a complete KR/DH/ER available.
  • the modular PKS systems permit a wide range of polyketides to be synthesized.
  • a wider range of starter units including aliphatic monomers (acetyl, propionyl, butyryl, isovaleryl, etc.), aromatics (aminohydroxybenzoyl), alicyclics (cyclohexanoyl), and heterocyclics (thiazolyl) are found in various macrocyclic polyketides.
  • aliphatic monomers acetyl, propionyl, butyryl, isovaleryl, etc.
  • aromatics aminohydroxybenzoyl
  • alicyclics cyclohexanoyl
  • heterocyclics thiazolyl
  • Modular PKSs also exhibit considerable variety with regard to the choice of extender units in each condensation cycle.
  • the degree of beta-ketoreduction following a condensation reaction has also been shown to be altered by genetic manipulation (Donadio et al., 1991 , Science, supra; Donadio et al., 1993 , Proc. Natl. Acad. Sci. USA 90: 7119-7123).
  • the size of the polyketide product can be varied by designing mutants with the appropriate number of modules (Kao et al., 1994 , J. Am. Chem. Soc. 116:11612-11613).
  • these enzymes are particularly well known for generating an impressive range of asymmetric centers in their products in a highly controlled manner.
  • the polyketides and antibiotics produced by the methods of the invention are typically single stereoisomeric forms. Although the compounds of the invention can occur as mixtures of stereoisomers, it may be beneficial in some instances to generate individual stereoisomers. Thus, the combinatorial potential within modular PKS pathways based on any naturally occurring modular, such as the narbonolide, PKS scaffold is virtually unlimited.
  • the combinatorial potential is increased even further when one considers that mutations in DNA encoding a polypeptide can be used to introduce, alter, or delete an activity in the encoded polypeptide.
  • Mutations can be made to the native sequences using conventional techniques.
  • the substrates for mutation can be an entire cluster of genes or only one or two of them; the substrate for mutation may also be portions of one or more of these genes.
  • Techniques for mutation include preparing synthetic oligonucleotides including the mutations and inserting the mutated sequence into the gene encoding a PKS subunit using restriction endonuclease digestion. See, e.g., Kunkel, 1985 , Proc. Natl. Acad. Sci.
  • the mutations can be effected using a mismatched primer (generally 10-20 nucleotides in length) that hybridizes to the native nucleotide sequence, at a temperature below the melting temperature of the mismatched duplex.
  • the primer can be made specific by keeping primer length and base composition within relatively narrow limits and by keeping the mutant base centrally located. See Zoller and Smith, 1983 , Methods Enzymol. 100:468.
  • Primer extension is effected using DNA polymerase, the product cloned, and clones containing the mutated DNA, derived by segregation of the primer extended strand, selected.
  • Identification can be accomplished using the mutant primer as a hybridization probe.
  • the technique is also applicable for generating multiple point mutations. See, e.g., Dalbie-McFarland et al., 1982 , Proc. Natl. Acad. Sci. USA 79: 6409. PCR mutagenesis can also be used to effect the desired mutations.
  • Random mutagenesis of selected portions of the nucleotide sequences encoding enzymatic activities can also be accomplished by several different techniques known in the art, e.g., by inserting an oligonucleotide linker randomly into a plasmid, by irradiation with X-rays or ultraviolet light, by incorporating incorrect nucleotides during in vitro DNA synthesis, by error-prone PCR mutagenesis, by preparing synthetic mutants, or by damaging plasmid DNA in vitro with chemicals.
  • Chemical mutagens include, for example, sodium bisulfite, nitrous acid, nitrosoguanidine, hydroxylamine, agents which damage or remove bases thereby preventing normal base-pairing such as hydrazine or formic acid, analogues of nucleotide precursors such as 5-bromouracil, 2-aminopurine, or acridine intercalating agents such as proflavine, acriflavine, quinacrine, and the like.
  • plasmid DNA or DNA fragments are treated with chemicals, transformed into E. coli and propagated as a pool or library of mutant plasmids.
  • regions encoding enzymatic activity i.e., regions encoding corresponding activities from different PKS synthases or from different locations in the same PKS, can be recovered, for example, using PCR techniques with appropriate primers.
  • corresponding activity encoding regions is meant those regions encoding the same general type of activity.
  • a KR activity encoded at one location of a gene cluster “corresponds” to a KR encoding activity in another location in the gene cluster or in a different gene cluster.
  • a complete reductase cycle could be considered corresponding.
  • KR/DH/ER corresponds to KR alone.
  • replacement of a particular target region in a host PKS is to be made, this replacement can be conducted in vitro using suitable restriction enzymes.
  • the replacement can also be effected in vivo using recombinant techniques involving homologous sequences framing the replacement gene in a donor plasmid and a receptor region in a recipient plasmid.
  • Such systems advantageously involving plasmids of differing temperature sensitivities are described, for example, in PCT publication No. WO 96/40968, incorporated herein by reference.
  • the vectors used to perform the various operations to replace the enzymatic activity in the host PKS genes or to support mutations in these regions of the host PKS genes can be chosen to contain control sequences operably linked to the resulting coding sequences in a manner such that expression of the coding sequences can be effected in an appropriate host.
  • nucleotide sequences are inserted into appropriate expression vectors. This need not be done individually, but a pool of isolated encoding nucleotide sequences can be inserted into expression vectors, the resulting vectors transformed or transfected into host cells, and the resulting cells plated out into individual colonies.
  • the various PKS nucleotide sequences can be cloned into one or more recombinant vectors as individual cassettes, with separate control elements, or under the control of, e.g., a single promoter.
  • the PKS subunit encoding regions can include flanking restriction sites to allow for the easy deletion and insertion of other PKS subunit encoding sequences so that hybrid PKSs can be generated.
  • the design of such unique restriction sites is known to those of skill in the art and can be accomplished using the techniques described above, such as site-directed mutagenesis and PCR.
  • the expression vectors containing nucleotide sequences encoding a variety of PKS enzymes for the production of different polyketides are then transformed into the appropriate host cells to construct the library.
  • a mixture of such vectors is transformed into the selected host cells and the resulting cells plated into individual colonies and selected to identify successful transformants.
  • Each individual colony has the ability to produce a particular PKS synthase and ultimately a particular polyketide.
  • the expression vectors can be used individually to transform hosts, which transformed hosts are then assembled into a library.
  • a variety of strategies are available to obtain a multiplicity of colonies each containing a PKS gene cluster derived from the naturally occurring host gene cluster so that each colony in the library produces a different PKS and ultimately a different polyketide.
  • the number of different polyketides that are produced by the library is typically at least four, more typically at least ten, and preferably at least 20, and more preferably at least 50, reflecting similar numbers of different altered PKS gene clusters and PKS gene products.
  • the number of members in the library is arbitrarily chosen; however, the degrees of freedom outlined above with respect to the variation of starter, extender units, stereochemistry, oxidation state, and chain length is quite large.
  • Methods for introducing the recombinant vectors of the invention into suitable hosts are known to those of skill, in the art and typically include the use of CaC12 or agents such as other divalent cations, lipofection, DMSO, protoplast transformation, infection, transfection, and electroporation.
  • the polyketide producing colonies can be identified and isolated using known techniques and the produced polyketides further characterized. The polyketides produced by these colonies can be used collectively in a panel to represent a library or may be assessed individually for activity.
  • the libraries of the invention can thus be considered at four levels: (1) a multiplicity of colonies each with a different PKS encoding sequence; (2) colonies that contain the proteins that are members of the PKS library produced by the coding sequences; (3) the polyketides produced; and (4) antibiotics or compounds with other desired activities derived from the polyketides.
  • combination libraries can also be constructed wherein members of a library derived, for example, from the narbonolide PKS can be considered as a part of the same library as those derived from, for example, the rapamycin PKS or DEBS.
  • Colonies in the library are induced to produce the relevant synthases and thus to produce the relevant polyketides to obtain a library of polyketides.
  • the polyketides secreted into the media can be screened for binding to desired targets, such as receptors, signaling proteins, and the like.
  • the supernatants per se can be used for screening, or partial or complete purification of the polyketides can first be effected.
  • screening methods involve detecting the binding of each member of the library to receptor or other target ligand. Binding can be detected either directly or through a competition assay. Means to screen such libraries for binding are well known in the art.
  • individual polyketide members of the library can be tested against a desired target.
  • screens wherein the biological response of the target is measured can more readily be included.
  • Antibiotic activity can be verified using typical screening assays such as those set forth in Lehrer et al., 1991 , J. Immunol. Meth. 137:167-173, incorporated herein by reference, and in the examples below.
  • the invention provides methods for the preparation of a large number of polyketides. These polyketides are useful intermediates in formation of compounds with antibiotic or other activity through hydroxylation and glycosylation reactions as described above. In general, the polyketide products of the PKS must be further modified, typically by hydroxylation and glycosylation, to exhibit antibiotic activity. Hydroxylation results in the novel polyketides of the invention that contain hydroxyl groups at C6, which can be accomplished using the hydroxylase encoded by the eryF gene, and/or C12, which can be accomplished using the hydroxylase encoded by the picK or eryK gene. The presence of hydroxyl-groups at these positions can enhance the antibiotic activity of the resulting compound relative to its unhydroxylated counterpart.
  • Gycosylation is important in conferring antibiotic activity to a polyketide as well.
  • Methods for glycosylating the polyketides are generally known in the art; the glycosylation may be effected intracellularly by providing the appropriate glycosylation enzymes or may be effected in vitro using chemical synthetic means as described herein and in PCT publication No. WO 98/49315, incorporated herein by reference.
  • glycosylation with desosamine is effected in accordance with the methods of the invention in recombinant host cells provided by the invention.
  • the approaches to effecting glycosylation mirror those described above with respect to hydroxylation.
  • the purified enzymes, isolated from native sources or recombinantly produced may be used in vitro.
  • glycosylation may be effected intracellularly using endogenous or recombinantly produced intracellular glycosylases.
  • synthetic chemical methods may be employed.
  • the antibiotic modular polyketides may contain any of a number of different sugars, although D-desosamine, or a close analog thereof, is most common.
  • Erythromycin, picromycin, narbomycin and methymycin contain desosamine.
  • Erythromycin also contains L-cladinose (3-O-methyl mycarose).
  • Tylosin contains mycaminose (4-hydroxy desosamine), mycarose and 6-deoxy-D-allose.
  • 2-acetyl-1-bromodesosamine has been used as a donor to glycosylate polyketides by Masamune et al., 1975 , J. Am. Chem. Soc. 97: 3512-3513.
  • Glycosylation can also be effected using the polyketide aglycones as starting materials and using Saccharopolyspora erythraea or Streptomyces venezuelae to make the conversion, preferably using mutants unable to synthesize macrolides.
  • narbonolide PKS a portion of the narbonolide PKS gene was fused to the DEBS genes.
  • This construct also allowed the examination of whether the TE domain of the narbonolide PKS (pikTE) could promote formation of 12-membered lactones in the context of a different PKS.
  • a construct was generated, plasmid pKOS039-18, in which the pikTE ORF was fused with the DEBS genes in place of the DEBS TE ORF (see FIG. 5).
  • the fusion junction was chosen between the AT and ACP to eliminate ketoreductase activity in DEBS extender module 6 (KR 6 ). This results in a hybrid PKS that presents the TE with a ⁇ -ketone heptaketide intermediate and a ⁇ -(S)-hydroxy hexaketide intermediate to cyclize, as in narbonolide and 10-deoxymethynolide biosynthesis.
  • the 12-membered intermediate can be formed by other recombinant PKS enzymes, see Kao et al., 1995, supra, the PIC TE domain appears incapable of forcing premature cyclization of the hexaketide intermediate generated by DEBS. This result, along with others reported herein, suggests that protein interactions between the narbonolide PKS modules play a role in formation of the 12 and 14-membered macrolides.
  • hybrid PKSs of the invention were constructed that yield this same compound. These constructs also illustrate the method of the invention in which hybrid PKSs are constructed at the protein, as opposed to the module, level.
  • the invention provides a method for constructing a hybrid PKS which comprises the coexpression of at least one gene from a first modular PKS gene cluster in a host cell that also expresses at least one gene from a second PKS gene cluster.
  • the invention also provides novel hybrid PKS enzymes prepared in accordance with the method. This method is not limited to hybrid PKS enzymes composed of at least one narbonolide PKS gene, although such constructs are illustrative and preferred.
  • the hybrid PKS enzymes are not limited to hybrids composed of unmodified proteins; as illustrated below, at least one of the genes can optionally be a hybrid PKS gene.
  • the eryAI and eryAII genes were coexpressed with picAIV and a gene encoding a hybrid extender module 5 composed of the KS and AT domains of extender module 5 of DEBS3 and the KR and ACP domains of extender module ⁇ 5 of the narbonolide PKS.
  • the picAIV coding sequence was fused to the hybrid extender module 5 coding sequence used in the first construct to yield a single protein.
  • Each of these constructs produced 3-deoxy-3-oxo-6-deoxyerythronolide B.
  • the coding sequence for extender module 5 of DEBS3 was fused to the picAIV coding sequence, but the levels of product produced were below the detection limits of the assay.
  • a variant of the first construct hybrid PKS was constructed that contained an inactivated DEBS1 extender module 1 KS domain.
  • host cells containing the resultant hybrid PKS were supplied the appropriate diketide precursor, the desired 13-desethyl-13-propyl compounds were obtained, as described in the examples below.
  • hybrid PKSs of the invention were made by coexpressing the picAI and picAII genes with genes encoding DEBS3 or DEBS3 variants. These constructs illustrate the method of the invention in which a hybrid PKS is produced from coexpression of PKS genes unmodified at the modular or domain level.
  • the eryAIII gene was coexpressed with the picAI and picAII genes, and the hybrid PKS produced 10-desmethyl-10,11-anhydro-6-deoxyerythronolide B in Streptomyces lividans.
  • Such a hybrid PKS could also be constructed in accordance with the method of the invention by transformation of S. venezuelae with an expression vector that produces the eryAIII gene product, DEBS3.
  • the S. venezuelae host cell has been modified to inactivate the picAIII gene.
  • the DEBS3 gene was a variant that had an inactive KR in extender module 5.
  • the hybrid PKS produced 5,6-dideoxy-5-oxo-10-desmethyl-10,11-anhydroerythronolide B in Streptomyces lividans.
  • the DEBS3 gene was a variant in which the KR domain of extender module 5 was replaced by the DH and KR domains of extender module 4 of the rapamycin PKS.
  • This construct produced 5,6-dideoxy-5-oxo-10-desmethyl-10,11-anhydroerythronolide B and 5,6-dideoxy-4,5-anhydro-10-desmethyl-10,11-anhydroerythronolide B in Streptomyces lividans, indicating that the rapamycin DH and KR domains functioned only inefficiently in this construct.
  • the DEBS3 gene was a variant in which the KR domain of extender module 5 was replaced by the DH, KR, and ER domains of extender module 1 of the rapamycin PKS.
  • This construct produced 5,6-dideoxy-5-oxo-10-desmethyl-10,11-anhydroerythronolide B as well as 5,6-dideoxy-10-desmethyl-10,11-anhydroerythronolide B in Streptomyces lividans, indicating that the rapamycin DH, KR, and ER domains functioned only inefficiently in this construct.
  • the DEBS3 gene was a variant in which the KR domain of extender module 6 was replaced by the DH and KR domains of extender module 4 of the rapamycin PKS.
  • This construct produced 3,6-dideoxy-2,3-anhydro-10-desmethyl-10,11-anhydroerythronolide B in Streptomyces lividans.
  • the DEBS3 gene was a variant in which the AT domain of extender module 6 was replaced by the AT domain of extender module 2 of the rapamycin PKS.
  • This construct produced 2,10-didesmethyl-10,11-anhydro-6-deoxyerythronolide B in Streptomyces lividans.
  • the methods and recombinant DNA compounds of the invention are useful in the production of polyketides.
  • the invention provides methods for making ketolides, polyketide compounds with significant antibiotic activity. See Griesgraber et al., 1996 , J. Antibiot. 49: 465-477, incorporated herein by reference. Most if not all of the ketolides prepared to date are synthesized using erythromycin A, a derivative of 6-dEB, as an intermediate. While the invention provides hybrid PKSs that produce a polyketide different in structure from 6-dEB, the invention also provides methods for making intermediates useful in preparing traditional, 6-dEB-derived ketolide compounds.
  • 6-dEB in part differs from narbonolide in that it comprises a 10-methyl group
  • the novel hybrid PKS genes of the invention based on the narbonolide PKS provide many novel ketolides that differ from the known ketolides only in that they lack a 10-methyl group.
  • the invention provides the 10-desmethyl analogues of the ketolides and intermediates and precursor compounds described in, for example, Griesgraber et al., supra; Agouridas et al., 1998 , J. Med. Chem. 41: 4080-4100, U.S. Pat. Nos.
  • a hybrid PKS of the invention that produces 10-methyl narbonolide is constructed by substituting the malonyl-specific AT domain of the narbonolide PKS extender module 2 with a methylmalonyl specific AT domain from a heterologous PKS.
  • a hybrid narbonolide PKS in which the AT of extender module 2 was replaced with the AT from DEBS extender module 2 was constructed using boundaries described in PCT publication No. WO 98/49315, incorporated herein by reference.
  • the hybrid PKS expression vector was introduced into Streptomyces venezuelae, detectable quantities of 10-methyl picromycin were not produced.
  • an AT domain from a module other than DEBS extender module 2 is preferred.
  • DEBS extender module 2 or another methylmalonyl specific AT but utilize instead different boundaries than those used for the substitution described above.
  • extension of extender module 2 of the narbonolide PKS is required, the extent of hybrid modules engineered need not be limited to module 2 to make 10-methyl narbonolide.
  • substitution of the KS domain of extender module 3 of the narbonolide PKS with a heterologous domain or module can result in more efficient processing of the intermediate generated by the hybrid extender module 2.
  • a heterologous TE domain may be more efficient in cyclizing 10-methyl narbonolide.
  • Substitution of the entire extender module 2 of the narbonolide PKS with a module encoding the correct enzymatic activities, i.e., a KS, a methylmalonyl specific AT, a KR, a DH, and an ACP, can also be used to create a hybrid PKS of the invention that produces a 10-methyl ketolide.
  • Modules useful for such whole module replacements include extender modules 4 and 10 from the rapamycin PKS, extender modules 1 and 5 from the FK506 PKS, extender module 2 of the tylosin PKS, and extender module 4 of the rifamycin PKS.
  • the invention provides many different hybrid PKSs that can be constructed starting from the narbonolide PKS that can be used to produce 10-methyl narbonolide. While 10-methyl narbonolide is referred to in describing these hybrid PKSs, those of skill recognize that the invention also therefore provides the corresponding derivatives produces by glycosylation and hydroxylation. For example, if the hybrid PKS is expressed in Streptomyces narbonensis or S. venezuelae, the compounds produced are 10-methyl narbomycin and picromycin, respectively. Alternatively, the PKS can be expressed in a host cell transformed with the vectors of the invention that encode the desosamine biosynthesis and desosaminyl transferase and picK hydroxylase genes.
  • 6-hydroxy ketolides include 3-deoxy-3-oxo erythronolide B, 6-hydroxy narbonolide, and 6-hydroxy-10-methyl narbonolide.
  • the invention provides a method for utilizing EryF to hydroxylate 3-ketolides that is applicable for the production of any 6-hydroxy-3-ketolide.
  • the hybrid PKS genes of the invention can be expressed in a host cell that contains the desosamine biosynthetic genes and desosaminyl transferase gene as well as the required hydroxylase gene(s), which may be either picK (for the C12 position) or eryK (for the C12 position) and/or eryF (for the C6 position).
  • the resulting compounds have antibiotic activity but can be further modified, as described in the patent publications referenced above, to yield a desired compound with improved or otherwise desired properties.
  • the aglycone compounds can be produced in the recombinant host cell, and the desired glycosylation and hydroxylation steps carried out in vitro or in vivo, in the latter case by supplying the converting cell with the aglycone.
  • the compounds of the invention are thus optionally glycosylated forms of the polyketide set forth in formula (2) below which are hydroxylated at either the C6 or the C12 or both.
  • the compounds of formula (2) can be prepared using the loading and the six extender modules of a modular PKS, modified or prepared in hybrid form as herein described. These polyketides have the formula:
  • R* is a straight chain, branched or cyclic, saturated or unsaturated substituted or unsubstituted hydrocarbyl of 1-15C;
  • each of R 1 -R 6 is independently H or alkyl (1-4C) wherein any alkyl at R 1 may optionally be substituted;
  • each of X 1 -X 5 is independently two H, H and OH, or ⁇ O; or
  • each of X 1 -X 5 is independently H and the compound of formula (2) contains a double-bond in the ring adjacent to the position of said X at 2-3, 4-5, 6-7, 8-9 and/or 10-11;
  • At least two of R 1 -R 6 are alkyl (1-4C).
  • Preferred compounds comprising formula 2 are those wherein at least three of R 1 -R 5 are alkyl (1-4C), preferably methyl or ethyl; more preferably wherein at least four of R 1 -R 5 are alkyl (1-4C), preferably methyl or ethyl. Also preferred are those wherein X 2 is two H, ⁇ O, or H and OH, and/or X 3 is H, and/or X 1 is OH and/or X 4 is OH and/or X 5 is OH. Also preferred are compounds with variable R* when R 1 -R 5 is methyl, X 2 is ⁇ O, and X 1 , X 4 and X 5 are OH. The glycosylated forms of the foregoing are also preferred.
  • the invention also provides the 12-membered macrolides corresponding to the compounds above but produced from a narbonolide-derived PKS lacking extender modules 5 and 6 of the narbonolide PKS.
  • R* is a straight chain, branched or cyclic, saturated or unsaturated substituted or unsubstituted hydrocarbyl of 1-15C;
  • each of R 1 -R 6 is independently H or alkyl (1-4C) wherein any alkyl at R 1 may optionally be substituted;
  • each of X 1 -X 5 is independently two H, H and OH, or ⁇ O; or
  • each of X 1 -X 5 is independently H and the compound of formula (2) contains a double-bond in the ring adjacent to the position of said X at 2-3, 4-5, 6-7, 8-9 and/or 10-11;
  • At least two of R 1 -R 6 are alkyl (1-4C).
  • Preferred compounds comprising formula 2 are those wherein at least three of R 1 -R 5 are alkyl (1-4C), preferably methyl or ethyl; more preferably wherein at least four of R 1 -R 5 are alkyl (1-4C), preferably methyl or ethyl. Also preferred are those wherein X 2 is two H, ⁇ O, or H and OH, and/or X 3 is H, and/or X 1 is OH and/or X 4 is OH and/or X 5 is OH. Also preferred are compounds with variable R* when R 1 -R 5 is methyl, X 2 is ⁇ O, and X 1 , X 4 and X 5 are OH. The glycosylated forms of the foregoing are also preferred.
  • the invention also provides the 12-membered macrolides corresponding to the compounds above but produced from a narbonolide-derived PKS lacking extender modules 5 and 6 of the narbonolide PKS.
  • the compounds of the invention can be produced by growing and fermenting the host cells of the invention under conditions known in the art for the production of other polyketides.
  • the compounds of the invention can be isolated from the fermentation broths of these cultured cells and purified by standard procedures.
  • the compounds can be readily formulated to provide the pharmaceutical compositions of the invention.
  • the pharmaceutical compositions of the invention can be used in the form of a pharmaceutical preparation, for example, in solid, semisolid, or liquid form. This preparation will contain one or more of the compounds of the invention as an active ingredient in admixture with an organic or inorganic carrier or excipient suitable for external, enteral, or parenteral application.
  • the active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, and any other form suitable for use.
  • the carriers which can be used include water, glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea, and other carriers suitable for use in manufacturing preparations, in solid, semi-solid, or liquefied form.
  • auxiliary stabilizing, thickening, and coloring agents and perfumes may be used.
  • the compounds of the invention may be utilized with hydroxypropyl methylcellulose essentially as described in U.S. Pat. No. 4,916,138, incorporated herein by reference, or with a surfactant essentially as described in EPO patent publication No. 428,169, incorporated herein by reference.
  • Oral dosage forms may be prepared essentially as described by Hondo et al., 1987 , Transplantation Proceedings XIX, Supp. 6: 17-22, incorporated herein by reference.
  • Dosage forms for external application may be prepared essentially as described in EPO patent publication No. 423,714, incorporated herein by reference.
  • the active compound is included in the pharmaceutical composition in an amount sufficient to produce the desired effect upon the disease process or condition.
  • a compound of the invention may be administered orally, topically, parenterally, by inhalation spray, or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvant, and vehicles.
  • parenteral includes subcutaneous injections, and intravenous, intramuscular, and intrastemal injection or infusion techniques.
  • Dosage levels of the compounds of the invention are of the order from about 0.01 mg to about 50 mg per kilogram of body weight per day, preferably from about 0.1 mg to about 10 mg per kilogram of body weight per day.
  • the dosage levels are useful in the treatment of the above-indicated conditions (from about 0.7 mg to about 3.5 mg per patient per day, assuming a 70 kg patient).
  • the compounds of the invention may be administered on an intermittent basis, i.e., at semi-weekly, weekly, semi-monthly, or monthly intervals.
  • the amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration.
  • a formulation intended for oral administration to humans may contain from 0.5 mg to 5 gm of active agent compounded with an appropriate and convenient amount of carrier material, which may vary from about 5 percent to about 95 percent of the total composition.
  • Dosage unit forms will generally contain from about 0.5 mg to about 500 mg of active ingredient.
  • the compounds of the invention may be formulated within the range of, for example, 0.00001% to 60% by weight, preferably from 0.001% to 10% by weight, and most preferably from about 0.005% to 0.8% by weight.
  • the specific dose level for any particular patient will depend on a variety of factors. These factors include the activity of the specific compound employed; the age, body weight, general health, sex, and diet of the subject; the time and route of administration and the rate of excretion of the drug; whether a drug combination is employed in the treatment; and the severity of the particular disease or condition for which therapy is sought.
  • E. coli ET12567 (dam dcm hsdS Cmr) (MacNeil, 1988 , J. Bacteriol. 170: 5607, incorporated herein by reference) to generate unmethylated DNA prior to transformation of S. coelicolor.
  • E. coli strains were grown under standard conditions.
  • S. coelicolor strains were grown on R 2 YE agar plates (Hopwood et al., Genetic manipulation of Streptomyces. A laboratory manual. The John Innes Foundation: Norwich, 1985, incorporated herein by reference).
  • plasmid pRM5 Many of the expression vectors of the invention illustrated in the examples are derived from plasmid pRM5, described in WO 95/08548, incorporated herein by reference.
  • This plasmid includes a colEI replicon, an appropriately truncated SCP2* Streptomyces replicon, two act-promoters to allow for bidirectional cloning, the gene encoding the actII-ORF4 activator which induces transcription from act promoters during the transition from growth phase to stationary phase, and appropriate marker genes.
  • Engineered restriction sites in the plasmid facilitate the combinatorial construction of PKS gene clusters starting from cassettes encoding individual domains of naturally occurring PKSs.
  • plasmid pRM5 When plasmid pRM5 is used for expression of a PKS, all relevant biosynthetic genes can be plasmid-borne and therefore amenable to facile manipulation and mutagenesis in E. coli. This plasmid is also suitable for use in Streptomyces host cells. Streptomyces is genetically and physiologically well-characterized and expresses the ancillary activities required for in vivo production of most polyketides. Plasmid pRM5 utilizes the act promoter for PKS gene expression, so polyketides are produced in a secondary metabolite-like manner, thereby alleviating the toxic effects of synthesizing potentially bioactive compounds in vivo.
  • PCR Polymerase chain reaction
  • Pfu polymerase (Stratagene; Taq polymerase from Perkin Elmer Cetus can also be used) under conditions recommended by the enzyme manufacturer.
  • Standard in vitro techniques were used for DNA manipulations (Sambrook et al. Molecular Cloning: A Laboratory Mantial (Current Edition)).
  • E. coli was transformed using standard calcium chloride-based methods; a Bio-Rad E. coli pulsing apparatus and protocols provided by Bio-Rad could also be used.
  • S. coelicolor was transformed by standard procedures (Hopwood et al. Genetic manipulation of Streptomyces. A laboratory manual.
  • transformants were selected using 1 mL of a 1.5 mg/mL thiostrepton overlay, 1 mL of a 2 mg/mL apramycin overlay, or both.
  • Genomic DNA 100 ⁇ g isolated from Streptomyces venezuelae ATCC15439 using standard procedures was partially digested with Sau3AI endonuclease to generate fragments ⁇ 40 kbp in length.
  • SuperCosI (Stratagene) DNA cosmid arms were prepared as directed by the manufacturer.
  • a cosmid library was prepared by ligating 2.5 ⁇ g of the digested genomic DNA with 1.5 ⁇ g of cosmid arms in a 20 ⁇ L reaction.
  • One microliter of the ligation mixture was propagated in E. coli XL 1-Blue MR (Stratagene) using a GigapackIII XL packaging extract kit (Stratagene). The resulting library of ⁇ 3000 colonies was plated on a 10 ⁇ 150 mm agar plate and replicated to a nylon membrane.
  • the library was initially screened by direct colony hybridization with a DNA probe specific for ketosynthase domain coding sequences of PKS genes. Colonies were alkaline lysed, and the DNA was crosslinked to the membrane using UV irradiation. After overnight incubation with the probe at 42° C., the membrane was washed twice at 25° C. in 2 ⁇ SSC buffer +0.1% SDS for 15 minutes, followed by two 15 minute washes with 2 ⁇ SSC buffer at 55° C. Approximately 30 colonies gave positive hybridization signals with the degenerate probe. Several cosmids were selected and divided into two classes based on restriction digestion patterns. A representative cosmid was selected from each class for further analysis.
  • the representative cosmids were designated pKOS023-26 and pKOS023-27. These cosmids were determined by DNA sequencing to comprise the narbonolide PKS genes, the desosamine biosynthesis and transferase genes, the beta-glucosidase gene, and the picK hydroxylase gene.
  • Cosmid pKOS023-26 was assigned accession number ATCC 203141, and cosmid pKOS023-27 was assigned accession number ATCC 203142.
  • narbonolide PKS genes had been cloned and to illustrate how the invention provides methods and reagents for constructing deletion variants of narbonolide PKS genes
  • a narbonolide PKS gene was deleted from the chromosome of Streptomyces venezuelae. This deletion is shown schematically in FIG. 4, parts B and C.
  • the ⁇ 4.5 kb HindIII-SpeI fragment from plasmid pKOS039-07 was ligated with the 2.5 kb HindIII-NheI fragment of integrating vector pSET52, available from the NRRL, which contains an E. coli origin of replication and an apramycin resistance-conferring gene to create plasmid pKOS039-16.
  • This vector was used to transform S. venezuelae, and apramycin-resistant transformants were selected.
  • the selected transformants were grown in TSB liquid medium without antibiotics for three transfers and then plated onto non-selective media to provide single colony isolates.
  • the isolated colonies were tested for sensitivity to apramycin, and the apramycin-sensitive colonies were then tested to determine if they produced picromycin.
  • the tests performed included a bioassay and LC/MS analysis of the fermentation media. Colonies determined not to produce picromycin (or methymycin or neomethymycin) were then analyzed using PCR to detect an amplification product diagnostic of the deletion. A colony designated K39-03 was identified, providing confirmation that the narbonolide PKS genes had been cloned.
  • Transformation of strain K39-03 with plasmid pKOS039-27 comprising an intact picA gene under the control of the ermE* promoter from plasmid pWHM3 was able to restore picromycin production.
  • each cosmid was probed by Southern hybridization using a labeled DNA fragment amplified by PCR from the Saccharopolyspora erythraea C12-hydroxylase gene, eryK.
  • the cosmids were digested with BamHI endonuclease and electrophoresed on a 1% agarose gel, and the resulting fragments were transferred to a nylon membrane.
  • the membrane was incubated with the eryK probe overnight at 42° C., washed twice at 25° C. in 2 ⁇ SSC buffer with 0.1% SDS for 15 minutes, followed by two 15 minute washes with 2 ⁇ SSC buffer at 50° C.
  • Cosmid pKOS023-26 produced an ⁇ 3 kb fragment that hybridized with the probe under these conditions. This fragment was subcloned into the PCRscriptTM (Stratagene) cloning vector to yield plasmid pKOS023-28 and sequenced. The ⁇ 1.2 kb gene designated picK above was thus identified. The picK gene product is homologous to eryK and other known macrolide cytochrome P450 hydroxylases.
  • the narbonolide PKS was transferred to the non-macrolide producing host Streptomyces lividans K4-114 (see Ziermann and Betlach, 1999 , Biotechniques 26, 106-110, and U.S. patent application Ser. No. 09/181,833, filed Oct. 28, 1998, each of which is incorporated herein by reference). This was accomplished by replacing the three DEBS ORFs on a modified version of pCK7 (see Kao et al., 1994 , Science 265, 509-512, and U.S. Pat. No.
  • pCK7′Kan' differs from pCK7 only in that it contains a kanamycin resistance conferring gene inserted at its HindIII restriction enzyme recognition site. Because the plasmid contains two selectable markers, one can select for both markers and so minimize contamination with cells containing rearranged, undesired vectors.
  • Protoplasts were transformed using standard procedures and transformants selected using overlays containing antibiotics.
  • the strains were grown in liquid R5 medium for growth/seed and production cultures at 30° C.
  • a 2 L shake flask culture of S. lividans K4-114/pKOS039-86 was grown for 7 days at 30° C.
  • the mycelia was filtered, and the aqueous layer was extracted with 2 ⁇ 2 L ethyl acetate.
  • the organic layers were combined, dried over MgSO4, filtered, and evaporated to dryness.
  • Polyketides were separated from the crude extract by silica gel chromatography (1:4 to 1:2 ethyl acetate:hexane gradient) to give an ⁇ 10 mg mixture of narbonolide and 10-deoxymethynolide, as indicated by LC/MS and 1H NMR. Purification of these two compounds was achieved by HPLC on a C-18 reverse phase column (20-80% acetonitrile in water over 45 minutes). This procedure yielded ⁇ 5 mg each of narbonolide and 10-deoxymethynolide. Polyketides produced in the host cells were analyzed by bioassay against Bacillus subtilis and by LC/MS analysis.
  • narbonolide in Streptomyces lividans represents the expression of an entire modular polyketide pathway in a heterologous host.
  • the combined yields of compounds 4 and 5 are similar to those obtained with expression of DEBS from pCK7 (see Kao et al., 1994, Science 265: 509-512, incorporated herein by reference).
  • the narbonolide PKS itself possesses an inherent ability to produce both 12 and 14-membered macrolactones without the requirement of additional activities unique to S. venezuelae.
  • the existence of a complementary enzyme present in S. lividans that provides this function is possible, it would be unusual to find-such a specific enzyme in an organism that does not produce any known macrolide.
  • the picB gene was integrated into the chromosome of Streptomyces lividans harboring plasmid pKOS039-86 to yield S. lividans K39-18/pKOS039-86.
  • the picB gene was cloned into the Streptomyces genome integrating vector pSET152 (see Bierman et al., 1992 , Gene 116, 43, incorporated herein by reference) under control of the same promoter (PactI) as the PKS on plasmid pKOS039-86.
  • Plasmid pKOS039-104 comprises the desosamine biosynthetic genes, the beta-glucosidase gene, and the desosaminyl transferase gene.
  • This plasmid was constructed by first inserting a polylinker oligonucleotide, containing a restriction enzyme recognition site for PacI, a Shine-Dalgarno sequence, and restriction enzyme recognition sites for NdeI, BglII, and HindIII, into a pUC 19 derivative, called pKOS24-47, to yield plasmid pKOS039-98.
  • An ⁇ 0.3 kb PCR fragment comprising the coding sequence for the N-terminus of the desI gene product and an ⁇ 0.12 kb PCR fragment comprising the coding sequence for the C-terminus of the desR gene product were amplified from cosmid pKOS23-26 (ATCC 203141) and inserted together into pLitmus28 treated with restriction enzymes NsiI and EcoRI to produce plasmid pKOS039-101.
  • the ⁇ 6 kb SphI-PstI restriction fragment of pKOS23-26 containing the desI, desII, desIII, desIV, and desV genes was inserted into plasmid pUC19 (Stratagene) to yield plasmid pKOS039-102.
  • the ⁇ 6 kb SphI-EcoRI restriction fragment from plasmid pKOS039-102 was inserted into pKOS039-101 to produce plasmid pKOS039-103.
  • the ⁇ 6 kb BglII-PstI fragment from pKOS23-26 that contains the desR, desVI, desVII, and desVIII genes was inserted into pKOS39-98 to yield pKOS39-100.
  • the ⁇ 6 kb PacI-PstI restriction fragment of pKOS39-100 and the ⁇ 6.4 kb NsiI-EcoRI fragment of pKOS39-103 were cloned into pKOS39-44 to yield pKOS39-104.
  • plasmid pKOS39-104 drives expression of the desosamine biosynthetic genes, the beta-glucosidase gene, and the desosaminyl transferase gene.
  • the glycosylated antibiotic narbomycin was produced in these host cells, and it is believed that YC17 was produced as well.
  • these host cells are transformed with vectors that drive expression of the picK gene, the antibiotics methymycin, neomethymycin, and picromycin are produced.
  • the invention provides expression vectors comprising all of the genes required for desosamine biosynthesis and transfer to a polyketide
  • the invention also provides expression vectors that encode any subset of those genes or any single gene.
  • the invention provides an expression vector for desosaminyl transferase. This vector is useful to desosaminylate polyketides in host cells that produce NDP-desosamine but lack a desosaminyl transferase gene or express a desosaminyl transferase that does not function as efficiently on the polyketide of interest as does the desosaminyl transferase of Streptomyces venezuelae.
  • This expression vector was constructed by first amplifying the desosaminyl transferase coding sequence from pKOS023-27 using the primers:
  • N3917 5′-CCCTGCAGCGGCAAGGAAGGACACGACGCCA-3′ (SEQ ID NO:25);
  • N3918 5′-AGGTCTAGAGCTCAGTGCCGGGCGTCGGCCGG-3′ (SEQ ID NO:26),
  • Plasmid pWHM110 described in Tang et al., 1996 , Molec. Microbiol. 22(5): 801-813, incorporated herein by reference, encodes the ermE* promoter.
  • Plasmid pKOS039-14 is constructed so that the desosaminyl transferase gene is placed under the control of the ermE* promoter and is suitable for expression of the desosaminyl transferase in Streptomyces, Saccharopolyspora erythraea, and other host cells in which the ermE* promoter functions.
  • the picK gene was PCR amplified from plasmid pKOS023-28 using the oligonucleotide primers:
  • Plasmid pKOS023-61 was digested with restriction enzymes SpeI and EcoRI, and a short linker fragment encoding 6 histidine residues and a stop codon (composed of oligonucleotides 30-85a: 5′-CTAGTATGCATCATCATCATCATCATTAA-3′ (SEQ ID NO:29); and 30-85b: 5′-AATTTTAATGATGATGATGATGATGCATA-3′ (SEQ ID NO:30) was inserted to obtain plasmid pKOS023-68. Both plasmid pKOS023-61 and pKOS023-68 produced active PicK enzyme in recombinant E. coli host cells.
  • Plasmid pKOS023-61 was transformed into E. coli BL21-DE3. Successful transformants were grown in LB-containing carbenicillin (100 ⁇ g/ml) at 37° C. to an OD600 of 0.6. Isopropyl-beta-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM, and the cells were grown for an additional 3 hours before harvesting. The cells were collected by centrifugation and frozen at ⁇ 80° C. A control culture of-BL21-DE3 containing the vector plasmid pET21c (Invitrogen) was prepared in parallel.
  • IPTG Isopropyl-beta-D-thiogalactopyranoside
  • the frozen BL21-DE3/pKOS023-61 cells were thawed, suspended in 2 ⁇ L of cold cell disruption buffer (5 mM imidazole, 500 mM NaCl, 20 mM Tris/HCl, pH 8.0) and sonicated to facilitate lysis. Cellular debris and supernatant were separated by centrifugation and subjected to SDS-PAGE on 10-15% gradient gels, with Coomassie Blue staining, using a Pharmacia Phast Gel Electrophoresis system.
  • the soluble crude extract from BL21-DE3/pKOS023-61 contained a Coomassie stained band of Mr ⁇ 46 kDa, which was absent in the control strain BL21-DE3/pET21c.
  • the hydroxylase activity of the picK protein was assayed as follows.
  • the crude supernatant (20 ⁇ L) was added to a reaction mixture (100 ⁇ L total volume) containing 50 mM Tris/HCl (pH 7.5), 20 ⁇ M spinach ferredoxin, 0.025 Unit of spinach ferredoxin:NADP+ oxidoreductase, 0.8 Unit of glucose-6-phosphate dehydrogenase, 1.4 mM NADP+, 7.6 mM glucose-6phosphate, and 20 nmol of narbomycin.
  • the reaction was allowed to proceed for 105 minutes at 30° C. Half of the reaction mixture was loaded onto an HPLC, and the effluent was analyzed by evaporative light scattering (ELSD) and mass spectrometry.
  • the control extract (BL21-DE3/pET21c) was processed identically.
  • the poly-histidine-linked PicK hydroxylase was prepared from pKOS023-68 transformed into E. coli BL21 (DE3) and cultured as described above. The cells were harvested and the PicK protein purified as follows. All purification steps were performed at 4° C. E. coli cell pellets were suspended in 32 ⁇ L of cold binding buffer (20 mM Tris/HCl, pH 8.0, 5 mM imidazole, 500 mM NaCl) per mL of culture and lysed by sonication. For analysis of E. coli cell-free extracts, the cellular debris was removed by low-speed centrifugation, and the supernatant was used directly in assays.
  • the supernatant was loaded (0.5 mL/min.) onto a 5 mL HiTrap Chelating column (Pharmacia, Piscataway, N.J.), equilibrated with binding buffer.
  • the column was washed with 25 ⁇ L of binding buffer and the protein was eluted with a ⁇ 35 ⁇ L linear gradient (5-500 mM imidazole in binding buffer).
  • Column effluent was monitored at 280 nm and 416 nm.
  • Narbomycin was purified as described above from a culture of Streptomyces narbonensis ATCC19790. Reactions for kinetic assays (100 ⁇ L) consisted of 50 mM Tris/HCl (pH 7.5), 100 ⁇ M spinach ferredoxin, 0.025 Unit of spinach ferredoxin:NADP+ oxidoreductase, 0.8 U glucose-6-phosphate dehydrogenase, 1.4 mM NADP+, 7.6 mM glucose-6-phosphate, 20-500 nM narbomycin substrate, and 50-500 nM of PicK enzyme. The reaction proceeded at 30° C., and samples were withdrawn for analysis at 5, 10, 15, and 90 minutes.
  • the picK gene was amplified from cosmid pKOS023-26 using the primers:
  • N3903 5′-TCCTCTAGACGTTTCCGT-3′ (SEQ ID NO:31);
  • N3904 5′-TGAAGCTTGAATTCAACCGGT-3′ (SEQ ID NO:32)
  • hybrid PKS contains portions of the narbonolide PKS and portions of rapamycin and/or DEBS PKS.
  • the hybrid PKS comprises the narbonolide PKS extender module 6 ACP and thioesterase domains and the DEBS loading module and extender modules 1-5 as well as the KS and AT domains of DEBS extender module 6 (but not the KR domain of extender module 6).
  • the hybrid PKS is identical except that the KS 1 domain is inactivated, i.e., the ketosynthase in extender module 1 is disabled.
  • the inactive DEBS KS1 domain and its construction are described in detail in PCT publication Nos. WO 97/02358 and WO 99/03986, each of which is incorporated herein by reference.
  • the primers used in the PCR were:
  • N3905 5′-TTTATGCATCCCGCGGGTCCCGGCGAG-3′ (SEQ ID NO:33);
  • N3906 5′-TCAGAATTCTGTCGGTCACTTGCCCGC-3′ (SEQ ID NO:34).
  • Plasmid pJRJ2 is described in PCT publication Nos. WO 99/03986 and WO 97/02358, incorporated herein by reference.
  • Certain compounds of the invention can be hydroxylated at the C6 position in a host cell that expresses the eryF gene. These compounds can also be hydroxylated in vitro, as illustrated by this example.
  • the 6-hydroxylase encoded by eryF was expressed in E. coli , and partially purified.
  • the hydroxylase (100 pmol in 10 ⁇ L) was added to a reaction mixture (100 ⁇ l total volume) containing 50 mM Tris/HCl (pH 7.5), 20 nM spinach ferredoxin, 0.025 Unit of spinach ferredoxin:NADP+ oxidoreductase, 0.8 Unit of glucose-6-phosphate dehydrogenase, 1.4 mM NADP+, 7.6 mM glucose-6-phosphate, and 10 nmol 6-deoxyerythronolide B.
  • the reaction was allowed to proceed for 90 minutes at 30° C.
  • Antibacterial activity was determined using either disk diffusion assays with Bacillus cereus as the test organism or by measurement of minimum inhibitory concentrations (MIC) in liquid culture against sensitive and resistant strains of Staphylococcus pneumoniae.

Abstract

Recombinant DNA compounds that encode all or a portion of the narbonolide polyketide synthase are used to express recombinant polyketide synthase genes in host cells for the production of narbonolide, narbonolide derivatives, and polyketides that are useful as antibiotics and as intermediates in the synthesis of compounds with pharmaceutical value.

Description

    CROSS-REFERENCE TO RELATED APPLICATIONS
  • This application claims priority under 35 U.S.C. §120 to and is a continuation-in-part of U.S. Ser. No. 09/141,908, filed Aug. 28, 1998, which is a continuation-in-part of U.S. Ser. No. 09/073,538, filed May 6, 1998, which is a continuation-in-part of U.S. Ser. No. 08/846,247, filed Apr. 30, 1997. This application also claims priority under 35 U.S.C. §119(e) to U.S. provisional application Serial No. 60/119,139, filed Feb. 8, 1999; No. 60/100,880, filed Sep. 22, 1998; and No. 60/087,080, filed May 28, 1998. Each of the above patent applications is incorporated herein by reference.[0001]
    • REFERENCE TO GOVERNMENT FUNDING
  • [0002] This invention was supported in part by SBIR grant 1R43-CA75792-01. The U.S. government has certain rights in this invention.
    • FIELD OF THE INVENTION
  • The present invention provides recombinant methods and materials for producing polyketides by recombinant DNA technology. More specifically, it relates to narbonolides and derivatives thereof. The invention relates to the fields of agriculture, animal husbandry, chemistry, medicinal chemistry, medicine, molecular biology, pharmacology, and veterinary technology. [0003]
    • BACKGROUND OF THE INVENTION
  • Polyketides represent a large family of diverse compounds synthesized from 2-carbon units through a series of condensations and subsequent modifications. Polyketides occur in many types of organisms, including fungi and mycelial bacteria, in particular, the actinomycetes. There is a wide variety of polyketide structures, and the class of polyketides encompasses numerous compounds with diverse activities. Tetracycline, erythromycin, FK506, FK520, narbomycin, picromycin, rapamycin, spinocyn, and tylosin, are examples of such compounds. Given the difficulty in producing polyketide compounds by traditional chemical methodology, and the typically low production of polyketides in wild-type cells, there has been considerable interest in finding improved or alternate means to produce polyketide compounds. See PCT publication Nos. WO 93/13663; WO 95/08548; WO 96/40968; WO 97/02358; and WO 98/27203; U.S. Pat. Nos. 4,874,748; 5,063,155; 5,098,837; 5,149,639; 5,672,491; and 5,712,146; Fu et al., 1994[0004] , Biochemistry 33: 9321-9326; McDaniel et al., 1993, Science 262: 1546-1550; and Rohr, 1995, Angew. Chem. Int. Ed. Engl. 34(8): 881-888, each of which is incorporated herein by reference.
  • Polyketides are synthesized in nature by polyketide synthase (PKS) enzymes. These enzymes, which are complexes of multiple large proteins, are similar to the synthases that catalyze condensation of 2-carbon units in the biosynthesis of fatty acids. PKS enzymes are encoded by PKS genes that usually consist of three or more open reading frames (ORFs). Two major types of PKS enzymes are known; these differ in their composition and mode of synthesis. These two major types of PKS enzymes are commonly referred to as Type I or “modular” and Type II “iterative” PKS enzymes. [0005]
  • Modular PKSs are responsible for producing a large number of 12, 14, and 16-membered macrolide antibiotics including methymycin, erythromycin, narbomycin, picromycin, and tylosin. These large multifunctional enzymes (>300,000 kDa) catalyze the biosynthesis of polyketide macrolactones through multistep pathways involving decarboxylative condensations between acyl thioesters followed by cycles of varying β-carbon processing activities (see O'Hagan, D. [0006] The polyketide metabolites; E. Horwood: New York, 1991, incorporated herein by reference). The modular PKS are generally encoded in multiple ORFs. Each ORF typically comprises two or more “modules” of ketosynthase activity, each module of which consists of at least two (if a loading module) and more typically three or more enzymatic activities or “domains.”
  • During the past half decade, the study of modular PKS function and specificity has been greatly facilitated by the plasmid-based [0007] Streptomyces coelicolor expression system developed with the 6-deoxyerythronolide B (6-dEB) synthase (DEBS) genes (see Kao et al., 1994, Science, 265: 509-512, McDaniel et al., 1993, Science 262: 1546-1557, and U.S. Pat. Nos. 5,672,491 and 5,712,146, each of which is incorporated herein by reference). The advantages to this plasmid-based genetic system for DEBS were that it overcame the tedious and limited techniques for manipulating the natural DEBS host organism, Saccharopolyspora erythraea, allowed more facile construction of recombinant PKSs, and reduced the complexity of PKS analysis by providing a “clean” host background. This system also expedited construction of the first combinatorial modular polyketide library in Streptomyces (see PCT publication No. WO 98/49315, incorporated herein by reference).
  • The ability to control aspects of polyketide biosynthesis, such as monomer selection and degree of 1-carbon processing, by genetic manipulation of PKSs has stimulated great interest in the combinatorial engineering of novel antibiotics (see Hutchinson, 1998[0008] , Curr. Opin. Microbiol. 1: 319-329; Carreras and Santi, 1998, Curr. Opin. Biotech. 9: 403-411; and U.S. Pat. Nos. 5,712,146 and 5,672,491, each of which is incorporated herein by reference). This interest has resulted in the cloning, analysis, and manipulation by recombinant DNA technology of genes that encode PKS enzymes. The resulting technology allows one to manipulate a known PKS gene cluster either to produce the polyketide synthesized by that PKS at higher levels than occur in nature or in hosts that otherwise do not produce the polyketide. The technology also allows one to produce molecules that are structurally related to, but distinct from, the polyketides produced from known PKS gene clusters. It has been possible to manipulate modular PKS genes other than the narbonolide PKS using generally known recombinant techniques to obtain altered and hybrid forms. See, e.g., U.S. Pat. Nos. 5,672,491 and 5,712,146 and PCT publication No. WO 98/49315. See Lau et al., 1999, “Dissecting the role of acyltransferase domains of modular polyketide synthases in the choice and stereochemical fate of extender units” Biochemistry 38(5):1643-1651, and Gokhale et al., April 16, 1999, Dissecting and Exploiting Intermodular Communication in Polyketide Synthases”, Science 284: 482-485.
  • The present invention provides methods and reagents relating to the modular PKS gene cluster for the polyketide antibiotics known as narbomycin and picromycin. Narbomycin is produced in [0009] Streptomyces narbonensis, and both narbomycin and picromycin are produced in S. venezuelae. These species are unique among macrolide producing organisms in that they produce, in addition to the 14-membered macrolides narbomycin and picromycin (picromycin is shown in FIG. 1, compound 1), the 12-membered macrolides neomethymycin and methymycin (methymycin is shown in FIG. 1, compound 2). Narbomycin differs from picromycin only by lacking the hydroxyl at position 12. Based on the structural similarities between picromycin and methymycin, it was speculated that methymycin would result from premature cyclization of a hexaketide intermediate in the picromycin pathway.
  • Glycosylation of the C5 hydroxyl group of the polyketide precursor, narbonolide, is achieved through an endogenous desosaminyl transferase to produce narbomycin. In [0010] Streptomyces venezuelae, narbomycin is then converted to picromycin by the endogenously produced narbomycin hydroxylase. (See FIG. 1) Thus, as in the case of other macrolide antibiotics, the macrolide product of the narbonolide PKS is further modified by hydroxylation and glycosylation. FIG. 1 also shows the metabolic relationships of the compounds discussed above.
  • Picromycin (FIG. 1, compound 1) is of particular interest because of its close structural relationship to ketolide compounds (e.g. HMR 3004, FIG. 1, compound 3). The ketolides are a new class of semi-synthetic macrolides with activity against pathogens resistant to erythromycin (see Agouridas et al., 1998[0011] , J. Med. Chem. 41: 4080-4100, incorporated herein by reference). Thus, genetic systems that allow rapid engineering of the narbonolide PKS would be valuable for creating novel ketolide analogs for pharmaceutical applications. Furthermore, the production of picromycin as well as novel compounds with useful activity could be accomplished if the heterologous expression of the narbonolide PKS in Streptomyces lividans and other host cells were possible. The present invention meets these and other needs.
    • DISCLOSURE OF THE INVENTION
  • The present invention provides recombinant methods and materials for expressing PKSs derived in whole and in part from the narbonolide PKS and other genes involved in narbomycin and picromycin biosynthesis in recombinant host cells. The invention also provides the polyketides derived from the narbonolide PKS. The invention provides the complete PKS gene cluster that ultimately results, in [0012] Streptomyces venezuelae, in the production of picromycin. The ketolide product of this PKS is narbonolide. Narbonolide is glycosylated to obtain narbomycin and then hydroxylated at C12 to obtain picromycin. The enzymes responsible for the glycosylation and hydroxylation are also provided in recombinant form by the invention.
  • Thus, in one embodiment, the invention is directed to recombinant materials that contain nucleotide sequences encoding at least one domain, module, or protein encoded by a narbonolide PKS gene. The recombinant materials may be “isolated.” The invention also provides recombinant materials useful for conversion of ketolides to antibiotics. These materials include recombinant DNA compounds that encode the C12hydroxylase (the picK gene), the desosamine biosynthesis and desosaminyl transferase enzymes, and the beta-glucosidase enzyme involved in picromycin biosynthesis in [0013] S. venezuelae and the recombinant proteins that can be produced from these nucleic acids in the recombinant host cells of the invention.
  • In one embodiment, the invention provides a recombinant expression system that comprises a heterologous promoter positioned to drive expression of the narbonolide PKS, including a “hybrid” narbonolide PKS. In a preferred embodiment, the promoter is derived from a PKS gene. In a related embodiment, the invention provides recombinant host cells comprising the vector that produces narbonolide. In a preferred embodiment, the host cell is [0014] Streptomyces lividans or S. coelicolor.
  • In another embodiment, the invention provides a recombinant expression system that comprises the desosamine biosynthetic genes as well as the desosaminyl transferase gene. In a related embodiment, the invention provides recombinant host cells comprising a vector that produces the desosamine biosynthetic gene products and desosaminyl transferase gene product. In a preferred embodiment, the host cell is [0015] Streptomyces lividans or S. coelicolor.
  • In another embodiment, the invention provides a method for desosaminylating polyketide compounds in recombinant host cells, which method comprises expressing the PKS for the polyketide and the desosaminyl transferase and desosamine biosynthetic genes in a host cell. In a preferred embodiment, the host cell expresses a beta-glucosidase gene as well. This preferred method is especially advantageous when producing desosaminylated polyketides in Streptomyces host cells, because such host cells typically glucosylate desosamine residues of polyketides, which can decrease desired activity, such as antibiotic activity. By coexpression of beta-glucosidase, the glucose residue is removed from the polyketide. [0016]
  • In another embodiment, the invention provides the picK hydroxylase gene in recombinant form and methods for hydroxylating polyketides with the recombinant gene product. The invention also provides polyketides thus produced and the antibiotics or other useful compounds derived therefrom. [0017]
  • In another embodiment, the invention provides a recombinant expression system that comprises a promoter positioned to drive expression of a “hybrid” PKS comprising all or part of the narbonolide PKS and at least a part of a second PKS, or comprising a narbonolide PKS modified by deletions, insertions and/or substitutions. In a related embodiment, the invention provides recombinant host cells comprising the vector that produces the hybrid PKS and its corresponding polyketide. In a preferred embodiment, the host cell is [0018] Streptomyces lividans or S. coelicolor.
  • In a related embodiment, the invention provides recombinant materials for the production of libraries of polyketides wherein the polyketide members of the library are synthesized by hybrid PKS enzymes of the invention. The resulting polyketides can be further modified to convert them to other useful compounds, such as antibiotics, typically through hydroxylation and/or glycosylation. Modified macrolides provided by the invention that are useful intermediates in the preparation of antibiotics are of particular benefit. [0019]
  • In another related embodiment, the invention provides a method to prepare a nucleic acid that encodes a modified PKS, which method comprises using the narbonolide PKS encoding sequence as a scaffold and modifying the portions of the nucleotide sequence that encode enzymatic activities, either by mutagenesis, inactivation, insertion, or replacement. The thus modified narbonolide PKS encoding nucleotide sequence can then be expressed in a suitable host cell and the cell employed to produce a polyketide different from that produced by the narbonolide PKS. In addition, portions of the narbonolide PKS coding sequence can be inserted into other PKS coding sequences to modify the products thereof. The narbonolide PKS can itself be manipulated, for example, by fusing two or more of its open reading frames, particularly those for [0020] extender modules 5 and 6, to make more efficient the production of 14-membered as opposed to 12-membered macrolides.
  • In another related embodiment, the invention is directed to a multiplicity of cell colonies, constituting a library of colonies, wherein each colony of the library contains an expression vector for the production of a modular PKS derived in whole or in part from the narbonolide PKS. Thus, at least a portion of the modular PKS is identical to that found in the PKS that produces narbonolide and is identifiable as such. The derived portion can be prepared synthetically or directly from DNA derived from organisms that produce narbonolide. In addition, the invention provides methods to screen the resulting polyketide and antibiotic libraries. [0021]
  • The invention also provides novel polyketides and antibiotics or other useful compounds derived therefrom. The compounds of the invention can be used in the manufacture of another compound. In a preferred embodiment, the antibiotic compounds of the invention are formulated in a mixture or solution for administration to an animal or human. [0022]
  • These and other embodiments of the invention are described in more detail in the following description, the examples, and claims set forth below.[0023]
    • BRIEF DESCRIPTION OF THE FIGURES
  • FIG. 1 shows the structures of picromycin (compound 1), methymycin (compound 2), and the ketolide HMR 3004 (compound 3) and the relationship of several compounds related to picromycin. [0024]
  • FIG. 2 shows a restriction site and function map of cosmid pKOS023-27. [0025]
  • FIG. 3 shows a restriction site and function map of cosmid pKOS023-26. [0026]
  • FIG. 4 has three parts. In Part A, the structures of picromycin (A(a)) and methymycin (A(b)) are shown, as well as the related structures of narbomycin, narbonolide, and methynolide. In the structures, the bolded lines indicate the two or three carbon chains produced by each module (loading and extender) of the narbonolide PKS. Part B shows the organization of the narbonolide PKS genes on the chromosome of [0027] Streptomyces venezuelae, including the location of the various module encoding sequences (the loading module domains are identified as sKS*, sAT, and sACP), as well as the picB thioesterase gene and two desosamine biosynthesis genes (picCII and picCII). Part C shows the engineering of the S. venezuelae host of the invention in which the picAI gene has been deleted. In the Figure, ACP is acyl carrier protein; AT is acyltransferase; DH is dehydratase; ER is enoylreductase; KR is ketoreductase; KS is ketosynthase; and TE is thioesterase.
  • FIG. 5 shows the narbonolide PKS genes encoded by plasmid pKOS039-86, the compounds synthesized by each module of that PKS and the narbonolide (compound 4) and 10-deoxymethynolide (compound 5) products produced in heterologous host cells transformed with the plasmid. The Figure also shows a hybrid PKS of the invention produced by plasmid pKOS038-18, which encodes a hybrid of DEBS and the narbonolide PKS. The Figure also shows the compound, 3,6-dideoxy-3-oxo-erythronolide B (compound 6), produced in heterologous host cells comprising the plasmid. [0028]
  • FIG. 6 shows a restriction site and function map of plasmid pKOS039-104, which contains the desosamine biosynthetic, beta-glucosidase, and desosaminyl transferase genes under transcriptional control of actII-4.[0029]
    • MODES OF CARRYING OUT THE INVENTION
  • The present invention provides useful compounds and methods for producing polyketides in recombinant host cells. As used herein, the term recombinant refers to a compound or composition produced by human intervention. The invention provides recombinant DNA compounds encoding all or a portion of the narbonolide PKS. The invention also provides recombinant DNA compounds encoding the enzymes that catalyze the further modification of the ketolides produced by the narbonolide PKS. The invention provides recombinant expression vectors useful in producing the narbonolide PKS and hybrid PKSs composed of a portion of the narbonolide PKS in recombinant host cells. Thus, the invention also provides the narbonolide PKS, hybrid PKSs, and polyketide modification enzymes in recombinant form. The invention provides the polyketides produced by the recombinant PKS and polyketide modification enzymes. In particular, the invention provides methods for producing the polyketides 10-deoxymethynolide, narbonolide, YC17, narbomycin, methymycin, neomethymycin, and picromycin in recombinant host cells. [0030]
  • To appreciate the many and diverse benefits and applications of the invention, the description of the invention below is organized as follows. First, a general description of polyketide biosynthesis and an overview of the synthesis of narbonolide and compounds derived therefrom in [0031] Streptomyces venezuelae are provided. This general description and overview are followed by a detailed description of the invention in six sections. In Section I, the recombinant narbonolide PKS provided by the invention is described. In Section II, the recombinant desosamine biosynthesis genes, the desosaminyl transferase gene, and the beta-glucosidase gene provided by the invention are described. In Section III, the recombinant picK hydroxylase gene provided by the invention is described. In Section IV, methods for heterologous expression of the narbonolide PKS and narbonolide modification enzymes provided by the invention are described. In Section V, the hybrid PKS genes provided by the invention and the polyketides produced thereby are described. In Section VI, the polyketide compounds provided by the invention and pharmaceutical compositions of those compounds are described. The detailed description is followed by a variety of working examples illustrating the invention.
  • The narbonolide synthase gene, like other PKS genes, is composed of coding sequences organized in a loading module, a number of extender modules, and a thioesterase domain. As described more fully below, each of these domains and modules is a polypeptide with one or more specific functions. Generally, the loading module is responsible for binding the first building block used to synthesize the polyketide and transferring it to the first extender module. The building blocks used to form complex polyketides are typically acylthioesters, most commonly acetyl, propionyl, malonyl, methylmalonyl, and ethylmalonyl CoA. Other building blocks include amino acid like acylthioesters. PKSs catalyze the biosynthesis of polyketides through repeated, decarboxylative Claisen condensations between the acylthioester building blocks. Each module is responsible for binding a building block, performing one or more functions on that building block, and transferring the resulting compound to the next module. The next module, in turn, is responsible for attaching the next building block and transferring the growing compound to the next module until synthesis is complete. At that point, an enzymatic thioesterase activity cleaves the polyketide from the PKS. See, generally, FIG. 5. [0032]
  • Such modular organization is characteristic of the modular class of PKS enzymes that synthesize complex polyketides and is well known in the art. The polyketide known as 6-deoxyerythronolide B is a classic example of this type of complex polyketide. The genes, known as eryAI, eryAII, and eryAIII (also referred to herein as the DEBS genes, for the proteins, known as DEBS1, DEBS2, and DEBS3, that comprise the 6-dEB synthase), that code for the multi-subunit protein known as DEBS that synthesizes 6-dEB, the precursor polyketide to erythromycin, are described in U.S. Pat. No. 5,824,513, incorporated herein by reference. Recombinant methods for manipulating modular PKS genes are described in U.S. Pat. Nos. 5,672,491; 5,843,718; 5,830,750; and 5,712,146; and in PCT publication Nos. WO 98/49315 and WO 97/02358, each of which is incorporated herein by reference. [0033]
  • The loading module of DEBS consists of two domains, an acyl-transferase (AT) domain and an acyl carrier protein (ACP) domain. Each extender module of DEBS, like those of other modular PKS enzymes, contains a ketosynthase (KS), AT, and ACP domains, and zero, one, two, or three domains for enzymatic activities that modify the beta-carbon of the growing polyketide chain. A module can also contain domains for other enzymatic activities, such as, for example, a methyltransferase or dimethyltransferase activity. Finally, the releasing domain contains a thioesterase and, often, a cyclase activity. [0034]
  • The AT domain of the loading module recognizes a particular acyl-CoA (usually acetyl or propionyl but sometimes butyryl) and transfers it as a thiol ester to the ACP of the loading module. Concurrently, the AT on each of the extender modules recognizes a particular extender-CoA (malonyl or alpha-substituted malonyl, i.e., methylmalonyl, ethylmalonyl, and carboxylglycolyl) and transfers it to the ACP of that module to form a thioester. Once the PKS is primed with acyl- and malonyl-ACPs, the acyl group of the loading module migrates to form a thiol ester (trans-esterification) at the KS of the first extender module; at this stage, [0035] extender module 1 possesses an acyl-KS adjacent to a malonyl (or substituted malonyl) ACP. The acyl group derived from the loading module is then covalently attached to the alpha-carbon of the malonyl group to form a carbon-carbon bond, driven by concomitant decarboxylation, and generating a new acyl-ACP that has a backbone two carbons longer than the loading unit (elongation or extension). The growing polyketide chain is transferred from the ACP to the KS of the next module, and the process continues.
  • The polyketide chain, growing by two carbons each module, is sequentially passed as covalently bound thiol esters from module to module, in an assembly line-like process. The carbon chain produced by this process alone would possess a ketone at every other carbon atom, producing a polyketone, from which the name polyketide arises. Most commonly, however, additional enzymatic activities modify the beta keto group of each two-carbon unit just after it has been added to the growing polyketide chain, but before it is transferred to the next module. Thus, in addition to the minimal module containing KS, AT, and ACP domains necessary to form the carbon-carbon bond, modules may contain a ketodreductase (KR) that reduces the keto group to an alcohol. Modules may also contain a KR plus a dehydratase (DH) that dehydrates the alcohol to a double bond. Modules may also contain a KR, a DH, and an enoylreductase (ER) that converts the double bond to a saturated single bond using the beta carbon as a methylene function. As noted above, modules may contain additional enzymatic activities as well. [0036]
  • Once a polyketide chain traverses the final extender module of a PKS, it encounters the releasing domain or thioesterase found at the carboxyl end of most PKSs. Here, the polyketide is cleaved from the enzyme and cyclyzed. The resulting polyketide can be modified further by tailoring enzymes; these enzymes add carbohydrate groups or methyl groups, or make other modifications, i.e., oxidation or reduction, on the polyketide core molecule. [0037]
  • While the above description applies generally to modular PKS enzymes, there are a number of variations that exist in nature. For example, some polyketides, such as epothilone, incorporate a building block that is derived from an amino acid. PKS enzymes for such polyketides include an activity that functions as an amino acid ligase or as a non-ribosomal peptide synthetase (NRPS). Another example of a variation, which is actually found more often than the two domain loading module construct found in DEBS, occurs when the loading module of the PKS is not composed of an AT and an ACP but instead utilizes an inactivated KS, an AT, and an ACP. This inactivated KS is in most instances called KS[0038] Q, where the superscript letter is the abbreviation for the amino acid, glutamine, that is present instead of the active site cysteine required for activity. For example, the narbonolide PKS loading module contains a KSQ. Yet another example of a variation has been mentioned above in the context of modules that include a methyltransferase or dimethyltransferase activity; modules can also include an epimerase activity. These variations will be described further below in specific reference to the narbonolide PKS and the various recombinant and hybrid PKSs provided by the invention.
  • With this general description of polyketide biosynthesis, one can better appreciate the biosynthesis of narbonolide related polyketides in [0039] Streptomyces venezuelae and S. narbonensis. The narbonolide PKS produces two polyketide products, narbonolide and 10-deoxymethynolide. Narbonolide is the polyketide product of all six extender modules of the narbonolide PKS. 10-deoxymethynolide is the polyketide product of only the first five extender modules of the narbonolide PKS. These two polyketides are desosaminylated to yield narbomycin and YC17, respectively. These two glycosylated polyketides are the final products produced in S. narbonensis. In S. venezuelae, these products are hydroxylated by the picK gene product to yield picromycin and either methymycin (hydroxylation at the C10 position of YC17) or neomethymycin (hydroxylation at the C12 position of YC17). (See FIG. 1) The present invention provides the genes required for the biosynthesis of all of these polyketides in recombinant form.
  • Section I. The Narbonolide PKS [0040]
  • The narbonolide PKS is composed of a loading module, six extender modules, and two thioesterase domains one of which is on a separate protein. FIG. 4, part B, shows the organization of the narbonolide PKS genes on the [0041] Streptomyces venezuelae chromosome, as well as the location of the module encoding sequences in those genes, and the various domains within those modules. In the Figure, the loading module is not numbered, and its domains are indicated as sKS*, sAT, and ACP. Also shown in the Figure, part A, are the structures of picromycin and methymycin.
  • The loading and six extender modules and the thioesterase domain of the narbonolide PKS reside on four proteins, designated PICAI, PICAII, PICAIII, and PICAIV. PICAI includes the loading module and [0042] extender modules 1 and 2 of the PKS. PICAII includes extender modules 3 and 4. PICAIII includes extender module 5. PICAIV includes extender module 6 and a thioesterase domain. There is a second thioesterase domain (TEII) on a separate protein, designated PICB. The amino acid sequences of these proteins are shown below.
  • Amino Acid Sequence of Narbonolide Synthase Subunit 1, PICAI (SEQ ID NO:1) [0043]
    (SEQ ID NO:1)
    1 MSTVSKSESE EFVSVSNDAG SAHGTAEPVA VVGISCRVPG ARDPREFWEL LAAGGQAVTD
    61 VPADRWNAGD FYDPDRSAPG RSNSRWGGFI EDVDRFDAAF FGISPREAAE MDPQQRLALE
    121 LGWEALERAG IDPSSLTGTR TGVFAGAIWD DYATLKHRQG GAAITPHTVT GLHRGIIANR
    181 LSYTLGLRGP SMVVDSGQSS SLVAVHLACE SLRRGESELA LAGGVSLNLV PDSIIGASKF
    241 GGLSPDGRAY TFDARANGYV RGEGGGFVVL KRLSRAVADG DPVLAVIRGS AVNNGGAAQG
    301 MTTPDAQAQE AVLREAHERA GTAPADVRYV ELHGTGTPVG DPIEAAALGA ALGTGRPAGQ
    361 PLLVGSVKTN IGHLEGAAGI AGLIKAVLAV RGRALPASLN YETPNPAIPF EELNLRVNTE
    421 YLPWEPEHDG QRMVVGVSSF GMGGTNAHVV LEEAPGVVEG ASVVESTVGG SAVGGGVVPW
    481 VVSAKSAAAL DAQIERLAAF ASRDRTDGVD AGAVDAGAVD AGAVARVLAG GRAQFEHRAV
    541 VVGSGPDDLA AALAAPEGLV RGVASGVGRV AFVFPGQGTQ WAGMGAELLD SSAVFAAAMA
    601 ECEAALSPYV DWSLEAVVRQ APGAPTLERV DVVQPVTFAV MVSLARVWQH HGVTPQAVVG
    661 HSQGEIAAAY VAGALSLDDA ARVVTLRSKS IAAHLAGKGG MLSLALSEDA VLERLAGFDG
    721 LSVAAVNGPT ATVVSGDPVQ IEELARACEA DGVRARVIPV DYASHSRQVE IIESELAEVL
    781 AGLSPQAPRV PFFSTLEGAW ITEPVLDGGY WYRNLRHRVG FAPAVETLAT DEGFTHFVEV
    841 SAHPVLTMAL PGTVTGLATL RRDNGGQDRL VASLAEAWAN GLAVDWSPLL PSATGHHSDL
    901 PTYAFQTERH WLGEIEALAP AGEPAVQPAV LRTEAAEPAE LDRDEQLRVI LDKVRAQTAQ
    961 VLGYATGGQI EVDRTFREAG CTSLTGVDLR NRINAAFGVR MAPSMIFDFP TPEALAEQLL
    1021 LVVHGEAAAN PAGAEPAPVA AAGAVDEPVA IVGMACRLPG GVASPEDLWR LVAGGGDAIS
    1081 EFPQDRGWDV EGLYHPDPEH PGTSYVRQGG FIENVAGEDA AFFGISPREA LAMDPQQRLL
    1141 LETSWEAVED AGIDPTSLRG RQVGVFTGAM THEYGPSLRD GGEGLDGYLL TGNTASVMSG
    1201 RVSYTLGLEG PALTVDTACS SSLVALHLAV QALRKGEVDM ALAGGVAVMP TPGMFVEFSR
    1261 QRGLAGDGRS KAFAASADGT SWSEGVGVLL VERLSDARRN GHQVLAVVRG SAVNQDGASN
    1321 GLTAPNGPSQ QRVIRRALAD ARLTTSDVDV VEAHGTGTRL GDPIEAQALI ATYGQGRDDE
    1381 QPLRLGSLKS NIGHTQAAAG VSGVIKMVQA MRHGLLPKTL HVDEPSDQID WSAGAVELLT
    1441 EAVDWPEKQD GGLRRAAVSS FGISGTNAHV VLEEAPVVVE GASVVEPSVG GSAVGGGVTP
    1501 WVVSAKSAAA LDAQIERLAA FASRDRTDDA DAGAVDAGAV AHVLADGRAQ FEHRAVALGA
    1561 GADDLVQALA DPDGLIRGTA SGVGRVAFVF PGQGTQWAGM GAELLDSSAV FAAAMAECEA
    1621 ALSPYVDWSL EAVVRQAPGA PTLERVDVVQ PVTFAVMVSL ARVWQHHGVT PQAVVGHSQG
    1681 EIAAAYVAGA LPLDDAARVV TLRSKSIAAH LAGKGGMLSL ALNEDAVLER LSDFDGLSVA
    1741 AVNGPTATVV SGDPVQIEEL AQACKADGFR ARIIPVDYAS HSRQVEIIES ELAQVLAGLS
    1801 PQAPRVPFFS TLEGTWITEP VLDGTYWYRN LRHRVGFAPA IETLAVDEGF THFVEVSAHP
    1861 VLTMTLPETV TGLGTLRREQ GGQERLVTSL AEAWVNGLPV AWTSLLPATA SRPGLPTYAF
    1921 QAERYWLENT PAALATGDDW RYRIDWKRLP AAEGSERTGL SGRWLAVTPE DHSAQAAAVL
    1981 TALVDAGAKV EVLTAGADDD REALAARLTA LTTGDGFTGV VSLLDGLVPQ VAWVQALGDA
    2041 GIKAPLWSVT QGAVSVGRLD TPADPDRAML WGLGRVVALE HPERWAGLVD LPAQPDAAAL
    2101 AHLVTALSGA TGEDQIAIRT TGLHARRLAR APLHGRRPTR DWQPHGTVLI TGGTGALGSH
    2161 AARWMAHHGA EHLLLVSRSG EQAPGATQLT AELTASGARV TIAACDVADP HAMRTLLDAI
    2221 PAETPLTAVV HTAGALDDGI VDTLTAEQVR RAHRAKAVGA SVLDELTRDL DLDAFVLFSS
    2281 VSSTLGIPGQ GNYAPHNAYL DALAARRRAT GRSAVSVAWG PWDGGGMAAG DGVAERLRNH
    2341 GVPGMDPELA LAALESALGR DETAITVADI DWDRFYLAYS SGRPQPLVEE LPEVRRIIDA
    2401 RDSATSGQGG SSAQGANPLA ERLAAAAPGE RTEILLGLVR AQAAAVLRMR SPEDVAADRA
    2461 FKDIGFDSLA GVELRNRLTR ATGLQLPATL VFDHPTPLAL VSLLRSEFLG DEETADARRS
    2521 AALPATVGAG AGAGAGTDAD DDPIAIVAMS CRYPGDIRSP EDLWRMLSEG GEGITPFPTD
    2581 RGWDLDGLYD ADPDALGRAY VREGGFLHDA AEFDAEFFGV SPREALAMDP QQRMLLTTSW
    2641 EAFERAGIEP ASLRGSSTGV FIGLSYQDYA ARVPNAPRGV EGYLLTGSTP SVASGRIAYT
    2701 FGLEGPATTV DTACSSSLTA LHLAVRALRS GECTMALAGG VAMMATPHMF VEFSRQRALA
    2761 PDGRSKAFSA DADGFGAAEG VGLLLVERLS DARRNGHPVL AVVRGTAVNQ DGASNGLTAP
    2821 NGPSQQRVIR QALADARLAP GDIDAVETHG TGTSLGDPIE AQGLQATYGK ERPAERPLAI
    2881 GSVKSNIGHT QAAAGAAGII KMVLAMRHGT LPKTLHADEP SPHVDWANSG LALVTEPIDW
    2941 PAGTGPRRAA VSSFGISGTN AHVVLEQAPD AAGEVLGADE VPEVSETVAM AGTAGTSEVA
    3001 EGSEASEAPA APGSREASLP GHLPWVLSAK DEQSLRGQAA ALHAWLSEPA ADLSDADGPA
    3061 RLRDVGYTLA TSRTAFAHRA AVTAADRDGF LDGLATLAQG GTSAHVHLDT ARDGTTAFLF
    3121 TGQGSQRPGA GRELYDRHPV FARALDEICA HLDGHLELPL LDVMFAAEGS AEAALLDETR
    3181 YTQCALFALE VALFRLVESW GMRPAALLGH SVGEIAAAHV AGVFSLADAA RLVAARGRLM
    3241 QELPAGGAML AVQAAEDEIR VWLETEERYA GRLDVAAVNG PEAAVLSGDA DAAREAEAYW
    3301 SGLGRRTRAL RVSHAFHSAH MDGMLDGFRA VLETVEFRRP SLTVVSNVTG LAAGPDDLCD
    3361 PEYWVRHVRG TVRFLDGVRV LRDLGVRTCL ELGPDGVLTA MAADGLADTP ADSAAGSPVG
    3421 SPAGSPADSA AGALRPRPLL VALLRRKRSE TETVADALGR AHAHGTGPDW HAWFAGSGAH
    3481 RVDLPTYSFR RDRYWLDAPA ADTAVDTAGL GLGTADHPLL GAVVSLPDRD GLLLTGRLSL
    3541 RTHPWLADHA VLGSVLLPGA AMVELAAHAA ESAGLRDVRE LTLLEPLVLP EHGGVELRVT
    3601 VGAPAGEPGG ESAGDGARPV SLHSRLADAP AGTAWSCHAT GLLATDRPEL PVAPDRAAMW
    3661 PPQGAEEVPL DGLYERLDGN GLAFGPLFQG LNAVWRYEGE VFADIALPAT TNATAPATAN
    3721 GGGSAAAAPY GIHPALLDAS LHAIAVGGLV DEPELVRVPF HWSGVTVHAA GAAAARVRLA
    3781 SAGTDAVSLS LTDGEGRPLV SVERLTLRPV TADQAAASRV GGLMHRVAWR PYALASSGEQ
    3841 DPHATSYGPT AVLGKDELKV AAALESAGVE VGLYPDLAAL SQDVAAGAPA PRTVLAPLPA
    3901 GPADGGAEGV RGTVARTLEL LQAWLADEHL AGTRLLLVTR GAVRDPEGSG ADDGGEDLSH
    3961 AAAWGLVRTA QTENPGRFGL LDLADDASSY RTLPSVLSDA GLRDEPQLAL HDGTIRLARL
    4021 ASVRPETGTA APALAPEGTV LLTGGTGGLG GLVARHVVGE WGVRRLLLVS RRGTDAPCAD
    4081 ELVHELEALG ADVSVAACDV ADREALTAVL DAIPAEHPLT AVVHTAGVLS DGTLPSMTTE
    4141 DVEHVLRPKV DAAFLLDELT STPAYDLAAF VMFSSAAAVF GGAGQGAYAA ANATLDALAW
    4201 RRRAAGLPAL SLGWGLWAET SGMTGELGQA DLRRNSRAGI GGISDAEGIA LLDAALRDDR
    4261 HPVLLPLRLD AAGLRDAAGN DPAGIPALFR DVVGARTVRA RPSAASASTT AGTAGTPCTA
    4321 DGAAETAAVT LADRAATVDG PARQRLLLEF VVGEVAEVLG HARGHRIDAE RGFLDLCFDS
    4381 LTAVELRNRL NSAGGLALPA TLVFDHPSPA ALASHLDAEL PRGASDQDGA GNRNGNENGT
    4441 TASRSTAETD ALLAQLTRLE GALVLTGLSD APGSEEVLEH LRSLRSMVTG ETGTGTASGA
    4501 PDGAGSGAED RPWAAGDGAG GGSEDGAGVP DFMNASAEEL FGLLDQDPST D
  • [0044]
    (SEQ ID NO:2)
    1 VSTVNEEKYL DYLRRATADL HEARGRLREL EAKAGEPVAI VGMACRLPGG VASPEDLWRL
    61 VAGGEDAISE FPQDRGWDVE GLYDPNPEAT GKSYAREAGF LYEAGEFDAD FFGISPREAL
    121 AMDPQQRLLL EASWEAFEHA GIPAATARGT SVGVFTGVMY HDYATRLTDV PEGIEGYLGT
    181 GNSGSVASGR VAYTLGLEGP AVTVDTACSS SLVALHLAVQ ALRKGEVDMA LAGGVTVMST
    241 PSTFVEFSRQ RGLAPDGRSK SFSSTADGTS WSEGVGVLLV ERLSDARRKG HRILAVVRGT
    301 AVNQDGASSG LTAPNGPSQQ RVIRRALADA RLTTSDVDVV EAHGTGTRLG DPIEAQAVIA
    361 TYGQGRDGEQ PLRLGSLKSN IGHTQAAAGV SGVTKMVQAM RHGVLPKTLH VEKPTDQVDW
    421 SAGAVELLTE AMDWPDKGDG GLRRAAVSSF GVSGTNAHVV LEEAPAAEET PASEATPAVE
    481 PSVGAGLVPW LVSAKTPAAL DAQIGRLAAF ASQGRTDAAD PGAVARVLAG GRAEFEHRAV
    541 VLGTCQDDFA QALTAPEGLI RGTPSDVGRV AFVFPGQGTQ WAGMGAELLD VSKEFAAAMA
    601 ECESALSRYV DWSLEAVVRQ APGAPTLERV DVVQPVTFAV MVSLAKVWQH HGVTPQAVVG
    661 HSQGEIAAAY VACALTLDDA ARVVTLRSKS IAAHLAGKGG MISLALSEEA TRQRIENLRG
    721 LSIAAVNGPT ATVVSGDPTQ IQELAQACEA DGVRARIIPV DYASHSAHVE TIESELAEVL
    781 AGLSPRTPEV PFFSTLEGAW ITEPVLDGTY WYRNLRHRVG FAPAVETLAT DEGFTHFIEV
    841 SAHPVLTMTL PETVTGLGTL RREQGGQERL VTSLAEAWTN GLTIDWAPVL PTATGHHPEL
    901 PTYAFQRRHY WLHDSPAVQG SVQDSWRYRI DWKRLAVADA SERAGLSGRW LVVVPEDRSA
    961 EAAPVLAALS GAGADPVQLD VSPLGDRQRL AATLGEALAA AGGAVDGVLS LLAWDESAHP
    1021 GHPAPFTRGT GATLTLVQAL EDAGVAAPLW CVTHGAVSVG RADHVTSPAQ AMVWGMGRVA
    1081 ALEHPERWGG LIDLPSDADR AALDRMTTVL AGGTGEDQVA VRASGLLARR LVRASLPAHG
    1141 TASPWWQADG TVLVTGAEEP AAAEAARRLA RDGAGHLLLH TTPSGSEGAE GTSGAAEDSG
    1201 LAGLVAELAD LGATATVVTC DLTDAEAAAR LLAGVSDAHP LSAVLHLPPT VDSEPLAATD
    1261 ADALARVVTA KATAALHLDR LLREAAAAGG RPPVLVLFSS VAAIWGGAGQ GAYAAGTAFL
    1321 DALAGQHRAD GPTVTSVAWS PWEGSRVTEG ATGERLRRLG LRPLAPATAL TALDTALGHG
    1381 DTAVTIADVD WSSFAPCFTT ARPGTLLADL PEARRALDEQ QSTTAADDTV LSRELGALTG
    1441 AEQQRRMQEL VREHLAVVLN HPSPEAVDTG RAFRDLGFDS LTAVELRNRL KNATGLALPA
    1501 TLVFDYPTPR TLAEFLLAET LGEQAGAGEQ LPVDGGVDDE PVAIVGMACR LPGGVASPED
    1561 LWRLVAGGED AISGFPQDRG WDVEGLYDPD PDASGRTYCR AGGFLDEAGE FDADFFGISP
    1621 REALAMDPQQ RLLLETSWEA VEDAGIDPTS LQGQQVGVFA GTNGPHYEPL LRNTAEDLEG
    1681 YVGTGNAASI MSGRVSYTLG LEGPAVTVDT ACSSSLVALH LAVQALRKGE CGLALAGGVT
    1741 VMSTPTTFVE FSRQRGLAED GRSKAFAASA DGFGPAEGVG MLLVERLSDA RRNGHRVLAV
    1801 VRGSAVNQDG ASNGLTAPNG PSQQRVIRRA LADARLTTAD VDVVEAHGTG TRLGDPTEAQ
    1861 ALIATYGQGR DTEQPLRLGS LKSNIGHTQA AAGVSGIIKM VQAMRHGVLP KTLHVDRPSD
    1921 QTDWSAGTVE LLTEAMDWPR KQEGGLRRAA VSSFGISGTN AHIVLEEAPV DEDAPADEPS
    1981 VGGVVPWLVS AKTPAALDAQ IGRLAAFASQ GRTDAADPGA VARVLAGGRA QFEHRAVALG
    2041 TGQDDLAAAL AAPEGLVRGV ASGVGRVAFV FPGQGTQWAG MGAELLDVSK EFAAAMAECE
    2101 AALAPYVDWS LEAVVRQAPG APTLERVDVV QPVTFAVMVS LAKVWQHHGV TPQAVVGHSQ
    2161 GEIAAAYVAG ALSLDDAARV VTLRSKSIGA HLAGQGGMLS LALSEAAVVE RLAGFDGLSV
    2221 AAVNGPTATV VSGDPTQTQE LAQACEADGV RARIIPVDYA SHSAHVETTE SELADVLAGL
    2281 SPQTPQVPFF STLEGAWTTE PALDOGYWYR NLRHRVGFAP AVETLATDEG FTHFVEVSAH
    2341 PVLTMALPET VTGLGTLRRD NGGQHRLTTS LAEAWANGLT VDWASLLPTT TTHPDLPTYA
    2401 FQTERYWPQP DLSAAGDITS AGLGAAEHPL LGAAVALADS DGCLLTGSLS LRTHPWLADH
    2461 AVAGTVLLPG TAFVELAFRA GDQVGCDLVE ELTLDAPLVL PRRGAVRVQL SVGASDESGR
    2521 RTFGLYAHPE DAPGEAEWTR HATGVLAARA DRTAPVADPE AWPPPGAEPV DVDGLYERFA
    2581 ANGYGYGPLF QGVRGVWRRG DEVFADVALP AEVAGAEGAR FGLHPALLDA AVQAAGAGGA
    2641 FGAGTRLPFA WSGISLYAVG ATALRVRLAP AGPDTVSVSA ADSSGQPVFA ADSLTVLPVD
    2701 PAQLAAFSDP TLDALHLLEW TAWDGAAQAL PGAVVLGGDA DGLAAALRAG GTEVLSFPDL
    2761 TDLVEAVDRG ETPAPATVLV ACPAAGPGGP EHVREALHGS LALMQAWLAD ERFTDGRLVL
    2821 VTRDAVAARS GDGLRSTGQA AVWGLGRSAQ TESPGRFVLL DLAGEARTAG DATAGDGLTT
    2881 GDATVGGTSG DAALGSALAT ALGSGEPQLA LRDGALLVPR LARAAAPAAA DGLAAADGLA
    2941 ALPLPAAPAL WRLEPGTDGS LESLTAAPGD AETLAPEPLG PGQVRIAIRA TGLNFRDVLI
    3001 ALGMYPDPAL MGTEGAGVVT ATGPGVTHLA PGDRVMGLLS GAYAPVVVAD ARTVARMPEG
    3061 WTFAQGASVP VVFLTAVYAL RDLADVKPGE RLLVHSAAGG VGMAAVQIAR HWGVEVHGTA
    3121 SHGKWDALRA LGLDDAHIAS SRTLDFESAF RAASGGAGMD VVLNSLAREF VDASLRLLGP
    3181 GGRFVEMGKT DVRDAERVAA DHPGVGYRAF DLGEAGPERI GEMLAEVIAL FEDGVLRHLP
    3241 VTTWDVRRAR DAFRHVSQAR HTGKVVLTMP SGLDPEGTVL LTGGTGALGG TVARHVVGEW
    3301 GVRRLLLVSR RGTDAPGAGE LVHELEALGA DVSVAACDVA DREALTAVLD SIPAEHPLTA
    3361 VVHTAGVLSD GTLPSMTAEO VEHVLRPKVD AAFLLDELTS TPGYDLAAFV MFSSAAAVFG
    3421 GAGQGAYAAA NATLDALAWR RRTAGLPALS LGWGLWAETS GMTGGLSDTD RSRLARSGAT
    3481 PMDSELTLSL LDAAMRRDDP ALVPIALDVA ALRAQQRDGM LAPLLSGLTR GSRVGGAPVN
    3541 QRRAAAGGAG EADTDLGGRL AAMTPDDRVA HLRDLVRTHV ATVLGHGTPS RVDLERAFRD
    3601 TGFDSLTAVE LRNRLNAATG LRLPATLVFD HPTPGELAGH LLDELATAAG GSWAEGTGSG
    3661 DTASATDRQT TAALAELDRL EGVLASLAPA AGGRPELAAR LRALAAALGD DGDDATDLDE
    3721 ASDDDLFSFI DKELGDSDF
  • Amino Acid Sequence of [0045] Narbonolide Synthase Subunit 2, PICAII (SEQ ID NO:2)
  • Amino Acid Sequence of [0046] Narbonolide Synthase Subunit 3, PICAIII (SEQ ID NO:3)
    (SEQ ID NO:3)
    1 MANNEDKLRD YLKRVTAELQ QNTRRLREIE GRTHEPVAIV GMACRLPGGV ASPEDLWQLV
    61 AGDGDAISEF PQDRGWDVEG LYDPDPDASG RTYCRSGGFL HDAGEFDADF FGISPREALA
    121 MDPQQRLSLT TAWEAIESAG IDPTALKGSG LGVFVGGWHT GYTSGQTTAV QSPELEGHLV
    181 SGAALGFLSG RIAYVLGTDG PALTVDTACS SSLVALHLAV QALRKGECDM ALAGGVTVMP
    241 NADLFVQFSR QRGLAADGRS KAFATSADGF GPAEGAGVLL VERLSDARRN GHRILAVVRG
    301 SAVNQDGASN GLTAPHGPSQ QRVIRRALAD ARLAPGDVDV VEAHGTGTRL GDPIEAQALI
    361 ATYGQEKSSE QPLRLGALKS NIGHTQAAAG VAGVIKMVQA MRHGLLPKTL HVDEPSDQID
    421 WSAGTVELLT EAVDWPEKQD GGLRRAAVSS FGISGTNAHV VLEEAPAVED SPAVEPPAGG
    481 GVVPWPVSAK TPAALDAQIG QLAAYADGRT DVDPAVAARA LVDSRTAMEH RAVAVGDSRE
    541 ALRDALRMPE GLVRGTSSDV GRVAFVFPGQ GTQWAGMGAE LLDSSPEFAA SMAECETALS
    601 RYVDWSLEAV VRQEPGAPTL DRVDVVQPVT EAVMVSLAKV WQHHGITPQA VVGHSQGEIA
    661 AAYVAGALTL DDAARVVTLR SKSIAAHLAG KGGMISLALD EAAVLKRLSD FDGLSVAAVN
    721 GPTATVVSGD PTQIEELART CEADGVRARI IPVDYASHSR QVEIIEKELA EVLAGLAPQA
    781 PHVPFFSTLE GTWITEPVLD GTYWYRNLRH RVGFAPAVET LAVDGFTHFI EVSAHPVLTM
    841 TLPETVTGLG TLRREQGGQE RLVTSLAEAW ANGLTIDWAP ILPTATGHHP ELPTYAFQTE
    901 RFWLQSSAPT SAADOWRYRV EWKPLTASGQ ADLSGRWIVA VGSEPEAELL GALKAAGAEV
    961 DVLEAGADDD REALAARLTA LTTGDGFTGV VSLLDDLVPQ VAWVQALGDA GTKAPLWSVT
    1021 QGAVSVGRLD TPADPDRAML WGLGRVVALE HPERWAGLVD LPAQPDAAAL AHLVTALSGA
    1081 TGEDQIAIRT TGLHARRLAR APLHGRRPTR DWQPHGTVLI TGGTGALGSH AARWMAHHGA
    1141 EHLLLVSRSG EQAPGATQLT AELTASGARV TTAACDVADP HAMRTLLDAI PAETPLTAVV
    1201 HTAGAPGGDP LDVTGPEDIA RILGAKTSGA EVLDDLLRGT PLDAFVLYSS NAGVWGSGSQ
    1261 GVYAAANAHL DALAARRRAR GETATSVAWG LWAGDGMGRG ADDAYWQRRG IRPMSPORAL
    1321 DELAKALSHD ETFVAVADVD WERFAPAFTV SRPSLLLDGV PEARQALAAP VGAPAPGDAA
    1381 VAPTGQSSAL AAITALPEPE RRPALLTLVR THAAAVLGHS SPDRVAPGRA FTELGFDSLT
    1441 AVQLRNQLST VVGNRLPATT VFDHPTPAAL AAHLHEAYLA PAEPAPTDWE GRVRRALAEL
    1501 PLDRLRDAGV LDTVLRLTGI EPEPGSGGSD GGAADPGAEP EASIDDLDAE ALIRMALGPR
    1561
  • Amino Acid Sequence of [0047] Narbonolide Synthase Subunit 4, PICAIV (SEQ ID NO:4)
    (SEQ ID NO:4)
    1 MTSSNEQLVD ALRASLKENE ELRKESRRRA DRRQEPMAIV GMSCRFAGGI RSPEDLWDAV
    61 AAGKDLVSEV PEERGWDIDS LYDPVPGRKG TTYVRNAAFL DDAAGFDAAF FGISPREALA
    121 MDPQQRQLLE ASWEVFERAG IDPASVRGTD VGVYVGCGYQ DYAPDIRVAP EGTGGYVVTG
    181 NSSAVASGRI AYSLGLEGPA VTVDTACSSS LVALHLALKG LRNGDCSTAL VGGVAVLATP
    241 GAFIEFSSQQ AMAADGRTKG FASAADGLAW GEGVAVLLLE RLSDARRKGH RVLAVVRGSA
    301 INQDGASNGL TAPHGPSQQR LIRQALADAR LTSSDVDVVE GHGTGTRLGD PTEAQALLAT
    361 YGQGRAPGQP LRLGTLKSNI GHTQAASGVA GVIKMVQALR HGVLPKTLHV DEPTDQVDWS
    421 AGSVELLTEA VDWPERPGRL RRAGVSAFGV GGTNAHVVLE EAPAVEESPA VEPPAGGGVV
    481 PWPVSAKTSA ALDAQIGQLA AYAEDRTDVD PAVAARALVD SRTAMEHRAV AVGDSREALR
    541 DALRMPEGLV RGTVTDPGRV AFVFPGQGTQ WAGMGAELLD SSPEFAAAMA ECETALSPYV
    601 DWSLEAVVRQ APSAPTLDRV DVVQPVTFAV MVSLAKVWQH HGITPEAVIG HSQGEIAAAY
    661 VAGALTLDDA ARVVTLRSKS IAAHLAGKGG MISLALSEEA TRQRIENLHG LSIAAVNGPT
    721 ATVVSGDPTQ IQELAQACEA DGIRARITPV DYASHSAHVE TIENELADVL AGLSPQTPQV
    781 PFFSTLEGTW ITEPALDGGY WYRNLRHRVG FAPAVETLAT DEGFTHFIEV SAHPVLTMTL
    841 PDKVTGLATL RREDGGQHRL TTSLAEAWAN GLALDWASLL PATGALSPAV PDLPTYAFQH
    901 RSYWISPAGP GEAPAHTASG REAVAETGLA WGPGAEDLDE EGRRSAVLAM VMRQAASVLR
    961 CDSPEEVPVD RPLREIGFDS LTAVDFRNRV NRLTGLQLPP TVVFEHPTPV ALAERISDEL
    1021 AERNWAVAEP SDHEQAEEEK AAAPAGARSG ADTGAGAGMF RALFRQAVED DRYGEFLDVL
    1081 AEASAFRPQF ASPEACSERL DPVLLAGGPT DRAEGRAVLV GCTGTAANGG PHEFLRLSTS
    1141 FQEERDFLAV PLPGYGTGTG TGTALLPADL DTALDAQARA ILRAAGDAPV VLLGHSGGAL
    1201 LAHELAFRLE RAHGAPPAGI VLVDPYPPGH QEPIEVWSRQ LGEGLFAGEL EPMSDARLLA
    1261 MGRYARFLAG PRPGRSSAPV LLVRASEPLG DWQEERGDWR AHWDLPHTVA DVPGDHFTMM
    1321 RDHAPAVAEA VLSWLDAIEG IEGAGK
  • Amino Acid Sequence of typeII Thioesterase, PICB (SEQ ID NO:5) [0048]
    (SEQ ID NO:5)
    1 VTDRPLNVDS GLWIRRFHPA PNSAVRLVCL PHAGGSASYF FRFSEELHPS VEALSVQYPG
    61 RQDRRAEPCL ESVEELAEHV VAATEPWWQE GRLAFFGHSL GASVAFETAR ILEQRHGVRP
    121 ECLYVSGRRA PSLAPDRLVH QLDDRAFLAE IRRLSGTDER FLQDDELLRL VLPALRSDYK
    181 AAETYLHRPS AKLTCPVMAL AGDRDPKAPL NEVAEWRRHT SGPFCLRAYS GGHFYLNDQW
    241 HEICNDISDH LLVTRGAPDA RVVQPPTSLI EGAAKRWQNP R
  • The DNA encoding the above proteins can be isolated in recombinant form from the recombinant cosmid pKOS023-27 of the invention, which was deposited with the American Type Culture Collection under the terms of the Budapest Treaty on Aug. 20, 1998 and is available under accession number ATCC 203141. Cosmid pKOS023-27 contains an insert of [0049] Streptomyces venezuelae DNA of 38506 nucleotides. The complete sequence of the insert from cosmid pKOS023-27 is shown below. The location of the various ORFs in the insert, as well as the boundaries of the sequences that encode the various domains of the multiple modules of the PKS, are summarized in the Table below. FIG. 2 shows a restriction site and function map of pKOS023-27, which contains the complete coding sequence for the four proteins that constitute narbonolide PKS and four additional ORFs. One of these additional ORFs encodes the picB gene product, the type II thioesterase mentioned above. PICB shows a high degree of similarity to other type II thioesterases, with an identity of 51%, 49%, 45% and 40% as compared to those of Amycolatopsis mediterranae, S. griseus, S. fradiae and Saccharopolyspora erythraea, respectively. The three additional ORFs in the cosmid pKOS023-27 insert DNA sequence, from the picCII, picCIII, and picCVI, genes, are involved in desosamine biosynthesis and transfer and described in the following section.
    From Nucleotide To Nucleotide Description
    70 13725 picAI
    70 13725 narbonolide synthase 1 (PICAI)
    148 3141 loading module
    148 1434 KS loading module
    1780 2802 AT loading module
    2869 3141 ACP loading module
    3208 7593 extender module 1
    3208 4497 KS1
    4828 5847 AT1
    6499 7257 KR1
    7336 7593 ACP1
    7693 13332 extender module 2
    7693 8974 KS2
    9418 10554 AT2
    10594 11160 DH2
    12175 12960 KR2
    13063 13332 ACP2
    13830 25049 picAII
    13830 25049 narbonolide synthase 2 (PICAII)
    13935 18392 extender module 3
    13935 15224 KS3
    15540 16562 AT3
    17271 18071 KR3 (inactive)
    18123 18392 ACP3
    18447 24767 extender module 4
    18447 19736 KS4
    20031 21050 AT4
    21093 21626 DH4
    22620 23588 ER4
    23652 24423 KR4
    24498 24765 ACP4
    25133 29821 picAIII
    25133 29821 narbonolide synthase 3 (PICAIII)
    25235 29567 extender module 5
    25235 26530 KS5
    26822 27841 AT5
    28474 29227 KR5
    29302 29569 ACP5
    29924 33964 picAIV
    29924 33964 narbonolide synthase 4 (PICAIV)
    30026 32986 extender module 6
    30026 31312 KS6
    31604 32635 AT6
    32708 32986 ACP6
    From Nucleotide To Nucleotide Description
    33068 33961 PKS thioesterase domain
    33961 34806 picB
    33961 34806 typeII thioesterase homolog
    34863 36011 picCII
    34863 36011 4-keto-6-deoxyglucose isomerase
    36159 37439 picCIII
    36159 37439 desosaminyl transferase
    37529 38242 picCVI
    37529 38242 3-amino dimethyltransferase
  • DNA Sequence of the Insert DNA in Cosmid pKOS023-27 (SEQ ID NO:19) [0050]
    (SEQ ID NO:19)
    1 GATCATGCGG AGCACTCCTT CTCTCGTGCT CCTACCGGTG ATGTGCGCGC CGAATTGATT
    61 CGTGGAGAGA TGTCGACAGT GTCCAAGAGT GAGTCCGAGG AATTCGTGTC CGTGTCGAAC
    121 GACGCCGGTT CCGCGCACGG CACAGCGGAA CCCGTCGCCG TCGTCGGCAT CTCCTGCCGG
    181 GTGCCCGGCG CCCGGGACCC GAGACAGTTC TGGGAACTCC TGGCGGCAGG CGGCCAGGCC
    241 GTCACCGACG TCCCCGCGGA CCGCTGGAAC GCCGGCGACT TCTACGACCC GGACCGCTCC
    301 GCCCCCGGCC GCTCGAACAG CCGGTGGGGC GGGTTCATCG ACGACGTCGA CCGGTTCGAC
    361 GCCGCCTTCT TCGGCATCTC CCCCCGCGAG GCCGCGGAGA TGGACCCGCA GCAGCGGCTC
    421 GCCCTGGAGC TGGGCTGGGA GGCCCTGGAG CGCGCCGGGA TCGACCCGTC CTCGCTCACC
    481 GGCACCCGCA CCGGCGTCTT CGCCGGCGCC ATCTGGGACG ACTACGCCAC CCTGAAGCAC
    541 CGCCAGGGCG GCGCCGCGAT CACCCCGCAC ACCGTCACCG GCCTCCACCG CGGCATCATC
    601 GCGAACCGAC TCTCGTACAC GCTCGGGCTC CGCGGCCCCA GCATGGTCGT CGACTCCGGC
    661 CAGTCCTCGT CGCTCGTCGC CGTCCACCTC GCGTGCGAGA GCCTGCGGCG CGGCGAGTCC
    721 GAGCTCGCCC TCGCCGGCGG CGTCTCGCTC AACCTGGTGC CGGACAGCAT CATCGGGGCG
    781 AGCAAGTTCG GCGGCCTCTC CCCCGACGGC CGCGCCTACA CCTTCGACGC GCGCGCCAAC
    841 GGCTACGTAC GCGGCGAGGG CGGCGGTTTC GTCGTCCTGA AGCGCCTCTC CCGGGCCGTC
    901 GCCGACGGCG ACCCGGTGCT CGCCGTGATC CGGGGCAGCG CCGTCAACAA CGGCGGCGCC
    961 GCCCAGGGCA TGACGACCCC CGACGCGCAG GCGCAGGAGG CCGTGCTCCG CGAGGCCCAC
    1021 GAGCGGGCCG GGACCGCGCC GGCCGACGTG CGGTACGTCG AGCTGCACGG CACCGGCACC
    1081 CCCGTGGGCG ACCCGATCGA GGCCGCTGCG CTCGGCGCCG CCCTCGGCAC CGGCCGCCCG
    1141 GCCGGACAGC CGCTCCTGGT CGGCTCGGTC AAGACGAACA TCGGCCACCT GGAGGGCGCG
    1201 GCCGGCATCG CCGGCCTCAT CAAGGCCGTC CTGGCGGTCC GCGGTCGCGC GCTGCCCGCC
    1261 AGCCTGAACT ACGAGACCCC GAACCCGGCG ATCCCGTTCG AGGAACTGAA CCTCCGGGTG
    1321 AACACGGAGT ACCTGCCGTG GGAGCCGGAG CACGACGGGC AGCGGATGGT CGTCGGCGTG
    1381 TCCTCGTTCG GCATGGGCGG CACGAACGCG CATGTCGTGC TCGAAGAGGC CCCGGGGGTT
    1441 GTCGAGGGTG CTTCGGTCGT GGAGTCGACG GTCGGCGGGT CGGCGGTCGG CGGCGGTGTG
    1501 GTGCCGTGGG TGGTGTCGGC GAAGTCCGCT GCCGCGCTGG ACGCGCAGAT CGAGCGGCTT
    1561 GCCGCGTTCG CCTCGCGGCA TCGTACGGAT GGTGTCGACG CGGGCGCTGT CGATGCGGGT
    1621 GCTGTCGATG CGGGTGCTGT CGCTCGCGTA CTGGCCGGCG GGCGTGCTCA GTTCGAGCAC
    1681 CGGGCCGTCG TCGTCGGCAG CGGGCCGGAC GATCTGGCGG CAGCGCTGGC CGCGCCTGAG
    1741 GGTCTGGTCC GGGGCGTGGC TTCCGGTGTC GGGCGAGTGG CGTTCGTGTT CCCCGGGCAG
    1801 GGCACGCAGT GGGCCGGCAT GGGTGCCGAA CTGCTGGACT CTTCCGCGGT GTTCGCGGCG
    1861 GCCATGGCCG AATGCGAGGC CGCACTCTCC CCGTACGTCG ACTGGTCGCT GGAGGCCGTC
    1921 GTACGGCAGG CCCCCGGTGC GCCCACGCTG GAGCGGGTCG ATGTCGTGCA GCCTGTGACG
    1981 TTCGCCGTCA TGGTCTCGCT GGCTCGCGTG TGGCAGCACC ACGGGGTGAC GCCCCAGGCG
    2041 GTCGTCGGCC ACTCGCAGGG CGAGATCGCC GCCGCGTACG TCGCCGGTGC CCTGAGCCTG
    2101 GACGACGCCG CTCGTGTCGT GACCCTGCGC AGCAAGTCCA TCGCCGCCCA CCTCGCCGGC
    2161 AAGGGCGGCA TGCTGTCCCT CGCGCTGAGC GAGGACGCCG TCCTGGAGCG ACTGGCCGGG
    2221 TTCGACGGGC TGTCCGTCGC CGCTGTGAAC GGGCCCACCG CCACCGTGGT CTCCGGTGAC
    2281 CCCGTACAGA TCGAAGAGCT TGCTCGGGCG TGTGAGGCCG ATGGGGTCCG TGCGCGGGTC
    2341 ATTCCCGTCG ACTACGCGTC CCACAGCCGG CAGGTCGAGA TCATCGAGAG CGAGCTCGCC
    2401 GAGGTCCTCG CCGGGCTCAG CCCGCAGGCT CCGCGCGTGC CGTTCTTCTC GACACTCGAA
    2461 GGCGCCTGGA TCACCGAGCC CGTGCTCGAC GGCGGCTACT GGTACCGCAA CCTGCGCCAT
    2521 CGTGTGGGCT TCGCCCCGGC CGTCGAGACC CTGGCCACCG ACGAGGGCTT CACCCACTTC
    2581 GTCGAGGTCA GCGCCCACCC CGTCCTCACC ATGGCCCTCC CCGGGACCGT CACCGGTCTG
    2641 GCGACCCTGC GTCGCGACAA CGGCGGTCAG GACCGCCTCG TCGCCTCCCT CGCCGAAGCA
    2701 TGGGCCAACG GACTCGCGGT CGACTGGAGC CCGCTCCTCC CCTCCGCGAC CGGCCACCAC
    2761 TCCGACCTCC CCACCTACGC GTTCCAGACC GAGCGCCACT GGCTGGGCGA GATCGAGGCG
    2821 CTCGCCCCGG CGGGCGAGCC GGCGGTGCAG CCCGCCGTCC TCCGCACGGA GGCGGCCGAG
    2881 CCGGCGGAGC TCGACCGGGA CGAGCAGCTG CGCGTGATCC TGGACAAGGT CCGGGCGCAG
    2941 ACGGCCCAGG TGCTGGGGTA CGCGACAGGC GGGCAGATCG AGCTCGACCG GACCTTCCGT
    3001 GAGGCCGGTT CCACCTCCCT GACCGGCGTG GACCTCCGCA ACCGGATCAA CGCCGCCTTC
    3061 GGCGTACGGA TGGCGCCGTC CATGATCTTC GACTTCCCCA CCCCCGAGGC TCTCGCGGAG
    3121 CAGCTGCTCC TCGTCGTGCA CGGGGAGGCG GCGGCGAACC CGGCCGGTGC GGAGCCGGCT
    3181 CCGGTGGCGG CGGCCGGTGC CGTCGACGAG CCGGTGGCGA TCGTCGGCAT CGCCTGCCGC
    3241 CTGCCCGGTG GGGTCGCCTC GCCGGAGGAC CTGTGGCGGC TGGTGGCCGG CGGCGGGGAC
    3301 GCGATCTCGG AGTTCCCGCA GGACCGCGGC TGGGACGTGG AGGGGCTGTA CCACCCGGAT
    3361 CCCGAGCACC CCGGCACGTC GTACGTCCGC CAGGGCGGTT TCATCGAGAA CGTCGCCGGC
    3421 TTCGACCCGG CCTTCTTCGG GATCTCGCCG CGCGAGGCCC TCGCCATGGA CCCCCAGCAG
    3481 CGGCTCCTCC TCGAAACCTC CTGGGAGGCC GTCGACGACG CCGGGATCGA CCCGACCTCC
    3541 CTGCGGGGAC GGCAGGTCGG CGTCTTCACT GGGGCGATGA CCCACGAGTA CGGGCCGAGC
    3601 CTGCGGGACG CCGGGGAAGG CCTCGACGGC TACCTGCTGA CCGGCAACAC GGCCAGCGTG
    3661 ATGTCGGGCC GCCTCTCGTA CACACTCGGC CTTGAGGGCC CCGCCCTGAC GGTGGACACG
    3721 GCCTGCTCGT CGTCGCTGGT CGCCCTGCAC CTCGCCGTGC AGGCCCTGCG CAAGGGCGAG
    3781 GTCGACATGG CGCTCGCCGG CGGCGTGGCC GTGATGCCCA CGCCCGGGAT GTTCGTCGAG
    3841 TTCAGCCGGC AGCGCGGGCT CGCCGGGGAC GGCCGGTCGA AGGCGTTCGC CGCGTCGGCG
    3901 GACGGCACCA GCTGGTCCGA GGGCGTCGGC GTCCTCCTCG TCGAGCGCCT GTCGGACGCC
    3961 CGCCGCAACG GACACCAGGT CCTCGCGCTC GTCCGCGGCA GCGCCGTGAA CCAGGACGGC
    4021 GCGAGCAACG GCCTCACGGC TCCGAACGGG CCCTCGCAGC AGCGCGTCAT CCGGCGCGCG
    4081 CTGGCGGACG CCCGGCTGAC GACCTCCGAC GTGGACGTCG TCGAGGCACA CGGCACGGGC
    4141 ACGCGACTCG GCGACCCGAT CGAGGCGCAG GCCCTGATCG CCACCTACGG CCAGGGCCGT
    4201 GACGACGAAC ACCCGCTGCG CCTCGGGTCG TTGAAGTCCA ACATCGGGCA CACCCAGGCC
    4261 GCGGCCGGCG TCTCCGGTGT CATCAAGATG GTCCAGGCGA TGCGCCACGG ACTGCTGCCG
    4321 AAGACGCTGC ACGTCGACGA GCCCTCGGAC CAGATCGACT GGTCGGCTGG CGCCGTGGAA
    4381 CTCCTCACCG AGGCCGTCGA CTGGCCGGAG AAGCAGGACG GCGGGCTGCG CCGGGCCGCC
    4441 GTCTCCTCCT TCGGGATCAG CGGCACCAAT GCGCATGTGG TGCTCGAAGA GGCCCCGGTG
    4501 GTTGTCGAGG GTGCTTCGGT CGTCGAGCCG TCGGTTGGCG GGTCGGCGGT CGGCGGCGGT
    4561 GTGACGCCTT GGGTGGTGTC GGCGAAGTCC GCTGCCGCGC TCGACGCGCA GATCGAGCGG
    4621 CTTGCCGCAT TCGCCTCGCG GGATCGTACG GATGACGCCG ACGCCGGTGC TGTCGACGCG
    4681 GGCGCTGTCG CTCACGTACT GGCTGACGGG CGTGCTCAGT TCGAGCACCG GGCCGTCGCG
    4741 CTCGGCGCCG GGGCGGACGA CCTCGTACAG GCGCTGGCCG ATCCGGACGG GCTGATACGC
    4801 GGAACGGCTT CCGGTGTCGG GCGAGTGGCG TTCGTGTTCC CCGGTCAGGG CACGCAGTGG
    4861 GCTGGCATGG GTGCCGAACT GCTGGACTCT TCCGCGGTGT TCGCGGCGGC CATGGCCGAG
    4921 TGTGAGGCCG CGCTGTCCCC GTACGTCGAC TGGTCGCTGG AGGCCGTCGT ACGGCAGGCC
    4981 CCCGGTGCGC CCACGCTGGA GCGGGTCGAT GTCGTGCAGC CTGTGACGTT CGCCGTCATG
    5041 GTCTCGCTGG CTCGCGTGTG GCAGCACCAC GGTGTGACGC CCCAGGCGGT CGTCGGCCAC
    5101 TCGCAGGGCG AGATCGCCGC CGCGTACGTC GCCGGAGCCC TGCCCCTGGA CGACGCCGCC
    5161 CGCGTCGTCA CCCTGCGCAG CAAGTCCATC GCCGCCCACC TCGCCGGCAA GGGCGGCATG
    5221 CTGTCCCTCG CGCTGAACGA GGACGCCGTC CTGGAGCGAC TGAGTGACTT CGACGGGCTG
    5281 TCCGTCGCCG CCGTCAACGG GCCCACCGCC ACTGTCGTGT CGGGTGACCC CGTACAGATC
    5341 GAAGAGCTTG CTCAGGCGTG CAAGGCGGAC GGATTCCGCG CGCGGATCAT TCCCGTCGAC
    5401 TACGCGTCCC ACAGCCGGCA GGTCGAGATC ATCGAGAGCG AGCTCGCCCA GGTCCTCGCC
    5461 GGTCTCAGCC CGCAGGCCCC GCGCGTGCCG TTCTTCTCGA CGCTCGAAGG CACCTGGATC
    5521 ACCGAGCCCG TCCTCGACGG CACCTACTGG TACCGCAACC TCCGTCACCG CGTCGGCTTC
    5581 GCCCCCGCCA TCGAGACCCT GGCCGTCGAC GAGGGCTTCA CGCACTTCGT CGAGGTCAGC
    5641 GCCCACCCCG TCCTCACCAT GACCCTCCCC GAGACCGTCA CCGGCCTCGG CACCCTCCGT
    5701 CGCGAACAGG GAGGCCAAGA GCGTCTGGTC ACCTCGCTCG CCGACGCGTG GGTCAACGGG
    5761 CTTCCCGTGG CATGGACTTC GCTCCTGCCC GCCACGGCCT CCCGCCCCGG TCTGCCCACC
    5821 TACGCCTTCC AGGCCGAGCG CTACTGGCTC GAGAACACTC CCGCCGCCCT GGCCACCGGC
    5881 GACGACTGGC GCTACCGCAT CGACTGGAAG CGCCTCCCGG CCGCCGAGGG GTCCGAGCGC
    5941 ACCGGCCTGT CCGGCCGCTG GCTCGCCGTC ACGCCGGAGG ACCACTCCGC GCAGGCCGCC
    6001 GCCGTGCTCA CCGCGCTGGT CGACGCCGGG GCGAAGGTCG AGGTGCTGAC GGCCGGGGCG
    6061 GACGACGACC GTGAGGCCCT CGCCGCCCGG CTCACCGCAC TGACGACCGG TGACGGCTTC
    6121 ACCGCCGTGG TCTCGCTCCT CGACGGACTC GTACCGCAGG TCGCCTGGGT CCAGGCGCTC
    6181 GGCGACGCCG GAATCAAGGC GCCCCTGTGG TCCGTCACCC AGGGCGCCGT CTCCGTCGGA
    6241 CGTCTCGACA CCCCCGCCGA CCCCGACCGG GCCATGCTCT GGGGCCTCGG CCGCCTCGTC
    6301 GCCCTTGAGC ACCCCGAACG CTGGGCCGGC CTCGTCGACC TCCCCGCCCA GCCCGATGCC
    6361 GCCGCCCTCG CCCACCTCGT CACCGCACTC TCCGGCGCCA CCGGCGAGGA CCAGATCGCC
    6421 ATCCGCACCA CCGGACTCCA CGCCCCCCGC CTCGCCCGCG CACCCCTCCA CGGACCTCGG
    6481 CCCACCCGCG ACTGGCAGCC CCACGGCACC GTCCTCATCA CCGGCGGCAC CGGAGCCCTC
    6541 GGCAGCCACG CCGCACGCTG GATGGCCCAC CACGGAGCCG AACACCTCCT CCTCGTCAGC
    6601 CGCAGCGGCG AACAAGCCCC CGGAGCCACC CAACTCACCG CCGAAGTCAC CGCATCGGGC
    6661 GCCCCCGTCA CCATCGCCGC CTGCGACGTC GCCGACCCCC ACGCCATGCG CACCCTCCTC
    6721 GACGCCATCC CCGCCGAGAC GCCCCTCACC GCCGTCGTCC ACACCGCCGG CGCGCTCCAC
    6781 CACGGCATCG TGGACACGCT GACCGCCGAG CAGGTCCGGC GGGCCCACCG TGCGAAGGCC
    6841 GTCGGCGCCT CGGTGCTCGA CGAGCTGACC CGGGACCTCG ACCTCGACGC GTTCGTGCTC
    6901 TTCTCGTCCG TGTCGAGCAC TCTGGGCATC CCCGGTCAGG GCAACTACGC CCCGCACAAC
    6961 GCCTACCTCG ACGCCCTCGC GGCTCGCCGC CGGGCCACCG GCCGGTCCGC CGTCTCGGTG
    7021 GCCTGGGGAC CGTGGGACCG TGGCGGCATG GCCGCCGGTC ACGGCGTGGC CGAGCGGCTG
    7081 CGCAACCACG GCGTGCCCGG CATGGACCCG GAACTCGCCC TGGCCGCACT GGAGTCCGCG
    7141 CTCGGCCGGG ACGAGACCGC GATCACCGTC GCGGACATCG ACTGGGACCG CTTCTACCTC
    7201 GCGTACTCCT CCGGTCGCCC GCAGCCCCTC GTCGAGGAGC TGCCCGAGGT GCGGCGCATC
    7261 ATCGACGCAC GGGACAGCGC CACGTCCGGA CAGGGCGGGA GCTCCGCCCA GGGCGCCAAC
    7321 CCCCTGGCCG AGCGGCTGGC CGCCGCGGCT CCCGGCGAGC GTACGGAGAT CCTCCTCGGT
    7381 CTCGTACGGG CGCAGGCCGC CGCCGTGCTC CGGATGCGTT CGCCGGAGGA CGTCGCCGCC
    7441 GACCGCGCCT TCAAGGACAT CGGCTTCGAC TCGCTCGCCG GTGTCGAGCT GCGCAACAGG
    7501 CTGACCCGGG CGACCGGGCT CCAGCTGCCC GCGACGCTCG TCTTCGACCA CCCGACGCCG
    7561 CTGGCCCTCG TGTCGCTGCT CCGCAGCGAG TTCCTCGGTG ACGAGGAGAC GGCGGACGCC
    7621 CGGCGGTCCG CGGCGCTGCC CGCGACTGTC GGTGCCGGTG CCGGCGCCGG CGCCGGCACC
    7681 GATGCCGACG ACGATCCGAT CGCGATCGTC GCGATGAGCT GCCGCTACCC CGGTGACATC
    7741 CGCAGCCCGG AGGACCTGTG GCGGATGCTG TCCGAGGGCG GCGAGGGCAT CACGCCGTTC
    7801 CCCACCGACC GCGGCTGGGA CCTCGACGGC CTGTACGACG CCGACCCGGA CGCGCTCGGC
    7861 AGGGCGTACG TCCGCGAGGG CGGGTTCCTG CACGACGCGG CCGAGTTCGA CGCGGAGTTC
    7921 TTCGGCGTCT CGCCGCGCGA GGCGCTGGCC ATGGACCCGC AGCAGCGGAT GCTCCTGACG
    7981 ACGTCCTGGG AGGCCTTCGA GCGGGCCGGC ATCGAGCCGG CATCCCTGCG CGGCAGCAGC
    8041 ACCGGTGTCT TCATCGGCCT CTCCTACCAG GACTACGCGG CCCGCGTCCC GAACGCCCCG
    8101 CGTGGCGTGG AGGGTTACCT GCTGACCGGC AGCACGCCGA GCGTCGCGTC GCGCCGTATC
    8161 GCGTACACCT TCGGTCTCGA AGGGCCCGCG ACGACCGTCG ACACCGCCTG CTCGTCGTCG
    8221 CTGACCGCCC TGCACCTGGC GGTGCGGGCG CTGCGCAGCG GCGAGTGCAC GATGGCGCTC
    8281 GCCGGTGGCG TGGCGATGAT GGCGACCCCG CACATGTTCG TGGAGTTCAG CCGTCAGCGG
    8341 GCGCTCGCCC CGGACGGCCG CAGCAAGGCC TTCTCGGCGG ACGCCGACGG GTTCGGCGCC
    8401 GCGGAGGGCG TCGGCCTGCT GCTCGTGGAG CGGCTCTCGG ACGCGCGGCG CAACGGTCAC
    8461 CCGGTGCTCG CCGTGGTCCG CGGTACCGCC GTCAACCAGG ACGGCGCCAG CAACGGGCTG
    8521 ACCGCGCCCA ACGGACCCTC GCAGCAGCGG GTGATCCGGC AGGCGCTCGC CGACGCCCGG
    8581 CTGGCACCCG GCGACATCGA CGCCGTCGAG ACGCACGGCA CGGGAACCTC GCTGGGCGAC
    8641 CCCATCGAGG CCCAGGGCCT CCAGGCCACG TACGGCAAGG AGCGGCCCGC GGAACGGCCG
    8701 CTCGCCATCG GCTCCGTGAA GTCCAACATC GGACACACCC AGGCCGCGGC CGGTGCGGCG
    8761 GGCATCATCA AGATGGTCCT CGCGATGCGC CACGGCACCC TGCCGAAGAC CCTCCACGCC
    8821 GACGAGCCGA GCCCGCACGT CGACTGGGCG AACAGCGGCC TGGCCCTCGT CACCGAGCCG
    8881 ATCGACTGGC CGGCCGGCAC CGGTCCGCGC CGCGCCGCCG TCTCCTCCTT CGCCATCAGC
    8941 GGGACGAACG CGCACGTCGT GCTGGAGCAG GCGCCGGATG CTGCTGGTGA GGTGCTTGGG
    9001 GCCGATGAGG TGCCTGAGGT GTCTGAGACG GTAGCGATGG CTGGGACGGC TGGGACCTCC
    9061 GAGGTCGCTG AGGGCTCTGA GGCCTCCGAG CCCCCCGCGG CCCCCGGCAG CCGTGAGGCG
    9121 TCCCTCCCCG GGCACCTGCC CTGGGTGCTG TCCGCCAAGG ACGAGCAGTC GCTGCGCGGC
    9181 CAGGCCGCCG CCCTGCACGC GTGGCTGTCC GAGCCCGCCG CCGACCTGTC GGACGCGGAC
    9241 GGACCGGCCC GCCTGCGGGA CGTCGGGTAC ACGCTCGCCA CGAGCCGTAC CGCCTTCGCG
    9301 CACCGCGCCG CCGTGACCGC CGCCGACCGG GACGGGTTCC TGGACGGGCT GGCCACGCTG
    9361 GCCCAGGGCG GCACCTCGGC CCACGTCCAC CTGGACACCG CCCGGGACGG CACCACCGCG
    9421 TTCCTCTTCA CCGGCCAGGG CAGTCAGCGC CCCGGCGCCG GCCGTGAGCT GTACGACCGG
    9481 CACCCCGTCT TCGCCCGGGC GCTCGACGAG ATCTGCGCCC ACCTCGACGG TCACCTCGAA
    9541 CTGCCCCTGC TCGACGTGAT GTTCGCGGCC GAGGGCAGCG CGGAGGCCGC GCTGCTCGAC
    9601 GAGACGCGGT ACACGCAGTG CGCGCTGTTC GCCCTGGAGG TCGCCCTCTT CCGGCTCGTC
    9661 GAGAGCTGGG GCATGCGGCC GGCCGCACTG CTCGGTCACT CGGTCGGCGA GATCGCCGCC
    9721 GCGCACGTCG CCGGTGTGTT CTCGCTCGCC GACGCCGCCC GCCTGGTCGC CGCGCGCGGC
    9781 CGGCTCATGC AGGAGCTGCC CGCCGGTGGC GCGATGCTCG CCGTCCAGGC CGCGGAGGAC
    9841 GAGATCCGCG TGTGGCTGGA GACGGAGGAG CGGTACGCGG GACGTCTGGA CGTCGCCGCC
    9901 GTCAACGGCC CCGAGGCCGC CGTCCTGTCC GGCGACGCGG ACGCGGCGCG GGAGGCGGAG
    9961 GCGTACTGGT CCGGGCTCGG CCGCAGGACC CGCGCGCTGC GGGTCAGCCA CGCCTTCCAC
    10021 TCCGCGCACA TGGACGGCAT GCTCGACGGG TTCCGCGCCG TCCTGGAGAC GGTGGAGTTC
    10081 CGGCGCCCCT CCCTGACCGT GGTCTCGAAC GTCACCGGCC TGGCCGCCGG CCCGGACCAC
    10141 CTGTGCGACC CCGAGTACTG GGTCCGGCAC GTCCGCGGCA CCGTCCGCTT CCTCGACGGC
    10201 GTCCGTGTCC TGCGCGACCT CGGCGTGCGG ACCTGCCTGG AGCTGGGCCC CGACGGGGTC
    10261 CTCACCGCCA TGGCGGCCGA CGGCCTCGCG GACACCCCCG CGGATTCCGC TGCCGGCTCC
    10321 CCCGTCGGCT CTCCCGCCGG CTCTCCCGCC GACTCCGCCG CCGGCGCGCT CCGGCCCCGG
    10381 CCGCTGCTCG TGGCGCTGCT GCGCCGCAAG CGGTCGGAGA CCGAGACCGT CGCGGACGCC
    10441 CTCGGCAGGG CGCACGCCCA CCGCACCGGA CCCGACTGGC ACGCCTGGTT CGCCGGCTCC
    10501 GGGGCGCACC GCGTGGACCT GCCCACGTAC TCCTTCCGGC GCGACCGCTA CTGGCTGGAC
    10561 GCCCCGGCGG CCGACACCGC GGTGGACACC GCCGGCCTCG GTCTCGGCAC CGCCGACCAC
    10621 CCGCTGCTCG GCGCCGTGGT CAGCCTTCCG GACCGGGACG GCCTGCTGCT CACCGGCCGC
    10681 CTCTCCCTGC GCACCCACCC GTGGCTCGCG GACCACGCCG TCCTGGGGAG CGTCCTGCTC
    10741 CCCGGCGCCG CGATGGTCGA ACTCGCCGCG CACGCTGCGG AGTCCGCCGG TCTGCGTGAC
    10801 GTGCGGGAGC TGACCCTCCT TGAACCGCTG GTACTGCCCG AGCACGGTGG CGTCGAGCTG
    10861 CGCGTGACGG TCGGGGCGCC GGCCGGAGAG CCCGGTGGCG AGTCGGCCGG GGACGGCGCA
    10921 CGGCCCGTCT CCCTCCACTC GCGGCTCGCC GACGCGCCCG CCGGTACCGC CTGGTCCTGC
    10981 CACGCCACCG GTCTGCTGGC CACCGACCGG CCCGAGCTTC CCGTCGCGCC CGACCGTGCG
    11041 GCCATGTGGC CGCCGCAGGG CGCCGAGGAG GTGCCGCTCG ACGGTCTCTA CGAGCGGCTC
    11101 GACGGGAACG GCCTCGCCTT CGGTCCGCTG TTCCAGGGGC TGAACGCGGT GTGGCGGTAC
    11161 GAGGGTGAGG TCTTCGCCGA CATCGCGCTC CCCGCCACCA CGAATGCGAC CGCGCCCGCG
    11221 ACCGCGAACG GCGGCGGGAG TGCGGCGGCG GCCCCCTACG GCATCCACCC CGCCCTGCTC
    11281 GACGCTTCGC TGCACGCCAT CGCGGTCGGC CGTCTCGTCG ACGAGCCCGA GCTCGTCCGC
    11341 GTCCCCTTCC ACTGGAGCGG TGTCACCGTG CACGCGGCCG GTGCCGCGGC GGCCCGGGTC
    11401 CGTCTCGCCT CCGCGGGGAC GGACGCCGTC TCGCTGTCCC TGACGGACGG CGAGGGACGC
    11461 CCGCTGGTCT CCGTGGAACG GCTCACGCTG CGCCCGGTCA CCGCCGATCA GGCGGCGGCG
    11521 AGCCGCGTCG GCGGGCTGAT GCACCGGGTG GCCTGGCGTC CGTACGCCCT CGCCTCGTCC
    11581 GGCGAACAGG ACCCGCACGC CACTTCGTAC GGGCCGACCG CCGTCCTCGG CAAGGACGAG
    11641 CTGAAGGTCG CCGCCGCCCT GGAGTCCGCG GGCGTCGAAG TCGGGCTCTA CCCCGACCTG
    11701 GCCGCGCTGT CCCAGGACGT GGCGGCCGGC GCCCCGGCGC CCCGTACCGT CCTTGCGCCG
    11761 CTGCCCGCGG GTCCCGCCGA CGGCGCCGCG GAGGGTGTAC GGGGCACGGT GGCCCGGACG
    11821 CTGGAGCTGC TCCAGGCCTG GCTGGCCGAC GAGCACCTCG CGGGCACCCG CCTGCTCCTG
    11881 GTCACCCGCG GTGCGGTGCG GGACCCCGAG GGGTCCGGCG CCGACGATGG CGGCGAGGAC
    11941 CTGTCGCACG CGGCCGCCTG GGGTCTCGTA CGGACCGCGC AGACCGAGAA CCCCGGCCGC
    12001 TTCGGCCTTC TCGACCTGGC CGACGACGCC TCGTCGTACC GGACCCTGCC GTCGGTGCTC
    12061 TCCGACGCGG GCCTGCGCGA CGAACCGCAG CTCGCCCTGC ACGACGGCAC CATCAGGCTG
    12121 GCCCGCCTGG CCTCCGTCCG GCCCGAGACC GGCACCGCCG CACCGGCGCT CGCCCCGGAG
    12181 GGCACGGTCC TGCTGACCGG CGGCACCGGC GGCCTGGGCG GACTGGTCGC CCGGCACGTG
    12241 GTGGGCGAGT GGGGCGTACG ACGCCTGCTG CTGGTGAGCC GGCGGGGCAC GGACGCCCCG
    12301 GGCGCCGACG AGCTCGTGCA CGAGCTGGAG GCCCTGGGAG CCGACGTCTC GGTGGCCGCG
    12361 TGCGACGTCG CCGACCGCGA AGCCCTCACC GCCGTACTCG ACGCCATCCC CGCCGAACAC
    12421 CCGCTCACCG CGGTCGTCCA CACGGCAGGC GTCCTCTCCG ACGGCACCCT CCCGTCCATG
    12481 ACGACGGAGC ACGTGGAACA CGTACTGCGG CCCAAGGTCG ACGCCGCGTT CCTCCTCGAC
    12541 GAACTCACCT CGACGCCCGC ATACGACCTG GCAGCGTTCG TCATGTTCTC CTCCGCCGCC
    12601 GCCGTCTTCG GTGGCGCGCG GCAGGGCGCC TACGCCGCCG CCAACGCCAC CCTCGACGCC
    12661 CTCGCCTGGC GCCGCCGGGC AGCCGGACTC CCCGCCCTCT CCCTCGGCTG GGGCCTCTGG
    12721 GCCGAGACCA GCGGCATGAC CGGCGAGCTC GGCCAGGCGG ACCTGCGCCG GATGAGCCGC
    12781 GCGGGCATCG GCGGGATCAG CGACGCCGAG GGCATCGCGC TCCTCGACGC CGCCCTCCGC
    12841 GACGACCGCC ACCCGGTCCT GCTGCCCCTG CGGCTCGACG CCGCCGGGCT GCGGGACGCG
    12901 GCCGGGAACG ACCCGGCCGG AATCCCGGCG CTCTTCCGGG ACGTCGTCGG CGCCAGGACC
    12961 GTCCGGGCCC GGCCGTCCGC GGCCTCCGCC TCGACGACAG CCGGGACGGC CGGCACGCCG
    13021 GGGACGGCGG ACGGCGCGGC GGAAACGGCG GCGGTCACGC TCGCCGACCC GGCCGCCACC
    13081 GTGGACGGGC CCGCACGGCA GCGCCTGCTG CTCGAGTTCG TCGTCGGCGA GGTCGCCGAA
    13141 GTACTCGGCC ACGCCCGCGG TCACCGGATC GACGCCGAAC GGGGCTTCCT CGACCTCGGC
    13201 TTCGACTCCC TGACCGCCGT CGAACTCCGC AACCGGCTCA ACTCCGCCGG TGGCCTCGCC
    13261 CTCCCGGCGA CCCTGGTCTT CGACCACCCA AGCCCGGCGG CACTCGCCTC CCACCTGGAC
    13321 GCCGAGCTGC CGCGCGGCGC CTCGGACCAG GACGGAGCCG GGAACCGGAA CGGGAACGAG
    13381 AACGGGACGA CGGCGTCCCG GAGCACCGCC GAGAGGGACG CGCTGCTGGC ACAACTGACC
    13441 CGCCTCGAAG GCGCCTTGGT GCTGACGGGC CTCTCGGACG CCCCCGGGAG CGAAGAAGTC
    13501 CTGGAGCACC TCCGGTCCCT GCGCTCGATG GTCACGGGCG AGACCGGGAC CGGGACCGCG
    13561 TCCGGAGCCC CGGACGGCGC CGGGTCCGGC GCCGAGGACC GGCCCTGGGC GGCCGGGGAC
    13621 GGAGCCGGGG GCGGGAGTGA GGACGGCGCG GGAGTGCCGG ACTTCATGAA CGCCTCGGCC
    13681 GAGGAACTCT TCGGCCTCCT CGACCAGGAC CCCAGCACGG ACTGATCCCT GCCGCACGGT
    13741 CGCCTCCCGC CCCGGACCCC GTCCCGGGCA CCTCGACTCG AATCACTTCA TGCGCGCCTC
    13801 GGGCGCCTCC AGGAACTCAA GGGGACAGCG TGTCCACGGT GAACGAAGAG AAGTACCTCG
    13861 ACTACCTGCG TCGTGCCACG GCGGACCTCC ACGAGGCCCG TGGCCGCCTC CGCGAGCTGG
    13921 AGGCGAAGGC GGGCGAGCCG GTGGCGATCG TCGGCATGGC CTGCCGCCTG CCCGGCGGCG
    13981 TCGCCTCGCC CGAGGACCTG TGGCGGCTGG TGGCCGGCGG CGAGGACGCG ATCTCGGAGT
    14041 TCCCCCAGGA CCGCGGCTGG GACGTGGAGG GCCTGTACGA CCCGAACCCG GAGGCCACGG
    14101 GCAAGAGTTA CGCCCGCGAG GCCGGATTCC TGTACGAGGC GGGCGAGTTC GACGCCGACT
    14161 TCTTCGGGAT CTCGCCGCGC GAGGCCCTCG CCATGGACCC GCAGCAGCGT CTCCTCCTGG
    14221 AGGCCTCCTG GGAGGCGTTC GAGCACGCCG GGATCCCGGC GGCCACCGCG CGCGGCACCT
    14281 CGGTCGGCGT CTTCACCGGC GTGATGTACC ACGACTACGC CACCCGTCTC ACCGATGTCC
    14341 CGGAGGGCAT CGAGGGCTAC CTGGGCACCG GCAACTCCGG CAGTGTCGCC TCGGGCCGCG
    14401 TCGCGTACAC GCTTGGCCTG GAGCGGCCGC CCGTCACGGT CGACACCGCC TGCTCGTCCT
    14461 CGCTGGTCGC CCTGCACCTC GCCGTGCAGG CCCTGCGCAA GGGCGAGGTC GACATGGCGC
    14521 TCGCCGGCGG CGTGACGGTC ATGTCGACGC CCAGCACCTT CGTCGAGTTC AGCCGTCAGC
    14581 GCGGGCTGGC GCCGGACGGC CGGTCGAAGT CCTTCTCGTC GACGGCCGAC GGCACCAGCT
    14641 GGTCCGAGGG CGTCGGCGTC CTCCTCGTCG AGCGCCTGTC CGACGCGCGT CGCAAGGGCC
    14701 ATCGGATCCT CGCCGTGGTC CGGGGCACCG CCGTCAACCA GGACGGCGCC AGCAGCGGCC
    14761 TCACGGCTCC GAACGGGCCG TCGCAGCAGC GCGTCATCCG ACGTGCCCTG GCGGACGCCC
    14821 GGCTCACGAC CTCCGACGTG GACGTCGTCG AGGCCCACGG CACGGGTACG CGACTCGGCG
    14881 ACCCGATCGA GGCGCAGGCC GTCATCGCCA CGTACGGGCA GGGCCGTGAC GGCGAACAGC
    14941 CGCTGCGCCT CGGGTCGTTG AAGTCGAACA TCGGACACAC CCAGGCCCCC GCCGGTGTCT
    15001 CCGGCGTGAT CAAGATGGTC CAGGCGATGC GCCACGGCGT CCTGCCGAAG ACGCTCCACG
    15061 TGGAGAAGCC GACGGACCAG GTGGACTGGT CCGCGGGCGC GGTCGAGCTG CTCACCGAGG
    15121 CCATGGACTG GCCGGACAAG GGCGACGGCG GACTGCGCAG GGCCGCGGTC TCCTCCTTCG
    15181 GCGTCAGCGG GACGAACGCG CACGTCGTGC TCGAAGAGGC CCCGGCGGCC GAGGAGACCC
    15241 CTGCCTCCGA GGCGACCCCG GCCGTCGAGC CGTCGGTCGG CGCCGGCCTG GTGCCGTGGC
    15301 TGGTGTCGGC GAAGACTCCG GCCGCGCTGG ACGCCCAGAT CGGACGCCTC GCCGCGTTCG
    15361 CCTCGCAGGG CCGTACGGAC GCCGCCGATC CGGGCGCGGT CGCTCGCGTA CTGGCCGGCG
    15421 GGCGCGCCGA GTTCGAGCAC CGGGCCGTCG TGCTCGGCAC CGGACAGGAC GATTTCGCGC
    15481 AGGCGCTGAC CGCTCCGGAA GGACTGATAC GCGGCACGCC CTCGGACGTG GGCCGGGTGG
    15541 CGTTCGTGTT CCCCGGTCAG GGCACGCAGT GGGCCGGGAT GGGCGCCGAA CTCCTCGACG
    15601 TGTCGAAGGA GTTCGCGGCG GCCATGGCCG AGTGCGAGAG CGCGCTCTCC CGCTATGTCG
    15661 ACTGGTCGCT GGAGGCCGTC GTCCGGCAGG CGCCGGGCGC GCCCACGCTG GAGCGGGTCG
    15721 ACGTCGTCCA GCCCGTGACC TTCGCTGTCA TGGTTTCGCT GGCGAAGGTC TGGCAGCACC
    15781 ACGGCGTGAC GCCGCAGGCC GTCGTCGGCC ACTCGCAGGG CGAGATCGCC GCCCCGTACG
    15841 TCGCCGGTGC CCTCACCCTC GACGACGCCG CCCGCGTCGT CACCCTGCGC AGCAAGTCCA
    15901 TCGCCGCCCA CCTCGCCGGC AAGGGCGGCA TGATCTCCCT CGCCCTCAGC GAGGAAGCCA
    15961 CCCGGCAGCG CATCGAGAAC CTCCACGGAC TGTCGATCGC CGCCGTCAAC GGCCCCACCG
    16021 CCACCGTGGT TTCGGGCGAC CCCACCCAGA TCCAAGAGCT CGCTCAGGCG TGTGAGGCCG
    16081 ACCGGGTCCG CGCACGGATC ATCCCCGTCG ACTACGCCTC CCACAGCGCC CACGTCGAGA
    16141 CCATCGAGAG CGAAGTCGCC GAGGTCCTCG CCGGGCTCAG CCCGCGGACA CCTGAGGTGC
    16201 CGTTCTTCTC GACACTCGAA GGCGCCTGGA TCACCGAGCC GGTGCTCGAC GGCACCTACT
    16261 GGTACCGCAA CCTCCGCCAC CGCGTCGGCT TCGCCCCCGC CGTCGAGACC CTCGCCACCG
    16321 ACGAAGGCTT CACCCACTTC ATCGAGGTCA GCGCCCACCC CGTCCTCACC ATGACCCTCC
    16381 CCGAGACCGT CACCGCCCTC GGCACCCTCC GCCGCGAACA GGGAGGCCAG GAGCGTCTGG
    16441 TCACCTCACT CGCCGAAGCC TGGACCAACG GCCTCACCAT CGACTGGGCG CCCGTCCTCC
    16501 CCACCGCAAC CGGCCACCAC CCCGAGCTCC CCACCTACGC CTTCCAGCGC CGTCACTACT
    16561 GGCTCCACGA CTCCCCCGCC GTCCAGGGCT CCGTGCAGGA CTCCTGGcGC TACCGCATCG
    16621 ACTGGAAGCG CCTCGCGGTC GCCGACGCGT CCGAGCGCGC CGGGCTGTCC GGGCGCTGGC
    16681 TCGTCGTCGT CCCCGAGGAC CGTTCCGCCG AGGCCGCCCC GGTGCTCGCC GCGCTGTCCG
    16741 GCGCCGGCGC CGACCCCGTA CAGCTGGACG TGTCCCCGCT GGGCGACCGG CAGCGGCTCG
    16801 CCGCGACCCT GGGCGAGGCC CTGGCGGCGG CCGGTGGAGC CGTCGACGGC GTCCTCTCGC
    16861 TGCTCGCGTG GGACGAGAGC GCGCACCCCG GCCACCCCGC CCCCTTCACC CGGGGCACCG
    16921 GCGCCACCCT CACCCTGGTG CAGGCGCTGG AGGACGCCGG CGTCGCCGCC CCGCTGTGGT
    16981 GCGTGACCCA CGGCGCGGTG TCCGTCGGCC GGGCCGACCA CGTCACCTCC CCCGCCCAGG
    17041 CCATGGTGTG GGGCATGGGC CGGGTCGCCG CCCTGGAGCA CCCCGAGCGG TGGGGCGGCC
    17101 TGATCGACCT GCCCTCGGAC GCCGACCGGG CGGCCCTGGA CCGCATGACC ACGGTCCTCG
    17161 CCGGCGGTAC GGGTGAGGAC CAGGTCGCGG TACGCGCCTC CGGGCTGCTC GCCCGCCGCC
    17221 TCGTCCGCGC CTCCCTCCCG GCGCACGGCA CGGCTTCGCC GTGGTGGCAG GCCGACGGCA
    17281 CGGTGCTCGT CACCGGTGCC GAGGAGCCTG CGGCCGCCGA GGCCGCACGC CGGCTGGCCC
    17341 GCGACGGCGC CGGACACCTC CTCCTCCACA CCACCCCCTC CGGCACCGAA GGCGCCGAAG
    17401 GCACCTCCGG TGCCGCCGAG GACTCCGGCC TCGCCGGGCT CGTCGCCGAA CTCGCGGACC
    17461 TGGGCGCGAC GGCCACCGTC GTGACCTGCG ACCTCACGGA CGCGGAGGCG GCCGCCCGGC
    17521 TGCTCGCCGG CGTCTCCGAC GCGCACCCGC TCAGCGCCGT CCTCCACCTG CCGCCCACCG
    17581 TCGACTCCGA GCCGCTCGCC GCGACCGACG CGGACGCGCT CGCCCGTGTC GTGACCGCGA
    17641 AGGCCACCGC CGCGCTCCAC CTGGACCGCC TCCTGCGGGA GGCCGCGGCT GCCGGAGGCC
    17701 GTCCGCCCGT CCTGGTCCTC TTCTCCTCGG TCGCCGCGAT CTGGGGCGGC GCCGGTCAGG
    17761 GCGCGTACGC CGCCGGTACG GCCTTCCTCG ACGCCCTCGC CGGTCAGCAC CGGGCCGACG
    17821 GCCCCACCGT GACCTCGGTG GCCTGGAGCC CCTGGGAGGG CAGCCGCGTC ACCGAGGGTG
    17881 CGACCGGGGA GCGGCTGCGC CGCCTCGGCC TGCGCCCCCT CGCCCCCGCG ACGGCGCTCA
    17941 CCGCCCTGGA CACCGCGCTC GGCCACGGCG ACACCGCCGT CACGATCGCC GACGTCGACT
    18001 GGTCGAGCTT CGCCCCCGGC TTCACCACGG CCCGGCCGGG CACCCTCCTC GCCGATCTGC
    18061 CCGAGGCGCG CCGCGCGCTC GACGAGCAGC AGTCGACGAC GGCCGCCGAC GACACCGTCC
    18121 TGAGCCGCGA GCTCGGTGCG CTCACCGGCC CCGAACAGCA GCGCCGTATG CAGGAGTTGG
    18181 TCCGCGAGCA CCTCGCCGTG GTCCTCAACC ACCCCTCCCC CGAGGCCGTC GACACGGGGC
    18241 GGGCCTTCCG TGACCTCGGA TTCGACTCGC TGACGGCGGT CGAGCTCCGC AACCGCCTCA
    18301 AGAACGCCAC CGGCCTGGCC CTCCCGGCCA CTCTGGTCTT CGACTACCCG ACCCCCCGGA
    18361 CGCTGGCGGA GTTCCTCCTC GCGGAGATCC TGGGCGAGCA GGCCGGTGCC GGCGAGCAGC
    18421 TTCCGGTGGA CGGCGGGGTC GACGACGAGC CCGTCGCGAT CCTCGGCATG GCGTGCCCCC
    18481 TGCCGGGCGG TGTCGCCTCG CCGGAGGACC TGTGGCGGCT GGTGGCCGGC GGCGAGGACG
    18541 CGATCTCCGG CTTCCCGCAG GACCGCGGCT GGGACGTGGA GGGGCTGTAC GACCCGGACC
    18601 CGGACGCGTC CGGGCGGACG TACTGCCGTG CCGGTGGCTT CCTCGACGAG CCGGGCGACT
    18661 TCGACGCCGA CTTCTTCGGG ATCTCGCCGC GCGAGGCCCT CGCCATGGAC CCGCAGCAGC
    18721 GGCTCCTCCT GGAGACCTCC TGGGAGGCCG TCGAGGACGC CGGGATCGAC CCGACCTCCC
    18781 TTCAGGGGCA GCAGGTCGGC GTGTTCGCGG GCACCAACGG CCCCCACTAC GAGCCGCTGC
    18841 TCCGCAACAC CGCCGAGGAT CTTGAGGGTT ACGTCGGGAC GGGCAACGCC GCCAGCATCA
    18901 TGTCGGGCCG TGTCTCGTAC ACCCTCGGCC TGGAGGGCCC GGCCGTCACG GTCGACACCG
    18961 CCTGCTCCTC CTCGCTGGTC GCCCTGCACC TCGCCGTGCA GGCCCTGCGC AAGGGCGAAT
    19021 GCGGACTGGC GCTCGCGGGC GGTGTGACGG TCATGTCGAC GCCCACGACG TTCGTGGAGT
    19081 TCAGCCGCCA GCGCGGGCTC GCGGAGGACG GCCGGTCGAA GGCGTTCGCC GCGTCGGCGG
    19141 ACGGCTTCGG CCCGGCGGAG GGCGTCGGCA TGCTCCTCGT CGAGCGCCTG TCGGACGCCC
    19201 GCCGCAACGG ACACCGTGTG CTGGCGGTCG TGCGCGGCAG CGCGGTCAAC CAGGACCGCG
    19261 CGAGCAACGG CCTGACCGCC CCGAACGGGC CCTCGCAGCA GCGCGTCATC CGGCGCGCGC
    19321 TCGCGGACGC CCGACTGACG ACCGCCGACG TGGACGTCGT CGAGGCCCAC GGCACGGGCA
    19381 CGCGACTCGG CGACCCGATC GAGGCACAGG CCCTCATCGC CACCTACGGC CAGGGGCGCG
    19441 ACACCGAACA GCCGCTGCGC CTGGGGTCGT TGAAGTCCAA CATCGGACAC ACCCAGGCCG
    19501 CCGCCGGTCT CTCCGGCATC ATCAAGATGG TCCAGGCGAT GCGCCACGGC GTCCTGCCGA
    19561 AGACGCTCCA CGTGGACCGG CCGTCGGACC AGATCGACTG GTCGGCGGGC ACGGTCGAGC
    19621 TGCTCACCGA GGCCATGGAC TGGCCGAGGA AGCAGGAGGG CGCGCTGCGC CGCGCGGCCG
    19681 TCTCCTCCTT CGGCATCAGC GGCACGAACG CGCACATCGT GCTCGAAGAA GCCCCGGTCG
    19741 ACGAGGACGC CCCGGCGGAC GAGCCGTCGG TCGGCGGTGT GGTGCCGTGG CTCGTGTCCG
    19801 CGAAGACTCC GGCCGCGCTG GACGCCCAGA TCGGACGCCT CGCCGCGTTC GCCTCGCAGG
    19861 GCCGTACGGA CGCCGCCGAT CCGGGCGCGG TCOCTCGCGT ACTGGCCGGC GGGCGTGCGC
    19921 AGTTCGAGCA CCGGGCCGTC GCGCTCGGCA CCGGACAGGA CGACCTGGCG GCCGCACTGG
    19981 CCGCGCCTGA GGGTCTGGTC CGGGGTGTGG CCTCCGGTGT GGGTCGAGTG GCGTTCGTGT
    20041 TCCCGGGACA GGGCACGCAG TGGGCCGGGA TCGGTGCCGA ACTCCTCGAC GTGTCGAAGG
    20101 AGTTCGCGGC GGCCATGGCC GAGTGCGAGG CCGCGCTCGC TCCGTACGTG GACTGGTCGC
    20161 TGGAGGCCGT CGTCCGACAG GCCCCCGGCG CGCCCACGCT GGAGCGGGTC GATGTCGTCC
    20221 AGCCCGTGAC GTTCGCCGTC ATGGTCTCGC TGGCGAAGGT CTGGCAGCAC CACGGGGTGA
    20281 CCCCGCAAGC CGTCGTCGGC CACTCGCAGG GCGAGATCGC CGCCGCGTAC GTCGCCGGTG
    20341 CCCTGAGCCT GGACGACGCC GCTCGTGTCG TGACCCTGCG CAGCAAGTCC ATCGGCGCGC
    20401 ACCTCGCGGG CCAGGGCGGC ATGCTOTCCC TCGCGCTGAG COAGGCGGCC GTTGTGGAGG
    20461 GACTGGCCGG GTTCGACGGG CTGTCCGTCG CCGCCGTCAA CGGGCCTACC GCCACCGTGG
    20521 TTTCGGGCGA CCCGACCCAG ATCCAAGAGC TCGCTCAGGC GTGTGAGGCC GACGGGGTCC
    20581 GCGCACGGAT CATCCCCGTC GACTACGCCT CCCACAGCGC CCACGTCGAG ACCATCGAGA
    20641 GCGAACTCGC CGACGTCCTG GCGGGGTTGT CCCCCCAGAC ACCCCAGGTC CCCTTCTTCT
    20701 CCACCCTCGA AGGCGCCTGG ATCACCGAAC CCGCCCTCGA CGGCGGCTAC TGGTACCGCA
    20761 ACCTCCGCCA TCGTGTGGGC TTCGCCCCGG CCGTCGAAAC CCTGGCCACC GACGAAGGCT
    20821 TCACCCACTT CGTCGAGGTC AGCGCCCACC CCGTCCTCAC CATGGCCCTG CCCGAGACCG
    20881 TCACCGGCCT CGGCACCCTC CGCCGTGACA ACGGCGGACA GCACCGCCTC ACCACCTCCC
    20941 TCGCCGAGGC CTGGGCCAAC GGCCTCACCG TCGACTGGGC CTCTCTCCTC CCCACCACGA
    21001 CCACCCACCC CGATCTGCCC ACCTACCCCT TCCAGACCGA GCGCTACTGG CCGCAGCCCG
    21061 ACCTCTCCGC CGCCGGTGAC ATCACCTCCG CCGGTCTCGG GGCGGCCGAG CACCCGCTGC
    21121 TCGGCGCGGC CGTGGCGCTC GCGGACTCCG ACGGCTGCCT GCTCACGGGG AGCCTCTCCC
    21181 TCCGTACGCA CCCCTGGCTG GCGGACCACG CGGTGGCCGG CACCGTGCTG CTGCCGGGAA
    21241 CCGCGTTCGT GGAGCTGGCG TTCCGAGCCG GGGACCAGCT CGGTTGCGAT CTGGTCGAGG
    21301 AGCTCACCCT CGACGCGCCG CTCGTGCTGC CCCGTCGTGG CGCGGTCCGT GTGCAGCTGT
    21361 CCGTCGGCGC GAGCGACGAC TCCGGGCGTC GTACCTTCGG GCTCTACGCG CACCCGGAGG
    21421 ACGCGCCGGG CGAGGCGGAG TGGACGCGGC ACGCCACCGG TGTGCTGGCC GCCCGTGCGG
    21481 ACCGCACCGC CCCCGTCGCC GACCCGGAGG CCTGGCCGCC GCCGGGCGCC GAGCCGGTGG
    21541 ACGTGGACGG TCTGTACGAG CGCTTCGCGG CGAACGGCTA CGGCTACGGC CCCCTCTTCC
    21601 AGGGCGTCCG TGGTGTCTGG CGGCGTGGCG ACGAGGTGTT CGCCGACGTG GCCCTGCCGG
    21661 CCGAGGTCGC CGGTGCCGAG GGCGCGCGGT TCGGCCTTCA CCCGGCGCTG CTCGACGCCG
    21721 CCGTGCAGGC GGCCGGTGCG GGCGGGGCGT TCGGCGCGGG CACGCGGCTG CCGTTCGCCT
    21781 GGAGCGGGAT CTCCCTGTAC GCGGTCGGCG CCACCGCCCT CCGCGTGCGG CTGGCCCCCG
    21841 CCGGCCCGGA CACGGTGTCC GTGAGCGCCG CCGACTCCTC CGCGCAGCCG GTGTTCGCCG
    21901 CGGACTCCCT CACGGTGCTG CCCGTCGACC CCGCGCAGCT GGCGGCCTTC AGCGACCCGA
    21961 CTCTGGACGC GCTGCACCTC CTGGAGTGGA CCGCCTGGGA CGGTGCCGCG CAGGCCCTGC
    22021 CCGGCGCGGT CGTGCTGGGC GGCGACGCCG ACGGTCTCGC CGCGGCGCTG CGCGCCGGTG
    22081 GCACCGAGGT CCTGTCCTTC CCGGACCTTA CGGACCTGGT GGAGGCCGTC GACCGGGGCG
    22141 AGACCCCGGC CCCGGCGACC GTCCTGGTGG CCTGCCCCGC CGCCGGCCCC GGTGGGCCGG
    22201 AGCATGTCCG CGAGGCCCTG CACGGGTCGC TCGCGCTGAT GCAGGCCTGG CTGGCCGACG
    22261 AGCGGTTCAC CGATGGGCGC CTGGTGCTCG TGACCCGCGA CGCGGTCGCC GCCCGTTCCG
    22321 GCGACGGCCT GCGGTCCACG GGACAGGCcG CCGTCTGGGG CCTCGGCCGG TCCGCGCAGA
    22381 CGGAGAGCCC GGGCCGGTTC GTCCTGCTCG ACCTCGCCGG GGAAGCCCGG ACGGCCGGGG
    22441 ACGCCACCGC CGGGGACGGC CTGACGACCG GGGACGCCAC CGTCGGCCGC ACCTCTGGAG
    22501 ACGCCGCCCT CGGCAGCGCC CTCGCGACCG CCCTCGGCTC GGGCGAGCCG CAGCTCGCCC
    22561 TCCGGGACGG GGCGCTCCTC GTACCCCGCC TGGCGCGGGC CGCCGCGCCC GCCGCGGCCG
    22621 ACGGCCTCGC CGCGGCCGAC GGCCTCGCCG CTCTGCCGCT GCCCGCCGCT CCGGCCCTCT
    22681 GGCGTCTGGA GCCCGGTACG GACGGCAGCC TGGAGAGCCT CAGGGCGGCG CCCGGCGACG
    22741 CCGAGACCCT CGCCCCGGAG CCGCTCGGCC CGGGACAGGT CCGCATCGCG ATCCGGGCCA
    22801 CCGGTCTCAA CTTCCGCGAC GTCCTGATCG CCCTCGGCAT GTACCCCGAT CCGGCGCTGA
    22861 TGGGCACCGA GGGAGCCGGC GTGGTCACCG CGACCGGCCC CGGCGTCACG CACCTCGCCC
    22921 CCGGCGACCG GGTCATGGGC CTGCTCTCCG GCGCGTACGC CCCGGTCGTC GTGGCGGACG
    22981 CGCGGACCGT CGCGCGGATG CCCGAGGGGT GGACGTTCGC CCAGGGCGCC TCCGTGCCGG
    23041 TGGTGTTCCT GACGGCCGTC TACGCCCTGC GCGACCTGGC GGACGTCAAG CCCGGCGAGC
    23101 GCCTCCTCGT CCACTCCGCC GCCGGTGGCG TGGGCATGGC CGCCGTGCAG CTCGCCCGGC
    23161 ACTGGGGCGT GGAGGTCCAC GGCACGGCGA CTCACGGGAA GTGGGACGCC CTGCGCGCGC
    23221 TCGGCCTGGA CGACGCGCAC ATCGCCTCCT CCCGCACCCT GGACTTCGAG TCCGCGTTCC
    23281 GTGCCGCTTC CGGCGGGGCG GGCATGGACG TCGTACTGAA CTCGCTCGCC CGCGAGTTCG
    23341 TCGACGCCTC GCTGCGCCTG CTCGGGCCGG GCGGCCGGTT CGTGGAGATG GGGAAGACCG
    23401 ACGTCCGCGA CGCGGAGCGG GTCGCCGCCG ACCACCCCGG TGTCGGCTAC CGCGCCTTCG
    23461 ACCTGGGCGA GGCCGGGCCG GAGCGGATCG GCGAGATGCT CGCCGAGGTC ATCGCCCTCT
    23521 TCGAGGACGG GGTGCTCCGG CACCTGCCCG TCACGACCTG GGACGTGCGC CGGGCCCGCG
    23581 ACGCCTTCCG GCACGTCAGC CAGGCCCGCC ACACGGGCAA GGTCGTCCTC ACGATGCCGT
    23641 CGGGCCTCGA CCCGGAGGGT ACGGTCCTGC TGACCGGCGG CACCGGTGCG CTGGGGGGCA
    23701 TCGTGGCCCG GCACGTGGTG GGCGAGTGGG GCGTACGACG CCTGCTGCTC GTGAGCCGGC
    23761 GGGGCACGGA CGCCCCGGGC GCCGGCGAGC TCGTGCACGA GCTGGAGGCC CTGGGAGCCG
    23821 ACGTCTCGGT GGCCGCGTGC GACGTCGCCG ACCGCGAAGC CCTCACCGCC GTACTCGACT
    23881 CGATCCCCGC CGAACACCCG CTCACCGCGG TCGTCCACAC GGCAGGCGTC CTCTCCGACG
    23941 GCACCCTCCC CTCGATGACA GCGGAGGATG TGGAACACGT ACTGCGTCCC AAGGTCGACG
    24001 CCGCGTTCCT CCTCGACGAA CTCACCTCGA CGCCCGGCTA CGACCTGGCA GCGTTCGTCA
    24061 TGTTCTCCTC CGCCGCCGCC GTCTTCGGTG GCGCGGGGCA GGGCGCCTAC GCCGCCGCCA
    24121 ACGCCACCCT CGACGCCCTC GCCTGGCGCC GCCGGACAGC CGGACTCCCC GCCCTCTCCC
    24181 TCGGCTGGGG CCTCTGGGCC GAGACCAGCG GCATGACCGG CGGACTCAGC GACACCGACC
    24241 GCTCGCGGCT GGCCCGTTCC GGGGCGACGC CCATGGACAG CGAGCTGACC CTCTCCCTCC
    24301 TGGACGCGGC CATGCGCCGC GACGACCCGG CGCTCGTCCC GATCGCCCTG GACGTCGCCG
    24361 CGCTCCGCGC CCAGCAGCGC GACGGCATGC TGGCGCCGCT GCTCAGCGGG CTCACCCGCG
    24421 GATCGCGGGT CGGCGCCGCG CCGGTCAACC AGCGCAGGGC AGCCGCCGGA GGCGCGGGCG
    24481 AGGCGGACAC GGACCTCGGC GGGCGGCTCG CCGCGATGAC ACCGGACGAC CGGGTCGCGC
    24541 ACCTGCGGGA CCTCGTCCGT ACGCACGTGG CGACCGTCCT GGGACACGGC ACCCCGAGCC
    24601 GGGTGGACCT GGAGCGGGCC TTCCGCGACA CCGGTTTCGA CTCGCTCACC GCCGTCGAAC
    24661 TCCGCAACCG TCTCAACGCC GCGACCGGGC TGCGGCTGCC GGCCACGCTG GTCTTCGACC
    24721 ACCCCACCCC GGGGGAGCTC GCCGGGCACC TGCTCGACGA ACTCGCCACG GCCGCGGGCG
    24781 GGTCCTGGGC CGAAGGCACC GGGTCCGGAG ACACGGCCTC GGCGACCGAT CGGCAGACCA
    24841 CGGCGGCCCT CGCCGAACTC GACCGCCTGG AAGGCGTGCT CGCCTCCCTC GCGCCCGCCG
    24901 CCGGCGGCCG TCCGGAGCTC GCCGCCCCGC TCAGGGCGCT GGCCGCGGCC CTGGGGGACG
    24961 ACGGCGACGA CGCCACCGAC CTGGACGAGG CGTCCGACGA CGACCTCTTC TCCTTCATCG
    25021 ACAAGGAGCT GGGCGACTCC GACTTCTGAC CTGCCCGACA CCACCGGCAC CACCGGCACC
    25081 ACCAGCCCCC CTCACACACG GAACACGGAA CGGACAGGCG AGAACGGGAG CCATGGCGAA
    25141 CAACGAAGAC AAGCTCCGCG ACTACCTCAA GCGCGTCACC GCCGAGCTGC AGCAGAACAC
    25201 CAGGCGTCTG CGCGAGATCG AGGGACGCAC GCACGAGCCG GTGGCGATCG TGGGCATGGC
    25261 CTGCCGCCTG CCGGGCGGTG TCGCCTCGCC CGAGGACCTG TGGCAGCTGG TGGCCGGGGA
    25321 CGGGGACGCG ATCTCGGAGT TCCCGCAGGA CCGCGGCTCG GACGTGGAGG GGCTGTACGA
    25381 CCCCGACCCG GACGCGTCCG GCAGGACGTA CTGCCGGTCC GGCGGATTCC TGCACGACGC
    25441 CGGCGAGTTC GACGCCGACT TCTTCGGGAT CTCGCCGCGC GAGGCCCTCG CCATGGACCC
    25501 GCAGCAGCGA CTGTCCCTCA CCACCGCGTG GGAGGCGATC GAGAGCGCGG GCATCGACCC
    25561 GACGGCCCTG AAGGGCAGCG GCCTCGGCGT CTTCGTCGGC GGCTGGCACA CCGGCTACAC
    25621 CTCGGGGCAG ACCACCGCCG TGCACTCGCC CGAGCTGGAG GGCCACCTGG TCAGCGGCGC
    25681 GGCGCTGGGC TTCCTGTCCG GCCGTATCGC GTACGTCCTC GGTACGGACG GACCGGCCCT
    25741 GACCGTGGAC ACGGCCTGCT CGTCCTCGCT GGTCGCCCTG CACCTCGCCG TGCAGGCCCT
    25801 CCGCAAGGGC GAGTGCGACA TGGCCCTCGC CGGTGGTGTC ACGGTCATGC CCAACGCGGA
    25861 CCTGTTCGTG CAGTTCAGCC GGCAGCGCGG GCTGGCCGCG GACGGCCGGT CGAAGGCGTT
    25921 CGCCACCTCG GCGGACGGCT TCGGCCCCGC GGAGGGCGCC GGAGTCCTGC TGGTGGAGCG
    25981 CCTGTCGGAC GCCCGCCGCA ACGGACACCG GATCCTCGCG GTCGTCCGCG GCAGCGCGGT
    26041 CAACCAGGAC GGCGCCAGCA ACGGCCTCAC GGCTCCGCAC GGGCCCTCCC AGCAGCGCGT
    26101 CATCCGACGG GCCCTGGCGG ACGCCCGGCT CGCGCCGGGT GACGTGGACG TCGTCGAGGC
    26161 GCACGGCACG GGCACGCGGC TCGGCCACCC GATCGAGGCG CAGGCCCTCA TCGCCACCTA
    26221 CGGCCAGGAG AAGAGCAGCG AACAGCCGCT GAGGCTGGGC GCGTTGAAGT CGAACATCGG
    26281 GCACACGCAG GCCGCGGCCG GTGTCGCAGG TGTCATCAAG ATGGTCCAGG CGATGCGCCA
    26341 CGGACTGCTG CCGAAGACGC TGCACGTCGA CGAGCCCTCG GACCAGATCG ACTGGTCGGC
    26401 GGGCACGGTG GAACTCCTCA CCGAGGCCGT CGACTGGCCG GAGAAGCAGG ACGGCGGGCT
    26461 CCGCCGCGCG GCTGTCTCCT CCTTCGGCAT CAGCGGGACG AACGCGCACG TCGTCCTGGA
    26521 GGAGGCCCCG GCGGTCGAGG ACTCCCCGGC CGTCGAGCCG CCCCCCGGTG GCGGTGTGGT
    26581 GCCGTGGCCG GTGTCCGCGA AGACTCCGGC CGCGCTGGAC GCCCAGATCG GGCAGCTCGC
    26641 CGCGTACGCG GACGGTCGTA CGGACGTGGA TCCGGCGGTG GCCGCCCGCG CCCTGGTCGA
    26701 CAGCCGTACG GCGATCGAGC ACCGCGCGGT CGCGGTCGGC GACAGCCGGG AGGCACTGCG
    26761 GGACGCCCTG CGGATGCCGG AAGGACTCGT ACGCGGCACG TCCTCGGACG TGGGCCCGGT
    26821 GGCGTTCGTC TTCCCCGGCC AGGGCACGCA GTGGGCCGGC ATGGGCGCCG AACTCCTTGA
    26881 CAGCTCACCG GAGTTCGCTG CCTCGATGGC CGAATGCGAG ACCGCGCTCT CCCGCTACGT
    26941 CGACTGGTCT CTTGAAGCCG TCGTCCGACA GGAACCCGGC GCACCCACGC TCGACCGCGT
    27001 CGACGTCGTC CAGCCCGTGA CCTTCGCTGT CATGGTCTCG CTGGCGAAGG TCTGGCAGCA
    27061 CCACGGCATC ACCCCCCAGG CCGTCGTCGC CCACTCGCAG GGCGAGATCG CCGCCGCGTA
    27121 CGTCGCCGGT GCACTCACCC TCGACGACGC CGCCCGCGTC GTCACCCTGC GCAGCAAGTC
    27181 CATCGCCGCC CACCTCGCCG GCAAGGGCGG CATGATCTCC CTCGCCCTCG ACGAGGCGGC
    27241 CGTCCTGAAG CGACTGAGCG ACTTCGACGG ACTCTCCGTC GCCGCCGTCA ACGGCCCCAC
    27301 CGCCACCGTC GTCTCCGGCG ACCCGACCCA GATCGAGGAA CTCGCCCGCA CCTGCGAGGC
    27361 CGACGGCGTC CGTGCGCGGA TCATCCCGGT CGACTACGCC TCCCACAGCC GGCAGGTCGA
    27421 GATCATCGAG AAGGAGCTGG CCGAGGTCCT CGCCGGACTC GCCCCGCAGG CTCCGCACGT
    27481 GCCGTTCTTC TCCACCCTCG AAGGCACCTG GATCACCGAG CCGGTGCTCG ACGGCACCTA
    27541 CTGGTACCGC AACCTGCGCC ATCGCGTGGG CTTCGCCCCC GCCGTGGAGA CCTTGGCGGT
    27601 TGACGGCTTC ACCCACTTCA TCGAGGTCAG CCCCCACCCC GTCCTCACCA TGACCCTCCC
    27661 CCAGACCGTC ACCGGCCTCG GCACCCTCCG CCGCGAACAG GGAGGCCAGG AGCGTCTGGT
    27721 CACCTCACTC GCCGAAGCCT GGGCCAACGG CCTCACCATC GACTGGGCGC CCATCCTCCC
    27781 CACCGCAACC GGCCACCACC CCGAGCTCCC CACCTACGCC TTCCAGACCG AGCCCTTCTG
    27841 GCTGCAGAGC TCCGCGCCCA CCAGCGCCGC CGACGACTGG CGTTACCGCG TCGAGTGGAA
    27901 GCCGCTGAcG GCCTCCGGCC AGGCGGACCT GTCCGGGCGG TGGATCGTCG CCGTCGGGAG
    27961 CGAGCCAGAA GCCGAGCTGC TGGGCGCGCT GAAGGCCGCG GGAGCGGAGG TCGACGTACT
    28021 GGAAGCCGGG GCGGACGACG ACCGTGAGGC CCTCGCCGCC CGGCTCACCG CACTGACGAC
    28081 CGGCGACGGC TTCACCGGCG TGGTCTCGCT CCTCGACGAC CTCGTGCCAC AGGTCGCCTG
    28141 GGTGCAGGCA CTCGGCGACG CCGGAATCAA GGCGCCCCTG TGGTCCGTCA CCCAGGGCGC
    28201 GGTCTCCGTC GGACGTCTCG ACACCCCCGC CGACCCCGAC CGGGCCATGC TCTGGGGCCT
    28261 CCGCCGCGTC GTCGCCCTTG AGCACCCCGA ACGCTGGGCC GGCCTCGTCG ACCTCCCCGC
    28321 CCAGCCCGAT GCCGCCGCCC TCGCCCACCT CGTCACCGCA CTCTCCGGCG CCACCGGCGA
    28381 GGACCAGATC GCCATCCGCA CCACCGGACT CCACGCCCGC CGCCTCGCCC GCGCACCCCT
    28441 CCACGGACGT CGGCCCACCC GCGACTGGCA GCCCCACGGC ACCGTCCTCA TCACCGGCGG
    28501 CACCGGAGCC CTCGGCAGCC ACGCCGCACG CTGGATGGCC CACCACGGAG CCGAACACCT
    28561 CCTCCTCGTC AGCCGCAGCG GCGAACAAGC CCCCGGAGCC ACCCAACTCA CCGCCGAACT
    28621 CACCGCATCG GGCGCCCGCG TCACCATCGC CGCCTGCGAC GTCGCCGACC CCCACGCCAT
    28681 GCGCACCCTC CTCGACGCCA TCCCCGCCGA GACGCCCCTC ACCGCCGTCG TCCACACCGC
    28741 CGGCGCACCG GGCGGCGATC CGCTGGACGT CACCGGCCCG GAGGACATCG CCCGCATCCT
    28801 GGGCGCGAAG ACGAGCGGCG CCGAGGTCCT CGACGACCTG CTCCGCGGCA CTCCGCTGGA
    28861 CGCCTTCGTC CTCTACTCCT CGAACGCCGG GGTCTGGGGC AGCGGCAGCC AGGGCGTCTA
    28921 CGCGGCGGCC AACGCCCACC TCGACGCGCT CGCCGCCCGG CGCCGCGCCC GGGGCGAGAC
    28981 GGCGACCTCG GTCGCCTGGG GCCTCTGGGC CGGCGACGGC ATGGGCCGGG GCGCCGACGA
    29041 CGCGTACTGG CAGCGTCGCG GCATCCGTCC GATGAGCCCC GACCGCGCCC TGGACGAACT
    29101 GGCCAAGGCC CTGAGCCACG ACGAGACCTT CGTCGCCGTG GCCGATGTCG ACTGGGAGCG
    29161 GTTCGCGCCC GCGTTCACGG TGTCCCGTCC CAGCCTTCTC CTCGACGGCG TCCCGGAGGC
    29221 CCGCCAGGCG CTCGCCGCAC CCGTCGGTGC CCCGGCTCCC GGCGACGCCG CCGTGGCGCC
    29281 GACCGGGCAG TCGTCGGCGC TGGCCGCGAT CACCGCGCTC CCCGAGCCCG AGCGCCGGCC
    29341 GGCCCTCCTC ACCCTCGTCC GTACCCACGC GGCGGCCGTA CTCGGCCATT CCTCCCCCGA
    29401 CCGGGTGGcc CCCGGCCGTG CCTTCACCGA GCTCGGCTTC GACTCGCTGA CGGCCGTGCA
    29461 GCTCCGCAAC CAGCTCTCCA CGGTGGTCGG CAACAGGCTC CCCGCCACCA CGGTCTTCGA
    29521 CCACCCCACG CCCGCCGCAC TCGCCGCGCA CCTCCACGAG GCGTACCTCG CACCGGCCGA
    29581 GCCGGCCCCG ACGGACTGGG AGGGGCGGGT GCGCCGGGCC CTGGCCGAAC TGCCCCTCGA
    29641 CCGGCTGCGG GACGCGGGGG TCCTCGACAC CGTCCTGCGC CTCACCGGCA TCGAGCCCGA
    29701 GCCGGGTTCC GGCGGTTCGG ACGGCGGCGC CGCCGACCCT GGTGCGGAGC CGGAGGCGTC
    29761 GATCGACGAC CTGGACGCCG AGGCCCTGAT CCGGATGGCT CTCGGCCCCC GTAACACCTG
    29821 ACCCGACCGC GGTCCTGCCC CACGCGCCGC ACCCCGCGCA TCCCGCGCAC CACCCGCCCC
    29881 CACACGCCCA CAACCCCATC CACGAGCGGA AGACCACACC CAGATGACGA GTTCCAACGA
    29941 ACAGTTGGTG GACGCTCTGC GCGCCTCTCT CAAGGAGAAC GAAGAACTCC GGAAAGAGAG
    30001 CCGTCGCCGG GCCGACCGTC GGCAGGAGCC CATGGCGATC GTCGGCATGA GCTGCCGGTT
    30061 CGCGGGCGGA ATCCGGTCCC CCGAGGACCT CTGGGACGGC GTCGCCGCGG GCAAGGACCT
    30121 GGTCTCCGAG GTACCGGAGG AGCGCGGCTG GGACATCGAC TCCCTCTACG ACCCGGTGCC
    30181 CGGGCGCAAG GGCACGACGT ACGTCCGCAA CGCCGCGTTC CTCGACGACG CCGCCGGATT
    30241 CGACGCGGCC TTCTTCGGGA TCTCGCCGCG CGAGGCCCTC GCCATGGACC CGCAGCAGCG
    30301 GCAGCTCCTC GAAGCCTCCT GGGAGGTCTT CGAGCGGGCC GGCATCGACC CCGCGTCGGT
    30361 CCGCGGCACC GACGTCGGCG TGTACGTGGG CTGTGGCTAC CAGGACTACG CGCCGGACAT
    30421 CCGGGTCGCC CCCGAAGGCA CCGGCGGTTA CGTCGTCACC GGCAACTCCT CCGCCGTGGC
    30481 CTCCGGGCGC ATCGCGTACT CCCTCGGCCT GGAGGGACCC GCCGTGACCG TGGACACGGC
    30541 GTGCTCCTCT TCGCTCGTCG CCCTGCACCT CGCCCTGAAG GGCCTGCGGA ACGGCGACTG
    30601 CTCGACGGCA CTCGTGGGCG GCGTGGCCGT CCTCGCGACG CCGGGCGCGT TCATCGAGTT
    30661 CAGCAGCCAG CAGGCCATGG CCGCCGACGG CCGGACCAAG GGCTTCGCCT CGGCGGCGCA
    30721 CGGCCTCGCC TGGGGCGAGG GCGTCGCCGT ACTCCTCCTC GAACGGCTCT CCGACGCGCG
    30781 GCGCAAGGGC CACCGGGTCC TGCCCGTCGT GCGCGGCAGC GCCATCAACC AGGACGGCGC
    30841 GAGCAACGGC CTCACGGCTC CGCACGGGCC CTCCCAGCAG CGCCTGATCC GCCAGGCCCT
    30901 GGCCGACGCG CGGCTCACGT CGAGCGACGT GGACGTCGTG GAGGGCCACG GCACGGGGAC
    30961 CCGTCTCGGC GACCCGATCG AGGCGCAGGC GCTCCTCGCC ACGTACGGGC AGGGGCGCGC
    31021 CCCGGGGCAG CCGCTGCGGC TGGGGACGCT GAAGTCGAAC ATCGGGCACA CGCAGGCCGC
    31081 TTCGGGTGTC GCCGGTGTCA TCAAGATCGT GCAGGCGCTG CGCCACGGGG TGCTGCCGAA
    31141 GACCCTGCAC GTGGACGAGC COACOGACCA GGTCGACTGG TCGGCCGGTT CGGTCGAGCT
    31201 GCTCACCGAG GCCGTGGACT GGCCGGAGCG CCCGGGCCGG CTCCGCCGGG CGGGCGTCTC
    31261 CGCGTTCGGC GTGGGCGGGA CGAACGCGCA CGTCGTCCTG GAGGAGGCCC CGGCGGTCGA
    31321 GGAGTCCCCT GCCGTCGAGC CGCCGGCCGG TGGCGGCGTG GTGCCGTGGC CGGTGTCCGC
    31381 GAAGACCTCG GCCGCACTGG ACGCCCAGAT CGGGCAGCTC GCCGCATACG CGGAAGACCG
    31441 CACGGACGTG GATCCGGCGG TCGCCGCCCG CGCCCTGGTC GACAGCCGTA CGGCGATGGA
    31501 GCACCGCGCG GTCGCGCTCG GCGACAGCCG GGAGCCACTG CGGGACGCCC TGCGGATCCC
    31561 GGAAGGACTG GTACGGGGCA CGGTCACCGA TCCGGGCCCC GTGGCGTTCG TCTTCCCCGG
    31621 CCAGGCCACG CAGTGGGCCG GCATGGGCGC CGAACTCCTC GACAGCTCAC CCGAATTCGC
    31681 CGCCGCCATG GCCGAATGCG ACACCGCACT CTCCCCGTAC CTCGACTGGT CTCTCGAAGC
    31741 CGTCGTCCGA CAGGCTCCCA GCGCACCGAC ACTCGACCGC GTCGACGTCG TCCAGCCCGT
    31801 CACCTTCGCC GTCATGGTCT CCCTCGCCAA GGTCTGGCAG CACCACGGCA TCACCCCCGA
    31861 GGCCGTCATC GGCCACTCCC AGGGCGAGAT CGCCGCCGCG TACGTCGCCG GTGCCCTCAC
    31921 CCTCGACGAC CCCGCTCGTG TCGTGACCCT CCGCAGCAAG TCCATCGCCG CCCACCTCGC
    31981 CGGCAAGGGC GGCATCATCT CCCTCGCCCT CAGCGAGGAA GCCACCCGGC AGCGCATCGA
    32041 CAACCTCCAC GGACTGTCGA TCGCCGCCGT CAACGGGCCT ACCGCCACCG TGGTTTCGGG
    32101 CGACCCCACC CAGATCCAAG AACTTGCTCA GGCGTGTGAG GCCGACGGCA TCCGCGCACG
    32161 GATCATCCCC GTCGACTACG CCTCCCACAG CGCCCACGTC GAGACCATCG AGAACGAACT
    32221 CGCCGACGTC CTGGCGGGGT TGTCCCCCCA GACACCCCAG GTCCCCTTCT TCTCCACCCT
    32281 CGAAGGCACC TGGATCACCG AACCCGCCCT CGACGGCGGC TACTGGTACC GCAACCTCCG
    32341 CCATCCTGTG GGCTTCGCCC CGGCCGTCGA GACCCTCGCC ACCGACGAAG GCTTCACCCA
    32401 CTTCATCGAG GTCAGCGCCC ACCCCGTCCT CACCATGACC CTCCCCGACA AGGTCACCGG
    32461 CCTCGCCACC CTCCCACGCG AGGACGGCGG ACAGCACCGC CTCACCACCT CCCTTGCCGA
    32521 GGCCTGGGCC AACGGCCTCG CCCTCGACTG GGCCTCCCTC CTGCCCGCCA CGGCCGCCCT
    32581 CAGCCCCGCC GTCCCCGACC TCCCCACGTA CGCCTTCCAG CACCGCTCGT ACTGGATCAG
    32641 CCCCGCGGGT CCCGGCGAGG CGCCCGCGCA CACCGCTTCC GGGCGCGAGG CCGTCGCCGA
    32701 GACGGGGCTC GCGTGGGGCC CGGGTGCCGA GGACCTCGAC GAGGAGGGCC CCCGCAGCGC
    32761 CGTACTCGCG ATGGTGATGC GGCAGGCGGC CTCCGTGCTC CGGTGCGACT CGCCCGAAGA
    32821 GGTCCCCGTC GACCGCCCGC TGCGGGAGAT CGGCTTCGAC TCGCTGACCG CCGTCGACTT
    32881 CCGCAACCGC GTCAACCCGC TGACCGCTCT CCAGCTGCCG CCCACCGTCG TGTTCGAGCA
    32941 CCCGACGCCC GTCGCGCTCG CCGAGCGCAT CAGCGACGAG CTGGCCGAGC GGAACTGGGC
    33001 CGTCGCCGAG CCGTCGGATC ACGAGCAGGC GGAGGAGGAG AAGGCCGCCG CTCCGGCGGG
    33061 GGCCCGCTCC GGGGCCGACA CCGGCGCCGG CGCCGGGATG TTCCGCGCCC TGTTCCGGCA
    33121 GGCCGTCCAG GACGACCGGT ACGCCGAGTT CCTCGACCTC CTCGCCGAAG CCTCCGCGTT
    33181 CCGCCCGCAG TTCGCCTCGC CCCAGGCCTG CTCGGAGCGG CTCGACCCGG TGCTGCTCGC
    33241 CGGCGGTCCG ACGGACCGGG CGGAAGGCCG TGCCGTTCTC GTCGGCTGCA CCGGCACCGC
    33301 GCCGAACGCC GCCCCGCACG AGTTCCTGCG GCTCAGCACC TCCTTCCAGG AGCAGCGGGA
    33361 CTTCCTCGCC GTACCTCTCC CCGGCTACGG CACGGCTACG GGCACCGGCA CCGCCCTCCT
    33421 CCCGGCCGAT CTCGACACCG CGCTCGACGC CCACGCCCGG GCGATCCTCC GGGCCGCCGG
    33481 GGACGCCCCG GTCGTCCTGC TCGCGCACTC CGGCGGCGCC CTGCTCGCGC ACGAGCTGGC
    33541 CTTCCGCCTG GAGCGGGCGC ACGGCGCGCC GCCGGCCGGG ATCGTCCTGG TCGACCCCTA
    33601 TCCGCCGGGC CATCAGGAGC CCATCGAGGT GTGGAGCAGG CAGCTGGGCG AGGGCCTGTT
    33661 CGCGGGCGAG CTCGAGCCGA TGTCCCATGC GCGGCTGCTG GCCATGGGCC GGTACGCGCG
    33721 GTTCCTCGCC GGCCCGCGGC CGGGCCGCAG CAGCGCGCCC GTGCTTCTGG TCCGTGCCTC
    33781 CGAACCGCTG GGCGACTGGC ACGAGGAGCG GGGCGACTGG CGTGCCCACT GGGACCTTCC
    33841 GCACACCGTC GCGGACGTGC CGCGCGACCA CTTCACGATG ATGCGGGACC ACGCGCCGGC
    33901 CGTCGCCGAG GCCGTCCTCT CCTGGCTCGA CGCCATCGAG GGCATCGAGG GGGCGGCCAA
    33961 CTGACCGACA GACCTCTGAA CGTGGACAGC GGACTGTGGA TCCGGCGCTT CCACCCCGCG
    34021 CCGAACAGCG CGGTGCGGCT GGTCTGCCTG CCGCACGCCG CCGGCTCCGC CAGCTACTTC
    34081 TTCCGCTTCT CGGAGGAGCT GCACCCCTCC GTCCAGGCCC TGTCGGTGCA GTATCCGGGC
    34141 CGCCAGGACC GGCGTGCCGA GCCCTGTCTG GAGAGCGTCG AGGAGCTCGC CGAGCATGTG
    34201 GTCGCGGCCA CCGAACCCTG GTGCCAGCAG GGCCCGCTCG CCTTCTTCGG GCACAGCCTC
    34261 GGCGCCTCCG TCGCCTTCGA GACGGCCCGC ATCCTGCAAC AGCGGCACGG GGTACGGCCC
    34321 GAGGGCCTGT ACGTCTCCGG TCGGCGCGCC CCGTCGCTCG CGCCGGACCG GCTCGTCCAC
    34381 CAGCTGGACG ACCGGGCGTT CCTGGCCCAG ATCCGGCGGC TCAGCGGCAC CGACGAGCGG
    34441 TTCCTCCAGG ACGACGAGCT GCTGCGGCTG GTGCTGCCCG CGCTGCGCAG CGACTACAAG
    34501 GCGGCGGAGA CGTACCTGCA CCGGCCGTCC GCCAAGCTCA CCTGCCCGGT GATGGCCCTG
    34561 GCCGGCGACC GTGACCCGAA GGCGCCGCTG AACGAGGTGG CCGAGTGGCG TCGGCACACC
    34621 AGCGCCCCGT TCTGCCTCCG GGCGTACTCC GGCGCCCACT TCTACCTCAA CGACCAGTGG
    34681 CACGAGATCT GCAACGACAT CTCCGACCAC CTGCTCGTCA CCCGCGGCGC GCCCGATGCC
    34741 CGCGTCGTGC AGCCCCCGAC CAGCCTTATC GAAGGAGCGG CGAAGAGATG GCAGAACCCA
    34801 CCGTCACCGA CGACCTGACG GGGGCCCTCA CGCAGCCCCC CCTGGCCCGC ACCGTCCGCG
    34861 CGGTGGCCGA CCGTGAACTC GGCACCCACC TCCTGGAGAC CCGCGGCATC CACTGGATCC
    34921 ACGCCGCGAA CGGCGACCCG TACGCCACCG TGCTGCGCGG CCAGGCGGAC GACCCGTATC
    34981 CCGCGTACGA GCGGGTGCGT GCCCGCCGCG CGCTCTCCTT CAGCCCGACG GGCAGCTGGG
    35041 TCACCGCCGA TCACGCCCTG GCCGCGAGCA TCCTCTGCTC GACGGACTTC GGGGTCTCCG
    35101 GCGCCGACGG CGTCCCGGTG CCGCAGCAGG TCCTCTCGTA CGGGGAGGGC TGTCCGCTGG
    35161 AGCGCGAGCA GGTGCTGCCG GCCCCCGGTG ACGTGCCGGA GGGCGGGCAG CGTGCCGTGG
    35221 TCGAGGGGAT CCACCGGGAG ACGCTGGACG GTCTCGCGCC GGACCCGTCG GCGTCGTACG
    35281 CCTTCGAGCT GCTGGGCOGT TTCGTCCGCC CGGCGGTGAC GGCCGCTGCC GCCGCCGTGC
    35341 TGGGTGTTCC CGCGGACCGG CGCGCGGACT TCGCGGATCT GCTGGAGCGG CTCCGGCCGC
    35401 TGTCCGACAG CCTGCTGGCC CCGCAGTCCC TGCGGACGGT ACGGGCGGCG GACGGCGCGC
    35461 TGGCCGAGCT CACGGCGCTG CTCGCCGATT CGGACGACTC CCCCGGGGCC CTGCTGTCGG
    35521 CGCTCGGGGT CACCGCAGCC GTCCACCTCA CCGGGAACGC CGTCCTCGCG CTCCTCGCGC
    35581 ATCCCGAGCA GTGGCGGGAG CTGTGCGACC GGCCCGGGCT CGCCGCGGCC GCCGTGGAGG
    35641 AGACCCTCCC CTACGACCCG CCGGTGCAGC TCGACGCCCG GGTGGTCCGC GGGCAGACGG
    35701 AGCTGGCGGG CCCGCGGCTG CCGGCCGGGG CGCATGTCGT CGTCCTGACC GCCGCGACCG
    35761 GCCGGGACCC GGAGGTCTTC ACGGACCCGG AGCGCTTCGA CCTCGCGCGC CCCGACGCCG
    35821 CCGCGCACCT CGCGCTGCAC CCCGCCGGTC CGTACGGCCC GGTGGCCTCC CTGGTCCGGC
    35881 TTCAGGCGGA GGTCGCGCTG CGGACCCTGG CCGGGCGTTT CCCCGGGCTG CGGCAGGCGG
    35941 GGGACCTGCT CCGCCCCCGC CGCGCGCCTG TCGGCCGCGG GCCGCTGAGC GTCCCGGTCA
    36001 GCAGCTCCTG AGACACCGGG GCCCCGGTCC GCCCGGCCCC CCTTCGGACG GACCGOACGG
    36061 CTCGGACCAC GGGGACGGCT CAGACCGTCC CGTGTGTCCC CGTCCGGCTC CCGTCCGCCC
    36121 CATCCCCCCC CTCCACCGGC AAGGAAGGAC ACGACGCCAT GCGCGTCCTG CTGACCTCGT
    36181 TCGCACATCA CACGCACTAC TACGGCCTGG TGCCCCTGGC CTGGGCGCTG CTCGCCGCCG
    36241 CGCACGAGGT CCGGGTCGCC AGCCAGCCCG CGCTCACGGA CACCATCACC GGGTCCGGGC
    36301 TCGCCGCGGT GCCGGTCGGC ACCGACCACC TCATCCACGA GTACCGGGTG CGGATGGCGG
    36361 GCGAGCCGCG CCCGAACCAT CCGGCGATCG CCTTCGACGA GGCCCGTCCC GAGCCGCTGG
    36421 ACTGGGACCA CGCCCTCGGC ATCGAGGCGA TCCTCGCCCC GTACTTCTAT CTGCTCGCCA
    36481 ACAACGACTC GATGGTCGAC GACCTCGTCG ACTTCGCCCG GTCCTGGCAG CCGGACCTGG
    36541 TGCTGTGGGA CCCGACGACC TACCCGGGCG CCGTCGCCGC CCAGGTCACC GGTGCCGCGC
    36601 ACGCCCGGGT CCTGTGGGGG CCCGACGTGA TGGGCAGCGC CCGCCGCAAG TTCGTCGCGC
    36661 TGCGGGACCG GCAGCCGCCC GAGCACCGCG AGGACCCCAC CGCGGAGTGG CTGACGTGGA
    36721 CGCTCGACCG GTACGGCGCC TCCTTCGAAG AGGAGCTGCT CACCGGCCAG TTCACGATCG
    36781 ACCCGACCCC GCCGAGCCTG CGCCTCGACA CGGGCCTGCC GACCGTCGGG ATGCGTTATG
    36841 TTCCGTACAA CGGCACGTCG GTCGTGCCGG ACTGGCTGAG TGAGCCGCCC GCGCGGCCCC
    36901 GGGTCTGCCT GACCCTCGGC GTCTCCGCGC GTGAGGTCCT CGGCGGCGAC GGCGTCTCGC
    36961 AGGGCGACAT CCTGGAGGCG CTCGCCGACC TCGACATCGA GCTCGTCGCC ACGCTCGACG
    37021 CGAGTCAGCG CGCCGAGATC CGCAACTACC CGAAGCACAC CCGGTTCACG GACTTCGTGC
    37081 CGATGCACGC GCTCCTGCCG AGCTGCTCGG CGATCATCCA CCACGGCGGG GCGGGCACCT
    37141 ACGCGACCGC CGTGATCAAC GCGGTGCCGC AGGTCATGCT CGCCGAGCTG TGGGACGCGC
    37201 CGGTCAAGGC GCGGGCCGTC GCCGAGCAGG GGGCGGGGTT CTTCCTGCCG CCGGCCGAGC
    37261 TCACGCCGCA GGCCGTGCGG GACGCCGTCG TCCGCATCCT CGACGACCCC TCGGTCGCCA
    37321 CCGCCGCGCA CCGGCTGCGC GAGGAGACOT TCGGCGACCC CACCCCGGCC GGGATCGTCC
    37381 CCGAGCTGGA GCGGCTCGCC GCGCAGCACC GCCGCCCGCC GGCCGACGCC CGGCACTGAG
    37441 CCGCACCCCT CGCCCCAGGC CTCACCCCTG TATCTGCGCC GGGGGACGCC CCCGGCCCAC
    37501 CCTCCGAAAG ACCGAAAGCA GGAGCACCGT GTACGAAGTC GACCACGCCG ACGTCTACGA
    37561 CCTCTTCTAC CTGGGTCGCG GCAAGGACTA CGCCGCCGAG GCCTCCGACA TCGCCGACCT
    37621 GGTGCGCTCC CGTACCCCCG AGGCCTCCTC GCTCCTGGAC GTGGCCTGCT GTACGGGCAC
    37681 GCATCTGGAG CACTTCACCA AGGAGTTCGG CGACACCGCC GGCCTGGAGC TGTCCGAGGA
    37741 CATGCTCACC CACGCCCGCA AGCGGCTGCC CGACGCCACG CTCCACCAGG GCGACATGCG
    37801 GGACTTCCGG CTCGGCCGGA AGTTCTCCGC CGTGGTCAGC ATGTTCAGCT CCGTCGGCTA
    37861 CCTGAAGACG ACCGAGGAAC TCGGCGCGGC CGTCGCCTCG TTCGCGGAGC ACCTGGAGCC
    37921 CGGTGGCGTC GTCGTCGTCG AGCCGTGGTG GTTCCCGGAG ACCTTCGCCG ACGGCTGGGT
    37981 CAGCGCCGAC GTCGTCCGCC GTGACGGGCG CACCGTGGCC CGTGTCTCGC ACTCGGTGCG
    38041 GGAGGGGAAC GCGACGCGCA TGGAGGTCCA CTTCACCGTG GCCGACCCGG GCAAGGGCGT
    38101 GCGGCACTTC TCCGACGTCC ATCTCATCAC CCTGTTCCAC CAGGCCGAGT ACGAGGCCGC
    38161 GTTCACGGCC GCCGGGCTGC GCGTCGAGTA CCTGGAGGGC GGCCCGTCGG GCCGTGGCCT
    38221 CTTCGTCGGC GTCCCCGCCT GAGCACCGCC CAAGACCCCC CGGGGCGGGA CGTCCCGGGT
    38281 GCACCAAGCA AAGAGAGAGA AACGAACCGT GACAGGTAAG ACCCGAATAC CGCGTGTCCG
    38341 CCGCGGCCGC ACCACGCCCA GGGCCTTCAC CCTGGCCGTC GTCGGCACCC TGCTGGCGGG
    38401 CACCACCGTG GCGGCCGCCG CTCCCGGCGC CGCCGACACG GCCAATGTTC AGTACACGAG
    38461 CCGGGCGGCG GAGCTCGTCG CCCAGATGAC GCTCGACGAG AAGATC
  • Those of skill in the art will recognize that, due to the degenerate nature of the genetic code, a variety of DNA compounds differing in their nucleotide sequences can be used to encode a given amino acid sequence of the invention. The native DNA sequence encoding the narbonolide PKS of [0051] Streptomyces venezuelae is shown herein merely to illustrate a preferred embodiment of the invention, and the invention includes DNA compounds of any sequence that encode the amino acid sequences of the polypeptides and proteins of the invention. In similar fashion, a polypeptide can typically tolerate one or more amino acid substitutions, deletions, and insertions in its amino acid sequence without loss or significant loss of a desired activity. The present invention includes such polypeptides with alternate amino acid sequences, and the amino acid sequences shown merely illustrate preferred embodiments of the invention.
  • The recombinant nucleic acids, proteins, and peptides of the invention are many and diverse. To facilitate an understanding of the invention and the diverse compounds and methods provided thereby, the following description of the various regions of the narbonolide PKS and corresponding coding sequences is provided. [0052]
  • The loading module of the narbonolide PKS contains an inactivated KS domain, an AT domain, and an ACP domain. The AT domain of the loading module binds propionyl CoA. Sequence analysis of the DNA encoding the KS domain indicates that this domain is enzymatically inactivated, as a critical cysteine residue in the motif TVDACSSSL, which is highly conserved among KS domains, is replaced by a glutamine so is referred to as a KS[0053] Q domain. Such inactivated KS domains are also found in the PKS enzymes that synthesize the 16-membered macrolides carbomycin, spiromycin, tylosin, and niddamycin. While the KS domain is inactive for its usual function in extender modules, it is believed to serve as a decarboxylase in the loading module.
  • The present invention provides recombinant DNA compounds that encode the loading module of the narbonolide PKS and useful portions thereof. These recombinant DNA compounds are useful in the construction of PKS coding sequences that encode all or a portion of the narbonolide PKS and in the construction of hybrid PKS encoding DNA compounds of the invention, as described in the section concerning hybrid PKSs below. To facilitate description of the invention, reference to a PKS, protein, module, or domain herein can also refer to DNA compounds comprising coding sequences therefor and vice versa. Also, reference to a heterologous PKS refers to a PKS or DNA compounds comprising coding sequences therefor from an organism other than [0054] Streptomyces venezuelae. In addition, reference to a PKS or its coding sequence includes reference to any portion thereof.
  • The present invention provides recombinant DNA compounds that encode one or more of the domains of each of the six extender modules (modules 1-6, inclusive) of the narbonolide PKS. [0055] Modules 1 and 5 of the narbonolide PKS are functionally similar. Each of these extender modules contains a KS domain, an AT domain specific for methylmalonyl CoA, a KR domain, and an ACP domain. Module 2 of the narbonolide PKS contains a KS domain, an AT domain specific for malonyl CoA, a KR domain, a DH domain, and an ACP domain. Module 3 differs from extender modules 1 and 5 only in that it contains an inactive ketoreductase domain. Module 4 of the narbonolide PKS contains a KS domain, an AT-domain specific for methylmalonyl CoA, a KR domain, a DH domain, an ER domain, and an ACP domain. Module 6 of the narbonolide PKS contains a KS domain, an AT domain specific for methylmalonyl CoA, and an ACP domain. The approximate boundaries of these “domains” is shown in Table 1.
  • In one important embodiment, the invention provides a recombinant narbonolide PKS that can be used to express only narbonolide (as opposed to the mixture of narbonolide and 10-deoxymethynolide that would otherwise be produced) in recombinant host cells. This recombinant narbonolide PKS results from a fusion of the coding sequences of the picAIII and picAIV genes so that [0056] extender modules 5 and 6 are present on a single protein. This recombinant PKS can be constructed on the Streptomyces venezuelae or S. narbonensis chromosome by homologous recombination. Alternatively, the recombinant PKS can be constructed on an expression vector and introduced into a heterologous host cell. This recombinant PKS is preferred for the expression of narbonolide and its glycosylated and/or hydroxylated derivatives, because a lesser amount or no 10-deoxymethynolide is produced from the recombinant PKS as compared to the native PKS. In a related embodiment, the invention provides a recombinant narbonolide PKS in which the picAIV gene has been rendered inactive by an insertion, deletion, or replacement. This recombinant PKS of the invention is useful in the production of 10-deoxymethynolide and its derivatives without production of narbonolide.
  • In similar fashion, the invention provides recombinant narbonolide PKS in which any of the domains of the native PKS have been deleted or rendered inactive to make the corresponding narbonolide or 10-deoxymethynolide derivative. Thus, the invention also provides recombinant narbonolide PKS genes that differ from the narbonolide PKS gene by one or more deletions. The deletions can encompass one or more modules and/or can be limited to a partial deletion within one or more modules. When a deletion encompasses an entire module, the resulting narbonolide derivative is at least two carbons shorter than the polyketide produced from the PKS encoded by the gene from which deleted PKS gene and corresponding polyketide were derived. When a deletion is within a module, the deletion typically encompasses a KR, DH, or ER domain, or both DH and ER domains, or both KR and DH domains, or all three KR, DH, and ER domains. [0057]
  • This aspect of the invention is illustrated in FIG. 4, parts B and C, which shows how a vector of the invention, plasmid pKOS039-16 (not shown), was used to delete or “knock out” the picAI gene from the [0058] Streptomyces venezuelae chromosome. Plasmid pKOS039-16 comprises two segments (shown as cross-hatched boxes in FIG. 4, part B) of DNA flanking the picAI gene and isolated from cosmid pKOS023-27 (shown as a linear segment in the Figure) of the invention. When plasmid pKOS039-16 was used to transform S. venezuelae and a double crossover homologous recombination event occurred, the picAI gene was deleted. The resulting host cell, designated K039-03 in the Figure, does not produce picromycin unless a functional picAI gene is introduced.
  • This [0059] Streptomyces venezuelae K039-03 host cell and corresponding host cells of the invention are especially useful for the production of polyketides produced from hybrid PKS or narbonolide PKS derivatives. Especially preferred for production in this host cell are narbonolide derivatives produced by PKS enzymes that differ from the narbonolide PKS only in the loading module and/or extender modules 1 and/or 2. These are especially preferred, because one need only introduce into the host cell the modified picAI gene or other corresponding gene to produce the desired PKS and corresponding polyketide. These host cells are also preferred for desosaninylating polyketides in accordance with the method of the invention in which a polyketide is provided to an S. venezuelae cell and desosaminylated by the endogenous desosamine biosynthesis and desosaminyl transferase gene products.
  • The recombinant DNA compounds of the invention that encode each of the domains of each of the modules of the narbonolide PKS are also useful in the construction of expression vectors for the heterologous expression of the narbonolide PKS and for the construction of hybrid PKS expression vectors, as described further below. [0060]
  • Section II: The Genes for Desosamine Biosynthesis and Transfer and for Beta-Glucosidase [0061]
  • Narbonolide and 10-deoxymethynolide are desosaminylated in [0062] Streptomyces venezuelae and S. narbonensis to yield narbomycin and YC-17, respectively. This conversion requires the biosynthesis of desosamine and the transfer of the desosamine to the substrate polyketides by the enzyme desosaminyl transferase. Like other Streptomyces, S. venezuelae and S. narbonensis produce glucose and a glucosyl transferase enzyme that glucosylates desosamine at the 2′ position. However, S. venezuelae and S. narbonensis also produce a beta-glucosidase, which removes the glucose residue from the desosamine. The present invention provides recombinant DNA compounds and expression vectors for each of the desosamine biosynthesis enzymes, desosaminyl transferase, and beta-glucosidase.
  • As noted above, cosmid pKOS023-27 contains three ORFs that encode proteins involved in desosamine biosynthesis and transfer. The first ORF is from the picCII gene, also known as desVIII, a homologue of eryCII, believed to encode a 4-keto-6-deoxyglucose isomerase. The second ORF is from the picCIII gene, also known as desVII, a homologue of eryCIII, which encodes a desosaminyl transferase. The third ORF is from the picCVI gene, also known as desVI, a homologue of eryCVI, which encodes a 3-amino dimethyltransferase. [0063]
  • The three genes above and the remaining desosamine biosynthetic genes can be isolated from cosmid pKOS023-26, which was deposited with the American Type Culture Collection on Aug. 20, 1998 under the Budapest Treaty and is available under the accession number ATCC 203141. FIG. 3 shows a restriction site and function map of cosmid pKOS023-26. This cosmid contains a region of overlap with cosmid pKOS023-27.representing nucleotides 14252 to nucleotides 38506 of pKOS023-27. [0064]
  • The remaining desosamine biosynthesis genes on cosmid pKOS023-26 include the following genes. ORF11, also known as desR, encodes beta-glucosidase and has no ery gene homologue. The picCI gene, also known as desV, is a homologue of eryCI. ORF14, also known as desIV, has no known ery gene homologue and encodes an [0065] NDP glucose 4,6-dehydratase. ORF13, also known as desIII, has no known ery gene homologue and encodes an NDP glucose synthase. The picCV gene, also known as desII, a homologue of eryCV is required for desosamine biosynthesis. The picCIV gene also known as desI, is a homologue of eryCIV, and its product is believed to be a 3,4-dehydratase. Other ORFs on cosmid pKOS023-26 include ORF12, believed to be a regulatory gene; ORF15, which encodes an S-adenosyl methionine synthase; and ORF16, which is a homolog of the M. tuberculosis cbhK gene. Cosmid pKOS023-26 also encodes the picK gene, which encodes the cytochrome P450 hydroxylase that hydroxylates the C12 of narbomycin and the C10 and C12 positions of YC-17. This gene is described in more detail in the following section.
  • Below, the amino acid sequences or partial amino acid sequences of the gene products of the desosamine biosynthesis and transfer and beta-glucosidase genes are shown. These amino acid sequences are followed by the DNA sequences that encode them. [0066]
  • Amino Acid Sequence of PICCI (desV) (SEQ ID NO:6) [0067]
    (SEQ ID NO:6)
    1 VSSRAETPRV PFLDLKAAYE ELRAETDAAI ARVLDSGRYL LGPELEGFEA EFAAYCETDH
    61 AVGVNSGMDA LQLALRGLGI GPGDEVIVPS HTYIASWLAV SATGATPVPV EPHEDHPTLD
    121 PLLVEKAITP RTRALLPVHL YCHPADMDAL RELADRHGLH IVEDAAQAHG ARYRGRRIGA
    181 GSSVAAFSFY PGKNLGCFGD GGAVVTGDPE LAERLRMLRN YGSRQKYSHE TKGTNSRLDE
    241 MQAAVLRIRL XHLDSWNGRR SALAAEYLSG LAGLPGIGLP VTAPDTDPVW HLFTVRTERR
    301 DELRSHLDAR GIDTLTHYPV PVHLSPAYAG EAPPEGSLPR AESFARQVLS LPIGPHLERP
    361 QALRVIDAVR EWAERVDQA
  • Amino Acid Sequence of 3-keto-6-deoxyglucose Isomerase, PICCII (desVIII) (SEQ ID NO:7) [0068]
    (SEQ ID NO:7)
    1 VADRELGTHL LETRGIHWIH AANGDPYATV LRGQADDPYP AYERVRARGA LSFSPTGSWV
    61 TADHALAASI LCSTDFGVSG ADGVPVPQQV LSYGEGCPLE REQVLPAAGD VPEGGQRAVV
    121 EGIRRETLEG LAPDPSASYA FELLGGFVRP AVTAAAAAVL GVPADRRADF ADLLERLRPL
    181 SDSLLAPQSL RTVRAADGAL AELTALLADS DDSPGALLSA LGVTAAVQLT GNAVLALLAH
    241 PEQWRELCDR PGLAAAAVEE TLRYDPPVQL DARVVRGETE LAGRRLPAGA HVVVLTAATG
    301 RDPEVFTDPE RFDLARPDAA AHLALHPAGP YGPVASLVRL QAEVALRTLA GRFPGLRQAG
    361 DVLRPRRAPV GRGPLSVPVS SS
  • Amino Acid Sequence of Desosaminyl Transferase, PICCIII (desVII) (SEQ ID NO:8) [0069]
    (SEQ ID NO:8)
    1 MRVLLTSFAH HTHYYGLVPL AWALLAAGHE VRVASQPALT DTITGSGLAA VPVGTDHLIH
    61 EYRVRMAGEP RPNHPAIAFD EARPEPLDWD HALGIEAILA PYFYLLANND SMVDDLVDFA
    121 RSWQPDLVLW EPTTYAGAVA AQVTGAAHAR VLWGPDVNGS ARRKEVALRD RQPPEHREDP
    181 TAEWLTWTLD RYGASFEEEL LTGQFTIDPT PPSLRLDTGL PTVGMRYVPY NGTSVVPDWL
    241 SEPPARFRVC LTLGVSAREV LGGDGVSQGD ILEALADLDI ELVATLDASQ PAEIRNYPKH
    301 TRFTDFVPMH ALLPSCSAII HHGGAGTYAT AVINAVPQVN LAELWDAPVK ARAVAEQGAG
    361 FFLPPAELTP QAVRDAVVRI LDDPSVATPA HRLREETFGD PTPAGIVPEL ERLAAQHRRP
    421 PADARH
  • Partial Amino Acid Sequence of Aminotransfetase-dehydrase, PICCIV (desI) (SEQ ID NO:9) [0070]
    (SEQ ID NO:9)
    1 VKSALSDLAF FGGPAAFDQP LLVGRPNRID RARLYERLDR ALDSQWLSNG GPLVREFEER
    61 VAGLAGVRHA VATCNATAGL QLLAHAAGLT GEVIMPSMTF AATPHALRWI GLTPVFADID
    121 PDTGNLDPDQ VAAAVTPRTS AVVCVHLWGR PCAADQLRKV ADEHGLRLYF DAAHALGCAV
    181 DGRPAGSLGD AEVESEHATK AVNAEEGGAV VTDDADLAAR IRALHNFGFD LPGGSPAGGT
    241 NAKMSEAAAA MGLTSLDAFP EVIDRNRRNH AXYREHLADL PGVLVADHDR HGLNNHQYVI
    301 VEIDEATTGI HRDLVMEVLK AEGVHTRAYF S
  • Amino Acid Sequence of PICCV (desII) (SEQ ID NO:10) [0071]
    1 MTAPALSATA PAERCAHPGA DLGAAVHAVG QTLAAGGLVP PDEAGTTARH LVRLAVRYGN (SEQ ID NO:10)
    61 SPFTPLEEAR HDLGVDRDAF RRLLALFGQV PELRTAVETG PAGAYWKNTL LPLEQRGVFD
    121 AALARKPVFP YSVGLYPGPT CMFRCHFCVR VTGARYDPSA LDAGNANFRS VIDEIPAGNP
    181 SAMYESGGLE FLTNPGLGSL AAHATDHGLR PTVYTNSFAL TERTLERQPG LWGLHAIRTS
    241 LYCLNDEEYE QTTGKKAAFR RVRENLRRFQ QLRAERESPI NLGFAYIVLP GRASRLLDLV
    301 DFIADLNDAG QGRTIDFVNI REDYSGRDDG KLPQEERAEL QEALNAFEER VRERTPGLHI
    361 DYGYALNSLR TGADAELLRI KPATMRPTAH PQVAVQVDLL GDVYLYREAG FPDLDGATRY
    421 IAGRVTPDTS LTEVVRDFVE RGGEVAAVDG DEYFMDGFDQ VVTARLNQLE RDAADGWEEA
    481 RGFLP
  • Amino Acid Sequence of 3-amino Dimethyl Transferase, PICCVI (desVI) (SEQ ID NO:11) [0072]
    1 VYEVDHADVY DLFYLGRGKD YAAEASDIAD LVRSRTPEAS SLLDVACGTG THLEHFTKEF (SEQ ID NO:11)
    61 GDTAGLELSE DMLTHARKRL PDATLHQGDM RDFRLGRKFS AVVSMFSSVG YLKTTEELGA
    121 AVASFAEHLE PGGVVVVEPW WFPETFADGW VSADVVRRDG RTVARVSHSV REGNATRMEV
    181 HFTVADPGKG VRHFSDVRLI TLFHQAEYEA AFTAAGLRVE YLEGGPSGRG LFVGVPA
  • Partial Amino Acid Sequence of Beta-Glucosidase, ORF11 (desR) (SEQ ID NO:12) [0073]
    1 MTLDEKISFV HWALDPDRQN VGYLPGVPRL GIPELRAADG PNGIRLVGQT ATALPAPVAL (SEQ ID NO:12)
    61 ASTFDDTMAD SYGKVMGRDG PALNQDMVLG PMMNNIRVPH GGRNYETFSE DPLVSSRTAV
    121 AQIKGIQGAG LMTTAKHFAA NNQENNRESV NANVDEQTLR EIEFPAFEAS SKAGAGSEMC
    181 AYNGLNGKPS CGNDELLNNV LRTQWGFQGW VNSDWLATPG TDAITKGLDQ EMGVELPGDV
    241 PKGEPSPPAK FFGEALKTAV LNGTVPEAAV TRSAERIVGQ MEKFGLLLAT PAPRPERDKA
    301 GAQAVSRKVA ENGAVLLRNE GQALPLAGDA CKSIAVIGPT AVDPKVTGLG SAHVVPDSAA
    361 APLDTIKARA GAGATVTYET GEETFCTQIP AGNLSPAFNQ GHQLEPGKAG ALYDGTLTVP
    421 ADGEYRIAVR ATGGYATVQL GSHTIEAGQV YGKVSSPLLK LTKGTHKLTI SGFAMSATPL
    481 SLELGWVTPA AADATIAKAV ESARKARTAV VEAYDDGTEG VDRPNLSLPG TQDKLISAVA
    541 DANPNTIVVL NTGSSVLMPW LSKTRAVLDM WYPGQAGAEA TAALLYGDVN PSGKLTQSFP
    601 AAENQHAVAG DPTSYPGVDN QQTYREGIHV GYRWFDKENV KPLFPFGHGL SYTSFTQSAP
    661 TVVRTSTGGL KVTVTVRNSG KRAGQEVVQA YLGASPNVTA PQAKKKLVGY TKVSLAAGEA
    721 KTVTVNVDRR QLQEWDAATD NWKTGTGNRL LQTGSSSADL RGSATVNVW
  • Amino Acid Sequence of Transcriptional Activator, ORF12 (Regulatory) (SEQ ID NO:13) [0074]
    1 MNLVERDGEI AHLRAVLDAS AAGDGTLLLV SGPAGSGKTE LLRSLRRLAA ERETPVWSVR (SEQ ID NO:13)
    61 ALPGDRDIPL GVLCQLLRSA EQHGADTSAV RDLLDAASRR AGTSPPPPTR RSASTRHTAC
    121 TTGCSPSPAG TPFLVAVDDL THADTASLRF LLYCAAHHDQ GGIGFVMTER ASQRAGYRVF
    181 RAELLRQPHC RNMWLSGLPP SGVRQLLAHY YGPEAAERRA PAYHATTGGN PLLLRALTQD
    241 RQASHTTLGA AGGDEPVHGD AFAQAVLDCL HRSAEGTLET ARWLAVLEQS DPLLVERLTG
    301 TTAAAVERHI QELAAIGLLD EDGTLGQPAI REAALQDLPA GERTELHRRA AEQLHRDGAD
    361 EDTVARHLLV GGAPDAPWAL PLLERGAQQA LFDDRLDDAF RILEFAVRSS TDNTQLARLA
    421 PHLVAASWRM NPHMTTRALA LFDRLLSGEL PPSHPVMALI RCLVWYGRLP EAADALSRLR
    481 PSSDNDALEL SLTRMWLAAL CPPLLESLPA TPEPERGPVP VRLAPRTTAL QAQAGVFQRG
    541 PDNASVAQAE QILQGCRLSE ETYEALETAL LVLVHADRLD RALFWSDALL AEAVERRSLG
    601 WEAVFAATRA MIAIRCGDLP TARERAELAL SHAAPESWGL AVGMPLSALL LACTEAGEYE
    661 QAERVLRQPV PDAMFDSRHG MEYMHARGRY WLAXGRLHAA LGEEMLCGEI LGSWNLDQPS
    721 IVPWRTSAAE VYLRLGNRQK ARALAEAQLA LVRPGRSRTR GLTLRVLAAA VDGQQAERLH
    781 AEAVDMLHDS GDRLEHARAL AGMSRHQQAQ GDNYRARMTA RLAGDMAWAC GAYPLAEEIV
    841 PGRGGRRAKA VSTELELPGG PDVGLLSEAE RRVAALAARC LTNRQIARRL CVTASTVEQH
    901 LTRVYRKLNV TRRADLPISL AQDKSVTA
  • Amino Acid Sequence of dNDP-Glucose Synthase (Glucose-1-phosphate Thymidyl Transferase), ORF13 (desIII) (SEQ ID NO:14) [0075]
    1 MKGIVLAGGS GTRLHPATSV ISKQILPVYN KPMIYYPLSV LMLGGIREIQ IISTPQHIEL (SEQ ID NO:14)
    61 FQSLLGNGRH LGIELDYAVQ KEPAGIADAL LVGAEHIGDD TCALILGDNI FHGPGLYTLL
    121 RDSIARLDGC VLFGYPVKDP ERYGVAEVDA TGRLTDLVEK PVKPRSNLAV TGLYLYDNDV
    181 VDIAKNIRPS PRGELEITDV NRVYLERGRA ELVNLGRGFA WLDTGTHDSL LRAAQYVQVL
    241 EERQGVWIAG LEEIAFRMGF IDAEACHGLG EGLSRTEYGS YLMEIAGREG AP
  • Amino Acid Sequence of dNDP-[0076] Glucose 4,6-dehydratase, ORF14 (desIV) (SEQ ID NO:15)
    1 VRLLVTGGAG FIGSHFVRQL LAGAYPDVPA DEVIVLDSLT YAGNRANLAP VDADPRLRFV (SEQ ID NO:15)
    61 HGDIRDAGLL ARELRGVDAI VHFAAESHVD RSIAGASVFT ETNVQGTQTL LQCAVDAGVG
    121 RVVHVSTDEV YGSIDSGSWT ESSPLEPNSP YAkSKAGSDL VARAYHRTYG LDVRITRCCN
    181 NYGPYQHPEK LIPLFVTNLL DGGTLPLYGD GANVREWVHT DDHCRGIALV LAGGRAGEIY
    241 HIGGGLELTN RELTGILLDS LGADWSSVRK VADRKGHDLR YSLDGGKIER ELGYRPQVSF
    301 ADGLARTVRW YRENRGWWEP LKATAPQLPA TAVEVSA
  • Partial Amino Acid Sequence of S-adenosylmethionine Synthase, ORF15 (SAM Synthase) (SEQ ID NO:16) [0077]
    1 IGYDSSKKGF DGASCGVSVS IGSQSPDIAQ GVDTAYEKRV EGASQRDEGD ELDKQGAGDQ (SEQ ID NO:16)
    61 GLMFGYASDE TPELMPLPIH LAHRLSRRLT EVRKNGTIPY LRPDGKTQVT IEYDGDRAVR
    121 LDTVVVSSQH ASDIDLESLL APDVRKFVVE HVLAQLVEDG IKLDTDGYRL LVNPTGRFEI
    181 GGPMGDAGLT GRKIIIDTYG GMARHGGGAF SGKDPSKVDR SAAYAMRWVA KNVVAAGLAS
    241 RCEVQVAYAI GKAEPVGLFV ETFGTHKIET EKIENAIGEV FDLRPAAIIR DLDLLRPIYS
    301 QTAAYGHFGR ELPDFTWERT DRVDALKKAA GL
  • Partial Amino Acid Sequence of ORF16 (Homologous to [0078] M. tuberculosis cbhK) (SEQ ID NO:17)
    1 MRIAVTGSIA TDHLMTFPGR FAEQILPDQL AHVSLSFLVD TLDIRHGGVA ANIAYGLGLL (SEQ ID NO:17)
    61 GRRPVLVGAV GKDFDGYGQL LRAAGVDTDS VRVSDRQHTA RFMCTTDEDG NQLASFYAGA
    121 MAEARDIDLG ETAGRPGGID LVLVGADDPE AMVRHTRVCR ELGLRRAADP SQQLARLEGD
    181 SVRELVDGAE LLFTNAYERA LLLSKTGWTE QEVLARVGTW ITTLGAKGCR
  • While not all of the insert DNA of cosmid pKOS023-26 has been sequenced, five large contigs shown of FIG. 3 have been assembled and provide sufficient sequence information to manipulate the genes therein in accordance with the methods of the invention. The sequences of each of these five contigs are shown below. [0079]
  • [0080] Contig 001 from cosmid pKOS023-26 contains 2401 nucleotides, the first 100 bases of which correspond to 100 bases of the insert sequence of cosmid pKOS023-27.
  • Nucleotides 80-2389 constitute ORF11, which encodes 1 beta Glucosidase. (SEQ ID NO:20) [0081]
    1 CGTGGCGGCC GCCGCTCCCG GCGCCGCCGA CACGGCCAAT GTTCAGTACA CGAGCCGGGC (SEQ ID NO:20)
    61 GGCGGAGCTC GTCGCCCAGA TGACGCTCGA CGAGAAGATC AGCTTCGTCC ACTGGGCGCT
    121 GGACCCCGAC CGGCAGAACG TCGGCTACCT TCCCGGCGTG CCGCGTCTGG GCATCCCGGA
    181 GCTGCGTGCC GCCGACGGCC CGAACGGCAT CCGCCTGGTG GGGCAGACCG CCACCGCGCT
    241 GCCCGCGCCG GTCGCCCTGG CCAGCACCTT CGACGACACC ATGGCCGACA GCTACGGCAA
    301 GGTCATGGGC CGCGACGGTC GCGCGCTCAA CCAGCACATG GTCCTGGGCC CGATGATGAA
    361 CAACATCCGG GTGCCGCACG GCGGCCGGAA CTACGAGACC TTCAGCGAGG ACCCCCTGGT
    421 CTCCTCGCGC ACCGCGGTCG CCCAGATCAA GGGCATCCAG GGTGCGGGTC TGATGACCAC
    481 GGCCAAGCAC TTCGCGGCCA ACAACCAGGA GAACAACCGC TTCTCCGTGA ACGCCAATGT
    541 CGACGAGCAG ACGCTCCGCG AGATCGAGTT CCCGGCGTTC GAGGCGTCCT CCAAGGCCGG
    601 CGCGGGCTCC TTCATGTGTG CCTACAACGG CCTCAACGGG AAGCCGTCCT GCGGCAACGA
    661 CGAGCTCCTC AACAACGTGC TGCGCACGCA GTGGGGCTTC CAGGGCTGGG TGATGTCCGA
    721 CTGGCTCGCC ACCCCGGGCA CCGACGCCAT CACCAAGGGC CTCCACCAGG AGATGGGCGT
    781 CGAGCTCCCC GGCGACGTCC CGAAGGGCCA GCCCTCGCCG CCGGCCAAGT TCTTCGGCGA
    841 CGCGCTGAAG ACGGCCGTCC TGAACGGCAC GGTCCCCGAG GCGGCCGTGA CGCGGTCGGC
    901 GGAGCGGATC GTCGGCCAGA TGGAGAAGTT CGGTCTGCTC CTCGCCACTC CGGCGCCGCG
    961 GCCCGAGCGC GACAAGGCGG GTGCCCAGGC GGTGTCCCGC AAGGTCGCCG AGAACGGCGC
    1021 GGTGCTCCTG CGCAACGAGG GCCAGGCCCT GCCGCTCGCC GGTGACGCCG GCAAGAGCAT
    1081 CGCGGTCATC GGCCCGACGG CCGTCGACCC CAAGGTCACC GGCCTGGGCA GCGCCCACGT
    1141 CGTCCCGGAC TCGGCGGCGG CGCCACTCGA CACCATCAAG GCCCGCGCGG GTGCGGGTGC
    1201 GACGGTGACG TACGAGACGG GTGAGGAGAC CTTCGGGACG CAGATCCCGG CGGGGAACCT
    1261 CAGCCCGGCG TTCAACCAGG GCCACCAGCT CGAGCCGGGC AAGGCGGGGG CGCTGTACGA
    1321 CGGCACGCTG ACCGTGCCCG CCGACGGCGA GTACCGCATC GCGGTCCGTG CCACCGGTGG
    1381 TTACGCCACG GTGCAGCTCG GCAGCCACAC CATCGAGGCC GGTCAGGTCT ACGGCAAGGT
    1441 GAGCAGCCCG CTCCTCAAGC TGACCAAGGG CACGCACAAG CTCACGATCT CGGGCTTCGC
    1501 GATGAGTGCC ACCCCGCTCT CCCTGGAGCT GGGCTGGGTN ACGCCGGCGG CGGCCGACGC
    1561 CACGATCGCG AAGGCCGTGG AGTCGGCGCG GAAGGCCCGT ACGGCGGTCG TCTTCGCCTA
    1621 CGACGACGGC ACCGAGGGCG TCGACCGTCC GAACCTGTCG CTGCCGGGTA CGCAGGACAA
    1681 GCTGATCTCG GCTCTCGCGG ACGCCAACCC GAACACCATC GTGGTCCTCA ACACCGGTTC
    1741 GTCGGTGCTG ATGCCGTGGC TGTCCAAGAC CCGCGCGGTC CTGGACATGT GGTACCCGGG
    1801 CCAGGCGGGC GCCGAGGCCA CCGCCGCGCT GCTCTACGGT GACGTCAACC CGAGCGGCAA
    1861 GCTCACGCAG AGCTTCCCGG CCGCCGAGAA CCAGCACGCG GTCGCCGGCG ACCCGACCAG
    1921 CTACCCGGGC GTCGACAACC AGCAGACGTA CCGCGAGGGC ATCCACGTCG GGTACCGCTG
    1981 GTTCGACAAG GAGAACGTCA AGCCGCTGTT CCCGTTCGGG CACGGCCTGT CGTACACCTC
    2041 GTTCACGCAG AGCGCCCCGA CCGTCGTGCG TACGTCCACC GGTGGTCTGA AGGTCACGGT
    2101 CACGGTCCGC AACAGCGGGA AGCGCGCCGG CCAGGAGGTC GTCCAGGCGT ACCTCGGTGC
    2161 CAGCCCGAAC GTGACGGCTC CGCAGGCGAA GAAGAAGCTC GTGGGCTACA CGAAGGTCTC
    2221 GCTCGCCGCG GGCGACGCGA AGACGGTGAC GGTGAACGTC GACCGCCGTC AGCTGCAGTT
    2281 CTGGGATCCC GCCACGGACA ACTGGAAGAC GGGAACGGGC AACCGCCTCC TGCAGACCCG
    2341 TTCGTCCTCC GCCGACCTGC GGGGCAGCGC CACGGTCAAC GTCTGGTGAC GTGACGCCGT
    2401 G
  • Contig 002 from cosmid pKOS023-26 contains 5970 nucleotides and the following ORFs: from nucleotide 995 to 1 is an ORF of picCIV that encodes a partial sequence of an amino transferase-dehydrase; from nucleotides 1356 to 2606 is an ORF of picK that encodes a cytochrome P450 hydroxylase; and from nucleotides 2739 to 5525 is ORF12, which encodes a transcriptional activator. (SEQ ID NO:21) [0082]
    1 GGCGAGAAGT AGGCGCGGGT GTGCACGCCT TCGGCCTTCA GGACCTCCAT GACGAGGTCG (SEQ ID NO:21)
    61 CGGTGGATGC CGGTGGTGGC CTCGTCGATC TCGACGATCA CGTACTGGTG CTTGTTGAGG
    121 CCGTGGCGCT CGTGGTCGGC GACGAGGACG CCGGGGAGGT CCGCGAGGTG CTCGCGGTAG
    181 SCGGCGTGGT TGCGCCGGTT CCGGTCGATG ACCTCGGGAA ACGCGTCGAG GGAGGTGAGG
    241 CCCATGGCGG CGGCGGCCTC GCTCATCTTG GCGTTGGTCC CGCCGGCGGG GCTGCCGCCG
    301 GGCAGGTCGA AGCCGAAGTT GTGGAGCCCG CGGATCCGGG CGGCGAGGTC GGCGTCGTCG
    361 GTGACGACGG CGCCGCCCTC GAAGGCGTTG ACGGCCTTGG TGGCGTGGAA GCTGAAGACC
    421 TCGGCGTCGC CGAGGCTGCC GGCGGGCCGG CCGTCCACCG CGCAGCCGAG GGCGTGCGCG
    481 GCGTCGAAGT ACAGCCGCAG GCCGTGCTCG TCGGCGACCT TCCGCAGCTG GTCGGCGGCG
    541 CAGGGGCGGC CCCAGAGGTG GACGCCGACG ACCGCCGAGG TGCGGGGTGT GACCGCGGCG
    601 GCCACCTGGT CCGGGTCGAG GTTGCCGGTG TCGGCCTCGA TGTCGGCGAA GACCGGGGTG
    661 AGGCCGATCC AGCGCAGTGC GTGCGGGGTG GCGGCGAACG TCATCGACGG CATGATCACT
    721 TCGCCGGTGA GGCCGGCGGC GTGCGCGAGG AGCTGGAGCC CGGCCGTGGC GTTGCAGGTG
    781 GCCACGGCAT GCCGGACCCC GGCGACCCCG GCGACGCGCT CCTCGAACTC GCGGACGAGC
    841 GGGCCGCCGT TGGACAGCCA CTGCCTGTCG AGGGCCCGGT CGAGCCGCTC GTACACCCTG
    901 GCGCGGTCGA TGCGGTTGGG CCGCCCCACG AGGAGCGGCT GGTCGAAAGC GGCGGGGCCG
    961 CCGAAGAATG CGAGGTCGGA TAAGGCGCTT TTCACGGATG TTCCCTCCGG GCCACCGTCA
    1021 CGAAATGATT CGCCGATCCG GGAATCCCGA ACGAGGTCGC CGCGCTCCAC CGTGACGTAC
    1081 GACGAGATGG TCGATTGTGG TGGTCGATTT CGGGGGGACT CTAATCCGCG CGGAACGGGA
    1141 CCGACAAGAG CACGCTATGC GCTCTCGATG TGCTTCGGAT CACATCCGCC TCCGGGGTAT
    1201 TCCATCGGCG GCCCGAATGT GATGATCCTT GACAGGATCC GGGAATCAGC CGAGCCGCCG
    1261 GGAGGGCCGG GGCGCGCTCC GCGGAAGAGT ACGTGTGAGA AGTCCCGTTC CTCTTCCCGT
    1321 TTCCGTTCCG CTTCCGGCCC GGTCTGGAGT TCTCCGTGCG CCGTACCCAG CAGGGAACGA
    1381 CCGCTTCTCC CCCGGTACTC GACCTCGGGG CCCTGGGGCA GGATTTCGCG GCCGATCCGT
    1441 ATCCGACGTA CGCGAGACTG CGTGCCGAGG GTCCGGCCCA CCGGGTGCGC ACCCCCGAGG
    1501 GGGACGAGGT GTGGCTGGTC GTCGGCTACG ACCGGGCGCG GGCGGTCCTC GCCGATCCCC
    1561 GGTTCAGCAA GGACTGGCGC AACTCCACGA CTCCCCTGAC CGAGGCCGAG GCCGCGCTCA
    1621 ACCACAACAT GCTGGAGTCC GACCCGCCGC GGCACACCCG GCTGCGCAAG CTGGTGGCCC
    1681 GTGAGTTCAC CATGCGCCGG GTCGAGTTGC TGCGGCCCCG GGTCCAGGAG ATCGTCGACG
    1741 GGCTCGTGGA CGCCATGCTG GCGGCGCCCG ACGGCCGCGC CGATCTGATG GAGTCCCTGG
    1801 CCTGGCCGCT GCCGATCACC GTGATCTCCG AACTCCTCGG CGTGCCCGAG CCGGACCGCG
    1861 CCGCCTTCCG CGTCTGGACC GACGCCTTCC TCTTCCCGGA CGATCCCGCC CAGGCCCAGA
    1921 CCGCCATGGC CGAGATGAGC GGCTATCTCT CCCGGCTCAT CGACTCCAAG CGCGGGCAGG
    1981 ACGGCGAGGA CCTGCTCAGC GCGCTCGTGC GGACCAGCGA CGAGGACGGC TCCCGGCTGA
    2041 CCTCCGAGGA GCTGCTCCGT ATGGCCCACA TCCTGCTCGT CGCGGGGCAC GAGACCACGG
    2101 TCAATCTGAT CGCCAACGGC ATGTACGCGC TGCTCTCGCA CCCCGACCAG CTGGCCGCCC
    2161 TGCGGGCCGA CATGACGCTC TTGGACGGCG CGGTGGAGGA GATGTTGCGC TACGAGGGCC
    2221 CGGTGGAATC CGCGACCTAC CGCTTCCCGG TCGAGCCCGT CGACCTGGAC GGCACGGTCA
    2281 TCCCGGCCGG TGACACGGTC CTCGTCGTCC TGGCCGACGC CCACCCCACC CCCGAGCGCT
    2341 TCCCGGACCC GCACCGCTTC GACATCCGCC GGGACACCGC CGGCCATCTC GCCTTCGGCC
    2401 ACGGCATCCA CTTCTGCATC GGCGCCCCCT TGGCCCGGTT GGAGCCCCGG ATCGCCGTCC
    2461 GCGCCCTTCT CGAACGCTGC CCGGACCTCG CCCTGGACGT CTCCCCCGGC GAACTCGTGT
    2521 GGTATCCGAA CCCGATGATC CGCGGGCTCA AGGCCCTCCC GATCCGCTGG CGGCGAGGAC
    2581 GGGAGGCGGG CCGCCGTACC GGTTGAACCC GCACGTCACC CATTACGACT CCTTGTCACG
    2641 GAAGCCCCGG ATCGGTCCCC CCTCGCCCTA ACAAGACCTG GTTAGAGTGA TGGACGACGA
    2701 CGAAGGCTTC GGCGCCCGGA CCAGGGGGCA CTTCCGCGAT GAATCTGGTG GAACGCGACG
    2761 GCGAGATAGC CCATCTCAGG GCCGTTCTTG ACGCATCCGC CGCAGGTCAC GGGACGCTCT
    2821 TACTCCTCTC CGCACCGGCC GGCAGCGGGA AGACGGAGCT GCTGCGGTCG CTCCGCCGGC
    2881 TGGCCCCCGA CCGGGAGACC CCCGTCTGGT CGGTCCGGGC GCTGCCGGGT GACCGCGACA
    2941 TCCCCCTGGG CGTCCTCTGC CACTTACTCC GCAGCGCCGA ACAACACGGT GCCGACACCT
    3001 CCGCCGTCCG CGACCTGCTG GACGCCGCCT CGCGGCGGGC CGGAACCTCA CCTCCCCCGC
    3061 CGACGCGCCG CTCCGCGTCG ACGAGACACA CCGCCTGCAC GACTGGCTGC TCTCCGTCTC
    3121 CCGCCGGCAC CCCGTTCCTC GTCGCCGTCG ACGACCTGAC CCACGCCGAC ACCGCGTCCC
    3181 TGAGGTTCCT CCTGTACTGC GCCGCCCACC ACGACCAGGG CGGCATCGGC TTCGTCATGA
    3241 CCGAGCGGGC CTCGCAGCGC GCCGGATACC GGGTGTTCCG CGCCGAGCTG CTCCGCCAGC
    3301 CGCACTGCCG CAACATGTGG CTCTCCGGGC TTCCCCCCAG CGGGGTACGC CAGTTACTCG
    3361 CCCACTACTA CGGCCCCGAG GCCGCCGAGC GGCGGGCCCC CGCGTACCAC GCGACGACCG
    3421 GCGGGAACCC GCTGCTCCTG CGGGCGCTGA CCCAGGACCG GCAGGCCTCC CACACCACCC
    3481 TCGGCGCGGC CGGCGGCGAC GAGCCCGTCC ACGGCGACGC CTTCGCCCAG GCCGTCCTCG
    3541 ACTGCCTGCA CCGCAGCGCC GAGGGCACAC TGGAGACCGC CCGCTGGCTC GCGGTCCTCG
    3601 AACAGTCCGA CCCGCTCCTG GTGGAGCGGC TCACGGGAAC GACCGCCGCC GCCGTCGAGC
    3661 GCCACATCCA GGAGCTCGCC GCCATCGGCC TCCTGGACGA GGACGGCACC CTGGGACAGC
    3721 CCGCGATCCG CGAGGCCGCC CTCCAGGACC TGCCGGCCGG CGAGCGCACC GAACTGCACC
    3781 GGCGCGCCGC GGAGCAGCTG CACCGGGACG GCGCCGACGA GGACACCCTG GCCCGCCACC
    3841 TGCTGGTCGG CGGCGCCCCC GACGCTCCCT GGGCGCTGCC CCTGCTCGAA CGGGGCGCGC
    3901 AGCAGGCCCT GTTCGACGAC CGACTCGACG ACGCCTTCCG GATCCTCGAG TTCGCCGTGC
    3961 GGTCGAGCAC CGACAACACC CAGCTGGCCC GCCTCGCCCC ACACCTGGTC GCGGCCTCCT
    4021 GGCGGATGAA CCCGCACATG ACGACCCGGG CCCTCGCACT CTTCGACCGG CTCCTGAGCG
    4081 GTGAACTGCC GCCCAGCCAC CCGGTCATGG CCCTGATCCG CTGCCTCGTC TGGTACGGNC
    4141 GGCTGCCCGA GGCCCCCGAC GCGCTGTCCC GGCTGCGGCC CAGCTCCGAC AACGATGCCT
    4201 TGGAGCTGTC GCTCACCCGG ATGTGGCTCG CGGCGCTGTG CCCGCCGCTC CTGGAGTCCC
    4261 TGCCGGCCAC GCCGGAGCCG GAGCGGGGTC CCCTCCCCGT ACGGCTCGCG CCGCGGACGA
    4321 CCGCGCTCCA GGCCCAGGCC GGCGTCTTCC AGCGGGGCCC GGACAACGCC TCGGTCGCGC
    4381 AGGCCGAACA GATCCTGCAG CGCTGCCGGC TGTCGGAGGA GACGTACGAG GCCCTGGAGA
    4441 CGGCCCTCTT GGTCCTCGTC CACGCCGACC GGCTCGACCG GGCGCTGTTC TGGTCGGACG
    4501 CCCTGCTCGC CCAGGCCGTG GACCGGCGGT CGCTCGGCTG GGAGGCGCTC TTCGCCGCGA
    4561 CCCGGGCGAT GATCGCGATC CGCTGCGGCG ACCTCCCGAC GGCGCGGGAG CGGGCCGAGC
    4621 TGGCGCTCTC CCACGCGGCG CCGGAGAGCT GGGGCCTCGC CGTGGGCATG CCCCTCTCCG
    4681 CGCTGCTGCT CGCCTGCACG GAGGCCGGCG AGTACGAACA GGCGGAGCGG GTCCTGCGGC
    4741 AGCCGGTGCC GGACGCGATG TTCGACTCGC GGCACGGCAT GGAGTACATG CACGCCCGGG
    4801 GCCGCTACTG GCTGGCGANC GGCCGGCTGC ACGCGGCGCT GGGCGAGTTC ATGCTCTGCG
    4861 GGGAGATCCT GGGCAGCTGG AACCTCGACC AGCCCTCGAT CGTGCCCTGG CGGACCTCCG
    4921 CCGCCGAGGT GTACCTGCGG CTCGGCAACC GCCAGAAGGC CAGGGCGCTG GCCGAGGCCC
    4981 AGCTCGCCCT GGTGCGGCCC GGGCGCTCCC GCACCCGGGG TCTCACCCTG CGGGTCCTGG
    5041 CGGCGGCGGT GGACGGCCAG CAGGCGGAGC GGCTGCACGC CGAGGCGGTC GACATGCTGC
    5101 ACGACAGCGG CGACCGGCTC GAACACGCCC GCGCGCTCGC CGGGATGAGC CGCCACCAGC
    5161 AGGCCCAGGG GGACAACTAC CGGGCGAGGA TGACGGCGCG GCTCGCCGGC GACATGGCGT
    5221 GGGCCTGCGG CGCGTACCCG CTGGCCGAGG AGATCGTGCC GGGCCGCGGC GGCCGCCGGG
    5281 CGAAGGCGGT GAGCACGGAG CTGGAACTGC CGGGCCGCCC GGACGTCGGC CTGCTCTCGG
    5341 AGGCCGAACG CCGGGTGGCG GCCCTGGCAG CCCGAGGATT GACGAACCGC CAGATAGCGC
    5401 GCCGGCTCTG CGTCACCGCG AGCACGGTCG AACAGCACCT GACGCGCGTC TACCGCAAAC
    5461 TGAACGTGAC CCGCCGAGCA GACCTCCCGA TCAGCCTCGC CCAGGACAAG TCCGTCACGG
    5521 CCTGAGCCAC CCCCGGTGTC CCCGTGCGAC GACCCGCCGC ACGGGCCACC GGGCCCGCCG
    5581 GGACACGCCG GTGCGACACG CGGGCGCGCC AGGTGCCATG GGGACCTCCG TGACCGCCCG
    5641 AGGCGCCCGA GGCGCCCGGT GCGGCACCCG GAGACGCCAG GACCGCCGGG ACCACCGGAG
    5701 ACGCCAGGGA CCGCTGGGGA CACCGGGACC TCAGGGACCG CCGGGACCGC CCGAGTTGCA
    5761 CCCGGTGCGC CCGGGGACAC CAGACCGCCG GGACCACCCG AGGGTGCCCG GTGTGGCCCC
    5821 GGCGGCCGGG GTCTCCTTCA TCGGTGGGCC TTCATCGGCA GGAGGAAGCG ACCGTGAGAC
    5881 CCGTCGTGCC GTCGGCGATC AGCCGCCTGT ACGGGCGTCG GACTCCCTGG CGGTCCCGGA
    5941 CCCGTCGTAC GGGCTCGCGG GACCCGGTGC
  • [0083] Contig 003 from cosmid pKOS023-26 contains 3292 nucleotides and the following ORFs: from nucleotide 104 to 982 is ORF13, which encodes dNDP glucose synthase (glucose-1-phosphate thymidyl transferase); from nucleotide 1114 to 2127 is ORF14, which encodes dNDP-glucose 4,6-dehydratase; and from nucleotide 2124 to 3263 is the picCI ORF. (SEQ ID NO:22)
    1 ACCCCCCAAA GGGGTGGTGA CACTCCCCCT GCGCAGCCCC TAGCGCCCCC CTAACTCGCC (SEQ ID NO:22)
    61 ACGCCGACCG TTATCACCGG CGCCCTGCTG CTAGTTTCCG AGAATGAAGG GAATAGTCCT
    121 GGCCGGCGGG AGCGGAACTC GGCTGCATCC GGCGACCTCG GTCATTTCGA AGCAGATTCT
    181 TCCGGTCTAC AACAAACCGA TGATCTACTA TCCGCTGTCG GTTCTCATGC TCGGCGGTAT
    241 TCGCGAGATT CAAATCATCT CGACCCCCCA GCACATCGAA CTCTTCCAGT CGCTTCTCGG
    301 AAACGGCAGG CACCTGGGAA TAGAACTCGA CTATGCGGTC CAGAAAGAGC CCGCAGGAAT
    361 CGCGGACGCA CTTCTCGTCG GAGCCGAGCA CATCGGCGAC GACACCTGCG CCCTGATCCT
    421 GGGCGACAAC ATCTTCCACG GGCCCGGCCT CTACACGCTC CTGCGGGACA GCATCGCGCG
    481 CCTCGACGGC TGCGTGCTCT TCGGCTACCC GGTCAAGGAC CCCGAGCGGT ACGGCGTCGC
    541 CGAGGTGGAC GCGACGGGCC GGCTGACCGA CCTCGTCGAG AAGCCCGTCA AGCCGCGCTC
    601 CAACCTCGCC GTCACCGGCC TCTACCTCTA CGACAACGAC GTCGTCGACA TCGCCAAGAA
    661 CATCCGGCCC TCGCCGCGCG GCGAGCTGGA GATCACCGAC GTCAACCGCG TCTACCTGGA
    721 GCGGGGCCGG GCCGAACTCG TCAACCTGGG CCGCGGCTTC GCCTGGCTGG ACACCGGCAC
    781 CCACGACTCG CTCCTGCGGG CCGCCCAGTA CGTCCAGGTC CTGGAGGAGC GGCAGGGCGT
    841 CTGGATCGCG GGCCTTGAGG AGATCGCCTT CCGCATGGGC TTCATCGACG CCGAGGCCTG
    901 TCACGGCCTG GGAGAAGGCC TCTCCCGCAC CGAGTACGGC AGCTATCTGA TGGAGATCGC
    961 CGGCCGCGAG GGAGCCCCGT GAGGGGACCT CGCGGCCGAC GCGTTCCCAC GACCGACAGC
    1021 GCCACCGACA GTGCGACCCA CACCGCGACC CGCACCGCCA CCGACAGTGC GACCCACACC
    1081 GCGACCTACA GCGCGACCGA AAGGAAGACG GCAGTGCGGC TTCTGGTGAC CGGAGGTGCG
    1141 GGCTTCATCG GCTCGCACTT CGTGCGGCAG CTCCTCGCCG GGGCGTACCC CGACGTGCCC
    1201 GCCGATGAGG TGATCGTCCT GGACAGCCTC ACCTACGCGG GCAACCGCGC CAACCTCGCC
    1261 CCGGTGGACG CGGACCCGCG ACTGCGCTTC GTCCACGGCG ACATCCGCGA CGCCGGCCTC
    1321 CTCGCCCGGG AACTGCGCGG CGTGGACGCC ATCGTCCACT TCGCGGCCGA GAGCCACGTG
    1381 GACCGCTCCA TCGCGGGCGC GTCCGTGTTC ACCGAGACCA ACGTGCAGGG CACGCAGACG
    1441 CTGCTCCAGT GCGCCGTCGA CGCCGGCGTC GGCCCGGTCG TGCACGTCTC CACCGACGAG
    1501 GTGTACGGGT CGATCGACTC CGGCTCCTGG ACCCAGAGCA GCCCGCTGGA GCCCAACTCG
    1561 CCCTACGCGG CGTCCAAGGC CGGCTCCGAC CTCGTTGCCC GCGCCTACCA CCGGACGTAC
    1621 GGCCTCGACG TACGGATCAC CCGCTGCTGC AACAACTACG GGCCGTACCA GCACCCCGAG
    1681 AAGCTCATCC CCCTCTTCGT GACGAACCTC CTCGACGGCG GGACGCTCCC GCTGTACGGC
    1741 GACGGCGCGA ACGTCCGCGA GTGGGTGCAC ACCGACGACC ACTGCCGGGG CATCGCGCTC
    1801 GTCCTCGCGG GCGGCCGGGC CGGCGAGATC TACCACATCG GCGGCGGCCT GGAGCTGACC
    1861 AACCGCGAAC TCACCGGCAT CCTCCTGGAC TCGCTCGGCG CCGACTGGTC CTCGGTCCGG
    1921 AAGGTCGCCG ACCGCAAGGG CCACGACCTG CGCTACTCCC TCGACGGCGG CAAGATCGAG
    1981 CGCGAGCTCG GCTACCGCCC GCAGGTCTCC TTCGCGGACG GCCTCGCGCG GACCGTCCGC
    2041 TGGTACCGGG AGAACCGCGG CTGGTGGGAG CCGCTCAAGG CGACCGCCCC GCAGCTGCCC
    2101 GCCACCGCCG TCGAGGTGTC CGCCTGAGCA GCCGCGCCGA GACCCCCCGC GTCCCCTTCC
    2161 TCGACCTCAA GGCCGCCTAC GAGGAGCTCC GCGCGGAGAC CGACGCCGCG ATCGCCCGCG
    2221 TCCTCGACTC GGGGCGCTAC CTCCTCGGAC CCGAACTCGA AGGATTCGAG GCGGAGTTCG
    2281 CCGCGTACTG CGAGACGGAC CACGCCGTCG GCGTGAACAG CGGGATGGAC GCCCTCCAGC
    2341 TCGCCCTCCG CGGCCTCGGC ATCGGACCCG GGGACGAGGT GATCGTCCCC TCGCACACGT
    2401 ACATCGCCAG CTGGCTCGCG GTGTCCGCCA CCGGCGCGAC CCCCGTGCCC GTCGAGCCGC
    2461 ACGAGGACCA CCCCACCCTG GACCCGCTGC TCGTCGAGAA GGCGATCACC CCCCGCACCC
    2521 GGGCGCTCCT CCCCGTCCAC CTCTACGGGC ACCCCGCCGA CATGGACGCC CTCCGCGAGC
    2581 TCGCGGACCG GCACGGCCTG CACATCGTCG AGGACGCCGC GCAGGCCCAC GGCGCCCGCT
    2641 ACCGGGGCCG GCGGATCGGC GCCGGGTCGT CGGTGGCCGC GTTCAGCTTC TACCCGGGCA
    2701 AGAACCTCGG CTGCTTCGGC GACGGCGGCG CCGTCGTCAC CGGCGACCCC GAGCTCGCCG
    2761 AACGGCTCCG GATGCTCCGC AACTACGGCT CGCGGCAGAA GTACAGCCAC GAGACGAAGG
    2821 GCACCAACTC CCGCCTGGAC GAGATGCAGG CCGCCGTGCT GCGGATCCGG CTCGNCCACC
    2881 TGGACAGCTG GAACGGCCGC AGGTCGGCGC TGGCCGCGGA GTACCTCTCC GGGCTCGCCG
    2941 GACTGCCCGG CATCGGCCTG CCGGTGACCG CGCCCGACAC CGACCCGGTC TGGCACCTCT
    3001 TCACCGTGCC CACCGAGCGC CGCGACGAGC TGCGCAGCCA CCTCGACGCC CGCGGCATCG
    3061 ACACCCTCAC GCACTACCCG GTACCCGTGC ACCTCTCGCC CGCCTACGCG GGCGAGGCAC
    3121 CGCCGGAAGG CTCGCTCCCG CGGGCCGAGA GCTTCGCGCG GCAGGTCCTC AGCCTGCCGA
    3181 TCGGCCCGCA CCTGGAGCGC CCGCAGGCGC TGCGGGTGAT CGACGCCGTG CGCGAATGGG
    3241 CCGAGCGGGT CGACCAGGCC TAGTCAGGTG GTCCGGTAGA CCCAGCAGGC CG
  • [0084] Contig 004 from cosmid pKOS023-26 contains 1693 nucleotides and the following ORFs: from nucleotide 1692 to 694 is ORF15, which encodes a part of S-adenosylmethionine synthetase; and from nucleotide 692 to 1 is ORF16, which encodes a part of a protein homologous to the M. tuberculosis cbhK gene. (SEQ ID NO:23)
    1 ATGCGGCACC CCTTGGCGCC GAGCGTGGTG ATCCAGGTGC CGACCCGGGC GAGCACCTCC (SEQ ID NO:23)
    61 TGCTCGGTCC AGCCCGTCTT GCTGAGCAGC AGCGCCCGCT CGTAGGCGTT CGTGAACAGC
    121 AGCTCGGCTC CGTCGACGAG CTCCCGGACG CTGTCGCCCT CCAGCCGGGC GAGCTGCTGC
    181 GAGGGGTCCG CGGCCCGGCG GAGGCCCAGC TCGCGGCAGA CCCGCGTGTG CCGCACCATC
    241 GCCTCGGGGT CGTCCGCGCC CACGAGGACG AGGTCGATCC CGCCGGGCCG GCCGGCCGTC
    301 TCGCCCAGGT CGATGTCGCG CGCCTCGGCC ATCGCGCCCG CGTAGAACGA GGCGAGCTGA
    361 TTGCCGTCCT CGTCGGTGGT GCACATGAAG CGGGCGGTGT GCTGACGGTC CGACACCCGC
    421 ACGGAGTCGG TGTCGACGCC CGCGGCGCGG AGCAGCTGCC CGTACCCGTC GAAGTCCTTG
    481 CCGACGGCGC CGACGAGGAC CGGGCGGCGA CCGAGCAGGC CGAGGCCGTA GGCGATGTTG
    541 GCGGCGACGC CGCCGTGCCG GATGTCCAGG GTGTCGACGA GGAACGACAG GGACACGTGG
    601 GCCAGCTGGT CCGGCAGGAT CTGCTCGGCG AAGCGGCCCG GGAAGGTCAT CAGGTGGTCG
    661 GTGGCGATCG ACCCGGTGAC GGCTATACGC ATGTCAGAGC CCCGCGGCCT TCTTCAGGGC
    721 CTCCACGCGG TCGGTGCGCT CCCAGGTGAA GTCCGGCAGC TCGCGGCCGA AGTGGCCGTA
    781 GGCCGCGGTC TGGGAGTAGA TCGGGCGGAG CAGGTCGAGG TCGCGGATGA TCGCGGCCGG
    841 GCGGAGGTCG AAGACCTCGC CGATGGCGTT CTCGATCTTC TCGGTCTCGA TCTTGTGGGT
    901 GCCGAAGGTC TCGACGAAGA GGCCGACGGG CTCGGCCTTG CCGATCGCGT ACGCGACCTG
    961 GACCTCGCAG CGCGAGGCGA GACCGGCGGC GACGACGTTC TTCGCCACCC AGCGCATCGC
    1021 GTACGCGGCG GAGCGGTCGA CCTTCGACGG GTCCTTGCCG GAGAAGGCGC CGCCACCGTG
    1081 GCGGGCCATG CCGCCGTAGG TGTCGATGAT GATCTTGCGG CCGGTGAGGC CGGCGTCGCC
    1141 CATCGGGCCG CCGATCTCGA AGCGACCGGT CGGGTTCACG AGCAGGCGGT AGCCGTCGGT
    1201 GTCGAGCTTG ATGCCGTCCT CGACGAGCTG CGCAAGCACG TGCTCGACGA CGAACTTCCG
    1261 CACGTCGGGG GCGAGCAGCG ACTCCAGGTC GATGTCCGAG GCGTGCTGCG ACGAGACGAC
    1321 GACCGTGTCG AGACGGACCG CCCTGTCGCC GTCGTACTCG ATGGTGACCT GGGTCTTGCC
    1381 GTCGGGACGC AGGTACGGGA TGGTCCCGTT CTTGCGGACC TCGGTCAGGC GGCGCGAGAG
    1441 ACCGTGCGCG AGGTGGATCG GCAGCGGCAT CAGCTCGGGC GTCTCGTCCG AGGCATAGCC
    1501 GAACATCAGG CCCTGGTCAC CGGCGCCCTG CTTGTCGAGC TCGTCCCCCT CGTCCCGCTG
    1561 GGAGGCACCC TCGACCCGCT TCTCGTACGC GGTGTCGACA CCCTGGGCGA TGTCCGGGGA
    1621 CTGCGACCCG ATGGACACCG ACACGCCGCA GGAGGCGCCG TCGAAGCCCT TCTTCGAGGA
    1681 GTCGTACCCG ATC
  • Contig 005 from cosmid pKOS023-26 contains 1565 nucleotides and contains the ORF of the picCV gene that encodes PICCV, involved in desosamine biosynthesis. (SEQ ID NO:24) [0085]
    1 CCCCGCTCGC GGCCCCCCAG ACATCCACGC CCACGATTGG ACGCTCCCGA TGACCGCCCC (SEQ ID NO:24)
    61 CGCCCTCTCC GCCACCGCCC CGGCCGAACG CTGCGCGCAC CCCGGAGCCG ATCTGGGGGC
    121 GGCGGTCCAC GCCGTCGGCC AGACCCTCGC CGCCGGCGGC CTCGTGCCGC CCGACGAGGC
    181 CGGAACGACC GCCCGCCACC TCGTCCGGCT CGCCCTGCGC TACGGCAACA GCCCCTTCAC
    241 CCCGCTGGAG GAGGCCCGCC ACGACCTGGG CGTCGACCGG GACGCCTTCC GGCGCCTCCT
    301 CGCCCTGTTC GGGCAGGTCC CGGAGCTCCG CACCGCGGTC GAGACCGGCC CCGCCGCCGC
    361 CTACTGGAAG AACACCCTGC TCCCGCTCGA ACAGCGCGGC GTCTTCGACC CGGCGCTCGC
    421 CAGGAAGCCC GTCTTCCCGT ACAGCGTCGG CCTCTACCCC GGCCCGACCT GCATGTTCCG
    481 CTGCCACTTC TGCGTCCGTG TGACCGCCGC CCGCTACGAC CCGTCCGCCC TCGACGCCGG
    541 CAACGCCATG TTCCGGTCGG TCATCGACGA GATACCCGCG GGCAACCCCT CGGCGATGTA
    601 CTTCTCCGGC GGCCTGGAGC CGCTCACCAA CCCCGGCCTC GGGAGCCTGG CCGCGCACGC
    661 CACCGACCAC GGCCTGCGGC CCACCGTCTA CACGAACTCC TTCGCGCTCA CCGAGCGCAC
    721 CCTGGAGCGC CAGCCCGGCC TCTGGGGCCT GCACGCCATC CGCACCTCGC TCTACGGCCT
    781 CAACGACGAG GAGTACGAGC AGACCACCGG CAAGAAGGCC GCCTTCCGCC GCGTCCGCGA
    841 GAACCTGCGC CGCTTCCAGC AGCTGCGCGC CGAGCGCGAG TCGCCGATCA ACCTCGGCTT
    901 CGCCTACATC GTGCTCCCGG GCCGTGCCTC CCGCCTGCTC GACCTGGTCG ACTTCATCGC
    961 CGACCTCAAC GACGCCGGGC AGGGCAGGAC GATCGACTTC GTCAACATTC GCGAGGACTA
    1021 CAGCGGCCGT GACGACGGCA AGCTGCCGCA GGAGGAGCGG GCCGAGCTCC AGGAGGCCCT
    1081 CAACGCCTTC GAGGAGCGGG TCCGCGAGCG CACCCCCGGA CTCCACATCG ACTACGGCTA
    1141 CGCCCTGAAC AGCCTGCGCA CCGGGGCCGA CGCCGAACTG CTGCGGATCA AGCCCGCCAC
    1201 CATGCGGCCC ACCGCGCACC CGCAGGTCCC GGTGCAGGTC GATCTCCTCG GCGACGTGTA
    1261 CCTGTACCGC GAGGCCGGCT TCCCCGACCT GGACGGCGCG ACCCGCTACA TCGCGGGCCG
    1321 CGTGACCCCC GACACCTCCC TCACCGAGGT CGTCAGGGAC TTCGTCGAGC GCGGCGGCGA
    1381 GCTGGCGGCC GTCGACGGCG ACGAGTACTT CATGGACGGC TTCGATCAGG TCGTCACCGC
    1441 CCGCCTGAAC CAGCTGGAGC GCGACGCCGC GGACGGCTGG GAGGAGGCCC GCGGCTTCCT
    1501 GCGCTGACCC GCACCCGCCC CGATCCCCCC GATCCCCCCC CCACGATCCC CCCACCTGAG
    1561 GGCCC
  • The recombinant desosamine biosynthesis and transfer and beta-glucosidase genes and proteins provided by the invention are useful in the production of glycosylated polyketides in a variety of host cells, as described in Section IV below. [0086]
  • Section III. The Genes for Macrolide Ring Modification: the picK Hydroxylase Gene [0087]
  • The present invention provides the picK gene in recombinant form as well as recombinant PicK protein. The availability of the hydroxylase encoded by the picK gene in recombinant form is of significant benefit in that the enzyme can convert narbomycin into picromycin and accepts in addition a variety of polyketide substrates, particularly those related to narbomycin in structure. The present invention also provides methods of hydroxylating polyketides, which method comprises contacting the polyketide with the recombinant PicK enzyme under conditions such that hydroxylation occurs. This methodology is applicable to large numbers of polyketides. [0088]
  • DNA encoding the picK gene can be isolated from cosmid pKOS023-26 of the invention. The DNA sequence of the picK gene is shown in the preceding section. This DNA sequence encodes one of the recombinant forms of the enzyme provided by the invention. The amino acid sequence of this form of the picK gene is shown below. The present invention also provides a recombinant picK gene that encodes a picK gene product in which the PicK protein is fused to a number of consecutive histidine residues, which facilitates purification from recombinant host cells. [0089]
  • Amino Acid Sequence of Picromycin/Methymycin Cytochrome P450 Hydroxylase, PicK (SEQ ID NO:18) [0090]
    1 VRRTQQGTTA SPPVLDLGAL GQDFAADPYP TYARLRAEGP AHRVRTPEGD EVWLVVGYDR (SEQ ID NO:18)
    61 ARAVLADPRF SKDWRNSTTP LTEAEAALNH NMLESDPPRH TRLRKLVARE FTMRRVELLR
    121 PRVQEIVDGL VDAMLAAPDG RADLMESLAW PLPITVISEL LGVPEPDRAA FRVWTDAFVF
    181 PDDPAQAQTA MAEMSGYLSR LIDSKRGQDG EDLLSALVRT SDEDGSRLTS EELLGMAHIL
    241 LVAGHETTVN LIANGMYALL SHPDQLAALR ADMTLLDGAV EEMLRYEGPV ESATYRFPVE
    301 PVDLDGTVIP AGDTVLVVLA DAHRTPERFP DPHRFDIRRD TAGHLAFGHG IHFCIGAPLA
    361 RLEARIAVRA LLERCPDLAL DVSPGELVWY PNPMIRGLKA LPIRWRRGRE AGRRTG
  • The recombinant PicK enzyme of the invention hydroxylates narbomycin at the C12 position and YC-17 at either the C10 or C12 position. Hydroxylation of these compounds at the respective positions increases the antibiotic activity of the compound relative to the unhydroxylated compound. Hydroxylation can be achieved by a number of methods. First, the hydroxylation may be performed in vitro using purified hydroxylase, or the relevant hydroxylase can be produced recombinantly and utilized directly in the cell that produces it. Thus, hydroxylation may be effected by supplying the nonhydroxylated precursor to a cell that expresses the hydroxylase. These and other details of this embodiment of the invention are described in additional detail below in Section IV and the examples. [0091]
  • Section IV: Heterologous Expression of the Narbonolide PKS; the Desosamine Biosynthetic and Transferase Genes; the Beta-Glucosidase Gene; and the picK Hydroxylase Gene [0092]
  • In one important embodiment, the invention provides methods for the heterologous expression of one or more of the genes involved in picromycin biosynthesis and recombinant DNA expression vectors useful in the method. Thus, included within the scope of the invention in addition to isolated nucleic acids encoding domains, modules, or proteins of the narbonolide PKS, glycosylation, and/or hydroxylation enzymes, are recombinant expression systems. These systems contain the coding sequences operably linked to promoters, enhancers, and/or termination sequences that operate to effect expression of the coding sequence in compatible host cells. The host cells are modified by transformation with the recombinant DNA expression vectors of the invention to contain these sequences either as extrachromosomal elements or integrated into the chromosome. The invention also provides methods to produce PKS and post-PKS tailoring enzymes as well as polyketides and antibiotics using these modified host cells. [0093]
  • As used herein, the term expression vector refers to a nucleic acid that can be introduced into a host cell or cell-free transcription and translation medium. An expression vector can be maintained stably or transiently in a cell, whether as part of the chromosomal or other DNA in the cell or in any cellular compartment, such as a replicating vector in the cytoplasm. An expression vector also comprises a gene that serves to produce RNA, which typically is translated into a polypeptide in the cell or cell extract. To drive production of the RNA, the expression vector typically comprises one or more promoter elements. Furthermore, expression vectors typically contain additional functional elements, such as, for example, a resistance-conferring gene that acts as a selectable marker. [0094]
  • The various components of an expression vector can vary widely, depending on the intended use of the vector. In particular, the components depend on the host cell(s) in which the vector will be introduced or in which it is intended to function. Components for expression and maintenance of vectors in [0095] E. coli are widely known and commercially available, are components for other commonly used organisms, such as yeast cells and Streptomyces cells.
  • One important component is the promoter, which can be referred to as, or can be included within, a control sequence or control element, which drives expression of the desired gene product in the heterologous host cell. Suitable promoters include those that function in eucaryotic or procaryotic host cells. In addition to a promoter, a control element can include, optionally, operator sequences, and other elements, such as ribosome binding sites, depending on the nature of the host. Regulatory sequences that allow for regulation of expression of the heterologous gene relative to the growth of the host cell may also be included. Examples of such regulatory sequences known to those of skill in the art are those that cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus. [0096]
  • Preferred host cells for purposes of selecting vector components include fungal host cells such as yeast and procaryotic, especially [0097] E. coli and Streptomyces, host cells, but single cell cultures of, for example, mammalian cells can also be used. In hosts such as yeasts, plants, or mammalian cells that ordinarily do not produce polyketides, it may be necessary to provide, also typically by recombinant means, suitable holo-ACP synthases to convert the recombinantly produced PKS to functionality. Provision of such enzymes is described, for example, in PCT publication Nos. WO 97/13845 and WO 98/27203, each of which is incorporated herein by reference. Control systems for expression in yeast, including controls that effect secretion are widely available and can be routinely used. For E. coli or other bacterial host cells, promoters such as those derived from sugar metabolizing enzymes, such as galactose, lactose (lac), and maltose, can be used. Additional examples include promoters derived from genes encoding biosynthetic enzymes, and the tryptophan (trp), the beta-lactamase (bla), bacteriophage lambda PL, and T5 promoters. In addition, synthetic promoters, such as the tac promoter (U.S. Pat. No. 4,551,433), can also be used.
  • Particularly preferred are control sequences compatible with Streptomyces spp. Particularly useful promoters for Streptomyces host cells include those from PKS gene clusters that result in the production of polyketides as secondary metabolites, including promoters from aromatic (Type II) PKS gene clusters. Examples of Type II PKS gene cluster promoters are act gene promoters and tcm gene promoters; an example of a Type I PKS gene cluster promoter is the spiramycin PKS gene promoter. [0098]
  • If a Streptomyces or other host ordinarily produces polyketides, it may be desirable to modify the host so as to prevent the production of endogenous polyketides prior to its use to express a recombinant PKS of the invention. Such hosts have been described, for example, in U.S. Pat. No. 5,672,491, incorporated herein by reference. In such hosts, it may not be necessary to provide enzymatic activities for all of the desired post-translational modifications of the enzymes that make up the recombinantly produced PKS, because the host naturally expresses such enzymes. In particular, these hosts generally contain holo-ACP synthases that provide the pantotheinyl residue needed for functionality of the PKS. [0099]
  • Thus, in one important embodiment, the vectors of the invention are used to transform Streptomyces host cells to provide the recombinant Streptomyces host cells of the invention. Streptomyces is a convenient host for expressing narbonolide or 10-deoxymethynolide or derivatives of those compounds, because narbonolide and 10-deoxymethynolide are naturally produced in certain Streptomyces species, and Streptomyces generally produce the precursors needed to form the desired polyketide. The present invention also provides the narbonolide PKS gene promoter in recombinant form, located upstream of the picAI gene on cosmid pKOS023-27. This promoter can be used to drive expression of the narbonolide PKS or any other coding sequence of interest in host cells in which the promoter functions, particularly [0100] S. venezuelae and generally any Streptomyces species. As described below, however, promoters other than the promoter of the narbonolide PKS genes will typically be used for heterologous expression.
  • For purposes of the invention, any host cell other than [0101] Streptomyces venezuelae is a heterologous host cell. Thus, S. narbonensis, which produces narbomycin but not picromycin is a heterologous host cell of the invention, although other host cells are generally preferred for purposes of heterologous expression. Those of skill in the art will recognize that, if a Streptomyces host that produces a picromycin or methymycin precursor is used as the host cell, the recombinant vector need drive expression of only a portion of the genes constituting the picromycin gene cluster. As used herein, the picromycin gene cluster includes the narbonolide PKS, the desosamine biosynthetic and transferase genes, the beta-glucosidase gene, and the picK hydroxylase gene. Thus, such a vector may comprise only a single ORF, with the desired remainder of the polypeptides encoded by the picromycin gene cluster provided by the genes on the host cell chromosomal DNA.
  • The present invention also provides compounds and recombinant DNA vectors useful for disrupting any gene in the picromycin gene cluster (as described above and illustrated in the examples below). Thus, the invention provides a variety of modified host cells (particularly, [0102] S. narbonensis and S. venezuelae) in which one or more of the genes in the picromycin gene cluster have been disrupted. These cells are especially useful when it is desired to replace the disrupted function with a gene product expressed by a recombinant DNA vector. Thus, the invention provides such Streptomyces host cells, which are preferred host cells for expressing narbonolide derivatives of the invention. Particularly preferred host-cells of this type include those in which the coding sequence for the loading module has been disrupted, those in which one or more of any of the PKS gene ORFs has been disrupted, and/or those in which the picK gene has been disrupted.
  • In a preferred embodiment, the expression vectors of the invention are used to construct a heterologous recombinant Streptomyces host cell that expresses a recombinant PKS of the invention. As noted above, a heterologous host cell for purposes of the present invention is any host cell other than [0103] S. venezuelae, and in most cases other than S. narbonensis as well. Particularly preferred heterologous host cells are those which lack endogenous functional PKS genes. Illustrative host cells of this type include the modified Streptomyces coelicolor CH999 and similarly modified S. lividans described in PCT publication No. WO 96/40968.
  • The invention provides a wide variety of expression vectors for use in Streptomyces. For replicating vectors, the origin of replication can be, for example and without limitation, a low copy number vector, such as SCP2* (see Hopwood et al., [0104] Genetic Manipulation of Streptomyces: A Laboratory manual (The John Innes Foundation, Norwich, U.K., 1985); Lydiate et al., 1985, Gene 35: 223-235; and Kieser and Melton, 1988, Gene 65: 83-91, each of which is incorporated herein by reference), SLP1.2 (Thompson et al., 1982, Gene 20: 51-62, incorporated herein by reference), and pSG5(ts) (Muth et al., 1989, Mol. Gen. Genet. 219: 341-348, and Bierman et al., 1992, Gene 116: 43-49, each of which is incorporated herein by reference), or a high copy number vector, such as pIJ101 and pJV1 (see Katz et al., 1983, J. Gen. Microbiol. 129: 2703-2714; Vara et al., 1989, J. Bacteriol. 171: 5782-5781; and Servin-Gonzalez, 1993, Plasmid 30: 131-140, each of which is incorporated herein by reference). High copy number vectors are generally, however, not preferred for expression of large genes or multiple genes. For non-replicating and integrating vectors and generally for any vector, it is useful to include at least an E. coli origin of replication, such as from pUC, p1P, p1I, and pBR. For phage based vectors, the phage phiC31 and its derivative KC515 can be employed (see Hopwood et al., supra). Also, plasmid pSET152, plasmid pSAM, plasmids pSE101 and pSE211, all of which integrate site-specifically in the chromosomal DNA of S. lividans, can be employed.
  • Preferred Streptomyces host cell/vector combinations of the invention include [0105] S. coelicolor CH999 and S. lividans K4-114 host cells, which do not produce actinorhodin, and expression vectors derived from the pRM1 and pRM5 vectors, as described in U.S. Pat. No. 5,830,750 and U.S. patent application Ser. No. 08/828,898, filed Mar. 31, 1997, and Ser. No. 09/181,833, filed Oct. 28, 1998, each of which is incorporated herein by reference.
  • As described above, particularly useful control sequences are those that alone or together with suitable regulatory systems activate expression during transition from growth to stationary phase in the vegetative mycelium. The system contained in the illustrative plasmid pRM5, i.e., the actI/actIII promoter pair and the actII-ORF4 activator gene, is particularly preferred. Other useful Streptomyces promoters include without limitation those from the ermE gene and the melC1 gene, which act constitutively, and the tipA gene and the merA gene, which can be induced at any growth stage. In addition, the T7 RNA polymerase system has been transferred to Streptomyces and can be employed in the vectors and host cells of the invention. In this system, the coding sequence for the T7 RNA polymerase is inserted into a neutral site of the chromosome or in a vector under the control of the inducible merA promoter, and the gene of interest is placed under the control of the T7 promoter. As noted above, one or more activator genes can also be employed to enhance the activity of a promoter. Activator genes in addition to the actII-ORF4 gene described above include dnrI, redD, and ptpA genes (see U.S. patent application Ser. No. 09/181,833, supra). [0106]
  • Typically, the expression vector will comprise one or more marker genes by which host cells containing the vector can be identified and/or selected. Selectable markers are often preferred for recombinant expression vectors. A variety of markers are known that are useful in selecting for transformed cell lines and generally comprise a gene that confers a selectable phenotype on transformed cells when the cells are grown in an appropriate selective medium. Such markers include, for example, genes that confer antibiotic resistance or sensitivity to the plasmid. Alternatively, several polyketides are naturally colored, and this characteristic can provide a built-in marker for identifying cells. Preferred selectable markers include antibiotic resistance conferring genes. Preferred for use in Streptomyces host cells are the ermE (confers resistance to erythromycin and lincomycin), tsr (confers resistance to thiostrepton), aadA (confers resistance to spectinomycin and streptomycin), aacC4 (confers resistance to apramycin, kanamycin, gentamicin, geneticin (G418), and neomycin), hyg (confers resistance to hygromycin), and vph (confers resistance to viomycin) resistance conferring genes. [0107]
  • To provide a preferred host cell and vector for purposes of the invention, the narbonolide PKS genes were placed on a recombinant expression vector that was transferred to the non-macrolide producing host [0108] Streptomyces lividans K4-114, as described in Example 3. Transformation of S. lividans K4-114 with this expression vector resulted in a strain which produced two compounds in similar yield (-5-10 mg/L each). Analysis of extracts by LC/MS followed by 1H-NMR spectroscopy of the purified compounds established their identity as narbonolide (FIG. 5, compound 4) and 10-deoxymethynolide (FIG. 5, compound 5), the respective 14 and 12-membered polyketide precursors of narbomycin and YC17. Foundation, Norwich, U.K., 1985); Lydiate et al., 1985, Gene 35: 223-235; and Kieser and Melton, 1988, Gene 65: 83-91, each of which is incorporated herein by reference), SLP1.2 (Thompson et al., 1982, Gene 20: 51-62, incorporated herein by reference), and pSG5(ts) (Muth et al., 1989, Mol. Gen. Genet. 219: 341-348, and Bierman et al., 1992, Gene 116: 43-49, each of which is incorporated herein by reference), or a high copy number vector, such as pIJ101 and pJV1 (see Katz et al., 1983, J. Gen. Microbiol. 129: 2703-2714; Vara et al., 1989, J. Bacteriol. 171: 5782-5781; and Servin-Gonzalez, 1993, Plasmid 30: 131-140, each of which is incorporated herein by reference). High copy number vectors are generally, however, not preferred for expression of large genes or multiple genes. For non-replicating and integrating vectors and generally for any vector, it is useful to include at least an E. coli origin of replication, such as from pUC, p1P, p1I, and pBR. For phage based vectors, the phage phiC31 and its derivative KC515 can be employed (see Hopwood et al., supra). Also, plasmid pSET152, plasmid pSAM, plasmids pSE101 and pSE211, all of which integrate site-specifically in the chromosomal DNA of S. lividans, can be employed.
  • Preferred Streptomyces host cell/vector combinations of the invention include [0109] S. coelicolor CH999 and S. lividans K4-114 host cells, which do not produce actinorhodin, and expression vectors derived from the pRM1 and pRM5 vectors, as described in U.S. Pat. No. 5,830,750 and U.S. patent application Ser. No. 08/828,898, filed Mar. 31, 1997, and Ser. No. 09/181,833, filed Oct. 28, 1998, each of which is incorporated herein by reference.
  • As described above, particularly useful control sequences are those that alone or together with suitable regulatory systems activate expression during transition from growth to stationary phase in the vegetative mycelium. The system contained in the illustrative plasmid pRM5, i.e., the actI/actIII promoter pair and the actII-ORF4 activator gene, is particularly preferred. Other useful Streptomyces promoters include without limitation those from the ermE gene and the melC1 gene, which act constitutively, and the tipA gene and the merA gene, which can be induced at any growth stage. In addition, the T7 RNA polymerase system has been transferred to Streptomyces and can be employed in the vectors and host cells of the invention. In this system, the coding [0110]
  • To provide a host cell of the invention that produces the narbonolide PKS as well as an additional narbonolide biosynthetic gene and to investigate the possible role of the PIC TEII in picromycin biosynthesis, the picB gene was integrated into the chromosome to provide the host cell of the invention [0111] Streptomyces lividans K39-18. The picB gene was cloned into the Streptomyces genome integrating vector pSET152 (see Bierman et al., 1992, Gene 116: 43, incorporated herein by reference) under control of the same promoter (PactI) as the PKS on plasmid pKOS039-86.
  • A comparison of strains [0112] Streptomyces lividans K39-18/pKOS039-86 and K4-114/pKOS039-86 grown under identical conditions indicated that the strain containing TEII produced 4-7 times more total polyketide. This increased production indicates that the enzyme is functional in this strain and is consistent with the observation that yields fall to below 5% for both picromycin and methymycin when picB is disrupted in S. venezuelae. Because the production levels of compound 4 and 5 from K39-18/pKOS03986 increased by the same relative amounts, TEII does not appear to influence the ratio of 12 and 14-membered lactone ring formation. Thus, the invention provides methods of coexpressing the picB gene product or any other type II thioesterase with the narbonolide PKS or any other PKS in heterologous host cells to increase polyketide production. However, transformation of a 6dEB-producing Streptomyces lividans/pCK7 strain with an expression vector of the invention that produces PIC TEII resulted in little or no increase in 6-dEB levels, indicating that TEII enzymes may have some specificity for their cognate PKS complexes and that use of homologous TEII enzymes will provide optimal activity.
  • In accordance with the methods of the invention, picromycin biosynthetic genes in addition to the genes encoding the PKS and PIC TEII can be introduced into heterologous host cells. In particular, the picK gene, desosamine biosynthetic genes, and the desosaminyl transferase gene can be expressed in the recombinant host cells of the invention to produce any and all of the polyketides in the picromycin biosynthetic pathway (or derivatives thereof). Those of skill will recognize that the present invention enables one to select whether only the 12-membered polyketides, or only the 14-membered polyketides, or both 12- and 14-membered polyketides will be produced. To produce only the 12-membered polyketides, the invention provides expression vectors in which the last module is deleted or the KS domain of that module is deleted or rendered inactive. If [0113] module 6 is deleted, then one preferably deletes only the non-TE domain portion of that module or one inserts a heterologous TE domain, as the TE domain facilitates cleavage of the polyketide from the PKS and cyclization and thus generally increases yields of the desired polyketide. To produce only the 14-membered polyketides, the invention provides expression vectors in which the coding sequences of extender modules 5 and 6 are fused to provide only a single polypeptide.
  • In one important embodiment, the invention provides methods for desosaminylating polyketides or other compounds. In this method, a host cell other than [0114] Streptomyces venezuelae is transformed with one or more recombinant vectors of the invention comprising the desosamine biosynthetic and desosaminyl transferase genes and control sequences positioned to express those genes. The host cells so transformed can either produce the polyketide to be desosaminylated naturally or can be transformed with expression vectors encoding the PKS that produces the desired polyketide. Alternatively, the polyketide can be supplied to the host cell containing those genes. Upon production of the polyketide and expression of the desosamine biosynthetic and desosaminyl transferase genes, the desired desosaminylated polyketide is produced. This method is especially useful in the production of polyketides to be used as antibiotics, because the presence of the desosamine residue is known to increase, relative to their undesosaminylated counterparts, the antibiotic activity of many polyketides significantly. The present invention also provides a method for desosaminylating a polyketide by transforming an S. venezuelae or S. narbonensis host cell with a recombinant vector that encodes a PKS that produces the polyketide and culturing the transformed cell under conditions such that said polyketide is produced and desosaminylated. In this method, use of an S. venezuelae or S. narbonensis host cell of the invention that does not produce a functional endogenous narbonolide PKS is preferred.
  • In a related aspect, the invention provides a method for improving the yield of a desired desosaminylated polyketide in a host cell, which method comprises transforming the host cell with a beta-glucosidase gene. This method is not limited to host cells that have been transformed with expression vectors of the invention encoding the desosamine biosynthetic and desosaminyl transferase genes of the invention but instead can be applied to any host cell that desosaminylates polyketides or other compounds. Moreover, while the beta-glucosidase gene from [0115] Streptomyces venezuelae provided by the invention is preferred for use in the method, any beta-glucosidase gene may be employed. In another embodiment, the beta-glucosidase treatment is conducted in a cell free extract.
  • Thus, the invention provides methods not only for producing narbonolide and 10-deoxymethynolide in heterologous host cells but also for producing narbomycin and YC-17 in heterologous host cells. In addition, the invention provides methods for expressing the picK gene product in heterologous host cells, thus providing a means to produce picromycin, methymycin, and neomethymycin in heterologous host cells. Moreover, because the recombinant expression vectors provided by the invention enable the artisan to provide for desosamine biosynthesis and transfer and/or C10 or C12 hydroxylation in any host cell, the invention provides methods and reagents for producing a very wide variety of glycosylated and/or hydroxylated polyketides. This variety of polyketides provided by the invention can be better appreciated upon consideration of the following section relating to the production of polyketides from heterologous or hybrid PKS enzymes provided by the invention. [0116]
  • Section V: Hybrid PKS Genes [0117]
  • The present invention provides recombinant DNA compounds encoding each of the domains of each of the modules of the narbonolide PKS, the proteins involved in desosamine biosynthesis and transfer to narbonolide, and the PicK protein. The availability of these compounds permits their use in recombinant procedures for production of desired portions of the narbonolide PKS fused to or expressed in conjunction with all or a portion of a heterologous PKS. The resulting hybrid PKS can then be expressed in a host cell, optionally with the desosamine biosynthesis and transfer genes and/or the picK hydroxylase gene to produce a desired polyketide. [0118]
  • Thus, in accordance with the methods of the invention, a portion of the narbonolide PKS coding sequence that encodes a particular activity can be isolated and manipulated, for example, to replace the corresponding region in a different modular PKS. In addition, coding sequences for individual modules of the PKS can be ligated into suitable expression systems and used to produce the portion of the protein encoded. The resulting protein can be isolated and purified or can may be employed in situ to effect polyketide synthesis. Depending on the host for the recombinant production of the domain, module, protein, or combination of proteins, suitable control sequences such as promoters, termination sequences, enhancers, and the like are ligated to the nucleotide sequence encoding the desired protein in the construction of the expression vector. [0119]
  • In one important embodiment, the invention thus provides a hybrid PKS and the corresponding recombinant DNA compounds that encode those hybrid PKS enzymes. For purposes of the invention, a hybrid PKS is a recombinant PKS that comprises all or part of one or more extender modules, loading module, and/or thioesterase/cyclase domain of a first PKS and all or part of one or more extender modules, loading module, and/or thioesterase/cyclase domain of a second PKS. In one preferred embodiment, the first PKS is most but not all of the narbonolide PKS, and the second PKS is only a portion or all of a non-narbonolide PKS. An illustrative example of such a hybrid PKS includes a narbonolide PKS in which the natural loading module has been replaced with a loading module of another PKS. Another example of such a hybrid PKS is a narbonolide PKS in which the AT domain of [0120] extender module 3 is replaced with an AT domain that binds only malonyl CoA.
  • In another preferred embodiment, the first PKS is most but not all of a non-narbonolide PKS, and the second PKS is only a portion or all of the narbonolide PKS. An illustrative example of such a hybrid PKS includes a DEBS PKS in which an AT specific for methylmalonyl CoA is replaced with the AT from the narbonolide PKS specific for malonyl CoA. [0121]
  • Those of skill in the art will recognize that all or part of either the first or second PKS in a hybrid PKS of the invention need not be isolated from a naturally occurring source. For example, only a small portion of an AT domain determines its specificity. See U.S. provisional patent application Serial No. 60/091,526, and Lau et al., infra, incorporated herein by reference. The state of the art in DNA synthesis allows the artisan to construct de novo DNA compounds of size sufficient to construct a useful portion of a PKS module or domain. Thus, the desired derivative coding sequences can be synthesized using standard solid phase synthesis methods such as those described by Jaye et al., 1984[0122] , J. Biol. Chem. 259: 6331, and instruments for automated synthesis are available commercially from, for example, Applied Biosystems, Inc. For purposes of the invention, such synthetic DNA compounds are deemed to be a portion of a PKS.
  • With this general background regarding hybrid PKSs of the invention, one can better appreciate the benefit provided by the DNA compounds of the invention that encode the individual domains, modules, and proteins that comprise the narbonolide PKS. As described above, the narbonolide PKS is comprised of a loading module, six extender modules composed of a KS, AT, ACP, and optional KR, DH, and ER domains, and a thioesterase domain. The DNA compounds of the invention that encode these domains individually or in combination are useful in the construction of the hybrid PKS encoding DNA compounds of the invention. [0123]
  • The recombinant DNA compounds of the invention that encode the loading module of the narbonolide PKS and the corresponding polypeptides encoded thereby are useful for a variety of applications. In one embodiment, a DNA compound comprising a sequence that encodes the narbonolide PKS loading module is inserted into a DNA compound that comprises the coding sequence for a heterologous PKS. The resulting construct, in which the coding sequence for the loading module of the heterologous PKS is replaced by that for the coding sequence of the narbonolide PKS loading module provides a novel PKS. Examples include the 6-deoxyerythronolide B, rapamycin, FK506, FK520, rifamycin, and avermectin PKS coding sequences. In another embodiment, a DNA compound comprising a sequence that encodes the narbonolide PKS loading module is inserted into a DNA compound that comprises the coding sequence for the narbonolide PKS or a recombinant narbonolide PKS that produces a narbonolide derivative in a different location in the modular system. [0124]
  • In another embodiment, a portion of the loading module coding sequence is utilized in conjunction with a heterologous coding sequence. In this embodiment, the invention provides, for example, replacing the propionyl CoA specific AT with an acetyl CoA, butyryl CoA, or other CoA specific AT. In addition, the KS[0125] Q and/or ACP can be replaced by another inactivated KS and/or another ACP. Alternatively, the KSQ, AT, and ACP of the loading module can be replaced by an AT and ACP of a loading module such as that of DEBS. The resulting heterologous loading module coding sequence can be utilized in conjunction with a coding sequence for a PKS that synthesizes narbonolide, a narbonolide derivative, or another polyketide.
  • The recombinant DNA compounds of the invention that encode the first extender module of the narbonolide PKS and the corresponding polypeptides encoded thereby are useful for a variety of applications. In one embodiment, a DNA compound comprising a sequence that encodes the narbonolide PKS first extender module is inserted into a DNA compound that comprises the coding sequence for a heterologous PKS. The resulting construct, in which the coding sequence for a module of the heterologous PKS is either replaced by that for the first extender module of the narbonolide PKS or the latter is merely added to coding sequences for modules of the heterologous PKS, provides a novel PKS coding sequence. In another embodiment, a DNA compound comprising a sequence that encodes the first extender module of the narbonolide PKS is inserted into a DNA compound that comprises coding sequences for the narbonolide PKS or a recombinant narbonolide PKS that produces a narbonolide derivative or into a different location in the modular system. [0126]
  • In another embodiment, a portion or all of the first extender module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module. In this embodiment, the invention provides, for example, replacing the methylmalonyl CoA specific AT with a malonyl CoA, ethylmalonyl CoA, or carboxyglycolyl CoA specific AT; deleting (which includes inactivating) the KR; inserting a DH or a DH and ER; and/or replacing the KR with another KR, a DH and KR, a DH, KR, and ER. In addition, the KS and/or ACP can be replaced with another KS and/or ACP. In each of these replacements or insertions, the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the narbonolide PKS, from a gene for a PKS that produces a polyketide other than narbonolide, or from chemical synthesis. The resulting heterologous first extender module coding sequence can be utilized in conjunction with a coding sequence for a PKS that synthesizes narbonolide, a narbonolide derivative, or another polyketide. [0127]
  • In an illustrative embodiment of this aspect of the invention, the invention provides recombinant PKSs and recombinant DNA compounds and vectors that encode such PKSs in which the KS domain of the first extender module has been inactivated. Such constructs are especially useful when placed in translational reading frame with the remaining modules and domains of a narbonolide PKS or narbonolide derivative PKS. The utility of these constructs is that host cells expressing, or cell free extracts containing, the PKS encoded thereby can be fed or supplied with N-acetylcysteamine thioesters of novel precursor molecules to prepare narbonolide derivatives. See U.S. patent application Serial No. 60/117,384, filed Jan. 27, 1999, and PCT publication Nos. WO 99/03986 and WO 97/02358, each of which is incorporated herein by reference. [0128]
  • The recombinant DNA compounds of the invention that encode the second extender module of the narbonolide PKS and the corresponding polypeptides encoded thereby are useful for a variety of applications. In one embodiment, a DNA compound comprising a sequence that encodes the narbonolide PKS second extender module is inserted into a DNA compound that comprises the coding sequence for a heterologous PKS. The resulting construct, in which the coding sequence for a module of the heterologous PKS is either replaced by that for the second extender module of the narbonolide PKS or the latter is merely added to coding sequences for the modules of the heterologous PKS, provides a novel PKS. In another embodiment, a DNA compound comprising a sequence that encodes the second extender module of the narbonolide PKS is inserted into a DNA compound that comprises the coding sequences for the narbonolide PKS or a recombinant narbonolide PKS that produces a narbonolide derivative. [0129]
  • In another embodiment, a portion or all of the second extender module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module. In this embodiment, the invention provides, for example, replacing the malonyl CoA specific AT with a methylmalonyl CoA, ethylmalonyl CoA, or carboxyglycolyl CoA specific AT; deleting (or inactivating) the KR, the DH, or both the DH and KR; replacing the KR or the KR and DH with a KR, a KR and a DH, or a KR, DH, and ER; and/or inserting an ER. In addition, the KS and/or ACP can be replaced with another KS and/or ACP. In each of these replacements or insertions, the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for-another module of the narbonolide PKS, from a coding sequence for a PKS that produces a polyketide other than narbonolide, or from chemical synthesis. The resulting heterologous second extender module coding sequence can be utilized in conjunction with a coding sequence from a PKS that synthesizes narbonolide, a narbonolide derivative, or another polyketide. [0130]
  • The recombinant DNA compounds of the invention that encode the third extender module of the narbonolide PKS and the corresponding polypeptides encoded thereby are useful for a variety of applications. In one embodiment, a DNA compound comprising a sequence that encodes the narbonolide PKS third extender module is inserted into a DNA compound that comprises the coding sequence for a heterologous PKS. The resulting construct, in which the coding sequence for a module of the heterologous PKS is either replaced by that for the third extender module of the narbonolide PKS or the latter is merely added to coding sequences for the modules of the heterologous PKS, provides a novel PKS. In another embodiment, a DNA compound comprising a sequence that encodes the third extender module of the narbonolide PKS is inserted into a DNA compound that comprises coding sequences for the narbonolide PKS or a recombinant narbonolide PKS that produces a narbonolide derivative. [0131]
  • In another embodiment, a portion or all of the third extender module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module. In this embodiment, the invention provides, for example, replacing the methylmalonyl CoA specific AT with a malonyl CoA, ethylmalonyl CoA, or carboxyglycolyl CoA specific AT; deleting the inactive KR; and/or inserting a KR, or a KR and DH, or a KR, DH, and ER. In addition, the KS and/or ACP can be replaced with another KS and/or ACP. In each of these replacements or insertions, the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the narbonolide PKS, from a gene for a PKS that produces a polyketide other than narbonolide, or from chemical synthesis. The resulting heterologous third extender module coding sequence can be utilized in conjunction with a coding sequence for a PKS that synthesizes narbonolide, a narbonolide derivative, or another polyketide. [0132]
  • The recombinant DNA compounds of the invention that encode the fourth extender module of the narbonolide PKS and the corresponding polypeptides encoded thereby are useful for a variety of applications. In one embodiment, a DNA compound comprising a sequence that encodes the narbonolide PKS fourth extender module is inserted into a DNA compound that comprises the coding sequence for a heterologous PKS. The resulting construct, in which the coding sequence for a module of the heterologous PKS is either replaced by that for the fourth extender module of the narbonolide PKS or the latter is merely added to coding sequences for the modules of the heterologous PKS, provides a novel PKS. In another embodiment, a DNA compound comprising a sequence that encodes the fourth extender module of the narbonolide PKS is inserted into a DNA compound that comprises coding sequences for the narbonolide PKS or a recombinant narbonolide PKS that produces a narbonolide derivative. [0133]
  • In another embodiment, a portion of the fourth extender module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module. In this embodiment, the invention provides, for example, replacing the methylmalonyl CoA specific AT with a malonyl CoA, ethylmalonyl CoA, or carboxyglycolyl CoA specific AT; deleting any one, two, or all three of the ER, DH, and KR; and/or replacing any one two, or all three of the ER, DH, and KR with either a KR, a DH and KR, or a KR, DH, and ER. In addition, the KS and/or ACP can be replaced with another KS and/or ACP. In each of these replacements or insertions, the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the narbonolide PKS, from a coding sequence for a PKS that produces a polyketide other than narbonolide, or from chemical synthesis. The resulting heterologous fourth extender module coding sequence can be utilized in conjunction with a coding sequence for a PKS that synthesizes narbonolide, a narbonolide derivative, or another polyketide. [0134]
  • The recombinant DNA compounds of the invention that encode the fifth extender module of the narbonolide PKS and the corresponding polypeptides encoded thereby are useful for a variety of applications. In one embodiment, a DNA compound comprising a sequence that encodes the narbonolide PKS fifth extender module is inserted into a DNA compound that comprises the coding sequence for a heterologous PKS. The resulting construct, in which the coding sequence for a module of the heterologous PKS is either replaced by that for the fifth extender module of the narbonolide PKS or the latter is merely added to coding sequences for the modules of the heterologous PKS, provides a novel PKS. In another embodiment, a DNA compound comprising a sequence that encodes the fifth extender module of the narbonolide PKS is inserted into a DNA compound that comprises the coding sequence for the narbonolide PKS or a recombinant narbonolide PKS that produces a narbonolide derivative. [0135]
  • In another embodiment, a portion or all of the fifth extender module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module. In this embodiment, the invention provides, for example, replacing the methylmalonyl CoA specific AT with a malonyl CoA, ethylmalonyl CoA, or carboxyglycolyl CoA specific AT; deleting (or inactivating) the KR, inserting a DH or a DH and ER; and/or replacing the KR with another KR, a DH and KR, or a DH, KR, and ER. In addition, the KS and/or ACP can be replaced with another KS and/or ACP. In each of these replacements or insertions, the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the narbonolide PKS, from a coding sequence for a PKS that produces a polyketide other than narbonolide, or from chemical synthesis. The resulting heterologous fifth extender module coding sequence can be utilized in conjunction with a coding sequence for a PKS that synthesizes narbonolide, a narbonolide derivative, or another polyketide. [0136]
  • The recombinant DNA compounds of the invention that encode the sixth extender module of the narbonolide PKS and the corresponding polypeptides encoded thereby are useful for a variety of applications. In one embodiment, a DNA compound comprising a sequence that encodes the narbonolide PKS sixth extender module is inserted into a DNA compound that comprises the coding sequence for a heterologous PKS. The resulting construct, in which the coding sequence for a module of the heterologous PKS is either replaced by that for the sixth extender module of the narbonolide PKS or the latter is merely added to coding sequences for the modules of the heterologous PKS, provides a novel PKS. In another embodiment, a DNA compound comprising a sequence that encodes the sixth extender module of the narbonolide PKS is inserted into a DNA compound that comprises the coding sequences for the narbonolide PKS or a recombinant narbonolide PKS that produces a narbonolide derivative. [0137]
  • In another embodiment, a portion or all of the sixth extender module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module. In this embodiment, the invention provides, for example, replacing the methylmalonyl CoA specific AT with a malonyl CoA, ethylmalonyl CoA, or carboxyglycolyl CoA specific AT; and/or inserting a KR, a KR and DH, or a KR, DH, and an ER. In addition, the KS and/or ACP can be replaced with another KS and/or ACP. In each of these replacements or insertions, the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the narbonolide PKS, from a coding sequence for a PKS that produces a polyketide other than narbonolide, or from chemical synthesis. The resulting heterologous sixth extender module coding sequence can be utilized in conjunction with a coding sequence for a PKS that synthesizes narbonolide, a narbonolide derivative, or another polyketide. [0138]
  • The sixth extender module of the narbonolide PKS is followed by a thioesterase domain. This domain is important in the cyclization of the polyketide and its cleavage from the PKS. The present invention provides recombinant DNA compounds that encode hybrid PKS enzymes in which the narbonolide PKS is fused to a heterologous thioesterase or a heterologous PKS is fused to the narbonolide synthase thioesterase. Thus, for example, a thioesterase domain coding sequence from another PKS gene can be inserted at the end of the sixth extender module coding sequence in recombinant DNA compounds of the invention. Recombinant DNA compounds encoding this thioesterase domain are therefore useful in constructing DNA compounds that encode the narbonolide PKS, a PKS that produces a narbonolide derivative, and a PKS that produces a polyketide other than narbonolide or a narbonolide derivative. [0139]
  • The following Table lists references describing illustrative PKS genes and corresponding enzymes that can be utilized in the construction of the recombinant hybrid PKSs and the corresponding DNA compounds that encode them of the invention. Also presented are various references describing tailoring enzymes and corresponding genes that can be employed in accordance with the methods of the invention. [0140]
  • Avermectin [0141]
  • U.S. Pat. No. 5,252,474 to Merck. [0142]
  • MacNeil et al., 1993[0143] , Industrial Microorganisms: Basic and Applied Molecular Genetics, Baltz, Hegeman, & Skatrud, eds. (ASM), pp. 245-256, A Comparison of the Genes Encoding the Polyketide Synthases for Avermectin, Erythromycin, and Nemadectin.
  • MacNeil et al., 1992[0144] , Gene 115: 119-125, Complex Organization of the Streptomyces avermitilis genes encoding the avermectin polyketide synthase.
  • Candicidin (FR008) [0145]
  • Hu et al., 1994[0146] , Mol. Microbiol. 14: 163-172.
  • Epothilone [0147]
  • U.S. patent application Serial No. 60/130,560, filed Apr. 22, 1999, and Serial No. 60/122,620, filed Mar. 3, 1999. [0148]
  • Erythromycin [0149]
  • PCT Pub. No. WO 93/13663 to Abbott. [0150]
  • U.S. Pat. No. 5,824,513 to Abbott. [0151]
  • Donadio et al., 1991[0152] , Science 252:675-9.
  • Cortes et al., Nov. 8, 1990[0153] , Nature 348:176-8, An unusually large multifunctional polypeptide in the erythromycin producing polyketide synthase of Saccharopolyspora erythraea.
  • Glycosylation Enzymes [0154]
  • PCT Pat. App. Pub. No. WO 97/23630 to Abbott. [0155]
  • FK506 [0156]
  • Motamedi et al., 1998, The biosynthetic gene cluster for the macrolactone ring of the immunosuppressant FK506[0157] , Eur. J. Biochem. 256: 528-534.
  • Motamedi et al., 1997, Structural organization of a multifunctional polyketide synthase involved in the biosynthesis of the macrolide immunosuppressant FK506[0158] , Eur. J. Biochem. 244: 74-80.
  • Methyltransferase [0159]
  • U.S. Pat. No. 5,264,355, issued Nov. 23, 1993, Methylating enzyme from Streptomyces MA6858. 31-O-desmethyl-FK506 methyltransferase. [0160]
  • Motamedi et al., 1996, Characterization of methyltransferase and hydroxylase genes involved in the biosynthesis of the immunosuppressants FK506 and FK520[0161] , J. Bacteriol. 178: 5243-5248.
  • FK520 [0162]
  • U.S. patent application Serial No. 60/123,800, filed Mar. 11, 1999. [0163]
  • Immunomycin [0164]
  • Nielsen et al., 1991[0165] , Biochem. 30:5789-96.
  • Lovastatin [0166]
  • U.S. Pat. No. 5,744,350 to Merck. [0167]
  • Nemadectin [0168]
  • MacNeil et al., 1993, supra. [0169]
  • Niddaymcin [0170]
  • Kakavas et al., 1997, Identification and characterization of the niddamycin polyketide synthase genes from [0171] Streptomyces caelestis, J. Bacteriol. 179: 7515-7522.
  • Oleandomycin [0172]
  • Swan et al., 1994, Characterization of a [0173] Streptomyces antibioticus gene encoding a type I polyketide synthase which has an unusual coding sequence, Mol. Gen. Genet. 242: 358-362.
  • Olano et al., 1998, Analysis of a [0174] Streptomyces antibioticus chromosomal region involved in oleandomycin biosynthesis, which encodes two glycosyltransferases responsible for glycosylation of the macrolactone ring, Mol. Gen. Genet. 259(3): 299-308.
  • U.S. patent application Serial No. 60/120,254, filed Feb. 16, 1999, and Serial No. 60/106,100, filed Oct. 29, 1998. [0175]
  • Platenolide [0176]
  • EP Pat. App. Pub. No. 791,656 to Lilly. [0177]
  • Pradimicin [0178]
  • PCT Pat. Pub. No. WO 98/11230 to Bristol-Myers Squibb. [0179]
  • Rapamycin [0180]
  • Schwecke et al., August 1995, The biosynthetic gene cluster for the polyketide rapamycin, [0181] Proc. Natl. Acad. Sci. USA 92:7839-7843.
  • Aparicio et al., 1996, Organization of the biosynthetic gene cluster for rapamycin in [0182] Streptomyces hygroscopicus: analysis of the enzymatic domains in the modular polyketide synthase, Gene 169: 9-16.
  • Rifamycin [0183]
  • August et al., Feb. 13, 1998, Biosynthesis of the ansamycin antibiotic rifamycin: deductions from the molecular analysis of the rif biosynthetic gene cluster of [0184] Amycolatopsis mediterranei S669, Chemistry & Biology, 5(2): 69-79.
  • Soraphen [0185]
  • U.S. Pat. No. 5,716,849 to Novartis. [0186]
  • Schupp et al., 1995[0187] , J. Bacteriology 177: 3673-3679. A Sorangium cellulosum (Myxobacterium) Gene Cluster for the Biosynthesis of the Macrolide Antibiotic Soraphen A: Cloning, Characterization, and Homology to Polyketide Synthase Genes from Actinomycetes.
  • Spiramycin [0188]
  • U.S. Pat. No. 5,098,837 to Lilly. [0189]
  • Activator Gene [0190]
  • U.S. Pat. No. 5,514,544 to Lilly. [0191]
  • Tylosin [0192]
  • EP Pub. No. 791,655 to Lilly. [0193]
  • Kuhstoss et al., 1996[0194] , Gene 183:231-6., Production of a novel polyketide through the construction of a hybrid polyketide synthase.
  • U.S. Pat. No. 5,876,991 to Lilly. [0195]
  • Tailoring Enzymes [0196]
  • Merson-Davies and Cundliffe, 1994[0197] , Mol. Microbiol. 13: 349-355. Analysis of five tylosin biosynthetic genes from the tylBA region of the Streptomyces fradiae genome.
  • As the above Table illustrates, there is a wide variety of PKS genes that serve as readily available sources of DNA and sequence information for use in constructing the hybrid PKS-encoding DNA compounds of the invention. Methods for constructing hybrid PKS-encoding DNA compounds are described without reference to the narbonolide PKS in U.S. Pat. Nos. 5,672,491 and 5,712,146 and PCT publication No. WO 98/49315, each of which is incorporated herein by reference. [0198]
  • In constructing hybrid PKSs of the invention, certain general methods may be helpful. For example, it is often beneficial to retain the framework of the module to be altered to make the hybrid PKS. Thus, if one desires to add DH and ER functionalities to a module, it is often preferred to replace the KR domain of the original module with a KR, DH, and ER domain-containing segment from another module, instead of merely inserting DH and ER domains. One can alter the stereochemical specificity of a module by replacement of the KS domain with a KS domain from a module that specifies a different stereochemistry. See Lau et al., 1999, “Dissecting the role of acyltransferase domains of modular polyketide synthases in the choice and stereochemical fate of extender units” [0199] Biochemistry 38(5):1643-1651, incorporated herein by reference. One can alter the specificity of an AT domain by changing only a small segment of the domain. See Lau et al., supra. One can also take advantage of known linker regions in PKS proteins to link modules from two different PKSs to create a hybrid PKS. See Gokhale et al., April 16, 1999, Dissecting and Exploiting Intermodular Communication in Polyketide Synthases”, Science 284: 482-485, incorporated herein by reference. the stereochemistry of the resulting polyketide is a function of three aspects of the synthase. The first aspect is related to the AT/KS specificity associated with substituted malonyls as extender units, which affects stereochemistry only when the reductive cycle is missing or when it contains only a ketoreductase, as the dehydratase would abolish chirality. Second, the specificity of the ketoreductase may determine the chirality of any beta-OH. Finally, the enoylreductase specificity for substituted malonyls as extender units may influence the result when there is a complete KR/DH/ER available.
  • Thus, the modular PKS systems, and in particular, the narbonolide PKS system, permit a wide range of polyketides to be synthesized. As compared to the aromatic PKS systems, a wider range of starter units including aliphatic monomers (acetyl, propionyl, butyryl, isovaleryl, etc.), aromatics (aminohydroxybenzoyl), alicyclics (cyclohexanoyl), and heterocyclics (thiazolyl) are found in various macrocyclic polyketides. Recent studies have shown that modular PKSs have relaxed specificity for their starter units (Kao et al., 1994[0200] , Science, supra). Modular PKSs also exhibit considerable variety with regard to the choice of extender units in each condensation cycle. The degree of beta-ketoreduction following a condensation reaction has also been shown to be altered by genetic manipulation (Donadio et al., 1991, Science, supra; Donadio et al., 1993, Proc. Natl. Acad. Sci. USA 90: 7119-7123). Likewise, the size of the polyketide product can be varied by designing mutants with the appropriate number of modules (Kao et al., 1994, J. Am. Chem. Soc. 116:11612-11613). Lastly, these enzymes are particularly well known for generating an impressive range of asymmetric centers in their products in a highly controlled manner. The polyketides and antibiotics produced by the methods of the invention are typically single stereoisomeric forms. Although the compounds of the invention can occur as mixtures of stereoisomers, it may be beneficial in some instances to generate individual stereoisomers. Thus, the combinatorial potential within modular PKS pathways based on any naturally occurring modular, such as the narbonolide, PKS scaffold is virtually unlimited.
  • The combinatorial potential is increased even further when one considers that mutations in DNA encoding a polypeptide can be used to introduce, alter, or delete an activity in the encoded polypeptide. Mutations can be made to the native sequences using conventional techniques. The substrates for mutation can be an entire cluster of genes or only one or two of them; the substrate for mutation may also be portions of one or more of these genes. Techniques for mutation include preparing synthetic oligonucleotides including the mutations and inserting the mutated sequence into the gene encoding a PKS subunit using restriction endonuclease digestion. See, e.g., Kunkel, 1985[0201] , Proc. Natl. Acad. Sci. USA 82: 448; Geisselsoder et al., 1987, BioTechniques 5:786. Alternatively, the mutations can be effected using a mismatched primer (generally 10-20 nucleotides in length) that hybridizes to the native nucleotide sequence, at a temperature below the melting temperature of the mismatched duplex. The primer can be made specific by keeping primer length and base composition within relatively narrow limits and by keeping the mutant base centrally located. See Zoller and Smith, 1983, Methods Enzymol. 100:468. Primer extension is effected using DNA polymerase, the product cloned, and clones containing the mutated DNA, derived by segregation of the primer extended strand, selected. Identification can be accomplished using the mutant primer as a hybridization probe. The technique is also applicable for generating multiple point mutations. See, e.g., Dalbie-McFarland et al., 1982, Proc. Natl. Acad. Sci. USA 79: 6409. PCR mutagenesis can also be used to effect the desired mutations.
  • Random mutagenesis of selected portions of the nucleotide sequences encoding enzymatic activities can also be accomplished by several different techniques known in the art, e.g., by inserting an oligonucleotide linker randomly into a plasmid, by irradiation with X-rays or ultraviolet light, by incorporating incorrect nucleotides during in vitro DNA synthesis, by error-prone PCR mutagenesis, by preparing synthetic mutants, or by damaging plasmid DNA in vitro with chemicals. Chemical mutagens include, for example, sodium bisulfite, nitrous acid, nitrosoguanidine, hydroxylamine, agents which damage or remove bases thereby preventing normal base-pairing such as hydrazine or formic acid, analogues of nucleotide precursors such as 5-bromouracil, 2-aminopurine, or acridine intercalating agents such as proflavine, acriflavine, quinacrine, and the like. Generally, plasmid DNA or DNA fragments are treated with chemicals, transformed into [0202] E. coli and propagated as a pool or library of mutant plasmids.
  • In constructing a hybrid PKS of the invention, regions encoding enzymatic activity, i.e., regions encoding corresponding activities from different PKS synthases or from different locations in the same PKS, can be recovered, for example, using PCR techniques with appropriate primers. By “corresponding” activity encoding regions is meant those regions encoding the same general type of activity. For example, a KR activity encoded at one location of a gene cluster “corresponds” to a KR encoding activity in another location in the gene cluster or in a different gene cluster. Similarly, a complete reductase cycle could be considered corresponding. For example, KR/DH/ER corresponds to KR alone. [0203]
  • If replacement of a particular target region in a host PKS is to be made, this replacement can be conducted in vitro using suitable restriction enzymes. The replacement can also be effected in vivo using recombinant techniques involving homologous sequences framing the replacement gene in a donor plasmid and a receptor region in a recipient plasmid. Such systems, advantageously involving plasmids of differing temperature sensitivities are described, for example, in PCT publication No. WO 96/40968, incorporated herein by reference. The vectors used to perform the various operations to replace the enzymatic activity in the host PKS genes or to support mutations in these regions of the host PKS genes can be chosen to contain control sequences operably linked to the resulting coding sequences in a manner such that expression of the coding sequences can be effected in an appropriate host. [0204]
  • However, simple cloning vectors may be used as well. If the cloning vectors employed to obtain PKS genes encoding derived PKS lack control sequences for expression operably linked to the encoding nucleotide sequences, the nucleotide sequences are inserted into appropriate expression vectors. This need not be done individually, but a pool of isolated encoding nucleotide sequences can be inserted into expression vectors, the resulting vectors transformed or transfected into host cells, and the resulting cells plated out into individual colonies. [0205]
  • The various PKS nucleotide sequences can be cloned into one or more recombinant vectors as individual cassettes, with separate control elements, or under the control of, e.g., a single promoter. The PKS subunit encoding regions can include flanking restriction sites to allow for the easy deletion and insertion of other PKS subunit encoding sequences so that hybrid PKSs can be generated. The design of such unique restriction sites is known to those of skill in the art and can be accomplished using the techniques described above, such as site-directed mutagenesis and PCR. [0206]
  • The expression vectors containing nucleotide sequences encoding a variety of PKS enzymes for the production of different polyketides are then transformed into the appropriate host cells to construct the library. In one straightforward approach, a mixture of such vectors is transformed into the selected host cells and the resulting cells plated into individual colonies and selected to identify successful transformants. Each individual colony has the ability to produce a particular PKS synthase and ultimately a particular polyketide. Typically, there will be duplications in some, most, or all of the colonies; the subset of the transformed colonies that contains a different PKS in each member colony can be considered the library. Alternatively, the expression vectors can be used individually to transform hosts, which transformed hosts are then assembled into a library. A variety of strategies are available to obtain a multiplicity of colonies each containing a PKS gene cluster derived from the naturally occurring host gene cluster so that each colony in the library produces a different PKS and ultimately a different polyketide. The number of different polyketides that are produced by the library is typically at least four, more typically at least ten, and preferably at least 20, and more preferably at least 50, reflecting similar numbers of different altered PKS gene clusters and PKS gene products. The number of members in the library is arbitrarily chosen; however, the degrees of freedom outlined above with respect to the variation of starter, extender units, stereochemistry, oxidation state, and chain length is quite large. [0207]
  • Methods for introducing the recombinant vectors of the invention into suitable hosts are known to those of skill, in the art and typically include the use of CaC12 or agents such as other divalent cations, lipofection, DMSO, protoplast transformation, infection, transfection, and electroporation. The polyketide producing colonies can be identified and isolated using known techniques and the produced polyketides further characterized. The polyketides produced by these colonies can be used collectively in a panel to represent a library or may be assessed individually for activity. [0208]
  • The libraries of the invention can thus be considered at four levels: (1) a multiplicity of colonies each with a different PKS encoding sequence; (2) colonies that contain the proteins that are members of the PKS library produced by the coding sequences; (3) the polyketides produced; and (4) antibiotics or compounds with other desired activities derived from the polyketides. Of course, combination libraries can also be constructed wherein members of a library derived, for example, from the narbonolide PKS can be considered as a part of the same library as those derived from, for example, the rapamycin PKS or DEBS. [0209]
  • Colonies in the library are induced to produce the relevant synthases and thus to produce the relevant polyketides to obtain a library of polyketides. The polyketides secreted into the media can be screened for binding to desired targets, such as receptors, signaling proteins, and the like. The supernatants per se can be used for screening, or partial or complete purification of the polyketides can first be effected. Typically, such screening methods involve detecting the binding of each member of the library to receptor or other target ligand. Binding can be detected either directly or through a competition assay. Means to screen such libraries for binding are well known in the art. Alternatively, individual polyketide members of the library can be tested against a desired target. In this event, screens wherein the biological response of the target is measured can more readily be included. Antibiotic activity can be verified using typical screening assays such as those set forth in Lehrer et al., 1991[0210] , J. Immunol. Meth. 137:167-173, incorporated herein by reference, and in the examples below.
  • The invention provides methods for the preparation of a large number of polyketides. These polyketides are useful intermediates in formation of compounds with antibiotic or other activity through hydroxylation and glycosylation reactions as described above. In general, the polyketide products of the PKS must be further modified, typically by hydroxylation and glycosylation, to exhibit antibiotic activity. Hydroxylation results in the novel polyketides of the invention that contain hydroxyl groups at C6, which can be accomplished using the hydroxylase encoded by the eryF gene, and/or C12, which can be accomplished using the hydroxylase encoded by the picK or eryK gene. The presence of hydroxyl-groups at these positions can enhance the antibiotic activity of the resulting compound relative to its unhydroxylated counterpart. [0211]
  • Gycosylation is important in conferring antibiotic activity to a polyketide as well. Methods for glycosylating the polyketides are generally known in the art; the glycosylation may be effected intracellularly by providing the appropriate glycosylation enzymes or may be effected in vitro using chemical synthetic means as described herein and in PCT publication No. WO 98/49315, incorporated herein by reference. Preferably, glycosylation with desosamine is effected in accordance with the methods of the invention in recombinant host cells provided by the invention. In general, the approaches to effecting glycosylation mirror those described above with respect to hydroxylation. The purified enzymes, isolated from native sources or recombinantly produced may be used in vitro. Alternatively and as noted, glycosylation may be effected intracellularly using endogenous or recombinantly produced intracellular glycosylases. In addition, synthetic chemical methods may be employed. [0212]
  • The antibiotic modular polyketides may contain any of a number of different sugars, although D-desosamine, or a close analog thereof, is most common. Erythromycin, picromycin, narbomycin and methymycin contain desosamine. Erythromycin also contains L-cladinose (3-O-methyl mycarose). Tylosin contains mycaminose (4-hydroxy desosamine), mycarose and 6-deoxy-D-allose. 2-acetyl-1-bromodesosamine has been used as a donor to glycosylate polyketides by Masamune et al., 1975[0213] , J. Am. Chem. Soc. 97: 3512-3513. Other, apparently more stable donors include glycosyl fluorides, thioglycosides, and trichloroacetimidates; see Woodward et al., 1981, J. Am. Chem. Soc. 103: 3215; Martin et al., 1997, J. Am. Chem. Soc. 119: 3193; Toshima et al., 1995, J. Am. Chem. Soc. 117: 3717; Matsumoto et al., 1988, Tetrahedron Lett. 29: 3575. Glycosylation can also be effected using the polyketide aglycones as starting materials and using Saccharopolyspora erythraea or Streptomyces venezuelae to make the conversion, preferably using mutants unable to synthesize macrolides.
  • To provide an illustrative hybrid PKS of the invention as well as an expression vector for that hybrid PKS and host cells comprising the vector and producing the hybrid polyketide, a portion of the narbonolide PKS gene was fused to the DEBS genes. This construct also allowed the examination of whether the TE domain of the narbonolide PKS (pikTE) could promote formation of 12-membered lactones in the context of a different PKS. A construct was generated, plasmid pKOS039-18, in which the pikTE ORF was fused with the DEBS genes in place of the DEBS TE ORF (see FIG. 5). To allow the TE to distinguish between substrates most closely resembling those generated by the narbonolide PKS, the fusion junction was chosen between the AT and ACP to eliminate ketoreductase activity in DEBS extender module 6 (KR[0214] 6). This results in a hybrid PKS that presents the TE with a β-ketone heptaketide intermediate and a β-(S)-hydroxy hexaketide intermediate to cyclize, as in narbonolide and 10-deoxymethynolide biosynthesis.
  • Analysis of this construct indicated the production of the 14-[0215] membered ketolide 3,6-dideoxy-3-oxo-erythronolide B (FIG. 5, compound 6). Extracts were analyzed by LC/MS. The identity of compound 6 was verified by comparison to a previously authenticated sample (see PCT publication No. WO 98/49315, incorporated herein by reference). The predicted 12-membered macrolactone, (8R,9S)-8,9-dihydro-8-methyl-9-hydroxy-10-deoxymethynolide (see Kao et al. J. Am. Chem. Soc. (1995) 117:9105-9106 incorporated herein by reference) was not detected. Because the 12-membered intermediate can be formed by other recombinant PKS enzymes, see Kao et al., 1995, supra, the PIC TE domain appears incapable of forcing premature cyclization of the hexaketide intermediate generated by DEBS. This result, along with others reported herein, suggests that protein interactions between the narbonolide PKS modules play a role in formation of the 12 and 14-membered macrolides.
  • The above example illustrates also how engineered PKSs can be improved for production of novel compounds. [0216] Compound 6 was originally produced by deletion of the KR6 domain in DEBS to create a 3-ketolide producing PKS (see U.S. patent application Ser. No. 09/073,538, filed May 6, 1998, and PCT publication No. WO 98/49315, each of which is incorporated herein by reference). Although the desired molecule was made, purification of compound 6 from this strain was hampered by the presence of 2-desmethyl ketolides that could not be easily separated. Extracts from Streptomyces lividans K4-114/pKOS039-18, however, do not contain the 2-desmethyl compounds, greatly simplifying purification. Thus, the invention provides a useful method of producing such compounds. The ability to combine the narbonolide PKS with DEBS and other modular PKSs provides a significant advantage in the production of macrolide antibiotics.
  • Two other hybrid PKSs of the invention were constructed that yield this same compound. These constructs also illustrate the method of the invention in which hybrid PKSs are constructed at the protein, as opposed to the module, level. Thus, the invention provides a method for constructing a hybrid PKS which comprises the coexpression of at least one gene from a first modular PKS gene cluster in a host cell that also expresses at least one gene from a second PKS gene cluster. The invention also provides novel hybrid PKS enzymes prepared in accordance with the method. This method is not limited to hybrid PKS enzymes composed of at least one narbonolide PKS gene, although such constructs are illustrative and preferred. Moreover, the hybrid PKS enzymes are not limited to hybrids composed of unmodified proteins; as illustrated below, at least one of the genes can optionally be a hybrid PKS gene. [0217]
  • In the first construct, the eryAI and eryAII genes were coexpressed with picAIV and a gene encoding a [0218] hybrid extender module 5 composed of the KS and AT domains of extender module 5 of DEBS3 and the KR and ACP domains of extender module ˜5 of the narbonolide PKS. In the second construct, the picAIV coding sequence was fused to the hybrid extender module 5 coding sequence used in the first construct to yield a single protein. Each of these constructs produced 3-deoxy-3-oxo-6-deoxyerythronolide B. In a third construct, the coding sequence for extender module 5 of DEBS3 was fused to the picAIV coding sequence, but the levels of product produced were below the detection limits of the assay.
  • A variant of the first construct hybrid PKS was constructed that contained an inactivated [0219] DEBS1 extender module 1 KS domain. When host cells containing the resultant hybrid PKS were supplied the appropriate diketide precursor, the desired 13-desethyl-13-propyl compounds were obtained, as described in the examples below.
  • Other illustrative hybrid PKSs of the invention were made by coexpressing the picAI and picAII genes with genes encoding DEBS3 or DEBS3 variants. These constructs illustrate the method of the invention in which a hybrid PKS is produced from coexpression of PKS genes unmodified at the modular or domain level. In the first construct, the eryAIII gene was coexpressed with the picAI and picAII genes, and the hybrid PKS produced 10-desmethyl-10,11-anhydro-6-deoxyerythronolide B in [0220] Streptomyces lividans. Such a hybrid PKS could also be constructed in accordance with the method of the invention by transformation of S. venezuelae with an expression vector that produces the eryAIII gene product, DEBS3. In a preferred embodiment, the S. venezuelae host cell has been modified to inactivate the picAIII gene.
  • In the second construct, the DEBS3 gene was a variant that had an inactive KR in [0221] extender module 5. The hybrid PKS produced 5,6-dideoxy-5-oxo-10-desmethyl-10,11-anhydroerythronolide B in Streptomyces lividans.
  • In the third construct, the DEBS3 gene was a variant in which the KR domain of [0222] extender module 5 was replaced by the DH and KR domains of extender module 4 of the rapamycin PKS. This construct produced 5,6-dideoxy-5-oxo-10-desmethyl-10,11-anhydroerythronolide B and 5,6-dideoxy-4,5-anhydro-10-desmethyl-10,11-anhydroerythronolide B in Streptomyces lividans, indicating that the rapamycin DH and KR domains functioned only inefficiently in this construct.
  • In the fourth construct, the DEBS3 gene was a variant in which the KR domain of [0223] extender module 5 was replaced by the DH, KR, and ER domains of extender module 1 of the rapamycin PKS. This construct produced 5,6-dideoxy-5-oxo-10-desmethyl-10,11-anhydroerythronolide B as well as 5,6-dideoxy-10-desmethyl-10,11-anhydroerythronolide B in Streptomyces lividans, indicating that the rapamycin DH, KR, and ER domains functioned only inefficiently in this construct.
  • In the fifth construct, the DEBS3 gene was a variant in which the KR domain of [0224] extender module 6 was replaced by the DH and KR domains of extender module 4 of the rapamycin PKS. This construct produced 3,6-dideoxy-2,3-anhydro-10-desmethyl-10,11-anhydroerythronolide B in Streptomyces lividans.
  • In the sixth construct, the DEBS3 gene was a variant in which the AT domain of [0225] extender module 6 was replaced by the AT domain of extender module 2 of the rapamycin PKS. This construct produced 2,10-didesmethyl-10,11-anhydro-6-deoxyerythronolide B in Streptomyces lividans.
  • These hybrid PKSs illustrate the wide variety of polyketides that can be produced by the methods and compounds of the invention. These polyketides are useful as antibiotics and as intermediates in the synthesis of other useful compounds, as described in the following section. [0226]
  • Section VI: Compounds [0227]
  • The methods and recombinant DNA compounds of the invention are useful in the production of polyketides. In one important aspect, the invention provides methods for making ketolides, polyketide compounds with significant antibiotic activity. See Griesgraber et al., 1996[0228] , J. Antibiot. 49: 465-477, incorporated herein by reference. Most if not all of the ketolides prepared to date are synthesized using erythromycin A, a derivative of 6-dEB, as an intermediate. While the invention provides hybrid PKSs that produce a polyketide different in structure from 6-dEB, the invention also provides methods for making intermediates useful in preparing traditional, 6-dEB-derived ketolide compounds.
  • Because 6-dEB in part differs from narbonolide in that it comprises a 10-methyl group, the novel hybrid PKS genes of the invention based on the narbonolide PKS provide many novel ketolides that differ from the known ketolides only in that they lack a 10-methyl group. Thus, the invention provides the 10-desmethyl analogues of the ketolides and intermediates and precursor compounds described in, for example, Griesgraber et al., supra; Agouridas et al., 1998[0229] , J. Med. Chem. 41: 4080-4100, U.S. Pat. Nos. 5,770,579; 5,760,233; 5,750,510; 5,747,467; 5,747,466; 5,656,607; 5,635,485; 5,614,614; 5,556,118; 5,543,400; 5,527,780; 5,444,051; 5,439,890; 5,439,889; and PCT publication Nos. WO 98/09978 and WO 98/28316, each of which is incorporated herein by reference. Because the invention also provides hybrid PKS genes that include a methylmalonyl-specific AT domain in extender module 2 of the narbonolide PKS, the invention also provides hybrid PKS that can be used to produce the 10-methyl-containing ketolides known in the art.
  • Thus, a hybrid PKS of the invention that produces 10-methyl narbonolide is constructed by substituting the malonyl-specific AT domain of the narbonolide [0230] PKS extender module 2 with a methylmalonyl specific AT domain from a heterologous PKS. A hybrid narbonolide PKS in which the AT of extender module 2 was replaced with the AT from DEBS extender module 2 was constructed using boundaries described in PCT publication No. WO 98/49315, incorporated herein by reference. However, when the hybrid PKS expression vector was introduced into Streptomyces venezuelae, detectable quantities of 10-methyl picromycin were not produced. Thus, to construct such a hybrid PKS of the invention, an AT domain from a module other than DEBS extender module 2 is preferred. One could also employ DEBS extender module 2 or another methylmalonyl specific AT but utilize instead different boundaries than those used for the substitution described above. In addition, one can construct such a hybrid PKS by substituting, in addition to the AT domain, additional extender module 2 domains, including the KS, the KR, and the DH, and/or additional extender module 3 domains.
  • Although modification of [0231] extender module 2 of the narbonolide PKS is required, the extent of hybrid modules engineered need not be limited to module 2 to make 10-methyl narbonolide. For example, substitution of the KS domain of extender module 3 of the narbonolide PKS with a heterologous domain or module can result in more efficient processing of the intermediate generated by the hybrid extender module 2. Likewise, a heterologous TE domain may be more efficient in cyclizing 10-methyl narbonolide.
  • Substitution of the [0232] entire extender module 2 of the narbonolide PKS with a module encoding the correct enzymatic activities, i.e., a KS, a methylmalonyl specific AT, a KR, a DH, and an ACP, can also be used to create a hybrid PKS of the invention that produces a 10-methyl ketolide. Modules useful for such whole module replacements include extender modules 4 and 10 from the rapamycin PKS, extender modules 1 and 5 from the FK506 PKS, extender module 2 of the tylosin PKS, and extender module 4 of the rifamycin PKS. Thus, the invention provides many different hybrid PKSs that can be constructed starting from the narbonolide PKS that can be used to produce 10-methyl narbonolide. While 10-methyl narbonolide is referred to in describing these hybrid PKSs, those of skill recognize that the invention also therefore provides the corresponding derivatives produces by glycosylation and hydroxylation. For example, if the hybrid PKS is expressed in Streptomyces narbonensis or S. venezuelae, the compounds produced are 10-methyl narbomycin and picromycin, respectively. Alternatively, the PKS can be expressed in a host cell transformed with the vectors of the invention that encode the desosamine biosynthesis and desosaminyl transferase and picK hydroxylase genes.
  • Other important compounds provided by the invention are the 6-hydroxy ketolides. These compounds include 3-deoxy-3-oxo erythronolide B, 6-hydroxy narbonolide, and 6-hydroxy-10-methyl narbonolide. In the examples below, the invention provides a method for utilizing EryF to hydroxylate 3-ketolides that is applicable for the production of any 6-hydroxy-3-ketolide. [0233]
  • Thus, the hybrid PKS genes of the invention can be expressed in a host cell that contains the desosamine biosynthetic genes and desosaminyl transferase gene as well as the required hydroxylase gene(s), which may be either picK (for the C12 position) or eryK (for the C12 position) and/or eryF (for the C6 position). The resulting compounds have antibiotic activity but can be further modified, as described in the patent publications referenced above, to yield a desired compound with improved or otherwise desired properties. Alternatively, the aglycone compounds can be produced in the recombinant host cell, and the desired glycosylation and hydroxylation steps carried out in vitro or in vivo, in the latter case by supplying the converting cell with the aglycone. [0234]
  • The compounds of the invention are thus optionally glycosylated forms of the polyketide set forth in formula (2) below which are hydroxylated at either the C6 or the C12 or both. The compounds of formula (2) can be prepared using the loading and the six extender modules of a modular PKS, modified or prepared in hybrid form as herein described. These polyketides have the formula: [0235]
    Figure US20030162262A1-20030828-C00001
  • including the glycosylated and isolated stereoisomeric forms thereof; [0236]
  • wherein R* is a straight chain, branched or cyclic, saturated or unsaturated substituted or unsubstituted hydrocarbyl of 1-15C; [0237]
  • each of R[0238] 1-R6 is independently H or alkyl (1-4C) wherein any alkyl at R1 may optionally be substituted;
  • each of X[0239] 1-X5 is independently two H, H and OH, or ═O; or
  • each of X[0240] 1-X5 is independently H and the compound of formula (2) contains a double-bond in the ring adjacent to the position of said X at 2-3, 4-5, 6-7, 8-9 and/or 10-11;
  • with the proviso that: [0241]
  • at least two of R[0242] 1-R6 are alkyl (1-4C).
  • Preferred [0243] compounds comprising formula 2 are those wherein at least three of R1-R5 are alkyl (1-4C), preferably methyl or ethyl; more preferably wherein at least four of R1-R5 are alkyl (1-4C), preferably methyl or ethyl. Also preferred are those wherein X2 is two H, ═O, or H and OH, and/or X3 is H, and/or X1 is OH and/or X4 is OH and/or X5 is OH. Also preferred are compounds with variable R* when R1-R5 is methyl, X2 is ═O, and X1, X4 and X5 are OH. The glycosylated forms of the foregoing are also preferred.
  • The invention also provides the 12-membered macrolides corresponding to the compounds above but produced from a narbonolide-derived PKS lacking [0244] extender modules 5 and 6 of the narbonolide PKS.
    Figure US20030162262A1-20030828-C00002
  • including the glycosylated and isolated stereoisomeric forms thereof; [0245]
  • wherein R* is a straight chain, branched or cyclic, saturated or unsaturated substituted or unsubstituted hydrocarbyl of 1-15C; [0246]
  • each of R[0247] 1-R6 is independently H or alkyl (1-4C) wherein any alkyl at R1 may optionally be substituted;
  • each of X[0248] 1-X5 is independently two H, H and OH, or ═O; or
  • each of X[0249] 1-X5 is independently H and the compound of formula (2) contains a double-bond in the ring adjacent to the position of said X at 2-3, 4-5, 6-7, 8-9 and/or 10-11;
  • with the proviso that: [0250]
  • at least two of R[0251] 1-R6 are alkyl (1-4C).
  • Preferred [0252] compounds comprising formula 2 are those wherein at least three of R1-R5 are alkyl (1-4C), preferably methyl or ethyl; more preferably wherein at least four of R1-R5 are alkyl (1-4C), preferably methyl or ethyl. Also preferred are those wherein X2 is two H, ═O, or H and OH, and/or X3 is H, and/or X1 is OH and/or X4 is OH and/or X5 is OH. Also preferred are compounds with variable R* when R1-R5 is methyl, X2 is ═O, and X1, X4 and X5 are OH. The glycosylated forms of the foregoing are also preferred.
  • The invention also provides the 12-membered macrolides corresponding to the compounds above but produced from a narbonolide-derived PKS lacking [0253] extender modules 5 and 6 of the narbonolide PKS.
  • The compounds of the invention can be produced by growing and fermenting the host cells of the invention under conditions known in the art for the production of other polyketides. The compounds of the invention can be isolated from the fermentation broths of these cultured cells and purified by standard procedures. The compounds can be readily formulated to provide the pharmaceutical compositions of the invention. The pharmaceutical compositions of the invention can be used in the form of a pharmaceutical preparation, for example, in solid, semisolid, or liquid form. This preparation will contain one or more of the compounds of the invention as an active ingredient in admixture with an organic or inorganic carrier or excipient suitable for external, enteral, or parenteral application. The active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, and any other form suitable for use. [0254]
  • The carriers which can be used include water, glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea, and other carriers suitable for use in manufacturing preparations, in solid, semi-solid, or liquefied form. In addition, auxiliary stabilizing, thickening, and coloring agents and perfumes may be used. For example, the compounds of the invention may be utilized with hydroxypropyl methylcellulose essentially as described in U.S. Pat. No. 4,916,138, incorporated herein by reference, or with a surfactant essentially as described in EPO patent publication No. 428,169, incorporated herein by reference. [0255]
  • Oral dosage forms may be prepared essentially as described by Hondo et al., 1987[0256] , Transplantation Proceedings XIX, Supp. 6: 17-22, incorporated herein by reference. Dosage forms for external application may be prepared essentially as described in EPO patent publication No. 423,714, incorporated herein by reference. The active compound is included in the pharmaceutical composition in an amount sufficient to produce the desired effect upon the disease process or condition.
  • For the treatment of conditions and diseases caused by infection, a compound of the invention may be administered orally, topically, parenterally, by inhalation spray, or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvant, and vehicles. The term parenteral, as used herein, includes subcutaneous injections, and intravenous, intramuscular, and intrastemal injection or infusion techniques. [0257]
  • Dosage levels of the compounds of the invention are of the order from about 0.01 mg to about 50 mg per kilogram of body weight per day, preferably from about 0.1 mg to about 10 mg per kilogram of body weight per day. The dosage levels are useful in the treatment of the above-indicated conditions (from about 0.7 mg to about 3.5 mg per patient per day, assuming a 70 kg patient). In addition, the compounds of the invention may be administered on an intermittent basis, i.e., at semi-weekly, weekly, semi-monthly, or monthly intervals. [0258]
  • The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, a formulation intended for oral administration to humans may contain from 0.5 mg to 5 gm of active agent compounded with an appropriate and convenient amount of carrier material, which may vary from about 5 percent to about 95 percent of the total composition. Dosage unit forms will generally contain from about 0.5 mg to about 500 mg of active ingredient. For external administration, the compounds of the invention may be formulated within the range of, for example, 0.00001% to 60% by weight, preferably from 0.001% to 10% by weight, and most preferably from about 0.005% to 0.8% by weight. [0259]
  • It will be understood, however, that the specific dose level for any particular patient will depend on a variety of factors. These factors include the activity of the specific compound employed; the age, body weight, general health, sex, and diet of the subject; the time and route of administration and the rate of excretion of the drug; whether a drug combination is employed in the treatment; and the severity of the particular disease or condition for which therapy is sought. [0260]
  • A detailed description of the invention having been provided above, the following examples are given for the purpose of illustrating the invention and shall not be construed as being a limitation on the scope of the invention or claims. [0261]
    • EXAMPLE 1 General Methodology
  • Bacterial strains, plasmids, and culture conditions. [0262] Streptomyces coeicolor CH999 described in WO 95/08548, published Mar. 30, 1995, or S. lividans K4-114, described in Ziermann and Betlach, January 99, Recombinant Polyketide Synthesis in Streptomyces: Engineering of Improved Host Strains, Bio Techniques 26:106-110, incorporated herein by reference, was used as an expression host. DNA manipulations were performed in Escherichia coli XL1-Blue, available from Stratagene. E. coli MC1061 is also suitable for use as a host for plasmid manipulation. Plasmids were passaged through E. coli ET12567 (dam dcm hsdS Cmr) (MacNeil, 1988, J. Bacteriol. 170: 5607, incorporated herein by reference) to generate unmethylated DNA prior to transformation of S. coelicolor. E. coli strains were grown under standard conditions. S. coelicolor strains were grown on R2YE agar plates (Hopwood et al., Genetic manipulation of Streptomyces. A laboratory manual. The John Innes Foundation: Norwich, 1985, incorporated herein by reference).
  • Many of the expression vectors of the invention illustrated in the examples are derived from plasmid pRM5, described in WO 95/08548, incorporated herein by reference. This plasmid includes a colEI replicon, an appropriately truncated SCP2* Streptomyces replicon, two act-promoters to allow for bidirectional cloning, the gene encoding the actII-ORF4 activator which induces transcription from act promoters during the transition from growth phase to stationary phase, and appropriate marker genes. Engineered restriction sites in the plasmid facilitate the combinatorial construction of PKS gene clusters starting from cassettes encoding individual domains of naturally occurring PKSs. When plasmid pRM5 is used for expression of a PKS, all relevant biosynthetic genes can be plasmid-borne and therefore amenable to facile manipulation and mutagenesis in [0263] E. coli. This plasmid is also suitable for use in Streptomyces host cells. Streptomyces is genetically and physiologically well-characterized and expresses the ancillary activities required for in vivo production of most polyketides. Plasmid pRM5 utilizes the act promoter for PKS gene expression, so polyketides are produced in a secondary metabolite-like manner, thereby alleviating the toxic effects of synthesizing potentially bioactive compounds in vivo.
  • Manipulation of DNA and organisms. Polymerase chain reaction (PCR) was performed using Pfu polymerase (Stratagene; Taq polymerase from Perkin Elmer Cetus can also be used) under conditions recommended by the enzyme manufacturer. Standard in vitro techniques were used for DNA manipulations (Sambrook et al. Molecular Cloning: A Laboratory Mantial (Current Edition)). [0264] E. coli was transformed using standard calcium chloride-based methods; a Bio-Rad E. coli pulsing apparatus and protocols provided by Bio-Rad could also be used. S. coelicolor was transformed by standard procedures (Hopwood et al. Genetic manipulation of Streptomyces. A laboratory manual. The John Innes Foundation: Norwich, 1985), and depending on what selectable marker was employed, transformants were selected using 1 mL of a 1.5 mg/mL thiostrepton overlay, 1 mL of a 2 mg/mL apramycin overlay, or both.
    • EXAMPLE 2 Cloning of the Picromycin Biosynthetic Gene Cluster from Streptomyces venezuelae
  • Genomic DNA (100 μg) isolated from [0265] Streptomyces venezuelae ATCC15439 using standard procedures was partially digested with Sau3AI endonuclease to generate fragments ˜40 kbp in length. SuperCosI (Stratagene) DNA cosmid arms were prepared as directed by the manufacturer. A cosmid library was prepared by ligating 2.5 μg of the digested genomic DNA with 1.5 μg of cosmid arms in a 20 μL reaction. One microliter of the ligation mixture was propagated in E. coli XL 1-Blue MR (Stratagene) using a GigapackIII XL packaging extract kit (Stratagene). The resulting library of ˜3000 colonies was plated on a 10×150 mm agar plate and replicated to a nylon membrane.
  • The library was initially screened by direct colony hybridization with a DNA probe specific for ketosynthase domain coding sequences of PKS genes. Colonies were alkaline lysed, and the DNA was crosslinked to the membrane using UV irradiation. After overnight incubation with the probe at 42° C., the membrane was washed twice at 25° C. in 2×SSC buffer +0.1% SDS for 15 minutes, followed by two 15 minute washes with 2×SSC buffer at 55° C. Approximately 30 colonies gave positive hybridization signals with the degenerate probe. Several cosmids were selected and divided into two classes based on restriction digestion patterns. A representative cosmid was selected from each class for further analysis. The representative cosmids were designated pKOS023-26 and pKOS023-27. These cosmids were determined by DNA sequencing to comprise the narbonolide PKS genes, the desosamine biosynthesis and transferase genes, the beta-glucosidase gene, and the picK hydroxylase gene. [0266]
  • These cosmids were deposited with the American Type Culture Collection in accordance with the terms of the Budapest Treaty. Cosmid pKOS023-26 was assigned accession number ATCC 203141, and cosmid pKOS023-27 was assigned accession number ATCC 203142. [0267]
  • To demonstrate that the narbonolide PKS genes had been cloned and to illustrate how the invention provides methods and reagents for constructing deletion variants of narbonolide PKS genes, a narbonolide PKS gene was deleted from the chromosome of [0268] Streptomyces venezuelae. This deletion is shown schematically in FIG. 4, parts B and C. A ˜2.4 kb EcoRI-KpnI fragment and a ˜2.1 kb KpnI-XhoI fragment, which together comprise both ends of the picAI gene (but lack a large portion of the coding sequence), were isolated from cosmid pKOS023-27 and ligated together into the commercially available vector pLitmus 28 (digested with restriction enzymes EcoRI and XhoI) to give plasmid pKOS039-07. The ˜4.5 kb HindIII-SpeI fragment from plasmid pKOS039-07 was ligated with the 2.5 kb HindIII-NheI fragment of integrating vector pSET52, available from the NRRL, which contains an E. coli origin of replication and an apramycin resistance-conferring gene to create plasmid pKOS039-16. This vector was used to transform S. venezuelae, and apramycin-resistant transformants were selected.
  • Then, to select for double-crossover mutants, the selected transformants were grown in TSB liquid medium without antibiotics for three transfers and then plated onto non-selective media to provide single colony isolates. The isolated colonies were tested for sensitivity to apramycin, and the apramycin-sensitive colonies were then tested to determine if they produced picromycin. The tests performed included a bioassay and LC/MS analysis of the fermentation media. Colonies determined not to produce picromycin (or methymycin or neomethymycin) were then analyzed using PCR to detect an amplification product diagnostic of the deletion. A colony designated K39-03 was identified, providing confirmation that the narbonolide PKS genes had been cloned. Transformation of strain K39-03 with plasmid pKOS039-27 comprising an intact picA gene under the control of the ermE* promoter from plasmid pWHM3 (see Vara et al., [0269] J. Bact. (1989) 171: 5872-5881, incorporated herein by reference) was able to restore picromycin production.
  • To determine that the cosmids also contained the picK hydroxylase gene, each cosmid was probed by Southern hybridization using a labeled DNA fragment amplified by PCR from the [0270] Saccharopolyspora erythraea C12-hydroxylase gene, eryK. The cosmids were digested with BamHI endonuclease and electrophoresed on a 1% agarose gel, and the resulting fragments were transferred to a nylon membrane. The membrane was incubated with the eryK probe overnight at 42° C., washed twice at 25° C. in 2×SSC buffer with 0.1% SDS for 15 minutes, followed by two 15 minute washes with 2×SSC buffer at 50° C. Cosmid pKOS023-26 produced an ˜3 kb fragment that hybridized with the probe under these conditions. This fragment was subcloned into the PCRscript™ (Stratagene) cloning vector to yield plasmid pKOS023-28 and sequenced. The ˜1.2 kb gene designated picK above was thus identified. The picK gene product is homologous to eryK and other known macrolide cytochrome P450 hydroxylases.
  • By such methodology, the complete set of picromycin biosynthetic genes were isolated and identified. DNA sequencing of the cloned DNA provided further confirmation that the correct genes had been cloned. In addition, and as described in the following example, the identity of the genes was confirmed by expression of narbomycin in heterologous host cells. [0271]
    • EXAMPLE 3 Heterologous Expression of the Narbonolide PKS and the Picromycin Biosynthetic Gene Cluster
  • To provide a preferred host cell and vector for purposes of the invention, the narbonolide PKS was transferred to the non-macrolide producing host [0272] Streptomyces lividans K4-114 (see Ziermann and Betlach, 1999, Biotechniques 26, 106-110, and U.S. patent application Ser. No. 09/181,833, filed Oct. 28, 1998, each of which is incorporated herein by reference). This was accomplished by replacing the three DEBS ORFs on a modified version of pCK7 (see Kao et al., 1994, Science 265, 509-512, and U.S. Pat. No. 5,672,491, each of which is incorporated herein by reference) with all four narbonolide PKS ORFs to generate plasmid pKOS039-86 (see FIG. 5). The pCK7 derivative employed, designated pCK7′Kan', differs from pCK7 only in that it contains a kanamycin resistance conferring gene inserted at its HindIII restriction enzyme recognition site. Because the plasmid contains two selectable markers, one can select for both markers and so minimize contamination with cells containing rearranged, undesired vectors.
  • Protoplasts were transformed using standard procedures and transformants selected using overlays containing antibiotics. The strains were grown in liquid R5 medium for growth/seed and production cultures at 30° C. A 2 L shake flask culture of [0273] S. lividans K4-114/pKOS039-86 was grown for 7 days at 30° C. The mycelia was filtered, and the aqueous layer was extracted with 2×2 L ethyl acetate. The organic layers were combined, dried over MgSO4, filtered, and evaporated to dryness. Polyketides were separated from the crude extract by silica gel chromatography (1:4 to 1:2 ethyl acetate:hexane gradient) to give an ˜10 mg mixture of narbonolide and 10-deoxymethynolide, as indicated by LC/MS and 1H NMR. Purification of these two compounds was achieved by HPLC on a C-18 reverse phase column (20-80% acetonitrile in water over 45 minutes). This procedure yielded ˜5 mg each of narbonolide and 10-deoxymethynolide. Polyketides produced in the host cells were analyzed by bioassay against Bacillus subtilis and by LC/MS analysis. Analysis of extracts by LC/MS followed by 1H-NMR spectroscopy of the purified compounds established their identity as narbonolide (FIG. 5, compound 4; see Kaiho et al., 1982, J. Org. Chem. 47: 1612-1614, incorporated herein by reference) and 10-deoxymethynolide (FIG. 5, compound 5; see Lambalot et al., 1992, J. Antibiotics 45, 1981-1982, incorporated herein by reference), the respective 14 and 12-membered polyketide aglycones of YC17, narbomycin, picromycin, and methymycin.
  • The production of narbonolide in [0274] Streptomyces lividans represents the expression of an entire modular polyketide pathway in a heterologous host. The combined yields of compounds 4 and 5 are similar to those obtained with expression of DEBS from pCK7 (see Kao et al., 1994, Science 265: 509-512, incorporated herein by reference). Furthermore, based on the relative ratios (˜1:1) of compounds 4 and 5 produced, it is apparent that the narbonolide PKS itself possesses an inherent ability to produce both 12 and 14-membered macrolactones without the requirement of additional activities unique to S. venezuelae. Although the existence of a complementary enzyme present in S. lividans that provides this function is possible, it would be unusual to find-such a specific enzyme in an organism that does not produce any known macrolide.
  • To provide a heterologous host cell of the invention that produces the narbonolide PKS and the picB gene, the picB gene was integrated into the chromosome of [0275] Streptomyces lividans harboring plasmid pKOS039-86 to yield S. lividans K39-18/pKOS039-86. To provide the integrating vector utilized, the picB gene was cloned into the Streptomyces genome integrating vector pSET152 (see Bierman et al., 1992, Gene 116, 43, incorporated herein by reference) under control of the same promoter (PactI) as the PKS on plasmid pKOS039-86.
  • A comparison of strains K39-18/pKOS039-86 and K4-114/pKOS039-86 grown under identical conditions indicated that the strain containing TEII produced 4-7 times more total polyketide. Each strain was grown in 30 mL of R[0276] 5 (see Hopwood et al., Genetic Manipulation of Streptomyces: A Laboratory Manual; John Innes Foundation: Norwich, UK, 1985, incorporated herein by reference) liquid (with 20 μg/mL thiostrepton) at 30° C. for 9 days. The fermentation broth was analyzed directly by reverse phase HPLC. Absorbance at 235 nm was used to monitor compounds and measure relative abundance. This increased production indicates that the enzyme is functional in this strain. As noted above, because the production levels of compound 4 and 5 from K39-18/pKOS03986 increased by the same relative amounts, TEII does not appear to influence the ratio of 12 and 14-membered lactone ring formation.
  • To express the glycosylated counterparts of narbonolide (narbomycin) and 10-deoxymethynolide (YC17) in heterologous host cells, the desosamine biosynthetic genes and desosaminyl transferase gene were transformed into the host cells harboring plasmid pKOS039-86 (and, optionally, the picB gene, which can be integrated into the chromosome as described above). [0277]
  • Plasmid pKOS039-104, see FIG. 6, comprises the desosamine biosynthetic genes, the beta-glucosidase gene, and the desosaminyl transferase gene. This plasmid was constructed by first inserting a polylinker oligonucleotide, containing a restriction enzyme recognition site for PacI, a Shine-Dalgarno sequence, and restriction enzyme recognition sites for NdeI, BglII, and HindIII, into a pUC 19 derivative, called pKOS24-47, to yield plasmid pKOS039-98. [0278]
  • An ˜0.3 kb PCR fragment comprising the coding sequence for the N-terminus of the desI gene product and an ˜0.12 kb PCR fragment comprising the coding sequence for the C-terminus of the desR gene product were amplified from cosmid pKOS23-26 (ATCC 203141) and inserted together into pLitmus28 treated with restriction enzymes NsiI and EcoRI to produce plasmid pKOS039-101. The ˜6 kb SphI-PstI restriction fragment of pKOS23-26 containing the desI, desII, desIII, desIV, and desV genes was inserted into plasmid pUC19 (Stratagene) to yield plasmid pKOS039-102. The ˜6 kb SphI-EcoRI restriction fragment from plasmid pKOS039-102 was inserted into pKOS039-101 to produce plasmid pKOS039-103. The ˜6 kb BglII-PstI fragment from pKOS23-26 that contains the desR, desVI, desVII, and desVIII genes was inserted into pKOS39-98 to yield pKOS39-100. The ˜6 kb PacI-PstI restriction fragment of pKOS39-100 and the ˜6.4 kb NsiI-EcoRI fragment of pKOS39-103 were cloned into pKOS39-44 to yield pKOS39-104. [0279]
  • When introduced into [0280] Streptomyces lividans host cells comprising the recombinant narbonolide PKS of the invention, plasmid pKOS39-104 drives expression of the desosamine biosynthetic genes, the beta-glucosidase gene, and the desosaminyl transferase gene. The glycosylated antibiotic narbomycin was produced in these host cells, and it is believed that YC17 was produced as well. When these host cells are transformed with vectors that drive expression of the picK gene, the antibiotics methymycin, neomethymycin, and picromycin are produced.
  • In similar fashion, when plasmid pKOS039-18, which encodes a hybrid PKS of the invention that produces 3-deoxy-3-oxo-6-deoxyerythronolide B was expressed in [0281] Streptomyces lividans host cells transformed with plasmid pKOS39-104, the 5-desosaminylated analog was produced. Likewise, when plasmid pCK7, which encodes DEBS, which produces 6-deoxyerythronolide B, was expressed in Streptomyces lividans host cells transformed with plasmid pKOS39-104, the 5-desosaminylated analog was produced. These compounds have antibiotic activity and are useful as intermediates in the synthesis of other antibiotics.
    • EXAMPLE 4 Expression Vector for Desosaminyl Transferase
  • While the invention provides expression vectors comprising all of the genes required for desosamine biosynthesis and transfer to a polyketide, the invention also provides expression vectors that encode any subset of those genes or any single gene. As one illustrative example, the invention provides an expression vector for desosaminyl transferase. This vector is useful to desosaminylate polyketides in host cells that produce NDP-desosamine but lack a desosaminyl transferase gene or express a desosaminyl transferase that does not function as efficiently on the polyketide of interest as does the desosaminyl transferase of [0282] Streptomyces venezuelae. This expression vector was constructed by first amplifying the desosaminyl transferase coding sequence from pKOS023-27 using the primers:
  • N3917: 5′-CCCTGCAGCGGCAAGGAAGGACACGACGCCA-3′ (SEQ ID NO:25); and [0283]
  • N3918: 5′-AGGTCTAGAGCTCAGTGCCGGGCGTCGGCCGG-3′ (SEQ ID NO:26), [0284]
  • to give a 1.5 kb product. This product was then treated with restriction enzymes PstI and XbaI and ligated with HindIII and XbaI digested plasmid pKOS039-06 together with the 7.6 kb PstI-HindIII restriction fragment of plasmid pWHM1104 to provide plasmid pKOS039-14. Plasmid pWHM1104, described in Tang et al., 1996[0285] , Molec. Microbiol. 22(5): 801-813, incorporated herein by reference, encodes the ermE* promoter. Plasmid pKOS039-14 is constructed so that the desosaminyl transferase gene is placed under the control of the ermE* promoter and is suitable for expression of the desosaminyl transferase in Streptomyces, Saccharopolyspora erythraea, and other host cells in which the ermE* promoter functions.
    • EXAMPLE 5 Heterologous Expression of the picK Gene Product in E. coli
  • The picK gene was PCR amplified from plasmid pKOS023-28 using the oligonucleotide primers: [0286]
  • N024-36B (forward): [0287]
  • 5′-TTGCATGCATATGCGCCGTACCCAGCAGGGAACGACC (SEQ ID NO:27); and [0288]
  • N024-37B (reverse): [0289]
  • 5′-TTGAATTCTCAACTAGTACGGCGGCCCGCCTCCCGTCC (SEQ ID NO:28). [0290]
  • These primers alter the Streptomyces GTG start codon to ATG and introduce a SpeI site at the C-terminal end of the gene, resulting in the substitution of a serine for the terminal glycine amino acid residue. The blunt-ended PCR product was subcloned into the commercially available vector pCRscript at the SrfI site to yield plasmid pKOS023-60. An ˜1.3 kb NdeI-XhoI fragment was then inserted into the NdeI/XhoI sites of the T7 expression vector pET22b (Novagen, Madison, Wis.) to generate pKOS023-61. Plasmid pKOS023-61 was digested with restriction enzymes SpeI and EcoRI, and a short [0291] linker fragment encoding 6 histidine residues and a stop codon (composed of oligonucleotides 30-85a: 5′-CTAGTATGCATCATCATCATCATCATTAA-3′ (SEQ ID NO:29); and 30-85b: 5′-AATTTTAATGATGATGATGATGATGCATA-3′ (SEQ ID NO:30) was inserted to obtain plasmid pKOS023-68. Both plasmid pKOS023-61 and pKOS023-68 produced active PicK enzyme in recombinant E. coli host cells.
  • Plasmid pKOS023-61 was transformed into [0292] E. coli BL21-DE3. Successful transformants were grown in LB-containing carbenicillin (100 μg/ml) at 37° C. to an OD600 of 0.6. Isopropyl-beta-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM, and the cells were grown for an additional 3 hours before harvesting. The cells were collected by centrifugation and frozen at −80° C. A control culture of-BL21-DE3 containing the vector plasmid pET21c (Invitrogen) was prepared in parallel.
  • The frozen BL21-DE3/pKOS023-61 cells were thawed, suspended in 2 μL of cold cell disruption buffer (5 mM imidazole, 500 mM NaCl, 20 mM Tris/HCl, pH 8.0) and sonicated to facilitate lysis. Cellular debris and supernatant were separated by centrifugation and subjected to SDS-PAGE on 10-15% gradient gels, with Coomassie Blue staining, using a Pharmacia Phast Gel Electrophoresis system. The soluble crude extract from BL21-DE3/pKOS023-61 contained a Coomassie stained band of Mr˜46 kDa, which was absent in the control strain BL21-DE3/pET21c. [0293]
  • The hydroxylase activity of the picK protein was assayed as follows. The crude supernatant (20 μL) was added to a reaction mixture (100 μL total volume) containing 50 mM Tris/HCl (pH 7.5), 20 μM spinach ferredoxin, 0.025 Unit of spinach ferredoxin:NADP+ oxidoreductase, 0.8 Unit of glucose-6-phosphate dehydrogenase, 1.4 mM NADP+, 7.6 mM glucose-6phosphate, and 20 nmol of narbomycin. The narbomycin was purified from a culture of [0294] Streptomyces narbonensis, and upon LC/MS analysis gave a single peak of [M+H]+=510. The reaction was allowed to proceed for 105 minutes at 30° C. Half of the reaction mixture was loaded onto an HPLC, and the effluent was analyzed by evaporative light scattering (ELSD) and mass spectrometry. The control extract (BL21-DE3/pET21c) was processed identically. The BL21-DE3/pKOS023-61 reaction contained a compound not present in the control having the same retention time, molecular weight and mass fragmentation pattern as picromycin ([M+H]+=526). The conversion of narbomycin to picromycin under these conditions was estimated to be greater than 90% by ELSD peak area.
  • The poly-histidine-linked PicK hydroxylase was prepared from pKOS023-68 transformed into [0295] E. coli BL21 (DE3) and cultured as described above. The cells were harvested and the PicK protein purified as follows. All purification steps were performed at 4° C. E. coli cell pellets were suspended in 32 μL of cold binding buffer (20 mM Tris/HCl, pH 8.0, 5 mM imidazole, 500 mM NaCl) per mL of culture and lysed by sonication. For analysis of E. coli cell-free extracts, the cellular debris was removed by low-speed centrifugation, and the supernatant was used directly in assays. For purification of PicK/6-His, the supernatant was loaded (0.5 mL/min.) onto a 5 mL HiTrap Chelating column (Pharmacia, Piscataway, N.J.), equilibrated with binding buffer. The column was washed with 25 μL of binding buffer and the protein was eluted with a −35 μL linear gradient (5-500 mM imidazole in binding buffer). Column effluent was monitored at 280 nm and 416 nm. Fractions corresponding to the 416 nm absorbance peak were pooled and dialyzed against storage buffer (45 mM Tris/HCl, pH 7.5, 0.1 mM EDTA, 0.2 mM DTT, 10% glycerol). The purified 46 kDa protein was analyzed by SDS-PAGE using Coomassie blue staining, and enzyme concentration and yield were determined.
  • Narbomycin was purified as described above from a culture of [0296] Streptomyces narbonensis ATCC19790. Reactions for kinetic assays (100 μL) consisted of 50 mM Tris/HCl (pH 7.5), 100 μM spinach ferredoxin, 0.025 Unit of spinach ferredoxin:NADP+ oxidoreductase, 0.8 U glucose-6-phosphate dehydrogenase, 1.4 mM NADP+, 7.6 mM glucose-6-phosphate, 20-500 nM narbomycin substrate, and 50-500 nM of PicK enzyme. The reaction proceeded at 30° C., and samples were withdrawn for analysis at 5, 10, 15, and 90 minutes. Reactions were stopped by heating to 100° C. for 1 minute, and denatured protein was removed by centrifugation. Depletion of narbomycin and formation of picromycin were determined by high performance liquid chromatography (HPLC, Beckman C-180.46×15 cm column) coupled to atmospheric pressure chemical ionization (APCI) mass spectroscopic detection (Perkin Elmer/Sciex API 100) and evaporative light scattering detection (Alltech 500 ELSD).
    • EXAMPLE 6 Expression of the picK Gene Encoding the Hydroxylase in Streptomyces narbonensis
  • To produce picromycin in [0297] Streptomyces narbonensis, a host that produces narbomycin but not picromycin, the methods and vectors of the invention were used to express the picK gene in this host.
  • The picK gene was amplified from cosmid pKOS023-26 using the primers: [0298]
  • N3903: 5′-TCCTCTAGACGTTTCCGT-3′ (SEQ ID NO:31); and [0299]
  • N3904: 5′-TGAAGCTTGAATTCAACCGGT-3′ (SEQ ID NO:32) [0300]
  • to obtain at ˜1.3 kb product. The product was treated with restriction enzymes XbaI and HindIII and ligated with the 7.6 kb XbaI-HindIII restriction fragment of plasmid pWHM1104 to provide plasmid pKOS039-01, placing the picK gene under the control of the ermE* promoter. The resulting plasmid was transformed into purified stocks of [0301] S. narbonensis by protoplast fusion and electroporation. The transformants were grown in suitable media and shown to convert narbomycin to picromycin at a yield of over 95%.
    • EXAMPLE 7 Construction of a Hybrid DEBS/Narbonolide PKS
  • This example describes the construction of illustrative hybrid PKS expression vectors of the invention. The hybrid PKS contains portions of the narbonolide PKS and portions of rapamycin and/or DEBS PKS. In the first constructs, pKOS039-18 and pKOS039-19, the hybrid PKS comprises the narbonolide [0302] PKS extender module 6 ACP and thioesterase domains and the DEBS loading module and extender modules 1-5 as well as the KS and AT domains of DEBS extender module 6 (but not the KR domain of extender module 6). In pKOS039-19, the hybrid PKS is identical except that the KS 1 domain is inactivated, i.e., the ketosynthase in extender module 1 is disabled. The inactive DEBS KS1 domain and its construction are described in detail in PCT publication Nos. WO 97/02358 and WO 99/03986, each of which is incorporated herein by reference. To construct pKOS039-18, the 2.33 kb BamHI-EcoRI fragment of pKOS023-27, which contains the desired sequence, was amplified by PCR and subcloned into plasmid pUC19. The primers used in the PCR were:
  • N3905: 5′-TTTATGCATCCCGCGGGTCCCGGCGAG-3′ (SEQ ID NO:33); and [0303]
  • N3906: 5′-TCAGAATTCTGTCGGTCACTTGCCCGC-3′ (SEQ ID NO:34). [0304]
  • The 1.6 kb PCR product was digested with PstI and EcoRI and cloned into the corresponding sites of plasmid pKOS015-52 (this plasmid contains the relevant portions of the coding sequence for the DEBS extender module 6) and commercially available plasmid pLitmus 28 to provide plasmids pKOS039-12 and pKOS039-13, respectively. The BglII-EcoRI fragment of plasmid pKOS039-12 was cloned into plasmid pKOS011-77, which contains the functional DEBS gene cluster and into plasmid pJRJ2, which contains the mutated DEBS gene that produces a DEBS PKS in which the KS domain of extender module I has been rendered inactive. Plasmid pJRJ2 is described in PCT publication Nos. WO 99/03986 and WO 97/02358, incorporated herein by reference. [0305]
  • Plasmids pKOS039-18 and pKOS039-19, respectively, were obtained. These two plasmids were transformed into [0306] Streptomyces coelicolor CH999 by protoplast fusion. The resulting cells were cultured under conditions such that expression of the PKS occurred. Cells transformed with plasmid pKOS039-18 produced the expected product 3-deoxy-3-oxo-6-deoxyerythronolide B. When cells transformed with plasmid pKOS039-19 were provided (2S,3R)-2-methyl-3-hydroxyhexanoate NACS, 13-desethyl-13-propyl-3-deoxy-3-oxo-6-deoxyerythronolide B was produced.
    • EXAMPLE 8 6-Hydroxylation of 3,6-dideoxy-3-oxoerythronolide B Using the eryF Hydroxylase
  • Certain compounds of the invention can be hydroxylated at the C6 position in a host cell that expresses the eryF gene. These compounds can also be hydroxylated in vitro, as illustrated by this example. [0307]
  • The 6-hydroxylase encoded by eryF was expressed in [0308] E. coli, and partially purified. The hydroxylase (100 pmol in 10 μL) was added to a reaction mixture (100 μl total volume) containing 50 mM Tris/HCl (pH 7.5), 20 nM spinach ferredoxin, 0.025 Unit of spinach ferredoxin:NADP+ oxidoreductase, 0.8 Unit of glucose-6-phosphate dehydrogenase, 1.4 mM NADP+, 7.6 mM glucose-6-phosphate, and 10 nmol 6-deoxyerythronolide B. The reaction was allowed to proceed for 90 minutes at 30° C. Half of the reaction mixture was loaded onto an HPLC, and the effluent was analyzed by mass spectrometry. The production of erythronolide B as evidenced by a new peak eluting earlier in the gradient and showing [M+H]+=401. Conversion was estimated at 50% based on relative total ion counts.
  • Those of skill in the art will recognize the potential for hemiketal formation in the above compound and compounds of similar structure. To reduce the amount of hemiketal formed, one can use more basic (as opposed to acidic) conditions or employ sterically hindered derivative compounds, such as 5-desosaminylated compounds. [0309]
    • EXAMPLE 9 Measurement of Antibacterial Activity
  • Antibacterial activity was determined using either disk diffusion assays with [0310] Bacillus cereus as the test organism or by measurement of minimum inhibitory concentrations (MIC) in liquid culture against sensitive and resistant strains of Staphylococcus pneumoniae.
  • The invention having now been described by way of written description and example, those of skill in the art will recognize that the invention can be practiced in a variety of embodiments and that the foregoing description and examples are for purposes of illustration and not limitation of the following claims. [0311]
  • 1 34 1 4551 PRT Streptomyces venezuelae 1 Met Ser Thr Val Ser Lys Ser Glu Ser Glu Glu Phe Val Ser Val Ser 1 5 10 15 Asn Asp Ala Gly Ser Ala His Gly Thr Ala Glu Pro Val Ala Val Val 20 25 30 Gly Ile Ser Cys Arg Val Pro Gly Ala Arg Asp Pro Arg Glu Phe Trp 35 40 45 Glu Leu Leu Ala Ala Gly Gly Gln Ala Val Thr Asp Val Pro Ala Asp 50 55 60 Arg Trp Asn Ala Gly Asp Phe Tyr Asp Pro Asp Arg Ser Ala Pro Gly 65 70 75 80 Arg Ser Asn Ser Arg Trp Gly Gly Phe Ile Glu Asp Val Asp Arg Phe 85 90 95 Asp Ala Ala Phe Phe Gly Ile Ser Pro Arg Glu Ala Ala Glu Met Asp 100 105 110 Pro Gln Gln Arg Leu Ala Leu Glu Leu Gly Trp Glu Ala Leu Glu Arg 115 120 125 Ala Gly Ile Asp Pro Ser Ser Leu Thr Gly Thr Arg Thr Gly Val Phe 130 135 140 Ala Gly Ala Ile Trp Asp Asp Tyr Ala Thr Leu Lys His Arg Gln Gly 145 150 155 160 Gly Ala Ala Ile Thr Pro His Thr Val Thr Gly Leu His Arg Gly Ile 165 170 175 Ile Ala Asn Arg Leu Ser Tyr Thr Leu Gly Leu Arg Gly Pro Ser Met 180 185 190 Val Val Asp Ser Gly Gln Ser Ser Ser Leu Val Ala Val His Leu Ala 195 200 205 Cys Glu Ser Leu Arg Arg Gly Glu Ser Glu Leu Ala Leu Ala Gly Gly 210 215 220 Val Ser Leu Asn Leu Val Pro Asp Ser Ile Ile Gly Ala Ser Lys Phe 225 230 235 240 Gly Gly Leu Ser Pro Asp Gly Arg Ala Tyr Thr Phe Asp Ala Arg Ala 245 250 255 Asn Gly Tyr Val Arg Gly Glu Gly Gly Gly Phe Val Val Leu Lys Arg 260 265 270 Leu Ser Arg Ala Val Ala Asp Gly Asp Pro Val Leu Ala Val Ile Arg 275 280 285 Gly Ser Ala Val Asn Asn Gly Gly Ala Ala Gln Gly Met Thr Thr Pro 290 295 300 Asp Ala Gln Ala Gln Glu Ala Val Leu Arg Glu Ala His Glu Arg Ala 305 310 315 320 Gly Thr Ala Pro Ala Asp Val Arg Tyr Val Glu Leu His Gly Thr Gly 325 330 335 Thr Pro Val Gly Asp Pro Ile Glu Ala Ala Ala Leu Gly Ala Ala Leu 340 345 350 Gly Thr Gly Arg Pro Ala Gly Gln Pro Leu Leu Val Gly Ser Val Lys 355 360 365 Thr Asn Ile Gly His Leu Glu Gly Ala Ala Gly Ile Ala Gly Leu Ile 370 375 380 Lys Ala Val Leu Ala Val Arg Gly Arg Ala Leu Pro Ala Ser Leu Asn 385 390 395 400 Tyr Glu Thr Pro Asn Pro Ala Ile Pro Phe Glu Glu Leu Asn Leu Arg 405 410 415 Val Asn Thr Glu Tyr Leu Pro Trp Glu Pro Glu His Asp Gly Gln Arg 420 425 430 Met Val Val Gly Val Ser Ser Phe Gly Met Gly Gly Thr Asn Ala His 435 440 445 Val Val Leu Glu Glu Ala Pro Gly Val Val Glu Gly Ala Ser Val Val 450 455 460 Glu Ser Thr Val Gly Gly Ser Ala Val Gly Gly Gly Val Val Pro Trp 465 470 475 480 Val Val Ser Ala Lys Ser Ala Ala Ala Leu Asp Ala Gln Ile Glu Arg 485 490 495 Leu Ala Ala Phe Ala Ser Arg Asp Arg Thr Asp Gly Val Asp Ala Gly 500 505 510 Ala Val Asp Ala Gly Ala Val Asp Ala Gly Ala Val Ala Arg Val Leu 515 520 525 Ala Gly Gly Arg Ala Gln Phe Glu His Arg Ala Val Val Val Gly Ser 530 535 540 Gly Pro Asp Asp Leu Ala Ala Ala Leu Ala Ala Pro Glu Gly Leu Val 545 550 555 560 Arg Gly Val Ala Ser Gly Val Gly Arg Val Ala Phe Val Phe Pro Gly 565 570 575 Gln Gly Thr Gln Trp Ala Gly Met Gly Ala Glu Leu Leu Asp Ser Ser 580 585 590 Ala Val Phe Ala Ala Ala Met Ala Glu Cys Glu Ala Ala Leu Ser Pro 595 600 605 Tyr Val Asp Trp Ser Leu Glu Ala Val Val Arg Gln Ala Pro Gly Ala 610 615 620 Pro Thr Leu Glu Arg Val Asp Val Val Gln Pro Val Thr Phe Ala Val 625 630 635 640 Met Val Ser Leu Ala Arg Val Trp Gln His His Gly Val Thr Pro Gln 645 650 655 Ala Val Val Gly His Ser Gln Gly Glu Ile Ala Ala Ala Tyr Val Ala 660 665 670 Gly Ala Leu Ser Leu Asp Asp Ala Ala Arg Val Val Thr Leu Arg Ser 675 680 685 Lys Ser Ile Ala Ala His Leu Ala Gly Lys Gly Gly Met Leu Ser Leu 690 695 700 Ala Leu Ser Glu Asp Ala Val Leu Glu Arg Leu Ala Gly Phe Asp Gly 705 710 715 720 Leu Ser Val Ala Ala Val Asn Gly Pro Thr Ala Thr Val Val Ser Gly 725 730 735 Asp Pro Val Gln Ile Glu Glu Leu Ala Arg Ala Cys Glu Ala Asp Gly 740 745 750 Val Arg Ala Arg Val Ile Pro Val Asp Tyr Ala Ser His Ser Arg Gln 755 760 765 Val Glu Ile Ile Glu Ser Glu Leu Ala Glu Val Leu Ala Gly Leu Ser 770 775 780 Pro Gln Ala Pro Arg Val Pro Phe Phe Ser Thr Leu Glu Gly Ala Trp 785 790 795 800 Ile Thr Glu Pro Val Leu Asp Gly Gly Tyr Trp Tyr Arg Asn Leu Arg 805 810 815 His Arg Val Gly Phe Ala Pro Ala Val Glu Thr Leu Ala Thr Asp Glu 820 825 830 Gly Phe Thr His Phe Val Glu Val Ser Ala His Pro Val Leu Thr Met 835 840 845 Ala Leu Pro Gly Thr Val Thr Gly Leu Ala Thr Leu Arg Arg Asp Asn 850 855 860 Gly Gly Gln Asp Arg Leu Val Ala Ser Leu Ala Glu Ala Trp Ala Asn 865 870 875 880 Gly Leu Ala Val Asp Trp Ser Pro Leu Leu Pro Ser Ala Thr Gly His 885 890 895 His Ser Asp Leu Pro Thr Tyr Ala Phe Gln Thr Glu Arg His Trp Leu 900 905 910 Gly Glu Ile Glu Ala Leu Ala Pro Ala Gly Glu Pro Ala Val Gln Pro 915 920 925 Ala Val Leu Arg Thr Glu Ala Ala Glu Pro Ala Glu Leu Asp Arg Asp 930 935 940 Glu Gln Leu Arg Val Ile Leu Asp Lys Val Arg Ala Gln Thr Ala Gln 945 950 955 960 Val Leu Gly Tyr Ala Thr Gly Gly Gln Ile Glu Val Asp Arg Thr Phe 965 970 975 Arg Glu Ala Gly Cys Thr Ser Leu Thr Gly Val Asp Leu Arg Asn Arg 980 985 990 Ile Asn Ala Ala Phe Gly Val Arg Met Ala Pro Ser Met Ile Phe Asp 995 1000 1005 Phe Pro Thr Pro Glu Ala Leu Ala Glu Gln Leu Leu Leu Val Val His 1010 1015 1020 Gly Glu Ala Ala Ala Asn Pro Ala Gly Ala Glu Pro Ala Pro Val Ala 1025 1030 1035 1040 Ala Ala Gly Ala Val Asp Glu Pro Val Ala Ile Val Gly Met Ala Cys 1045 1050 1055 Arg Leu Pro Gly Gly Val Ala Ser Pro Glu Asp Leu Trp Arg Leu Val 1060 1065 1070 Ala Gly Gly Gly Asp Ala Ile Ser Glu Phe Pro Gln Asp Arg Gly Trp 1075 1080 1085 Asp Val Glu Gly Leu Tyr His Pro Asp Pro Glu His Pro Gly Thr Ser 1090 1095 1100 Tyr Val Arg Gln Gly Gly Phe Ile Glu Asn Val Ala Gly Phe Asp Ala 1105 1110 1115 1120 Ala Phe Phe Gly Ile Ser Pro Arg Glu Ala Leu Ala Met Asp Pro Gln 1125 1130 1135 Gln Arg Leu Leu Leu Glu Thr Ser Trp Glu Ala Val Glu Asp Ala Gly 1140 1145 1150 Ile Asp Pro Thr Ser Leu Arg Gly Arg Gln Val Gly Val Phe Thr Gly 1155 1160 1165 Ala Met Thr His Glu Tyr Gly Pro Ser Leu Arg Asp Gly Gly Glu Gly 1170 1175 1180 Leu Asp Gly Tyr Leu Leu Thr Gly Asn Thr Ala Ser Val Met Ser Gly 1185 1190 1195 1200 Arg Val Ser Tyr Thr Leu Gly Leu Glu Gly Pro Ala Leu Thr Val Asp 1205 1210 1215 Thr Ala Cys Ser Ser Ser Leu Val Ala Leu His Leu Ala Val Gln Ala 1220 1225 1230 Leu Arg Lys Gly Glu Val Asp Met Ala Leu Ala Gly Gly Val Ala Val 1235 1240 1245 Met Pro Thr Pro Gly Met Phe Val Glu Phe Ser Arg Gln Arg Gly Leu 1250 1255 1260 Ala Gly Asp Gly Arg Ser Lys Ala Phe Ala Ala Ser Ala Asp Gly Thr 1265 1270 1275 1280 Ser Trp Ser Glu Gly Val Gly Val Leu Leu Val Glu Arg Leu Ser Asp 1285 1290 1295 Ala Arg Arg Asn Gly His Gln Val Leu Ala Val Val Arg Gly Ser Ala 1300 1305 1310 Val Asn Gln Asp Gly Ala Ser Asn Gly Leu Thr Ala Pro Asn Gly Pro 1315 1320 1325 Ser Gln Gln Arg Val Ile Arg Arg Ala Leu Ala Asp Ala Arg Leu Thr 1330 1335 1340 Thr Ser Asp Val Asp Val Val Glu Ala His Gly Thr Gly Thr Arg Leu 1345 1350 1355 1360 Gly Asp Pro Ile Glu Ala Gln Ala Leu Ile Ala Thr Tyr Gly Gln Gly 1365 1370 1375 Arg Asp Asp Glu Gln Pro Leu Arg Leu Gly Ser Leu Lys Ser Asn Ile 1380 1385 1390 Gly His Thr Gln Ala Ala Ala Gly Val Ser Gly Val Ile Lys Met Val 1395 1400 1405 Gln Ala Met Arg His Gly Leu Leu Pro Lys Thr Leu His Val Asp Glu 1410 1415 1420 Pro Ser Asp Gln Ile Asp Trp Ser Ala Gly Ala Val Glu Leu Leu Thr 1425 1430 1435 1440 Glu Ala Val Asp Trp Pro Glu Lys Gln Asp Gly Gly Leu Arg Arg Ala 1445 1450 1455 Ala Val Ser Ser Phe Gly Ile Ser Gly Thr Asn Ala His Val Val Leu 1460 1465 1470 Glu Glu Ala Pro Val Val Val Glu Gly Ala Ser Val Val Glu Pro Ser 1475 1480 1485 Val Gly Gly Ser Ala Val Gly Gly Gly Val Thr Pro Trp Val Val Ser 1490 1495 1500 Ala Lys Ser Ala Ala Ala Leu Asp Ala Gln Ile Glu Arg Leu Ala Ala 1505 1510 1515 1520 Phe Ala Ser Arg Asp Arg Thr Asp Asp Ala Asp Ala Gly Ala Val Asp 1525 1530 1535 Ala Gly Ala Val Ala His Val Leu Ala Asp Gly Arg Ala Gln Phe Glu 1540 1545 1550 His Arg Ala Val Ala Leu Gly Ala Gly Ala Asp Asp Leu Val Gln Ala 1555 1560 1565 Leu Ala Asp Pro Asp Gly Leu Ile Arg Gly Thr Ala Ser Gly Val Gly 1570 1575 1580 Arg Val Ala Phe Val Phe Pro Gly Gln Gly Thr Gln Trp Ala Gly Met 1585 1590 1595 1600 Gly Ala Glu Leu Leu Asp Ser Ser Ala Val Phe Ala Ala Ala Met Ala 1605 1610 1615 Glu Cys Glu Ala Ala Leu Ser Pro Tyr Val Asp Trp Ser Leu Glu Ala 1620 1625 1630 Val Val Arg Gln Ala Pro Gly Ala Pro Thr Leu Glu Arg Val Asp Val 1635 1640 1645 Val Gln Pro Val Thr Phe Ala Val Met Val Ser Leu Ala Arg Val Trp 1650 1655 1660 Gln His His Gly Val Thr Pro Gln Ala Val Val Gly His Ser Gln Gly 1665 1670 1675 1680 Glu Ile Ala Ala Ala Tyr Val Ala Gly Ala Leu Pro Leu Asp Asp Ala 1685 1690 1695 Ala Arg Val Val Thr Leu Arg Ser Lys Ser Ile Ala Ala His Leu Ala 1700 1705 1710 Gly Lys Gly Gly Met Leu Ser Leu Ala Leu Asn Glu Asp Ala Val Leu 1715 1720 1725 Glu Arg Leu Ser Asp Phe Asp Gly Leu Ser Val Ala Ala Val Asn Gly 1730 1735 1740 Pro Thr Ala Thr Val Val Ser Gly Asp Pro Val Gln Ile Glu Glu Leu 1745 1750 1755 1760 Ala Gln Ala Cys Lys Ala Asp Gly Phe Arg Ala Arg Ile Ile Pro Val 1765 1770 1775 Asp Tyr Ala Ser His Ser Arg Gln Val Glu Ile Ile Glu Ser Glu Leu 1780 1785 1790 Ala Gln Val Leu Ala Gly Leu Ser Pro Gln Ala Pro Arg Val Pro Phe 1795 1800 1805 Phe Ser Thr Leu Glu Gly Thr Trp Ile Thr Glu Pro Val Leu Asp Gly 1810 1815 1820 Thr Tyr Trp Tyr Arg Asn Leu Arg His Arg Val Gly Phe Ala Pro Ala 1825 1830 1835 1840 Ile Glu Thr Leu Ala Val Asp Glu Gly Phe Thr His Phe Val Glu Val 1845 1850 1855 Ser Ala His Pro Val Leu Thr Met Thr Leu Pro Glu Thr Val Thr Gly 1860 1865 1870 Leu Gly Thr Leu Arg Arg Glu Gln Gly Gly Gln Glu Arg Leu Val Thr 1875 1880 1885 Ser Leu Ala Glu Ala Trp Val Asn Gly Leu Pro Val Ala Trp Thr Ser 1890 1895 1900 Leu Leu Pro Ala Thr Ala Ser Arg Pro Gly Leu Pro Thr Tyr Ala Phe 1905 1910 1915 1920 Gln Ala Glu Arg Tyr Trp Leu Glu Asn Thr Pro Ala Ala Leu Ala Thr 1925 1930 1935 Gly Asp Asp Trp Arg Tyr Arg Ile Asp Trp Lys Arg Leu Pro Ala Ala 1940 1945 1950 Glu Gly Ser Glu Arg Thr Gly Leu Ser Gly Arg Trp Leu Ala Val Thr 1955 1960 1965 Pro Glu Asp His Ser Ala Gln Ala Ala Ala Val Leu Thr Ala Leu Val 1970 1975 1980 Asp Ala Gly Ala Lys Val Glu Val Leu Thr Ala Gly Ala Asp Asp Asp 1985 1990 1995 2000 Arg Glu Ala Leu Ala Ala Arg Leu Thr Ala Leu Thr Thr Gly Asp Gly 2005 2010 2015 Phe Thr Gly Val Val Ser Leu Leu Asp Gly Leu Val Pro Gln Val Ala 2020 2025 2030 Trp Val Gln Ala Leu Gly Asp Ala Gly Ile Lys Ala Pro Leu Trp Ser 2035 2040 2045 Val Thr Gln Gly Ala Val Ser Val Gly Arg Leu Asp Thr Pro Ala Asp 2050 2055 2060 Pro Asp Arg Ala Met Leu Trp Gly Leu Gly Arg Val Val Ala Leu Glu 2065 2070 2075 2080 His Pro Glu Arg Trp Ala Gly Leu Val Asp Leu Pro Ala Gln Pro Asp 2085 2090 2095 Ala Ala Ala Leu Ala His Leu Val Thr Ala Leu Ser Gly Ala Thr Gly 2100 2105 2110 Glu Asp Gln Ile Ala Ile Arg Thr Thr Gly Leu His Ala Arg Arg Leu 2115 2120 2125 Ala Arg Ala Pro Leu His Gly Arg Arg Pro Thr Arg Asp Trp Gln Pro 2130 2135 2140 His Gly Thr Val Leu Ile Thr Gly Gly Thr Gly Ala Leu Gly Ser His 2145 2150 2155 2160 Ala Ala Arg Trp Met Ala His His Gly Ala Glu His Leu Leu Leu Val 2165 2170 2175 Ser Arg Ser Gly Glu Gln Ala Pro Gly Ala Thr Gln Leu Thr Ala Glu 2180 2185 2190 Leu Thr Ala Ser Gly Ala Arg Val Thr Ile Ala Ala Cys Asp Val Ala 2195 2200 2205 Asp Pro His Ala Met Arg Thr Leu Leu Asp Ala Ile Pro Ala Glu Thr 2210 2215 2220 Pro Leu Thr Ala Val Val His Thr Ala Gly Ala Leu Asp Asp Gly Ile 2225 2230 2235 2240 Val Asp Thr Leu Thr Ala Glu Gln Val Arg Arg Ala His Arg Ala Lys 2245 2250 2255 Ala Val Gly Ala Ser Val Leu Asp Glu Leu Thr Arg Asp Leu Asp Leu 2260 2265 2270 Asp Ala Phe Val Leu Phe Ser Ser Val Ser Ser Thr Leu Gly Ile Pro 2275 2280 2285 Gly Gln Gly Asn Tyr Ala Pro His Asn Ala Tyr Leu Asp Ala Leu Ala 2290 2295 2300 Ala Arg Arg Arg Ala Thr Gly Arg Ser Ala Val Ser Val Ala Trp Gly 2305 2310 2315 2320 Pro Trp Asp Gly Gly Gly Met Ala Ala Gly Asp Gly Val Ala Glu Arg 2325 2330 2335 Leu Arg Asn His Gly Val Pro Gly Met Asp Pro Glu Leu Ala Leu Ala 2340 2345 2350 Ala Leu Glu Ser Ala Leu Gly Arg Asp Glu Thr Ala Ile Thr Val Ala 2355 2360 2365 Asp Ile Asp Trp Asp Arg Phe Tyr Leu Ala Tyr Ser Ser Gly Arg Pro 2370 2375 2380 Gln Pro Leu Val Glu Glu Leu Pro Glu Val Arg Arg Ile Ile Asp Ala 2385 2390 2395 2400 Arg Asp Ser Ala Thr Ser Gly Gln Gly Gly Ser Ser Ala Gln Gly Ala 2405 2410 2415 Asn Pro Leu Ala Glu Arg Leu Ala Ala Ala Ala Pro Gly Glu Arg Thr 2420 2425 2430 Glu Ile Leu Leu Gly Leu Val Arg Ala Gln Ala Ala Ala Val Leu Arg 2435 2440 2445 Met Arg Ser Pro Glu Asp Val Ala Ala Asp Arg Ala Phe Lys Asp Ile 2450 2455 2460 Gly Phe Asp Ser Leu Ala Gly Val Glu Leu Arg Asn Arg Leu Thr Arg 2465 2470 2475 2480 Ala Thr Gly Leu Gln Leu Pro Ala Thr Leu Val Phe Asp His Pro Thr 2485 2490 2495 Pro Leu Ala Leu Val Ser Leu Leu Arg Ser Glu Phe Leu Gly Asp Glu 2500 2505 2510 Glu Thr Ala Asp Ala Arg Arg Ser Ala Ala Leu Pro Ala Thr Val Gly 2515 2520 2525 Ala Gly Ala Gly Ala Gly Ala Gly Thr Asp Ala Asp Asp Asp Pro Ile 2530 2535 2540 Ala Ile Val Ala Met Ser Cys Arg Tyr Pro Gly Asp Ile Arg Ser Pro 2545 2550 2555 2560 Glu Asp Leu Trp Arg Met Leu Ser Glu Gly Gly Glu Gly Ile Thr Pro 2565 2570 2575 Phe Pro Thr Asp Arg Gly Trp Asp Leu Asp Gly Leu Tyr Asp Ala Asp 2580 2585 2590 Pro Asp Ala Leu Gly Arg Ala Tyr Val Arg Glu Gly Gly Phe Leu His 2595 2600 2605 Asp Ala Ala Glu Phe Asp Ala Glu Phe Phe Gly Val Ser Pro Arg Glu 2610 2615 2620 Ala Leu Ala Met Asp Pro Gln Gln Arg Met Leu Leu Thr Thr Ser Trp 2625 2630 2635 2640 Glu Ala Phe Glu Arg Ala Gly Ile Glu Pro Ala Ser Leu Arg Gly Ser 2645 2650 2655 Ser Thr Gly Val Phe Ile Gly Leu Ser Tyr Gln Asp Tyr Ala Ala Arg 2660 2665 2670 Val Pro Asn Ala Pro Arg Gly Val Glu Gly Tyr Leu Leu Thr Gly Ser 2675 2680 2685 Thr Pro Ser Val Ala Ser Gly Arg Ile Ala Tyr Thr Phe Gly Leu Glu 2690 2695 2700 Gly Pro Ala Thr Thr Val Asp Thr Ala Cys Ser Ser Ser Leu Thr Ala 2705 2710 2715 2720 Leu His Leu Ala Val Arg Ala Leu Arg Ser Gly Glu Cys Thr Met Ala 2725 2730 2735 Leu Ala Gly Gly Val Ala Met Met Ala Thr Pro His Met Phe Val Glu 2740 2745 2750 Phe Ser Arg Gln Arg Ala Leu Ala Pro Asp Gly Arg Ser Lys Ala Phe 2755 2760 2765 Ser Ala Asp Ala Asp Gly Phe Gly Ala Ala Glu Gly Val Gly Leu Leu 2770 2775 2780 Leu Val Glu Arg Leu Ser Asp Ala Arg Arg Asn Gly His Pro Val Leu 2785 2790 2795 2800 Ala Val Val Arg Gly Thr Ala Val Asn Gln Asp Gly Ala Ser Asn Gly 2805 2810 2815 Leu Thr Ala Pro Asn Gly Pro Ser Gln Gln Arg Val Ile Arg Gln Ala 2820 2825 2830 Leu Ala Asp Ala Arg Leu Ala Pro Gly Asp Ile Asp Ala Val Glu Thr 2835 2840 2845 His Gly Thr Gly Thr Ser Leu Gly Asp Pro Ile Glu Ala Gln Gly Leu 2850 2855 2860 Gln Ala Thr Tyr Gly Lys Glu Arg Pro Ala Glu Arg Pro Leu Ala Ile 2865 2870 2875 2880 Gly Ser Val Lys Ser Asn Ile Gly His Thr Gln Ala Ala Ala Gly Ala 2885 2890 2895 Ala Gly Ile Ile Lys Met Val Leu Ala Met Arg His Gly Thr Leu Pro 2900 2905 2910 Lys Thr Leu His Ala Asp Glu Pro Ser Pro His Val Asp Trp Ala Asn 2915 2920 2925 Ser Gly Leu Ala Leu Val Thr Glu Pro Ile Asp Trp Pro Ala Gly Thr 2930 2935 2940 Gly Pro Arg Arg Ala Ala Val Ser Ser Phe Gly Ile Ser Gly Thr Asn 2945 2950 2955 2960 Ala His Val Val Leu Glu Gln Ala Pro Asp Ala Ala Gly Glu Val Leu 2965 2970 2975 Gly Ala Asp Glu Val Pro Glu Val Ser Glu Thr Val Ala Met Ala Gly 2980 2985 2990 Thr Ala Gly Thr Ser Glu Val Ala Glu Gly Ser Glu Ala Ser Glu Ala 2995 3000 3005 Pro Ala Ala Pro Gly Ser Arg Glu Ala Ser Leu Pro Gly His Leu Pro 3010 3015 3020 Trp Val Leu Ser Ala Lys Asp Glu Gln Ser Leu Arg Gly Gln Ala Ala 3025 3030 3035 3040 Ala Leu His Ala Trp Leu Ser Glu Pro Ala Ala Asp Leu Ser Asp Ala 3045 3050 3055 Asp Gly Pro Ala Arg Leu Arg Asp Val Gly Tyr Thr Leu Ala Thr Ser 3060 3065 3070 Arg Thr Ala Phe Ala His Arg Ala Ala Val Thr Ala Ala Asp Arg Asp 3075 3080 3085 Gly Phe Leu Asp Gly Leu Ala Thr Leu Ala Gln Gly Gly Thr Ser Ala 3090 3095 3100 His Val His Leu Asp Thr Ala Arg Asp Gly Thr Thr Ala Phe Leu Phe 3105 3110 3115 3120 Thr Gly Gln Gly Ser Gln Arg Pro Gly Ala Gly Arg Glu Leu Tyr Asp 3125 3130 3135 Arg His Pro Val Phe Ala Arg Ala Leu Asp Glu Ile Cys Ala His Leu 3140 3145 3150 Asp Gly His Leu Glu Leu Pro Leu Leu Asp Val Met Phe Ala Ala Glu 3155 3160 3165 Gly Ser Ala Glu Ala Ala Leu Leu Asp Glu Thr Arg Tyr Thr Gln Cys 3170 3175 3180 Ala Leu Phe Ala Leu Glu Val Ala Leu Phe Arg Leu Val Glu Ser Trp 3185 3190 3195 3200 Gly Met Arg Pro Ala Ala Leu Leu Gly His Ser Val Gly Glu Ile Ala 3205 3210 3215 Ala Ala His Val Ala Gly Val Phe Ser Leu Ala Asp Ala Ala Arg Leu 3220 3225 3230 Val Ala Ala Arg Gly Arg Leu Met Gln Glu Leu Pro Ala Gly Gly Ala 3235 3240 3245 Met Leu Ala Val Gln Ala Ala Glu Asp Glu Ile Arg Val Trp Leu Glu 3250 3255 3260 Thr Glu Glu Arg Tyr Ala Gly Arg Leu Asp Val Ala Ala Val Asn Gly 3265 3270 3275 3280 Pro Glu Ala Ala Val Leu Ser Gly Asp Ala Asp Ala Ala Arg Glu Ala 3285 3290 3295 Glu Ala Tyr Trp Ser Gly Leu Gly Arg Arg Thr Arg Ala Leu Arg Val 3300 3305 3310 Ser His Ala Phe His Ser Ala His Met Asp Gly Met Leu Asp Gly Phe 3315 3320 3325 Arg Ala Val Leu Glu Thr Val Glu Phe Arg Arg Pro Ser Leu Thr Val 3330 3335 3340 Val Ser Asn Val Thr Gly Leu Ala Ala Gly Pro Asp Asp Leu Cys Asp 3345 3350 3355 3360 Pro Glu Tyr Trp Val Arg His Val Arg Gly Thr Val Arg Phe Leu Asp 3365 3370 3375 Gly Val Arg Val Leu Arg Asp Leu Gly Val Arg Thr Cys Leu Glu Leu 3380 3385 3390 Gly Pro Asp Gly Val Leu Thr Ala Met Ala Ala Asp Gly Leu Ala Asp 3395 3400 3405 Thr Pro Ala Asp Ser Ala Ala Gly Ser Pro Val Gly Ser Pro Ala Gly 3410 3415 3420 Ser Pro Ala Asp Ser Ala Ala Gly Ala Leu Arg Pro Arg Pro Leu Leu 3425 3430 3435 3440 Val Ala Leu Leu Arg Arg Lys Arg Ser Glu Thr Glu Thr Val Ala Asp 3445 3450 3455 Ala Leu Gly Arg Ala His Ala His Gly Thr Gly Pro Asp Trp His Ala 3460 3465 3470 Trp Phe Ala Gly Ser Gly Ala His Arg Val Asp Leu Pro Thr Tyr Ser 3475 3480 3485 Phe Arg Arg Asp Arg Tyr Trp Leu Asp Ala Pro Ala Ala Asp Thr Ala 3490 3495 3500 Val Asp Thr Ala Gly Leu Gly Leu Gly Thr Ala Asp His Pro Leu Leu 3505 3510 3515 3520 Gly Ala Val Val Ser Leu Pro Asp Arg Asp Gly Leu Leu Leu Thr Gly 3525 3530 3535 Arg Leu Ser Leu Arg Thr His Pro Trp Leu Ala Asp His Ala Val Leu 3540 3545 3550 Gly Ser Val Leu Leu Pro Gly Ala Ala Met Val Glu Leu Ala Ala His 3555 3560 3565 Ala Ala Glu Ser Ala Gly Leu Arg Asp Val Arg Glu Leu Thr Leu Leu 3570 3575 3580 Glu Pro Leu Val Leu Pro Glu His Gly Gly Val Glu Leu Arg Val Thr 3585 3590 3595 3600 Val Gly Ala Pro Ala Gly Glu Pro Gly Gly Glu Ser Ala Gly Asp Gly 3605 3610 3615 Ala Arg Pro Val Ser Leu His Ser Arg Leu Ala Asp Ala Pro Ala Gly 3620 3625 3630 Thr Ala Trp Ser Cys His Ala Thr Gly Leu Leu Ala Thr Asp Arg Pro 3635 3640 3645 Glu Leu Pro Val Ala Pro Asp Arg Ala Ala Met Trp Pro Pro Gln Gly 3650 3655 3660 Ala Glu Glu Val Pro Leu Asp Gly Leu Tyr Glu Arg Leu Asp Gly Asn 3665 3670 3675 3680 Gly Leu Ala Phe Gly Pro Leu Phe Gln Gly Leu Asn Ala Val Trp Arg 3685 3690 3695 Tyr Glu Gly Glu Val Phe Ala Asp Ile Ala Leu Pro Ala Thr Thr Asn 3700 3705 3710 Ala Thr Ala Pro Ala Thr Ala Asn Gly Gly Gly Ser Ala Ala Ala Ala 3715 3720 3725 Pro Tyr Gly Ile His Pro Ala Leu Leu Asp Ala Ser Leu His Ala Ile 3730 3735 3740 Ala Val Gly Gly Leu Val Asp Glu Pro Glu Leu Val Arg Val Pro Phe 3745 3750 3755 3760 His Trp Ser Gly Val Thr Val His Ala Ala Gly Ala Ala Ala Ala Arg 3765 3770 3775 Val Arg Leu Ala Ser Ala Gly Thr Asp Ala Val Ser Leu Ser Leu Thr 3780 3785 3790 Asp Gly Glu Gly Arg Pro Leu Val Ser Val Glu Arg Leu Thr Leu Arg 3795 3800 3805 Pro Val Thr Ala Asp Gln Ala Ala Ala Ser Arg Val Gly Gly Leu Met 3810 3815 3820 His Arg Val Ala Trp Arg Pro Tyr Ala Leu Ala Ser Ser Gly Glu Gln 3825 3830 3835 3840 Asp Pro His Ala Thr Ser Tyr Gly Pro Thr Ala Val Leu Gly Lys Asp 3845 3850 3855 Glu Leu Lys Val Ala Ala Ala Leu Glu Ser Ala Gly Val Glu Val Gly 3860 3865 3870 Leu Tyr Pro Asp Leu Ala Ala Leu Ser Gln Asp Val Ala Ala Gly Ala 3875 3880 3885 Pro Ala Pro Arg Thr Val Leu Ala Pro Leu Pro Ala Gly Pro Ala Asp 3890 3895 3900 Gly Gly Ala Glu Gly Val Arg Gly Thr Val Ala Arg Thr Leu Glu Leu 3905 3910 3915 3920 Leu Gln Ala Trp Leu Ala Asp Glu His Leu Ala Gly Thr Arg Leu Leu 3925 3930 3935 Leu Val Thr Arg Gly Ala Val Arg Asp Pro Glu Gly Ser Gly Ala Asp 3940 3945 3950 Asp Gly Gly Glu Asp Leu Ser His Ala Ala Ala Trp Gly Leu Val Arg 3955 3960 3965 Thr Ala Gln Thr Glu Asn Pro Gly Arg Phe Gly Leu Leu Asp Leu Ala 3970 3975 3980 Asp Asp Ala Ser Ser Tyr Arg Thr Leu Pro Ser Val Leu Ser Asp Ala 3985 3990 3995 4000 Gly Leu Arg Asp Glu Pro Gln Leu Ala Leu His Asp Gly Thr Ile Arg 4005 4010 4015 Leu Ala Arg Leu Ala Ser Val Arg Pro Glu Thr Gly Thr Ala Ala Pro 4020 4025 4030 Ala Leu Ala Pro Glu Gly Thr Val Leu Leu Thr Gly Gly Thr Gly Gly 4035 4040 4045 Leu Gly Gly Leu Val Ala Arg His Val Val Gly Glu Trp Gly Val Arg 4050 4055 4060 Arg Leu Leu Leu Val Ser Arg Arg Gly Thr Asp Ala Pro Gly Ala Asp 4065 4070 4075 4080 Glu Leu Val His Glu Leu Glu Ala Leu Gly Ala Asp Val Ser Val Ala 4085 4090 4095 Ala Cys Asp Val Ala Asp Arg Glu Ala Leu Thr Ala Val Leu Asp Ala 4100 4105 4110 Ile Pro Ala Glu His Pro Leu Thr Ala Val Val His Thr Ala Gly Val 4115 4120 4125 Leu Ser Asp Gly Thr Leu Pro Ser Met Thr Thr Glu Asp Val Glu His 4130 4135 4140 Val Leu Arg Pro Lys Val Asp Ala Ala Phe Leu Leu Asp Glu Leu Thr 4145 4150 4155 4160 Ser Thr Pro Ala Tyr Asp Leu Ala Ala Phe Val Met Phe Ser Ser Ala 4165 4170 4175 Ala Ala Val Phe Gly Gly Ala Gly Gln Gly Ala Tyr Ala Ala Ala Asn 4180 4185 4190 Ala Thr Leu Asp Ala Leu Ala Trp Arg Arg Arg Ala Ala Gly Leu Pro 4195 4200 4205 Ala Leu Ser Leu Gly Trp Gly Leu Trp Ala Glu Thr Ser Gly Met Thr 4210 4215 4220 Gly Glu Leu Gly Gln Ala Asp Leu Arg Arg Met Ser Arg Ala Gly Ile 4225 4230 4235 4240 Gly Gly Ile Ser Asp Ala Glu Gly Ile Ala Leu Leu Asp Ala Ala Leu 4245 4250 4255 Arg Asp Asp Arg His Pro Val Leu Leu Pro Leu Arg Leu Asp Ala Ala 4260 4265 4270 Gly Leu Arg Asp Ala Ala Gly Asn Asp Pro Ala Gly Ile Pro Ala Leu 4275 4280 4285 Phe Arg Asp Val Val Gly Ala Arg Thr Val Arg Ala Arg Pro Ser Ala 4290 4295 4300 Ala Ser Ala Ser Thr Thr Ala Gly Thr Ala Gly Thr Pro Gly Thr Ala 4305 4310 4315 4320 Asp Gly Ala Ala Glu Thr Ala Ala Val Thr Leu Ala Asp Arg Ala Ala 4325 4330 4335 Thr Val Asp Gly Pro Ala Arg Gln Arg Leu Leu Leu Glu Phe Val Val 4340 4345 4350 Gly Glu Val Ala Glu Val Leu Gly His Ala Arg Gly His Arg Ile Asp 4355 4360 4365 Ala Glu Arg Gly Phe Leu Asp Leu Gly Phe Asp Ser Leu Thr Ala Val 4370 4375 4380 Glu Leu Arg Asn Arg Leu Asn Ser Ala Gly Gly Leu Ala Leu Pro Ala 4385 4390 4395 4400 Thr Leu Val Phe Asp His Pro Ser Pro Ala Ala Leu Ala Ser His Leu 4405 4410 4415 Asp Ala Glu Leu Pro Arg Gly Ala Ser Asp Gln Asp Gly Ala Gly Asn 4420 4425 4430 Arg Asn Gly Asn Glu Asn Gly Thr Thr Ala Ser Arg Ser Thr Ala Glu 4435 4440 4445 Thr Asp Ala Leu Leu Ala Gln Leu Thr Arg Leu Glu Gly Ala Leu Val 4450 4455 4460 Leu Thr Gly Leu Ser Asp Ala Pro Gly Ser Glu Glu Val Leu Glu His 4465 4470 4475 4480 Leu Arg Ser Leu Arg Ser Met Val Thr Gly Glu Thr Gly Thr Gly Thr 4485 4490 4495 Ala Ser Gly Ala Pro Asp Gly Ala Gly Ser Gly Ala Glu Asp Arg Pro 4500 4505 4510 Trp Ala Ala Gly Asp Gly Ala Gly Gly Gly Ser Glu Asp Gly Ala Gly 4515 4520 4525 Val Pro Asp Phe Met Asn Ala Ser Ala Glu Glu Leu Phe Gly Leu Leu 4530 4535 4540 Asp Gln Asp Pro Ser Thr Asp 4545 4550 2 3739 PRT Streptomyces venezuelae 2 Val Ser Thr Val Asn Glu Glu Lys Tyr Leu Asp Tyr Leu Arg Arg Ala 1 5 10 15 Thr Ala Asp Leu His Glu Ala Arg Gly Arg Leu Arg Glu Leu Glu Ala 20 25 30 Lys Ala Gly Glu Pro Val Ala Ile Val Gly Met Ala Cys Arg Leu Pro 35 40 45 Gly Gly Val Ala Ser Pro Glu Asp Leu Trp Arg Leu Val Ala Gly Gly 50 55 60 Glu Asp Ala Ile Ser Glu Phe Pro Gln Asp Arg Gly Trp Asp Val Glu 65 70 75 80 Gly Leu Tyr Asp Pro Asn Pro Glu Ala Thr Gly Lys Ser Tyr Ala Arg 85 90 95 Glu Ala Gly Phe Leu Tyr Glu Ala Gly Glu Phe Asp Ala Asp Phe Phe 100 105 110 Gly Ile Ser Pro Arg Glu Ala Leu Ala Met Asp Pro Gln Gln Arg Leu 115 120 125 Leu Leu Glu Ala Ser Trp Glu Ala Phe Glu His Ala Gly Ile Pro Ala 130 135 140 Ala Thr Ala Arg Gly Thr Ser Val Gly Val Phe Thr Gly Val Met Tyr 145 150 155 160 His Asp Tyr Ala Thr Arg Leu Thr Asp Val Pro Glu Gly Ile Glu Gly 165 170 175 Tyr Leu Gly Thr Gly Asn Ser Gly Ser Val Ala Ser Gly Arg Val Ala 180 185 190 Tyr Thr Leu Gly Leu Glu Gly Pro Ala Val Thr Val Asp Thr Ala Cys 195 200 205 Ser Ser Ser Leu Val Ala Leu His Leu Ala Val Gln Ala Leu Arg Lys 210 215 220 Gly Glu Val Asp Met Ala Leu Ala Gly Gly Val Thr Val Met Ser Thr 225 230 235 240 Pro Ser Thr Phe Val Glu Phe Ser Arg Gln Arg Gly Leu Ala Pro Asp 245 250 255 Gly Arg Ser Lys Ser Phe Ser Ser Thr Ala Asp Gly Thr Ser Trp Ser 260 265 270 Glu Gly Val Gly Val Leu Leu Val Glu Arg Leu Ser Asp Ala Arg Arg 275 280 285 Lys Gly His Arg Ile Leu Ala Val Val Arg Gly Thr Ala Val Asn Gln 290 295 300 Asp Gly Ala Ser Ser Gly Leu Thr Ala Pro Asn Gly Pro Ser Gln Gln 305 310 315 320 Arg Val Ile Arg Arg Ala Leu Ala Asp Ala Arg Leu Thr Thr Ser Asp 325 330 335 Val Asp Val Val Glu Ala His Gly Thr Gly Thr Arg Leu Gly Asp Pro 340 345 350 Ile Glu Ala Gln Ala Val Ile Ala Thr Tyr Gly Gln Gly Arg Asp Gly 355 360 365 Glu Gln Pro Leu Arg Leu Gly Ser Leu Lys Ser Asn Ile Gly His Thr 370 375 380 Gln Ala Ala Ala Gly Val Ser Gly Val Ile Lys Met Val Gln Ala Met 385 390 395 400 Arg His Gly Val Leu Pro Lys Thr Leu His Val Glu Lys Pro Thr Asp 405 410 415 Gln Val Asp Trp Ser Ala Gly Ala Val Glu Leu Leu Thr Glu Ala Met 420 425 430 Asp Trp Pro Asp Lys Gly Asp Gly Gly Leu Arg Arg Ala Ala Val Ser 435 440 445 Ser Phe Gly Val Ser Gly Thr Asn Ala His Val Val Leu Glu Glu Ala 450 455 460 Pro Ala Ala Glu Glu Thr Pro Ala Ser Glu Ala Thr Pro Ala Val Glu 465 470 475 480 Pro Ser Val Gly Ala Gly Leu Val Pro Trp Leu Val Ser Ala Lys Thr 485 490 495 Pro Ala Ala Leu Asp Ala Gln Ile Gly Arg Leu Ala Ala Phe Ala Ser 500 505 510 Gln Gly Arg Thr Asp Ala Ala Asp Pro Gly Ala Val Ala Arg Val Leu 515 520 525 Ala Gly Gly Arg Ala Glu Phe Glu His Arg Ala Val Val Leu Gly Thr 530 535 540 Gly Gln Asp Asp Phe Ala Gln Ala Leu Thr Ala Pro Glu Gly Leu Ile 545 550 555 560 Arg Gly Thr Pro Ser Asp Val Gly Arg Val Ala Phe Val Phe Pro Gly 565 570 575 Gln Gly Thr Gln Trp Ala Gly Met Gly Ala Glu Leu Leu Asp Val Ser 580 585 590 Lys Glu Phe Ala Ala Ala Met Ala Glu Cys Glu Ser Ala Leu Ser Arg 595 600 605 Tyr Val Asp Trp Ser Leu Glu Ala Val Val Arg Gln Ala Pro Gly Ala 610 615 620 Pro Thr Leu Glu Arg Val Asp Val Val Gln Pro Val Thr Phe Ala Val 625 630 635 640 Met Val Ser Leu Ala Lys Val Trp Gln His His Gly Val Thr Pro Gln 645 650 655 Ala Val Val Gly His Ser Gln Gly Glu Ile Ala Ala Ala Tyr Val Ala 660 665 670 Gly Ala Leu Thr Leu Asp Asp Ala Ala Arg Val Val Thr Leu Arg Ser 675 680 685 Lys Ser Ile Ala Ala His Leu Ala Gly Lys Gly Gly Met Ile Ser Leu 690 695 700 Ala Leu Ser Glu Glu Ala Thr Arg Gln Arg Ile Glu Asn Leu His Gly 705 710 715 720 Leu Ser Ile Ala Ala Val Asn Gly Pro Thr Ala Thr Val Val Ser Gly 725 730 735 Asp Pro Thr Gln Ile Gln Glu Leu Ala Gln Ala Cys Glu Ala Asp Gly 740 745 750 Val Arg Ala Arg Ile Ile Pro Val Asp Tyr Ala Ser His Ser Ala His 755 760 765 Val Glu Thr Ile Glu Ser Glu Leu Ala Glu Val Leu Ala Gly Leu Ser 770 775 780 Pro Arg Thr Pro Glu Val Pro Phe Phe Ser Thr Leu Glu Gly Ala Trp 785 790 795 800 Ile Thr Glu Pro Val Leu Asp Gly Thr Tyr Trp Tyr Arg Asn Leu Arg 805 810 815 His Arg Val Gly Phe Ala Pro Ala Val Glu Thr Leu Ala Thr Asp Glu 820 825 830 Gly Phe Thr His Phe Ile Glu Val Ser Ala His Pro Val Leu Thr Met 835 840 845 Thr Leu Pro Glu Thr Val Thr Gly Leu Gly Thr Leu Arg Arg Glu Gln 850 855 860 Gly Gly Gln Glu Arg Leu Val Thr Ser Leu Ala Glu Ala Trp Thr Asn 865 870 875 880 Gly Leu Thr Ile Asp Trp Ala Pro Val Leu Pro Thr Ala Thr Gly His 885 890 895 His Pro Glu Leu Pro Thr Tyr Ala Phe Gln Arg Arg His Tyr Trp Leu 900 905 910 His Asp Ser Pro Ala Val Gln Gly Ser Val Gln Asp Ser Trp Arg Tyr 915 920 925 Arg Ile Asp Trp Lys Arg Leu Ala Val Ala Asp Ala Ser Glu Arg Ala 930 935 940 Gly Leu Ser Gly Arg Trp Leu Val Val Val Pro Glu Asp Arg Ser Ala 945 950 955 960 Glu Ala Ala Pro Val Leu Ala Ala Leu Ser Gly Ala Gly Ala Asp Pro 965 970 975 Val Gln Leu Asp Val Ser Pro Leu Gly Asp Arg Gln Arg Leu Ala Ala 980 985 990 Thr Leu Gly Glu Ala Leu Ala Ala Ala Gly Gly Ala Val Asp Gly Val 995 1000 1005 Leu Ser Leu Leu Ala Trp Asp Glu Ser Ala His Pro Gly His Pro Ala 1010 1015 1020 Pro Phe Thr Arg Gly Thr Gly Ala Thr Leu Thr Leu Val Gln Ala Leu 1025 1030 1035 1040 Glu Asp Ala Gly Val Ala Ala Pro Leu Trp Cys Val Thr His Gly Ala 1045 1050 1055 Val Ser Val Gly Arg Ala Asp His Val Thr Ser Pro Ala Gln Ala Met 1060 1065 1070 Val Trp Gly Met Gly Arg Val Ala Ala Leu Glu His Pro Glu Arg Trp 1075 1080 1085 Gly Gly Leu Ile Asp Leu Pro Ser Asp Ala Asp Arg Ala Ala Leu Asp 1090 1095 1100 Arg Met Thr Thr Val Leu Ala Gly Gly Thr Gly Glu Asp Gln Val Ala 1105 1110 1115 1120 Val Arg Ala Ser Gly Leu Leu Ala Arg Arg Leu Val Arg Ala Ser Leu 1125 1130 1135 Pro Ala His Gly Thr Ala Ser Pro Trp Trp Gln Ala Asp Gly Thr Val 1140 1145 1150 Leu Val Thr Gly Ala Glu Glu Pro Ala Ala Ala Glu Ala Ala Arg Arg 1155 1160 1165 Leu Ala Arg Asp Gly Ala Gly His Leu Leu Leu His Thr Thr Pro Ser 1170 1175 1180 Gly Ser Glu Gly Ala Glu Gly Thr Ser Gly Ala Ala Glu Asp Ser Gly 1185 1190 1195 1200 Leu Ala Gly Leu Val Ala Glu Leu Ala Asp Leu Gly Ala Thr Ala Thr 1205 1210 1215 Val Val Thr Cys Asp Leu Thr Asp Ala Glu Ala Ala Ala Arg Leu Leu 1220 1225 1230 Ala Gly Val Ser Asp Ala His Pro Leu Ser Ala Val Leu His Leu Pro 1235 1240 1245 Pro Thr Val Asp Ser Glu Pro Leu Ala Ala Thr Asp Ala Asp Ala Leu 1250 1255 1260 Ala Arg Val Val Thr Ala Lys Ala Thr Ala Ala Leu His Leu Asp Arg 1265 1270 1275 1280 Leu Leu Arg Glu Ala Ala Ala Ala Gly Gly Arg Pro Pro Val Leu Val 1285 1290 1295 Leu Phe Ser Ser Val Ala Ala Ile Trp Gly Gly Ala Gly Gln Gly Ala 1300 1305 1310 Tyr Ala Ala Gly Thr Ala Phe Leu Asp Ala Leu Ala Gly Gln His Arg 1315 1320 1325 Ala Asp Gly Pro Thr Val Thr Ser Val Ala Trp Ser Pro Trp Glu Gly 1330 1335 1340 Ser Arg Val Thr Glu Gly Ala Thr Gly Glu Arg Leu Arg Arg Leu Gly 1345 1350 1355 1360 Leu Arg Pro Leu Ala Pro Ala Thr Ala Leu Thr Ala Leu Asp Thr Ala 1365 1370 1375 Leu Gly His Gly Asp Thr Ala Val Thr Ile Ala Asp Val Asp Trp Ser 1380 1385 1390 Ser Phe Ala Pro Gly Phe Thr Thr Ala Arg Pro Gly Thr Leu Leu Ala 1395 1400 1405 Asp Leu Pro Glu Ala Arg Arg Ala Leu Asp Glu Gln Gln Ser Thr Thr 1410 1415 1420 Ala Ala Asp Asp Thr Val Leu Ser Arg Glu Leu Gly Ala Leu Thr Gly 1425 1430 1435 1440 Ala Glu Gln Gln Arg Arg Met Gln Glu Leu Val Arg Glu His Leu Ala 1445 1450 1455 Val Val Leu Asn His Pro Ser Pro Glu Ala Val Asp Thr Gly Arg Ala 1460 1465 1470 Phe Arg Asp Leu Gly Phe Asp Ser Leu Thr Ala Val Glu Leu Arg Asn 1475 1480 1485 Arg Leu Lys Asn Ala Thr Gly Leu Ala Leu Pro Ala Thr Leu Val Phe 1490 1495 1500 Asp Tyr Pro Thr Pro Arg Thr Leu Ala Glu Phe Leu Leu Ala Glu Ile 1505 1510 1515 1520 Leu Gly Glu Gln Ala Gly Ala Gly Glu Gln Leu Pro Val Asp Gly Gly 1525 1530 1535 Val Asp Asp Glu Pro Val Ala Ile Val Gly Met Ala Cys Arg Leu Pro 1540 1545 1550 Gly Gly Val Ala Ser Pro Glu Asp Leu Trp Arg Leu Val Ala Gly Gly 1555 1560 1565 Glu Asp Ala Ile Ser Gly Phe Pro Gln Asp Arg Gly Trp Asp Val Glu 1570 1575 1580 Gly Leu Tyr Asp Pro Asp Pro Asp Ala Ser Gly Arg Thr Tyr Cys Arg 1585 1590 1595 1600 Ala Gly Gly Phe Leu Asp Glu Ala Gly Glu Phe Asp Ala Asp Phe Phe 1605 1610 1615 Gly Ile Ser Pro Arg Glu Ala Leu Ala Met Asp Pro Gln Gln Arg Leu 1620 1625 1630 Leu Leu Glu Thr Ser Trp Glu Ala Val Glu Asp Ala Gly Ile Asp Pro 1635 1640 1645 Thr Ser Leu Gln Gly Gln Gln Val Gly Val Phe Ala Gly Thr Asn Gly 1650 1655 1660 Pro His Tyr Glu Pro Leu Leu Arg Asn Thr Ala Glu Asp Leu Glu Gly 1665 1670 1675 1680 Tyr Val Gly Thr Gly Asn Ala Ala Ser Ile Met Ser Gly Arg Val Ser 1685 1690 1695 Tyr Thr Leu Gly Leu Glu Gly Pro Ala Val Thr Val Asp Thr Ala Cys 1700 1705 1710 Ser Ser Ser Leu Val Ala Leu His Leu Ala Val Gln Ala Leu Arg Lys 1715 1720 1725 Gly Glu Cys Gly Leu Ala Leu Ala Gly Gly Val Thr Val Met Ser Thr 1730 1735 1740 Pro Thr Thr Phe Val Glu Phe Ser Arg Gln Arg Gly Leu Ala Glu Asp 1745 1750 1755 1760 Gly Arg Ser Lys Ala Phe Ala Ala Ser Ala Asp Gly Phe Gly Pro Ala 1765 1770 1775 Glu Gly Val Gly Met Leu Leu Val Glu Arg Leu Ser Asp Ala Arg Arg 1780 1785 1790 Asn Gly His Arg Val Leu Ala Val Val Arg Gly Ser Ala Val Asn Gln 1795 1800 1805 Asp Gly Ala Ser Asn Gly Leu Thr Ala Pro Asn Gly Pro Ser Gln Gln 1810 1815 1820 Arg Val Ile Arg Arg Ala Leu Ala Asp Ala Arg Leu Thr Thr Ala Asp 1825 1830 1835 1840 Val Asp Val Val Glu Ala His Gly Thr Gly Thr Arg Leu Gly Asp Pro 1845 1850 1855 Ile Glu Ala Gln Ala Leu Ile Ala Thr Tyr Gly Gln Gly Arg Asp Thr 1860 1865 1870 Glu Gln Pro Leu Arg Leu Gly Ser Leu Lys Ser Asn Ile Gly His Thr 1875 1880 1885 Gln Ala Ala Ala Gly Val Ser Gly Ile Ile Lys Met Val Gln Ala Met 1890 1895 1900 Arg His Gly Val Leu Pro Lys Thr Leu His Val Asp Arg Pro Ser Asp 1905 1910 1915 1920 Gln Ile Asp Trp Ser Ala Gly Thr Val Glu Leu Leu Thr Glu Ala Met 1925 1930 1935 Asp Trp Pro Arg Lys Gln Glu Gly Gly Leu Arg Arg Ala Ala Val Ser 1940 1945 1950 Ser Phe Gly Ile Ser Gly Thr Asn Ala His Ile Val Leu Glu Glu Ala 1955 1960 1965 Pro Val Asp Glu Asp Ala Pro Ala Asp Glu Pro Ser Val Gly Gly Val 1970 1975 1980 Val Pro Trp Leu Val Ser Ala Lys Thr Pro Ala Ala Leu Asp Ala Gln 1985 1990 1995 2000 Ile Gly Arg Leu Ala Ala Phe Ala Ser Gln Gly Arg Thr Asp Ala Ala 2005 2010 2015 Asp Pro Gly Ala Val Ala Arg Val Leu Ala Gly Gly Arg Ala Gln Phe 2020 2025 2030 Glu His Arg Ala Val Ala Leu Gly Thr Gly Gln Asp Asp Leu Ala Ala 2035 2040 2045 Ala Leu Ala Ala Pro Glu Gly Leu Val Arg Gly Val Ala Ser Gly Val 2050 2055 2060 Gly Arg Val Ala Phe Val Phe Pro Gly Gln Gly Thr Gln Trp Ala Gly 2065 2070 2075 2080 Met Gly Ala Glu Leu Leu Asp Val Ser Lys Glu Phe Ala Ala Ala Met 2085 2090 2095 Ala Glu Cys Glu Ala Ala Leu Ala Pro Tyr Val Asp Trp Ser Leu Glu 2100 2105 2110 Ala Val Val Arg Gln Ala Pro Gly Ala Pro Thr Leu Glu Arg Val Asp 2115 2120 2125 Val Val Gln Pro Val Thr Phe Ala Val Met Val Ser Leu Ala Lys Val 2130 2135 2140 Trp Gln His His Gly Val Thr Pro Gln Ala Val Val Gly His Ser Gln 2145 2150 2155 2160 Gly Glu Ile Ala Ala Ala Tyr Val Ala Gly Ala Leu Ser Leu Asp Asp 2165 2170 2175 Ala Ala Arg Val Val Thr Leu Arg Ser Lys Ser Ile Gly Ala His Leu 2180 2185 2190 Ala Gly Gln Gly Gly Met Leu Ser Leu Ala Leu Ser Glu Ala Ala Val 2195 2200 2205 Val Glu Arg Leu Ala Gly Phe Asp Gly Leu Ser Val Ala Ala Val Asn 2210 2215 2220 Gly Pro Thr Ala Thr Val Val Ser Gly Asp Pro Thr Gln Ile Gln Glu 2225 2230 2235 2240 Leu Ala Gln Ala Cys Glu Ala Asp Gly Val Arg Ala Arg Ile Ile Pro 2245 2250 2255 Val Asp Tyr Ala Ser His Ser Ala His Val Glu Thr Ile Glu Ser Glu 2260 2265 2270 Leu Ala Asp Val Leu Ala Gly Leu Ser Pro Gln Thr Pro Gln Val Pro 2275 2280 2285 Phe Phe Ser Thr Leu Glu Gly Ala Trp Ile Thr Glu Pro Ala Leu Asp 2290 2295 2300 Gly Gly Tyr Trp Tyr Arg Asn Leu Arg His Arg Val Gly Phe Ala Pro 2305 2310 2315 2320 Ala Val Glu Thr Leu Ala Thr Asp Glu Gly Phe Thr His Phe Val Glu 2325 2330 2335 Val Ser Ala His Pro Val Leu Thr Met Ala Leu Pro Glu Thr Val Thr 2340 2345 2350 Gly Leu Gly Thr Leu Arg Arg Asp Asn Gly Gly Gln His Arg Leu Thr 2355 2360 2365 Thr Ser Leu Ala Glu Ala Trp Ala Asn Gly Leu Thr Val Asp Trp Ala 2370 2375 2380 Ser Leu Leu Pro Thr Thr Thr Thr His Pro Asp Leu Pro Thr Tyr Ala 2385 2390 2395 2400 Phe Gln Thr Glu Arg Tyr Trp Pro Gln Pro Asp Leu Ser Ala Ala Gly 2405 2410 2415 Asp Ile Thr Ser Ala Gly Leu Gly Ala Ala Glu His Pro Leu Leu Gly 2420 2425 2430 Ala Ala Val Ala Leu Ala Asp Ser Asp Gly Cys Leu Leu Thr Gly Ser 2435 2440 2445 Leu Ser Leu Arg Thr His Pro Trp Leu Ala Asp His Ala Val Ala Gly 2450 2455 2460 Thr Val Leu Leu Pro Gly Thr Ala Phe Val Glu Leu Ala Phe Arg Ala 2465 2470 2475 2480 Gly Asp Gln Val Gly Cys Asp Leu Val Glu Glu Leu Thr Leu Asp Ala 2485 2490 2495 Pro Leu Val Leu Pro Arg Arg Gly Ala Val Arg Val Gln Leu Ser Val 2500 2505 2510 Gly Ala Ser Asp Glu Ser Gly Arg Arg Thr Phe Gly Leu Tyr Ala His 2515 2520 2525 Pro Glu Asp Ala Pro Gly Glu Ala Glu Trp Thr Arg His Ala Thr Gly 2530 2535 2540 Val Leu Ala Ala Arg Ala Asp Arg Thr Ala Pro Val Ala Asp Pro Glu 2545 2550 2555 2560 Ala Trp Pro Pro Pro Gly Ala Glu Pro Val Asp Val Asp Gly Leu Tyr 2565 2570 2575 Glu Arg Phe Ala Ala Asn Gly Tyr Gly Tyr Gly Pro Leu Phe Gln Gly 2580 2585 2590 Val Arg Gly Val Trp Arg Arg Gly Asp Glu Val Phe Ala Asp Val Ala 2595 2600 2605 Leu Pro Ala Glu Val Ala Gly Ala Glu Gly Ala Arg Phe Gly Leu His 2610 2615 2620 Pro Ala Leu Leu Asp Ala Ala Val Gln Ala Ala Gly Ala Gly Gly Ala 2625 2630 2635 2640 Phe Gly Ala Gly Thr Arg Leu Pro Phe Ala Trp Ser Gly Ile Ser Leu 2645 2650 2655 Tyr Ala Val Gly Ala Thr Ala Leu Arg Val Arg Leu Ala Pro Ala Gly 2660 2665 2670 Pro Asp Thr Val Ser Val Ser Ala Ala Asp Ser Ser Gly Gln Pro Val 2675 2680 2685 Phe Ala Ala Asp Ser Leu Thr Val Leu Pro Val Asp Pro Ala Gln Leu 2690 2695 2700 Ala Ala Phe Ser Asp Pro Thr Leu Asp Ala Leu His Leu Leu Glu Trp 2705 2710 2715 2720 Thr Ala Trp Asp Gly Ala Ala Gln Ala Leu Pro Gly Ala Val Val Leu 2725 2730 2735 Gly Gly Asp Ala Asp Gly Leu Ala Ala Ala Leu Arg Ala Gly Gly Thr 2740 2745 2750 Glu Val Leu Ser Phe Pro Asp Leu Thr Asp Leu Val Glu Ala Val Asp 2755 2760 2765 Arg Gly Glu Thr Pro Ala Pro Ala Thr Val Leu Val Ala Cys Pro Ala 2770 2775 2780 Ala Gly Pro Gly Gly Pro Glu His Val Arg Glu Ala Leu His Gly Ser 2785 2790 2795 2800 Leu Ala Leu Met Gln Ala Trp Leu Ala Asp Glu Arg Phe Thr Asp Gly 2805 2810 2815 Arg Leu Val Leu Val Thr Arg Asp Ala Val Ala Ala Arg Ser Gly Asp 2820 2825 2830 Gly Leu Arg Ser Thr Gly Gln Ala Ala Val Trp Gly Leu Gly Arg Ser 2835 2840 2845 Ala Gln Thr Glu Ser Pro Gly Arg Phe Val Leu Leu Asp Leu Ala Gly 2850 2855 2860 Glu Ala Arg Thr Ala Gly Asp Ala Thr Ala Gly Asp Gly Leu Thr Thr 2865 2870 2875 2880 Gly Asp Ala Thr Val Gly Gly Thr Ser Gly Asp Ala Ala Leu Gly Ser 2885 2890 2895 Ala Leu Ala Thr Ala Leu Gly Ser Gly Glu Pro Gln Leu Ala Leu Arg 2900 2905 2910 Asp Gly Ala Leu Leu Val Pro Arg Leu Ala Arg Ala Ala Ala Pro Ala 2915 2920 2925 Ala Ala Asp Gly Leu Ala Ala Ala Asp Gly Leu Ala Ala Leu Pro Leu 2930 2935 2940 Pro Ala Ala Pro Ala Leu Trp Arg Leu Glu Pro Gly Thr Asp Gly Ser 2945 2950 2955 2960 Leu Glu Ser Leu Thr Ala Ala Pro Gly Asp Ala Glu Thr Leu Ala Pro 2965 2970 2975 Glu Pro Leu Gly Pro Gly Gln Val Arg Ile Ala Ile Arg Ala Thr Gly 2980 2985 2990 Leu Asn Phe Arg Asp Val Leu Ile Ala Leu Gly Met Tyr Pro Asp Pro 2995 3000 3005 Ala Leu Met Gly Thr Glu Gly Ala Gly Val Val Thr Ala Thr Gly Pro 3010 3015 3020 Gly Val Thr His Leu Ala Pro Gly Asp Arg Val Met Gly Leu Leu Ser 3025 3030 3035 3040 Gly Ala Tyr Ala Pro Val Val Val Ala Asp Ala Arg Thr Val Ala Arg 3045 3050 3055 Met Pro Glu Gly Trp Thr Phe Ala Gln Gly Ala Ser Val Pro Val Val 3060 3065 3070 Phe Leu Thr Ala Val Tyr Ala Leu Arg Asp Leu Ala Asp Val Lys Pro 3075 3080 3085 Gly Glu Arg Leu Leu Val His Ser Ala Ala Gly Gly Val Gly Met Ala 3090 3095 3100 Ala Val Gln Leu Ala Arg His Trp Gly Val Glu Val His Gly Thr Ala 3105 3110 3115 3120 Ser His Gly Lys Trp Asp Ala Leu Arg Ala Leu Gly Leu Asp Asp Ala 3125 3130 3135 His Ile Ala Ser Ser Arg Thr Leu Asp Phe Glu Ser Ala Phe Arg Ala 3140 3145 3150 Ala Ser Gly Gly Ala Gly Met Asp Val Val Leu Asn Ser Leu Ala Arg 3155 3160 3165 Glu Phe Val Asp Ala Ser Leu Arg Leu Leu Gly Pro Gly Gly Arg Phe 3170 3175 3180 Val Glu Met Gly Lys Thr Asp Val Arg Asp Ala Glu Arg Val Ala Ala 3185 3190 3195 3200 Asp His Pro Gly Val Gly Tyr Arg Ala Phe Asp Leu Gly Glu Ala Gly 3205 3210 3215 Pro Glu Arg Ile Gly Glu Met Leu Ala Glu Val Ile Ala Leu Phe Glu 3220 3225 3230 Asp Gly Val Leu Arg His Leu Pro Val Thr Thr Trp Asp Val Arg Arg 3235 3240 3245 Ala Arg Asp Ala Phe Arg His Val Ser Gln Ala Arg His Thr Gly Lys 3250 3255 3260 Val Val Leu Thr Met Pro Ser Gly Leu Asp Pro Glu Gly Thr Val Leu 3265 3270 3275 3280 Leu Thr Gly Gly Thr Gly Ala Leu Gly Gly Ile Val Ala Arg His Val 3285 3290 3295 Val Gly Glu Trp Gly Val Arg Arg Leu Leu Leu Val Ser Arg Arg Gly 3300 3305 3310 Thr Asp Ala Pro Gly Ala Gly Glu Leu Val His Glu Leu Glu Ala Leu 3315 3320 3325 Gly Ala Asp Val Ser Val Ala Ala Cys Asp Val Ala Asp Arg Glu Ala 3330 3335 3340 Leu Thr Ala Val Leu Asp Ser Ile Pro Ala Glu His Pro Leu Thr Ala 3345 3350 3355 3360 Val Val His Thr Ala Gly Val Leu Ser Asp Gly Thr Leu Pro Ser Met 3365 3370 3375 Thr Ala Glu Asp Val Glu His Val Leu Arg Pro Lys Val Asp Ala Ala 3380 3385 3390 Phe Leu Leu Asp Glu Leu Thr Ser Thr Pro Gly Tyr Asp Leu Ala Ala 3395 3400 3405 Phe Val Met Phe Ser Ser Ala Ala Ala Val Phe Gly Gly Ala Gly Gln 3410 3415 3420 Gly Ala Tyr Ala Ala Ala Asn Ala Thr Leu Asp Ala Leu Ala Trp Arg 3425 3430 3435 3440 Arg Arg Thr Ala Gly Leu Pro Ala Leu Ser Leu Gly Trp Gly Leu Trp 3445 3450 3455 Ala Glu Thr Ser Gly Met Thr Gly Gly Leu Ser Asp Thr Asp Arg Ser 3460 3465 3470 Arg Leu Ala Arg Ser Gly Ala Thr Pro Met Asp Ser Glu Leu Thr Leu 3475 3480 3485 Ser Leu Leu Asp Ala Ala Met Arg Arg Asp Asp Pro Ala Leu Val Pro 3490 3495 3500 Ile Ala Leu Asp Val Ala Ala Leu Arg Ala Gln Gln Arg Asp Gly Met 3505 3510 3515 3520 Leu Ala Pro Leu Leu Ser Gly Leu Thr Arg Gly Ser Arg Val Gly Gly 3525 3530 3535 Ala Pro Val Asn Gln Arg Arg Ala Ala Ala Gly Gly Ala Gly Glu Ala 3540 3545 3550 Asp Thr Asp Leu Gly Gly Arg Leu Ala Ala Met Thr Pro Asp Asp Arg 3555 3560 3565 Val Ala His Leu Arg Asp Leu Val Arg Thr His Val Ala Thr Val Leu 3570 3575 3580 Gly His Gly Thr Pro Ser Arg Val Asp Leu Glu Arg Ala Phe Arg Asp 3585 3590 3595 3600 Thr Gly Phe Asp Ser Leu Thr Ala Val Glu Leu Arg Asn Arg Leu Asn 3605 3610 3615 Ala Ala Thr Gly Leu Arg Leu Pro Ala Thr Leu Val Phe Asp His Pro 3620 3625 3630 Thr Pro Gly Glu Leu Ala Gly His Leu Leu Asp Glu Leu Ala Thr Ala 3635 3640 3645 Ala Gly Gly Ser Trp Ala Glu Gly Thr Gly Ser Gly Asp Thr Ala Ser 3650 3655 3660 Ala Thr Asp Arg Gln Thr Thr Ala Ala Leu Ala Glu Leu Asp Arg Leu 3665 3670 3675 3680 Glu Gly Val Leu Ala Ser Leu Ala Pro Ala Ala Gly Gly Arg Pro Glu 3685 3690 3695 Leu Ala Ala Arg Leu Arg Ala Leu Ala Ala Ala Leu Gly Asp Asp Gly 3700 3705 3710 Asp Asp Ala Thr Asp Leu Asp Glu Ala Ser Asp Asp Asp Leu Phe Ser 3715 3720 3725 Phe Ile Asp Lys Glu Leu Gly Asp Ser Asp Phe 3730 3735 3 1562 PRT Streptomyces venezuelae 3 Met Ala Asn Asn Glu Asp Lys Leu Arg Asp Tyr Leu Lys Arg Val Thr 1 5 10 15 Ala Glu Leu Gln Gln Asn Thr Arg Arg Leu Arg Glu Ile Glu Gly Arg 20 25 30 Thr His Glu Pro Val Ala Ile Val Gly Met Ala Cys Arg Leu Pro Gly 35 40 45 Gly Val Ala Ser Pro Glu Asp Leu Trp Gln Leu Val Ala Gly Asp Gly 50 55 60 Asp Ala Ile Ser Glu Phe Pro Gln Asp Arg Gly Trp Asp Val Glu Gly 65 70 75 80 Leu Tyr Asp Pro Asp Pro Asp Ala Ser Gly Arg Thr Tyr Cys Arg Ser 85 90 95 Gly Gly Phe Leu His Asp Ala Gly Glu Phe Asp Ala Asp Phe Phe Gly 100 105 110 Ile Ser Pro Arg Glu Ala Leu Ala Met Asp Pro Gln Gln Arg Leu Ser 115 120 125 Leu Thr Thr Ala Trp Glu Ala Ile Glu Ser Ala Gly Ile Asp Pro Thr 130 135 140 Ala Leu Lys Gly Ser Gly Leu Gly Val Phe Val Gly Gly Trp His Thr 145 150 155 160 Gly Tyr Thr Ser Gly Gln Thr Thr Ala Val Gln Ser Pro Glu Leu Glu 165 170 175 Gly His Leu Val Ser Gly Ala Ala Leu Gly Phe Leu Ser Gly Arg Ile 180 185 190 Ala Tyr Val Leu Gly Thr Asp Gly Pro Ala Leu Thr Val Asp Thr Ala 195 200 205 Cys Ser Ser Ser Leu Val Ala Leu His Leu Ala Val Gln Ala Leu Arg 210 215 220 Lys Gly Glu Cys Asp Met Ala Leu Ala Gly Gly Val Thr Val Met Pro 225 230 235 240 Asn Ala Asp Leu Phe Val Gln Phe Ser Arg Gln Arg Gly Leu Ala Ala 245 250 255 Asp Gly Arg Ser Lys Ala Phe Ala Thr Ser Ala Asp Gly Phe Gly Pro 260 265 270 Ala Glu Gly Ala Gly Val Leu Leu Val Glu Arg Leu Ser Asp Ala Arg 275 280 285 Arg Asn Gly His Arg Ile Leu Ala Val Val Arg Gly Ser Ala Val Asn 290 295 300 Gln Asp Gly Ala Ser Asn Gly Leu Thr Ala Pro His Gly Pro Ser Gln 305 310 315 320 Gln Arg Val Ile Arg Arg Ala Leu Ala Asp Ala Arg Leu Ala Pro Gly 325 330 335 Asp Val Asp Val Val Glu Ala His Gly Thr Gly Thr Arg Leu Gly Asp 340 345 350 Pro Ile Glu Ala Gln Ala Leu Ile Ala Thr Tyr Gly Gln Glu Lys Ser 355 360 365 Ser Glu Gln Pro Leu Arg Leu Gly Ala Leu Lys Ser Asn Ile Gly His 370 375 380 Thr Gln Ala Ala Ala Gly Val Ala Gly Val Ile Lys Met Val Gln Ala 385 390 395 400 Met Arg His Gly Leu Leu Pro Lys Thr Leu His Val Asp Glu Pro Ser 405 410 415 Asp Gln Ile Asp Trp Ser Ala Gly Thr Val Glu Leu Leu Thr Glu Ala 420 425 430 Val Asp Trp Pro Glu Lys Gln Asp Gly Gly Leu Arg Arg Ala Ala Val 435 440 445 Ser Ser Phe Gly Ile Ser Gly Thr Asn Ala His Val Val Leu Glu Glu 450 455 460 Ala Pro Ala Val Glu Asp Ser Pro Ala Val Glu Pro Pro Ala Gly Gly 465 470 475 480 Gly Val Val Pro Trp Pro Val Ser Ala Lys Thr Pro Ala Ala Leu Asp 485 490 495 Ala Gln Ile Gly Gln Leu Ala Ala Tyr Ala Asp Gly Arg Thr Asp Val 500 505 510 Asp Pro Ala Val Ala Ala Arg Ala Leu Val Asp Ser Arg Thr Ala Met 515 520 525 Glu His Arg Ala Val Ala Val Gly Asp Ser Arg Glu Ala Leu Arg Asp 530 535 540 Ala Leu Arg Met Pro Glu Gly Leu Val Arg Gly Thr Ser Ser Asp Val 545 550 555 560 Gly Arg Val Ala Phe Val Phe Pro Gly Gln Gly Thr Gln Trp Ala Gly 565 570 575 Met Gly Ala Glu Leu Leu Asp Ser Ser Pro Glu Phe Ala Ala Ser Met 580 585 590 Ala Glu Cys Glu Thr Ala Leu Ser Arg Tyr Val Asp Trp Ser Leu Glu 595 600 605 Ala Val Val Arg Gln Glu Pro Gly Ala Pro Thr Leu Asp Arg Val Asp 610 615 620 Val Val Gln Pro Val Thr Phe Ala Val Met Val Ser Leu Ala Lys Val 625 630 635 640 Trp Gln His His Gly Ile Thr Pro Gln Ala Val Val Gly His Ser Gln 645 650 655 Gly Glu Ile Ala Ala Ala Tyr Val Ala Gly Ala Leu Thr Leu Asp Asp 660 665 670 Ala Ala Arg Val Val Thr Leu Arg Ser Lys Ser Ile Ala Ala His Leu 675 680 685 Ala Gly Lys Gly Gly Met Ile Ser Leu Ala Leu Asp Glu Ala Ala Val 690 695 700 Leu Lys Arg Leu Ser Asp Phe Asp Gly Leu Ser Val Ala Ala Val Asn 705 710 715 720 Gly Pro Thr Ala Thr Val Val Ser Gly Asp Pro Thr Gln Ile Glu Glu 725 730 735 Leu Ala Arg Thr Cys Glu Ala Asp Gly Val Arg Ala Arg Ile Ile Pro 740 745 750 Val Asp Tyr Ala Ser His Ser Arg Gln Val Glu Ile Ile Glu Lys Glu 755 760 765 Leu Ala Glu Val Leu Ala Gly Leu Ala Pro Gln Ala Pro His Val Pro 770 775 780 Phe Phe Ser Thr Leu Glu Gly Thr Trp Ile Thr Glu Pro Val Leu Asp 785 790 795 800 Gly Thr Tyr Trp Tyr Arg Asn Leu Arg His Arg Val Gly Phe Ala Pro 805 810 815 Ala Val Glu Thr Leu Ala Val Asp Gly Phe Thr His Phe Ile Glu Val 820 825 830 Ser Ala His Pro Val Leu Thr Met Thr Leu Pro Glu Thr Val Thr Gly 835 840 845 Leu Gly Thr Leu Arg Arg Glu Gln Gly Gly Gln Glu Arg Leu Val Thr 850 855 860 Ser Leu Ala Glu Ala Trp Ala Asn Gly Leu Thr Ile Asp Trp Ala Pro 865 870 875 880 Ile Leu Pro Thr Ala Thr Gly His His Pro Glu Leu Pro Thr Tyr Ala 885 890 895 Phe Gln Thr Glu Arg Phe Trp Leu Gln Ser Ser Ala Pro Thr Ser Ala 900 905 910 Ala Asp Asp Trp Arg Tyr Arg Val Glu Trp Lys Pro Leu Thr Ala Ser 915 920 925 Gly Gln Ala Asp Leu Ser Gly Arg Trp Ile Val Ala Val Gly Ser Glu 930 935 940 Pro Glu Ala Glu Leu Leu Gly Ala Leu Lys Ala Ala Gly Ala Glu Val 945 950 955 960 Asp Val Leu Glu Ala Gly Ala Asp Asp Asp Arg Glu Ala Leu Ala Ala 965 970 975 Arg Leu Thr Ala Leu Thr Thr Gly Asp Gly Phe Thr Gly Val Val Ser 980 985 990 Leu Leu Asp Asp Leu Val Pro Gln Val Ala Trp Val Gln Ala Leu Gly 995 1000 1005 Asp Ala Gly Ile Lys Ala Pro Leu Trp Ser Val Thr Gln Gly Ala Val 1010 1015 1020 Ser Val Gly Arg Leu Asp Thr Pro Ala Asp Pro Asp Arg Ala Met Leu 1025 1030 1035 1040 Trp Gly Leu Gly Arg Val Val Ala Leu Glu His Pro Glu Arg Trp Ala 1045 1050 1055 Gly Leu Val Asp Leu Pro Ala Gln Pro Asp Ala Ala Ala Leu Ala His 1060 1065 1070 Leu Val Thr Ala Leu Ser Gly Ala Thr Gly Glu Asp Gln Ile Ala Ile 1075 1080 1085 Arg Thr Thr Gly Leu His Ala Arg Arg Leu Ala Arg Ala Pro Leu His 1090 1095 1100 Gly Arg Arg Pro Thr Arg Asp Trp Gln Pro His Gly Thr Val Leu Ile 1105 1110 1115 1120 Thr Gly Gly Thr Gly Ala Leu Gly Ser His Ala Ala Arg Trp Met Ala 1125 1130 1135 His His Gly Ala Glu His Leu Leu Leu Val Ser Arg Ser Gly Glu Gln 1140 1145 1150 Ala Pro Gly Ala Thr Gln Leu Thr Ala Glu Leu Thr Ala Ser Gly Ala 1155 1160 1165 Arg Val Thr Ile Ala Ala Cys Asp Val Ala Asp Pro His Ala Met Arg 1170 1175 1180 Thr Leu Leu Asp Ala Ile Pro Ala Glu Thr Pro Leu Thr Ala Val Val 1185 1190 1195 1200 His Thr Ala Gly Ala Pro Gly Gly Asp Pro Leu Asp Val Thr Gly Pro 1205 1210 1215 Glu Asp Ile Ala Arg Ile Leu Gly Ala Lys Thr Ser Gly Ala Glu Val 1220 1225 1230 Leu Asp Asp Leu Leu Arg Gly Thr Pro Leu Asp Ala Phe Val Leu Tyr 1235 1240 1245 Ser Ser Asn Ala Gly Val Trp Gly Ser Gly Ser Gln Gly Val Tyr Ala 1250 1255 1260 Ala Ala Asn Ala His Leu Asp Ala Leu Ala Ala Arg Arg Arg Ala Arg 1265 1270 1275 1280 Gly Glu Thr Ala Thr Ser Val Ala Trp Gly Leu Trp Ala Gly Asp Gly 1285 1290 1295 Met Gly Arg Gly Ala Asp Asp Ala Tyr Trp Gln Arg Arg Gly Ile Arg 1300 1305 1310 Pro Met Ser Pro Asp Arg Ala Leu Asp Glu Leu Ala Lys Ala Leu Ser 1315 1320 1325 His Asp Glu Thr Phe Val Ala Val Ala Asp Val Asp Trp Glu Arg Phe 1330 1335 1340 Ala Pro Ala Phe Thr Val Ser Arg Pro Ser Leu Leu Leu Asp Gly Val 1345 1350 1355 1360 Pro Glu Ala Arg Gln Ala Leu Ala Ala Pro Val Gly Ala Pro Ala Pro 1365 1370 1375 Gly Asp Ala Ala Val Ala Pro Thr Gly Gln Ser Ser Ala Leu Ala Ala 1380 1385 1390 Ile Thr Ala Leu Pro Glu Pro Glu Arg Arg Pro Ala Leu Leu Thr Leu 1395 1400 1405 Val Arg Thr His Ala Ala Ala Val Leu Gly His Ser Ser Pro Asp Arg 1410 1415 1420 Val Ala Pro Gly Arg Ala Phe Thr Glu Leu Gly Phe Asp Ser Leu Thr 1425 1430 1435 1440 Ala Val Gln Leu Arg Asn Gln Leu Ser Thr Val Val Gly Asn Arg Leu 1445 1450 1455 Pro Ala Thr Thr Val Phe Asp His Pro Thr Pro Ala Ala Leu Ala Ala 1460 1465 1470 His Leu His Glu Ala Tyr Leu Ala Pro Ala Glu Pro Ala Pro Thr Asp 1475 1480 1485 Trp Glu Gly Arg Val Arg Arg Ala Leu Ala Glu Leu Pro Leu Asp Arg 1490 1495 1500 Leu Arg Asp Ala Gly Val Leu Asp Thr Val Leu Arg Leu Thr Gly Ile 1505 1510 1515 1520 Glu Pro Glu Pro Gly Ser Gly Gly Ser Asp Gly Gly Ala Ala Asp Pro 1525 1530 1535 Gly Ala Glu Pro Glu Ala Ser Ile Asp Asp Leu Asp Ala Glu Ala Leu 1540 1545 1550 Ile Arg Met Ala Leu Gly Pro Arg Asn Thr 1555 1560 4 1346 PRT Streptomyces venezuelae 4 Met Thr Ser Ser Asn Glu Gln Leu Val Asp Ala Leu Arg Ala Ser Leu 1 5 10 15 Lys Glu Asn Glu Glu Leu Arg Lys Glu Ser Arg Arg Arg Ala Asp Arg 20 25 30 Arg Gln Glu Pro Met Ala Ile Val Gly Met Ser Cys Arg Phe Ala Gly 35 40 45 Gly Ile Arg Ser Pro Glu Asp Leu Trp Asp Ala Val Ala Ala Gly Lys 50 55 60 Asp Leu Val Ser Glu Val Pro Glu Glu Arg Gly Trp Asp Ile Asp Ser 65 70 75 80 Leu Tyr Asp Pro Val Pro Gly Arg Lys Gly Thr Thr Tyr Val Arg Asn 85 90 95 Ala Ala Phe Leu Asp Asp Ala Ala Gly Phe Asp Ala Ala Phe Phe Gly 100 105 110 Ile Ser Pro Arg Glu Ala Leu Ala Met Asp Pro Gln Gln Arg Gln Leu 115 120 125 Leu Glu Ala Ser Trp Glu Val Phe Glu Arg Ala Gly Ile Asp Pro Ala 130 135 140 Ser Val Arg Gly Thr Asp Val Gly Val Tyr Val Gly Cys Gly Tyr Gln 145 150 155 160 Asp Tyr Ala Pro Asp Ile Arg Val Ala Pro Glu Gly Thr Gly Gly Tyr 165 170 175 Val Val Thr Gly Asn Ser Ser Ala Val Ala Ser Gly Arg Ile Ala Tyr 180 185 190 Ser Leu Gly Leu Glu Gly Pro Ala Val Thr Val Asp Thr Ala Cys Ser 195 200 205 Ser Ser Leu Val Ala Leu His Leu Ala Leu Lys Gly Leu Arg Asn Gly 210 215 220 Asp Cys Ser Thr Ala Leu Val Gly Gly Val Ala Val Leu Ala Thr Pro 225 230 235 240 Gly Ala Phe Ile Glu Phe Ser Ser Gln Gln Ala Met Ala Ala Asp Gly 245 250 255 Arg Thr Lys Gly Phe Ala Ser Ala Ala Asp Gly Leu Ala Trp Gly Glu 260 265 270 Gly Val Ala Val Leu Leu Leu Glu Arg Leu Ser Asp Ala Arg Arg Lys 275 280 285 Gly His Arg Val Leu Ala Val Val Arg Gly Ser Ala Ile Asn Gln Asp 290 295 300 Gly Ala Ser Asn Gly Leu Thr Ala Pro His Gly Pro Ser Gln Gln Arg 305 310 315 320 Leu Ile Arg Gln Ala Leu Ala Asp Ala Arg Leu Thr Ser Ser Asp Val 325 330 335 Asp Val Val Glu Gly His Gly Thr Gly Thr Arg Leu Gly Asp Pro Ile 340 345 350 Glu Ala Gln Ala Leu Leu Ala Thr Tyr Gly Gln Gly Arg Ala Pro Gly 355 360 365 Gln Pro Leu Arg Leu Gly Thr Leu Lys Ser Asn Ile Gly His Thr Gln 370 375 380 Ala Ala Ser Gly Val Ala Gly Val Ile Lys Met Val Gln Ala Leu Arg 385 390 395 400 His Gly Val Leu Pro Lys Thr Leu His Val Asp Glu Pro Thr Asp Gln 405 410 415 Val Asp Trp Ser Ala Gly Ser Val Glu Leu Leu Thr Glu Ala Val Asp 420 425 430 Trp Pro Glu Arg Pro Gly Arg Leu Arg Arg Ala Gly Val Ser Ala Phe 435 440 445 Gly Val Gly Gly Thr Asn Ala His Val Val Leu Glu Glu Ala Pro Ala 450 455 460 Val Glu Glu Ser Pro Ala Val Glu Pro Pro Ala Gly Gly Gly Val Val 465 470 475 480 Pro Trp Pro Val Ser Ala Lys Thr Ser Ala Ala Leu Asp Ala Gln Ile 485 490 495 Gly Gln Leu Ala Ala Tyr Ala Glu Asp Arg Thr Asp Val Asp Pro Ala 500 505 510 Val Ala Ala Arg Ala Leu Val Asp Ser Arg Thr Ala Met Glu His Arg 515 520 525 Ala Val Ala Val Gly Asp Ser Arg Glu Ala Leu Arg Asp Ala Leu Arg 530 535 540 Met Pro Glu Gly Leu Val Arg Gly Thr Val Thr Asp Pro Gly Arg Val 545 550 555 560 Ala Phe Val Phe Pro Gly Gln Gly Thr Gln Trp Ala Gly Met Gly Ala 565 570 575 Glu Leu Leu Asp Ser Ser Pro Glu Phe Ala Ala Ala Met Ala Glu Cys 580 585 590 Glu Thr Ala Leu Ser Pro Tyr Val Asp Trp Ser Leu Glu Ala Val Val 595 600 605 Arg Gln Ala Pro Ser Ala Pro Thr Leu Asp Arg Val Asp Val Val Gln 610 615 620 Pro Val Thr Phe Ala Val Met Val Ser Leu Ala Lys Val Trp Gln His 625 630 635 640 His Gly Ile Thr Pro Glu Ala Val Ile Gly His Ser Gln Gly Glu Ile 645 650 655 Ala Ala Ala Tyr Val Ala Gly Ala Leu Thr Leu Asp Asp Ala Ala Arg 660 665 670 Val Val Thr Leu Arg Ser Lys Ser Ile Ala Ala His Leu Ala Gly Lys 675 680 685 Gly Gly Met Ile Ser Leu Ala Leu Ser Glu Glu Ala Thr Arg Gln Arg 690 695 700 Ile Glu Asn Leu His Gly Leu Ser Ile Ala Ala Val Asn Gly Pro Thr 705 710 715 720 Ala Thr Val Val Ser Gly Asp Pro Thr Gln Ile Gln Glu Leu Ala Gln 725 730 735 Ala Cys Glu Ala Asp Gly Ile Arg Ala Arg Ile Ile Pro Val Asp Tyr 740 745 750 Ala Ser His Ser Ala His Val Glu Thr Ile Glu Asn Glu Leu Ala Asp 755 760 765 Val Leu Ala Gly Leu Ser Pro Gln Thr Pro Gln Val Pro Phe Phe Ser 770 775 780 Thr Leu Glu Gly Thr Trp Ile Thr Glu Pro Ala Leu Asp Gly Gly Tyr 785 790 795 800 Trp Tyr Arg Asn Leu Arg His Arg Val Gly Phe Ala Pro Ala Val Glu 805 810 815 Thr Leu Ala Thr Asp Glu Gly Phe Thr His Phe Ile Glu Val Ser Ala 820 825 830 His Pro Val Leu Thr Met Thr Leu Pro Asp Lys Val Thr Gly Leu Ala 835 840 845 Thr Leu Arg Arg Glu Asp Gly Gly Gln His Arg Leu Thr Thr Ser Leu 850 855 860 Ala Glu Ala Trp Ala Asn Gly Leu Ala Leu Asp Trp Ala Ser Leu Leu 865 870 875 880 Pro Ala Thr Gly Ala Leu Ser Pro Ala Val Pro Asp Leu Pro Thr Tyr 885 890 895 Ala Phe Gln His Arg Ser Tyr Trp Ile Ser Pro Ala Gly Pro Gly Glu 900 905 910 Ala Pro Ala His Thr Ala Ser Gly Arg Glu Ala Val Ala Glu Thr Gly 915 920 925 Leu Ala Trp Gly Pro Gly Ala Glu Asp Leu Asp Glu Glu Gly Arg Arg 930 935 940 Ser Ala Val Leu Ala Met Val Met Arg Gln Ala Ala Ser Val Leu Arg 945 950 955 960 Cys Asp Ser Pro Glu Glu Val Pro Val Asp Arg Pro Leu Arg Glu Ile 965 970 975 Gly Phe Asp Ser Leu Thr Ala Val Asp Phe Arg Asn Arg Val Asn Arg 980 985 990 Leu Thr Gly Leu Gln Leu Pro Pro Thr Val Val Phe Glu His Pro Thr 995 1000 1005 Pro Val Ala Leu Ala Glu Arg Ile Ser Asp Glu Leu Ala Glu Arg Asn 1010 1015 1020 Trp Ala Val Ala Glu Pro Ser Asp His Glu Gln Ala Glu Glu Glu Lys 1025 1030 1035 1040 Ala Ala Ala Pro Ala Gly Ala Arg Ser Gly Ala Asp Thr Gly Ala Gly 1045 1050 1055 Ala Gly Met Phe Arg Ala Leu Phe Arg Gln Ala Val Glu Asp Asp Arg 1060 1065 1070 Tyr Gly Glu Phe Leu Asp Val Leu Ala Glu Ala Ser Ala Phe Arg Pro 1075 1080 1085 Gln Phe Ala Ser Pro Glu Ala Cys Ser Glu Arg Leu Asp Pro Val Leu 1090 1095 1100 Leu Ala Gly Gly Pro Thr Asp Arg Ala Glu Gly Arg Ala Val Leu Val 1105 1110 1115 1120 Gly Cys Thr Gly Thr Ala Ala Asn Gly Gly Pro His Glu Phe Leu Arg 1125 1130 1135 Leu Ser Thr Ser Phe Gln Glu Glu Arg Asp Phe Leu Ala Val Pro Leu 1140 1145 1150 Pro Gly Tyr Gly Thr Gly Thr Gly Thr Gly Thr Ala Leu Leu Pro Ala 1155 1160 1165 Asp Leu Asp Thr Ala Leu Asp Ala Gln Ala Arg Ala Ile Leu Arg Ala 1170 1175 1180 Ala Gly Asp Ala Pro Val Val Leu Leu Gly His Ser Gly Gly Ala Leu 1185 1190 1195 1200 Leu Ala His Glu Leu Ala Phe Arg Leu Glu Arg Ala His Gly Ala Pro 1205 1210 1215 Pro Ala Gly Ile Val Leu Val Asp Pro Tyr Pro Pro Gly His Gln Glu 1220 1225 1230 Pro Ile Glu Val Trp Ser Arg Gln Leu Gly Glu Gly Leu Phe Ala Gly 1235 1240 1245 Glu Leu Glu Pro Met Ser Asp Ala Arg Leu Leu Ala Met Gly Arg Tyr 1250 1255 1260 Ala Arg Phe Leu Ala Gly Pro Arg Pro Gly Arg Ser Ser Ala Pro Val 1265 1270 1275 1280 Leu Leu Val Arg Ala Ser Glu Pro Leu Gly Asp Trp Gln Glu Glu Arg 1285 1290 1295 Gly Asp Trp Arg Ala His Trp Asp Leu Pro His Thr Val Ala Asp Val 1300 1305 1310 Pro Gly Asp His Phe Thr Met Met Arg Asp His Ala Pro Ala Val Ala 1315 1320 1325 Glu Ala Val Leu Ser Trp Leu Asp Ala Ile Glu Gly Ile Glu Gly Ala 1330 1335 1340 Gly Lys 1345 5 281 PRT Streptomyces venezuelae 5 Val Thr Asp Arg Pro Leu Asn Val Asp Ser Gly Leu Trp Ile Arg Arg 1 5 10 15 Phe His Pro Ala Pro Asn Ser Ala Val Arg Leu Val Cys Leu Pro His 20 25 30 Ala Gly Gly Ser Ala Ser Tyr Phe Phe Arg Phe Ser Glu Glu Leu His 35 40 45 Pro Ser Val Glu Ala Leu Ser Val Gln Tyr Pro Gly Arg Gln Asp Arg 50 55 60 Arg Ala Glu Pro Cys Leu Glu Ser Val Glu Glu Leu Ala Glu His Val 65 70 75 80 Val Ala Ala Thr Glu Pro Trp Trp Gln Glu Gly Arg Leu Ala Phe Phe 85 90 95 Gly His Ser Leu Gly Ala Ser Val Ala Phe Glu Thr Ala Arg Ile Leu 100 105 110 Glu Gln Arg His Gly Val Arg Pro Glu Gly Leu Tyr Val Ser Gly Arg 115 120 125 Arg Ala Pro Ser Leu Ala Pro Asp Arg Leu Val His Gln Leu Asp Asp 130 135 140 Arg Ala Phe Leu Ala Glu Ile Arg Arg Leu Ser Gly Thr Asp Glu Arg 145 150 155 160 Phe Leu Gln Asp Asp Glu Leu Leu Arg Leu Val Leu Pro Ala Leu Arg 165 170 175 Ser Asp Tyr Lys Ala Ala Glu Thr Tyr Leu His Arg Pro Ser Ala Lys 180 185 190 Leu Thr Cys Pro Val Met Ala Leu Ala Gly Asp Arg Asp Pro Lys Ala 195 200 205 Pro Leu Asn Glu Val Ala Glu Trp Arg Arg His Thr Ser Gly Pro Phe 210 215 220 Cys Leu Arg Ala Tyr Ser Gly Gly His Phe Tyr Leu Asn Asp Gln Trp 225 230 235 240 His Glu Ile Cys Asn Asp Ile Ser Asp His Leu Leu Val Thr Arg Gly 245 250 255 Ala Pro Asp Ala Arg Val Val Gln Pro Pro Thr Ser Leu Ile Glu Gly 260 265 270 Ala Ala Lys Arg Trp Gln Asn Pro Arg 275 280 6 379 PRT Streptomyces venezuelae 251 unsure unsure of amino acid at this position 6 Val Ser Ser Arg Ala Glu Thr Pro Arg Val Pro Phe Leu Asp Leu Lys 1 5 10 15 Ala Ala Tyr Glu Glu Leu Arg Ala Glu Thr Asp Ala Ala Ile Ala Arg 20 25 30 Val Leu Asp Ser Gly Arg Tyr Leu Leu Gly Pro Glu Leu Glu Gly Phe 35 40 45 Glu Ala Glu Phe Ala Ala Tyr Cys Glu Thr Asp His Ala Val Gly Val 50 55 60 Asn Ser Gly Met Asp Ala Leu Gln Leu Ala Leu Arg Gly Leu Gly Ile 65 70 75 80 Gly Pro Gly Asp Glu Val Ile Val Pro Ser His Thr Tyr Ile Ala Ser 85 90 95 Trp Leu Ala Val Ser Ala Thr Gly Ala Thr Pro Val Pro Val Glu Pro 100 105 110 His Glu Asp His Pro Thr Leu Asp Pro Leu Leu Val Glu Lys Ala Ile 115 120 125 Thr Pro Arg Thr Arg Ala Leu Leu Pro Val His Leu Tyr Gly His Pro 130 135 140 Ala Asp Met Asp Ala Leu Arg Glu Leu Ala Asp Arg His Gly Leu His 145 150 155 160 Ile Val Glu Asp Ala Ala Gln Ala His Gly Ala Arg Tyr Arg Gly Arg 165 170 175 Arg Ile Gly Ala Gly Ser Ser Val Ala Ala Phe Ser Phe Tyr Pro Gly 180 185 190 Lys Asn Leu Gly Cys Phe Gly Asp Gly Gly Ala Val Val Thr Gly Asp 195 200 205 Pro Glu Leu Ala Glu Arg Leu Arg Met Leu Arg Asn Tyr Gly Ser Arg 210 215 220 Gln Lys Tyr Ser His Glu Thr Lys Gly Thr Asn Ser Arg Leu Asp Glu 225 230 235 240 Met Gln Ala Ala Val Leu Arg Ile Arg Leu Xaa His Leu Asp Ser Trp 245 250 255 Asn Gly Arg Arg Ser Ala Leu Ala Ala Glu Tyr Leu Ser Gly Leu Ala 260 265 270 Gly Leu Pro Gly Ile Gly Leu Pro Val Thr Ala Pro Asp Thr Asp Pro 275 280 285 Val Trp His Leu Phe Thr Val Arg Thr Glu Arg Arg Asp Glu Leu Arg 290 295 300 Ser His Leu Asp Ala Arg Gly Ile Asp Thr Leu Thr His Tyr Pro Val 305 310 315 320 Pro Val His Leu Ser Pro Ala Tyr Ala Gly Glu Ala Pro Pro Glu Gly 325 330 335 Ser Leu Pro Arg Ala Glu Ser Phe Ala Arg Gln Val Leu Ser Leu Pro 340 345 350 Ile Gly Pro His Leu Glu Arg Pro Gln Ala Leu Arg Val Ile Asp Ala 355 360 365 Val Arg Glu Trp Ala Glu Arg Val Asp Gln Ala 370 375 7 382 PRT Streptomyces venezuelae 7 Val Ala Asp Arg Glu Leu Gly Thr His Leu Leu Glu Thr Arg Gly Ile 1 5 10 15 His Trp Ile His Ala Ala Asn Gly Asp Pro Tyr Ala Thr Val Leu Arg 20 25 30 Gly Gln Ala Asp Asp Pro Tyr Pro Ala Tyr Glu Arg Val Arg Ala Arg 35 40 45 Gly Ala Leu Ser Phe Ser Pro Thr Gly Ser Trp Val Thr Ala Asp His 50 55 60 Ala Leu Ala Ala Ser Ile Leu Cys Ser Thr Asp Phe Gly Val Ser Gly 65 70 75 80 Ala Asp Gly Val Pro Val Pro Gln Gln Val Leu Ser Tyr Gly Glu Gly 85 90 95 Cys Pro Leu Glu Arg Glu Gln Val Leu Pro Ala Ala Gly Asp Val Pro 100 105 110 Glu Gly Gly Gln Arg Ala Val Val Glu Gly Ile His Arg Glu Thr Leu 115 120 125 Glu Gly Leu Ala Pro Asp Pro Ser Ala Ser Tyr Ala Phe Glu Leu Leu 130 135 140 Gly Gly Phe Val Arg Pro Ala Val Thr Ala Ala Ala Ala Ala Val Leu 145 150 155 160 Gly Val Pro Ala Asp Arg Arg Ala Asp Phe Ala Asp Leu Leu Glu Arg 165 170 175 Leu Arg Pro Leu Ser Asp Ser Leu Leu Ala Pro Gln Ser Leu Arg Thr 180 185 190 Val Arg Ala Ala Asp Gly Ala Leu Ala Glu Leu Thr Ala Leu Leu Ala 195 200 205 Asp Ser Asp Asp Ser Pro Gly Ala Leu Leu Ser Ala Leu Gly Val Thr 210 215 220 Ala Ala Val Gln Leu Thr Gly Asn Ala Val Leu Ala Leu Leu Ala His 225 230 235 240 Pro Glu Gln Trp Arg Glu Leu Cys Asp Arg Pro Gly Leu Ala Ala Ala 245 250 255 Ala Val Glu Glu Thr Leu Arg Tyr Asp Pro Pro Val Gln Leu Asp Ala 260 265 270 Arg Val Val Arg Gly Glu Thr Glu Leu Ala Gly Arg Arg Leu Pro Ala 275 280 285 Gly Ala His Val Val Val Leu Thr Ala Ala Thr Gly Arg Asp Pro Glu 290 295 300 Val Phe Thr Asp Pro Glu Arg Phe Asp Leu Ala Arg Pro Asp Ala Ala 305 310 315 320 Ala His Leu Ala Leu His Pro Ala Gly Pro Tyr Gly Pro Val Ala Ser 325 330 335 Leu Val Arg Leu Gln Ala Glu Val Ala Leu Arg Thr Leu Ala Gly Arg 340 345 350 Phe Pro Gly Leu Arg Gln Ala Gly Asp Val Leu Arg Pro Arg Arg Ala 355 360 365 Pro Val Gly Arg Gly Pro Leu Ser Val Pro Val Ser Ser Ser 370 375 380 8 426 PRT Streptomyces venezuelae 8 Met Arg Val Leu Leu Thr Ser Phe Ala His His Thr His Tyr Tyr Gly 1 5 10 15 Leu Val Pro Leu Ala Trp Ala Leu Leu Ala Ala Gly His Glu Val Arg 20 25 30 Val Ala Ser Gln Pro Ala Leu Thr Asp Thr Ile Thr Gly Ser Gly Leu 35 40 45 Ala Ala Val Pro Val Gly Thr Asp His Leu Ile His Glu Tyr Arg Val 50 55 60 Arg Met Ala Gly Glu Pro Arg Pro Asn His Pro Ala Ile Ala Phe Asp 65 70 75 80 Glu Ala Arg Pro Glu Pro Leu Asp Trp Asp His Ala Leu Gly Ile Glu 85 90 95 Ala Ile Leu Ala Pro Tyr Phe Tyr Leu Leu Ala Asn Asn Asp Ser Met 100 105 110 Val Asp Asp Leu Val Asp Phe Ala Arg Ser Trp Gln Pro Asp Leu Val 115 120 125 Leu Trp Glu Pro Thr Thr Tyr Ala Gly Ala Val Ala Ala Gln Val Thr 130 135 140 Gly Ala Ala His Ala Arg Val Leu Trp Gly Pro Asp Val Met Gly Ser 145 150 155 160 Ala Arg Arg Lys Phe Val Ala Leu Arg Asp Arg Gln Pro Pro Glu His 165 170 175 Arg Glu Asp Pro Thr Ala Glu Trp Leu Thr Trp Thr Leu Asp Arg Tyr 180 185 190 Gly Ala Ser Phe Glu Glu Glu Leu Leu Thr Gly Gln Phe Thr Ile Asp 195 200 205 Pro Thr Pro Pro Ser Leu Arg Leu Asp Thr Gly Leu Pro Thr Val Gly 210 215 220 Met Arg Tyr Val Pro Tyr Asn Gly Thr Ser Val Val Pro Asp Trp Leu 225 230 235 240 Ser Glu Pro Pro Ala Arg Pro Arg Val Cys Leu Thr Leu Gly Val Ser 245 250 255 Ala Arg Glu Val Leu Gly Gly Asp Gly Val Ser Gln Gly Asp Ile Leu 260 265 270 Glu Ala Leu Ala Asp Leu Asp Ile Glu Leu Val Ala Thr Leu Asp Ala 275 280 285 Ser Gln Arg Ala Glu Ile Arg Asn Tyr Pro Lys His Thr Arg Phe Thr 290 295 300 Asp Phe Val Pro Met His Ala Leu Leu Pro Ser Cys Ser Ala Ile Ile 305 310 315 320 His His Gly Gly Ala Gly Thr Tyr Ala Thr Ala Val Ile Asn Ala Val 325 330 335 Pro Gln Val Met Leu Ala Glu Leu Trp Asp Ala Pro Val Lys Ala Arg 340 345 350 Ala Val Ala Glu Gln Gly Ala Gly Phe Phe Leu Pro Pro Ala Glu Leu 355 360 365 Thr Pro Gln Ala Val Arg Asp Ala Val Val Arg Ile Leu Asp Asp Pro 370 375 380 Ser Val Ala Thr Ala Ala His Arg Leu Arg Glu Glu Thr Phe Gly Asp 385 390 395 400 Pro Thr Pro Ala Gly Ile Val Pro Glu Leu Glu Arg Leu Ala Ala Gln 405 410 415 His Arg Arg Pro Pro Ala Asp Ala Arg His 420 425 9 331 PRT Streptomyces venezuelae 272 unsure unsure of amino acid at this position 9 Val Lys Ser Ala Leu Ser Asp Leu Ala Phe Phe Gly Gly Pro Ala Ala 1 5 10 15 Phe Asp Gln Pro Leu Leu Val Gly Arg Pro Asn Arg Ile Asp Arg Ala 20 25 30 Arg Leu Tyr Glu Arg Leu Asp Arg Ala Leu Asp Ser Gln Trp Leu Ser 35 40 45 Asn Gly Gly Pro Leu Val Arg Glu Phe Glu Glu Arg Val Ala Gly Leu 50 55 60 Ala Gly Val Arg His Ala Val Ala Thr Cys Asn Ala Thr Ala Gly Leu 65 70 75 80 Gln Leu Leu Ala His Ala Ala Gly Leu Thr Gly Glu Val Ile Met Pro 85 90 95 Ser Met Thr Phe Ala Ala Thr Pro His Ala Leu Arg Trp Ile Gly Leu 100 105 110 Thr Pro Val Phe Ala Asp Ile Asp Pro Asp Thr Gly Asn Leu Asp Pro 115 120 125 Asp Gln Val Ala Ala Ala Val Thr Pro Arg Thr Ser Ala Val Val Gly 130 135 140 Val His Leu Trp Gly Arg Pro Cys Ala Ala Asp Gln Leu Arg Lys Val 145 150 155 160 Ala Asp Glu His Gly Leu Arg Leu Tyr Phe Asp Ala Ala His Ala Leu 165 170 175 Gly Cys Ala Val Asp Gly Arg Pro Ala Gly Ser Leu Gly Asp Ala Glu 180 185 190 Val Phe Ser Phe His Ala Thr Lys Ala Val Asn Ala Phe Glu Gly Gly 195 200 205 Ala Val Val Thr Asp Asp Ala Asp Leu Ala Ala Arg Ile Arg Ala Leu 210 215 220 His Asn Phe Gly Phe Asp Leu Pro Gly Gly Ser Pro Ala Gly Gly Thr 225 230 235 240 Asn Ala Lys Met Ser Glu Ala Ala Ala Ala Met Gly Leu Thr Ser Leu 245 250 255 Asp Ala Phe Pro Glu Val Ile Asp Arg Asn Arg Arg Asn His Ala Xaa 260 265 270 Tyr Arg Glu His Leu Ala Asp Leu Pro Gly Val Leu Val Ala Asp His 275 280 285 Asp Arg His Gly Leu Asn Asn His Gln Tyr Val Ile Val Glu Ile Asp 290 295 300 Glu Ala Thr Thr Gly Ile His Arg Asp Leu Val Met Glu Val Leu Lys 305 310 315 320 Ala Glu Gly Val His Thr Arg Ala Tyr Phe Ser 325 330 10 485 PRT Streptomyces venezuelae 10 Met Thr Ala Pro Ala Leu Ser Ala Thr Ala Pro Ala Glu Arg Cys Ala 1 5 10 15 His Pro Gly Ala Asp Leu Gly Ala Ala Val His Ala Val Gly Gln Thr 20 25 30 Leu Ala Ala Gly Gly Leu Val Pro Pro Asp Glu Ala Gly Thr Thr Ala 35 40 45 Arg His Leu Val Arg Leu Ala Val Arg Tyr Gly Asn Ser Pro Phe Thr 50 55 60 Pro Leu Glu Glu Ala Arg His Asp Leu Gly Val Asp Arg Asp Ala Phe 65 70 75 80 Arg Arg Leu Leu Ala Leu Phe Gly Gln Val Pro Glu Leu Arg Thr Ala 85 90 95 Val Glu Thr Gly Pro Ala Gly Ala Tyr Trp Lys Asn Thr Leu Leu Pro 100 105 110 Leu Glu Gln Arg Gly Val Phe Asp Ala Ala Leu Ala Arg Lys Pro Val 115 120 125 Phe Pro Tyr Ser Val Gly Leu Tyr Pro Gly Pro Thr Cys Met Phe Arg 130 135 140 Cys His Phe Cys Val Arg Val Thr Gly Ala Arg Tyr Asp Pro Ser Ala 145 150 155 160 Leu Asp Ala Gly Asn Ala Met Phe Arg Ser Val Ile Asp Glu Ile Pro 165 170 175 Ala Gly Asn Pro Ser Ala Met Tyr Phe Ser Gly Gly Leu Glu Pro Leu 180 185 190 Thr Asn Pro Gly Leu Gly Ser Leu Ala Ala His Ala Thr Asp His Gly 195 200 205 Leu Arg Pro Thr Val Tyr Thr Asn Ser Phe Ala Leu Thr Glu Arg Thr 210 215 220 Leu Glu Arg Gln Pro Gly Leu Trp Gly Leu His Ala Ile Arg Thr Ser 225 230 235 240 Leu Tyr Gly Leu Asn Asp Glu Glu Tyr Glu Gln Thr Thr Gly Lys Lys 245 250 255 Ala Ala Phe Arg Arg Val Arg Glu Asn Leu Arg Arg Phe Gln Gln Leu 260 265 270 Arg Ala Glu Arg Glu Ser Pro Ile Asn Leu Gly Phe Ala Tyr Ile Val 275 280 285 Leu Pro Gly Arg Ala Ser Arg Leu Leu Asp Leu Val Asp Phe Ile Ala 290 295 300 Asp Leu Asn Asp Ala Gly Gln Gly Arg Thr Ile Asp Phe Val Asn Ile 305 310 315 320 Arg Glu Asp Tyr Ser Gly Arg Asp Asp Gly Lys Leu Pro Gln Glu Glu 325 330 335 Arg Ala Glu Leu Gln Glu Ala Leu Asn Ala Phe Glu Glu Arg Val Arg 340 345 350 Glu Arg Thr Pro Gly Leu His Ile Asp Tyr Gly Tyr Ala Leu Asn Ser 355 360 365 Leu Arg Thr Gly Ala Asp Ala Glu Leu Leu Arg Ile Lys Pro Ala Thr 370 375 380 Met Arg Pro Thr Ala His Pro Gln Val Ala Val Gln Val Asp Leu Leu 385 390 395 400 Gly Asp Val Tyr Leu Tyr Arg Glu Ala Gly Phe Pro Asp Leu Asp Gly 405 410 415 Ala Thr Arg Tyr Ile Ala Gly Arg Val Thr Pro Asp Thr Ser Leu Thr 420 425 430 Glu Val Val Arg Asp Phe Val Glu Arg Gly Gly Glu Val Ala Ala Val 435 440 445 Asp Gly Asp Glu Tyr Phe Met Asp Gly Phe Asp Gln Val Val Thr Ala 450 455 460 Arg Leu Asn Gln Leu Glu Arg Asp Ala Ala Asp Gly Trp Glu Glu Ala 465 470 475 480 Arg Gly Phe Leu Arg 485 11 237 PRT Streptomyces venezuelae 11 Val Tyr Glu Val Asp His Ala Asp Val Tyr Asp Leu Phe Tyr Leu Gly 1 5 10 15 Arg Gly Lys Asp Tyr Ala Ala Glu Ala Ser Asp Ile Ala Asp Leu Val 20 25 30 Arg Ser Arg Thr Pro Glu Ala Ser Ser Leu Leu Asp Val Ala Cys Gly 35 40 45 Thr Gly Thr His Leu Glu His Phe Thr Lys Glu Phe Gly Asp Thr Ala 50 55 60 Gly Leu Glu Leu Ser Glu Asp Met Leu Thr His Ala Arg Lys Arg Leu 65 70 75 80 Pro Asp Ala Thr Leu His Gln Gly Asp Met Arg Asp Phe Arg Leu Gly 85 90 95 Arg Lys Phe Ser Ala Val Val Ser Met Phe Ser Ser Val Gly Tyr Leu 100 105 110 Lys Thr Thr Glu Glu Leu Gly Ala Ala Val Ala Ser Phe Ala Glu His 115 120 125 Leu Glu Pro Gly Gly Val Val Val Val Glu Pro Trp Trp Phe Pro Glu 130 135 140 Thr Phe Ala Asp Gly Trp Val Ser Ala Asp Val Val Arg Arg Asp Gly 145 150 155 160 Arg Thr Val Ala Arg Val Ser His Ser Val Arg Glu Gly Asn Ala Thr 165 170 175 Arg Met Glu Val His Phe Thr Val Ala Asp Pro Gly Lys Gly Val Arg 180 185 190 His Phe Ser Asp Val His Leu Ile Thr Leu Phe His Gln Ala Glu Tyr 195 200 205 Glu Ala Ala Phe Thr Ala Ala Gly Leu Arg Val Glu Tyr Leu Glu Gly 210 215 220 Gly Pro Ser Gly Arg Gly Leu Phe Val Gly Val Pro Ala 225 230 235 12 769 PRT Streptomyces venezuelae 12 Met Thr Leu Asp Glu Lys Ile Ser Phe Val His Trp Ala Leu Asp Pro 1 5 10 15 Asp Arg Gln Asn Val Gly Tyr Leu Pro Gly Val Pro Arg Leu Gly Ile 20 25 30 Pro Glu Leu Arg Ala Ala Asp Gly Pro Asn Gly Ile Arg Leu Val Gly 35 40 45 Gln Thr Ala Thr Ala Leu Pro Ala Pro Val Ala Leu Ala Ser Thr Phe 50 55 60 Asp Asp Thr Met Ala Asp Ser Tyr Gly Lys Val Met Gly Arg Asp Gly 65 70 75 80 Arg Ala Leu Asn Gln Asp Met Val Leu Gly Pro Met Met Asn Asn Ile 85 90 95 Arg Val Pro His Gly Gly Arg Asn Tyr Glu Thr Phe Ser Glu Asp Pro 100 105 110 Leu Val Ser Ser Arg Thr Ala Val Ala Gln Ile Lys Gly Ile Gln Gly 115 120 125 Ala Gly Leu Met Thr Thr Ala Lys His Phe Ala Ala Asn Asn Gln Glu 130 135 140 Asn Asn Arg Phe Ser Val Asn Ala Asn Val Asp Glu Gln Thr Leu Arg 145 150 155 160 Glu Ile Glu Phe Pro Ala Phe Glu Ala Ser Ser Lys Ala Gly Ala Gly 165 170 175 Ser Phe Met Cys Ala Tyr Asn Gly Leu Asn Gly Lys Pro Ser Cys Gly 180 185 190 Asn Asp Glu Leu Leu Asn Asn Val Leu Arg Thr Gln Trp Gly Phe Gln 195 200 205 Gly Trp Val Met Ser Asp Trp Leu Ala Thr Pro Gly Thr Asp Ala Ile 210 215 220 Thr Lys Gly Leu Asp Gln Glu Met Gly Val Glu Leu Pro Gly Asp Val 225 230 235 240 Pro Lys Gly Glu Pro Ser Pro Pro Ala Lys Phe Phe Gly Glu Ala Leu 245 250 255 Lys Thr Ala Val Leu Asn Gly Thr Val Pro Glu Ala Ala Val Thr Arg 260 265 270 Ser Ala Glu Arg Ile Val Gly Gln Met Glu Lys Phe Gly Leu Leu Leu 275 280 285 Ala Thr Pro Ala Pro Arg Pro Glu Arg Asp Lys Ala Gly Ala Gln Ala 290 295 300 Val Ser Arg Lys Val Ala Glu Asn Gly Ala Val Leu Leu Arg Asn Glu 305 310 315 320 Gly Gln Ala Leu Pro Leu Ala Gly Asp Ala Gly Lys Ser Ile Ala Val 325 330 335 Ile Gly Pro Thr Ala Val Asp Pro Lys Val Thr Gly Leu Gly Ser Ala 340 345 350 His Val Val Pro Asp Ser Ala Ala Ala Pro Leu Asp Thr Ile Lys Ala 355 360 365 Arg Ala Gly Ala Gly Ala Thr Val Thr Tyr Glu Thr Gly Glu Glu Thr 370 375 380 Phe Gly Thr Gln Ile Pro Ala Gly Asn Leu Ser Pro Ala Phe Asn Gln 385 390 395 400 Gly His Gln Leu Glu Pro Gly Lys Ala Gly Ala Leu Tyr Asp Gly Thr 405 410 415 Leu Thr Val Pro Ala Asp Gly Glu Tyr Arg Ile Ala Val Arg Ala Thr 420 425 430 Gly Gly Tyr Ala Thr Val Gln Leu Gly Ser His Thr Ile Glu Ala Gly 435 440 445 Gln Val Tyr Gly Lys Val Ser Ser Pro Leu Leu Lys Leu Thr Lys Gly 450 455 460 Thr His Lys Leu Thr Ile Ser Gly Phe Ala Met Ser Ala Thr Pro Leu 465 470 475 480 Ser Leu Glu Leu Gly Trp Val Thr Pro Ala Ala Ala Asp Ala Thr Ile 485 490 495 Ala Lys Ala Val Glu Ser Ala Arg Lys Ala Arg Thr Ala Val Val Phe 500 505 510 Ala Tyr Asp Asp Gly Thr Glu Gly Val Asp Arg Pro Asn Leu Ser Leu 515 520 525 Pro Gly Thr Gln Asp Lys Leu Ile Ser Ala Val Ala Asp Ala Asn Pro 530 535 540 Asn Thr Ile Val Val Leu Asn Thr Gly Ser Ser Val Leu Met Pro Trp 545 550 555 560 Leu Ser Lys Thr Arg Ala Val Leu Asp Met Trp Tyr Pro Gly Gln Ala 565 570 575 Gly Ala Glu Ala Thr Ala Ala Leu Leu Tyr Gly Asp Val Asn Pro Ser 580 585 590 Gly Lys Leu Thr Gln Ser Phe Pro Ala Ala Glu Asn Gln His Ala Val 595 600 605 Ala Gly Asp Pro Thr Ser Tyr Pro Gly Val Asp Asn Gln Gln Thr Tyr 610 615 620 Arg Glu Gly Ile His Val Gly Tyr Arg Trp Phe Asp Lys Glu Asn Val 625 630 635 640 Lys Pro Leu Phe Pro Phe Gly His Gly Leu Ser Tyr Thr Ser Phe Thr 645 650 655 Gln Ser Ala Pro Thr Val Val Arg Thr Ser Thr Gly Gly Leu Lys Val 660 665 670 Thr Val Thr Val Arg Asn Ser Gly Lys Arg Ala Gly Gln Glu Val Val 675 680 685 Gln Ala Tyr Leu Gly Ala Ser Pro Asn Val Thr Ala Pro Gln Ala Lys 690 695 700 Lys Lys Leu Val Gly Tyr Thr Lys Val Ser Leu Ala Ala Gly Glu Ala 705 710 715 720 Lys Thr Val Thr Val Asn Val Asp Arg Arg Gln Leu Gln Phe Trp Asp 725 730 735 Ala Ala Thr Asp Asn Trp Lys Thr Gly Thr Gly Asn Arg Leu Leu Gln 740 745 750 Thr Gly Ser Ser Ser Ala Asp Leu Arg Gly Ser Ala Thr Val Asn Val 755 760 765 Trp 13 928 PRT Streptomyces venezuelae 694 unsure unsure of amino acid at this position 13 Met Asn Leu Val Glu Arg Asp Gly Glu Ile Ala His Leu Arg Ala Val 1 5 10 15 Leu Asp Ala Ser Ala Ala Gly Asp Gly Thr Leu Leu Leu Val Ser Gly 20 25 30 Pro Ala Gly Ser Gly Lys Thr Glu Leu Leu Arg Ser Leu Arg Arg Leu 35 40 45 Ala Ala Glu Arg Glu Thr Pro Val Trp Ser Val Arg Ala Leu Pro Gly 50 55 60 Asp Arg Asp Ile Pro Leu Gly Val Leu Cys Gln Leu Leu Arg Ser Ala 65 70 75 80 Glu Gln His Gly Ala Asp Thr Ser Ala Val Arg Asp Leu Leu Asp Ala 85 90 95 Ala Ser Arg Arg Ala Gly Thr Ser Pro Pro Pro Pro Thr Arg Arg Ser 100 105 110 Ala Ser Thr Arg His Thr Ala Cys Thr Thr Gly Cys Ser Pro Ser Pro 115 120 125 Ala Gly Thr Pro Phe Leu Val Ala Val Asp Asp Leu Thr His Ala Asp 130 135 140 Thr Ala Ser Leu Arg Phe Leu Leu Tyr Cys Ala Ala His His Asp Gln 145 150 155 160 Gly Gly Ile Gly Phe Val Met Thr Glu Arg Ala Ser Gln Arg Ala Gly 165 170 175 Tyr Arg Val Phe Arg Ala Glu Leu Leu Arg Gln Pro His Cys Arg Asn 180 185 190 Met Trp Leu Ser Gly Leu Pro Pro Ser Gly Val Arg Gln Leu Leu Ala 195 200 205 His Tyr Tyr Gly Pro Glu Ala Ala Glu Arg Arg Ala Pro Ala Tyr His 210 215 220 Ala Thr Thr Gly Gly Asn Pro Leu Leu Leu Arg Ala Leu Thr Gln Asp 225 230 235 240 Arg Gln Ala Ser His Thr Thr Leu Gly Ala Ala Gly Gly Asp Glu Pro 245 250 255 Val His Gly Asp Ala Phe Ala Gln Ala Val Leu Asp Cys Leu His Arg 260 265 270 Ser Ala Glu Gly Thr Leu Glu Thr Ala Arg Trp Leu Ala Val Leu Glu 275 280 285 Gln Ser Asp Pro Leu Leu Val Glu Arg Leu Thr Gly Thr Thr Ala Ala 290 295 300 Ala Val Glu Arg His Ile Gln Glu Leu Ala Ala Ile Gly Leu Leu Asp 305 310 315 320 Glu Asp Gly Thr Leu Gly Gln Pro Ala Ile Arg Glu Ala Ala Leu Gln 325 330 335 Asp Leu Pro Ala Gly Glu Arg Thr Glu Leu His Arg Arg Ala Ala Glu 340 345 350 Gln Leu His Arg Asp Gly Ala Asp Glu Asp Thr Val Ala Arg His Leu 355 360 365 Leu Val Gly Gly Ala Pro Asp Ala Pro Trp Ala Leu Pro Leu Leu Glu 370 375 380 Arg Gly Ala Gln Gln Ala Leu Phe Asp Asp Arg Leu Asp Asp Ala Phe 385 390 395 400 Arg Ile Leu Glu Phe Ala Val Arg Ser Ser Thr Asp Asn Thr Gln Leu 405 410 415 Ala Arg Leu Ala Pro His Leu Val Ala Ala Ser Trp Arg Met Asn Pro 420 425 430 His Met Thr Thr Arg Ala Leu Ala Leu Phe Asp Arg Leu Leu Ser Gly 435 440 445 Glu Leu Pro Pro Ser His Pro Val Met Ala Leu Ile Arg Cys Leu Val 450 455 460 Trp Tyr Gly Arg Leu Pro Glu Ala Ala Asp Ala Leu Ser Arg Leu Arg 465 470 475 480 Pro Ser Ser Asp Asn Asp Ala Leu Glu Leu Ser Leu Thr Arg Met Trp 485 490 495 Leu Ala Ala Leu Cys Pro Pro Leu Leu Glu Ser Leu Pro Ala Thr Pro 500 505 510 Glu Pro Glu Arg Gly Pro Val Pro Val Arg Leu Ala Pro Arg Thr Thr 515 520 525 Ala Leu Gln Ala Gln Ala Gly Val Phe Gln Arg Gly Pro Asp Asn Ala 530 535 540 Ser Val Ala Gln Ala Glu Gln Ile Leu Gln Gly Cys Arg Leu Ser Glu 545 550 555 560 Glu Thr Tyr Glu Ala Leu Glu Thr Ala Leu Leu Val Leu Val His Ala 565 570 575 Asp Arg Leu Asp Arg Ala Leu Phe Trp Ser Asp Ala Leu Leu Ala Glu 580 585 590 Ala Val Glu Arg Arg Ser Leu Gly Trp Glu Ala Val Phe Ala Ala Thr 595 600 605 Arg Ala Met Ile Ala Ile Arg Cys Gly Asp Leu Pro Thr Ala Arg Glu 610 615 620 Arg Ala Glu Leu Ala Leu Ser His Ala Ala Pro Glu Ser Trp Gly Leu 625 630 635 640 Ala Val Gly Met Pro Leu Ser Ala Leu Leu Leu Ala Cys Thr Glu Ala 645 650 655 Gly Glu Tyr Glu Gln Ala Glu Arg Val Leu Arg Gln Pro Val Pro Asp 660 665 670 Ala Met Phe Asp Ser Arg His Gly Met Glu Tyr Met His Ala Arg Gly 675 680 685 Arg Tyr Trp Leu Ala Xaa Gly Arg Leu His Ala Ala Leu Gly Glu Phe 690 695 700 Met Leu Cys Gly Glu Ile Leu Gly Ser Trp Asn Leu Asp Gln Pro Ser 705 710 715 720 Ile Val Pro Trp Arg Thr Ser Ala Ala Glu Val Tyr Leu Arg Leu Gly 725 730 735 Asn Arg Gln Lys Ala Arg Ala Leu Ala Glu Ala Gln Leu Ala Leu Val 740 745 750 Arg Pro Gly Arg Ser Arg Thr Arg Gly Leu Thr Leu Arg Val Leu Ala 755 760 765 Ala Ala Val Asp Gly Gln Gln Ala Glu Arg Leu His Ala Glu Ala Val 770 775 780 Asp Met Leu His Asp Ser Gly Asp Arg Leu Glu His Ala Arg Ala Leu 785 790 795 800 Ala Gly Met Ser Arg His Gln Gln Ala Gln Gly Asp Asn Tyr Arg Ala 805 810 815 Arg Met Thr Ala Arg Leu Ala Gly Asp Met Ala Trp Ala Cys Gly Ala 820 825 830 Tyr Pro Leu Ala Glu Glu Ile Val Pro Gly Arg Gly Gly Arg Arg Ala 835 840 845 Lys Ala Val Ser Thr Glu Leu Glu Leu Pro Gly Gly Pro Asp Val Gly 850 855 860 Leu Leu Ser Glu Ala Glu Arg Arg Val Ala Ala Leu Ala Ala Arg Gly 865 870 875 880 Leu Thr Asn Arg Gln Ile Ala Arg Arg Leu Cys Val Thr Ala Ser Thr 885 890 895 Val Glu Gln His Leu Thr Arg Val Tyr Arg Lys Leu Asn Val Thr Arg 900 905 910 Arg Ala Asp Leu Pro Ile Ser Leu Ala Gln Asp Lys Ser Val Thr Ala 915 920 925 14 292 PRT Streptomyces venezuelae 14 Met Lys Gly Ile Val Leu Ala Gly Gly Ser Gly Thr Arg Leu His Pro 1 5 10 15 Ala Thr Ser Val Ile Ser Lys Gln Ile Leu Pro Val Tyr Asn Lys Pro 20 25 30 Met Ile Tyr Tyr Pro Leu Ser Val Leu Met Leu Gly Gly Ile Arg Glu 35 40 45 Ile Gln Ile Ile Ser Thr Pro Gln His Ile Glu Leu Phe Gln Ser Leu 50 55 60 Leu Gly Asn Gly Arg His Leu Gly Ile Glu Leu Asp Tyr Ala Val Gln 65 70 75 80 Lys Glu Pro Ala Gly Ile Ala Asp Ala Leu Leu Val Gly Ala Glu His 85 90 95 Ile Gly Asp Asp Thr Cys Ala Leu Ile Leu Gly Asp Asn Ile Phe His 100 105 110 Gly Pro Gly Leu Tyr Thr Leu Leu Arg Asp Ser Ile Ala Arg Leu Asp 115 120 125 Gly Cys Val Leu Phe Gly Tyr Pro Val Lys Asp Pro Glu Arg Tyr Gly 130 135 140 Val Ala Glu Val Asp Ala Thr Gly Arg Leu Thr Asp Leu Val Glu Lys 145 150 155 160 Pro Val Lys Pro Arg Ser Asn Leu Ala Val Thr Gly Leu Tyr Leu Tyr 165 170 175 Asp Asn Asp Val Val Asp Ile Ala Lys Asn Ile Arg Pro Ser Pro Arg 180 185 190 Gly Glu Leu Glu Ile Thr Asp Val Asn Arg Val Tyr Leu Glu Arg Gly 195 200 205 Arg Ala Glu Leu Val Asn Leu Gly Arg Gly Phe Ala Trp Leu Asp Thr 210 215 220 Gly Thr His Asp Ser Leu Leu Arg Ala Ala Gln Tyr Val Gln Val Leu 225 230 235 240 Glu Glu Arg Gln Gly Val Trp Ile Ala Gly Leu Glu Glu Ile Ala Phe 245 250 255 Arg Met Gly Phe Ile Asp Ala Glu Ala Cys His Gly Leu Gly Glu Gly 260 265 270 Leu Ser Arg Thr Glu Tyr Gly Ser Tyr Leu Met Glu Ile Ala Gly Arg 275 280 285 Glu Gly Ala Pro 290 15 337 PRT Streptomyces venezuelae 15 Val Arg Leu Leu Val Thr Gly Gly Ala Gly Phe Ile Gly Ser His Phe 1 5 10 15 Val Arg Gln Leu Leu Ala Gly Ala Tyr Pro Asp Val Pro Ala Asp Glu 20 25 30 Val Ile Val Leu Asp Ser Leu Thr Tyr Ala Gly Asn Arg Ala Asn Leu 35 40 45 Ala Pro Val Asp Ala Asp Pro Arg Leu Arg Phe Val His Gly Asp Ile 50 55 60 Arg Asp Ala Gly Leu Leu Ala Arg Glu Leu Arg Gly Val Asp Ala Ile 65 70 75 80 Val His Phe Ala Ala Glu Ser His Val Asp Arg Ser Ile Ala Gly Ala 85 90 95 Ser Val Phe Thr Glu Thr Asn Val Gln Gly Thr Gln Thr Leu Leu Gln 100 105 110 Cys Ala Val Asp Ala Gly Val Gly Arg Val Val His Val Ser Thr Asp 115 120 125 Glu Val Tyr Gly Ser Ile Asp Ser Gly Ser Trp Thr Glu Ser Ser Pro 130 135 140 Leu Glu Pro Asn Ser Pro Tyr Ala Ala Ser Lys Ala Gly Ser Asp Leu 145 150 155 160 Val Ala Arg Ala Tyr His Arg Thr Tyr Gly Leu Asp Val Arg Ile Thr 165 170 175 Arg Cys Cys Asn Asn Tyr Gly Pro Tyr Gln His Pro Glu Lys Leu Ile 180 185 190 Pro Leu Phe Val Thr Asn Leu Leu Asp Gly Gly Thr Leu Pro Leu Tyr 195 200 205 Gly Asp Gly Ala Asn Val Arg Glu Trp Val His Thr Asp Asp His Cys 210 215 220 Arg Gly Ile Ala Leu Val Leu Ala Gly Gly Arg Ala Gly Glu Ile Tyr 225 230 235 240 His Ile Gly Gly Gly Leu Glu Leu Thr Asn Arg Glu Leu Thr Gly Ile 245 250 255 Leu Leu Asp Ser Leu Gly Ala Asp Trp Ser Ser Val Arg Lys Val Ala 260 265 270 Asp Arg Lys Gly His Asp Leu Arg Tyr Ser Leu Asp Gly Gly Lys Ile 275 280 285 Glu Arg Glu Leu Gly Tyr Arg Pro Gln Val Ser Phe Ala Asp Gly Leu 290 295 300 Ala Arg Thr Val Arg Trp Tyr Arg Glu Asn Arg Gly Trp Trp Glu Pro 305 310 315 320 Leu Lys Ala Thr Ala Pro Gln Leu Pro Ala Thr Ala Val Glu Val Ser 325 330 335 Ala 16 332 PRT Streptomyces venezuelae 16 Ile Gly Tyr Asp Ser Ser Lys Lys Gly Phe Asp Gly Ala Ser Cys Gly 1 5 10 15 Val Ser Val Ser Ile Gly Ser Gln Ser Pro Asp Ile Ala Gln Gly Val 20 25 30 Asp Thr Ala Tyr Glu Lys Arg Val Glu Gly Ala Ser Gln Arg Asp Glu 35 40 45 Gly Asp Glu Leu Asp Lys Gln Gly Ala Gly Asp Gln Gly Leu Met Phe 50 55 60 Gly Tyr Ala Ser Asp Glu Thr Pro Glu Leu Met Pro Leu Pro Ile His 65 70 75 80 Leu Ala His Arg Leu Ser Arg Arg Leu Thr Glu Val Arg Lys Asn Gly 85 90 95 Thr Ile Pro Tyr Leu Arg Pro Asp Gly Lys Thr Gln Val Thr Ile Glu 100 105 110 Tyr Asp Gly Asp Arg Ala Val Arg Leu Asp Thr Val Val Val Ser Ser 115 120 125 Gln His Ala Ser Asp Ile Asp Leu Glu Ser Leu Leu Ala Pro Asp Val 130 135 140 Arg Lys Phe Val Val Glu His Val Leu Ala Gln Leu Val Glu Asp Gly 145 150 155 160 Ile Lys Leu Asp Thr Asp Gly Tyr Arg Leu Leu Val Asn Pro Thr Gly 165 170 175 Arg Phe Glu Ile Gly Gly Pro Met Gly Asp Ala Gly Leu Thr Gly Arg 180 185 190 Lys Ile Ile Ile Asp Thr Tyr Gly Gly Met Ala Arg His Gly Gly Gly 195 200 205 Ala Phe Ser Gly Lys Asp Pro Ser Lys Val Asp Arg Ser Ala Ala Tyr 210 215 220 Ala Met Arg Trp Val Ala Lys Asn Val Val Ala Ala Gly Leu Ala Ser 225 230 235 240 Arg Cys Glu Val Gln Val Ala Tyr Ala Ile Gly Lys Ala Glu Pro Val 245 250 255 Gly Leu Phe Val Glu Thr Phe Gly Thr His Lys Ile Glu Thr Glu Lys 260 265 270 Ile Glu Asn Ala Ile Gly Glu Val Phe Asp Leu Arg Pro Ala Ala Ile 275 280 285 Ile Arg Asp Leu Asp Leu Leu Arg Pro Ile Tyr Ser Gln Thr Ala Ala 290 295 300 Tyr Gly His Phe Gly Arg Glu Leu Pro Asp Phe Thr Trp Glu Arg Thr 305 310 315 320 Asp Arg Val Asp Ala Leu Lys Lys Ala Ala Gly Leu 325 330 17 230 PRT Streptomyces venezuelae 17 Met Arg Ile Ala Val Thr Gly Ser Ile Ala Thr Asp His Leu Met Thr 1 5 10 15 Phe Pro Gly Arg Phe Ala Glu Gln Ile Leu Pro Asp Gln Leu Ala His 20 25 30 Val Ser Leu Ser Phe Leu Val Asp Thr Leu Asp Ile Arg His Gly Gly 35 40 45 Val Ala Ala Asn Ile Ala Tyr Gly Leu Gly Leu Leu Gly Arg Arg Pro 50 55 60 Val Leu Val Gly Ala Val Gly Lys Asp Phe Asp Gly Tyr Gly Gln Leu 65 70 75 80 Leu Arg Ala Ala Gly Val Asp Thr Asp Ser Val Arg Val Ser Asp Arg 85 90 95 Gln His Thr Ala Arg Phe Met Cys Thr Thr Asp Glu Asp Gly Asn Gln 100 105 110 Leu Ala Ser Phe Tyr Ala Gly Ala Met Ala Glu Ala Arg Asp Ile Asp 115 120 125 Leu Gly Glu Thr Ala Gly Arg Pro Gly Gly Ile Asp Leu Val Leu Val 130 135 140 Gly Ala Asp Asp Pro Glu Ala Met Val Arg His Thr Arg Val Cys Arg 145 150 155 160 Glu Leu Gly Leu Arg Arg Ala Ala Asp Pro Ser Gln Gln Leu Ala Arg 165 170 175 Leu Glu Gly Asp Ser Val Arg Glu Leu Val Asp Gly Ala Glu Leu Leu 180 185 190 Phe Thr Asn Ala Tyr Glu Arg Ala Leu Leu Leu Ser Lys Thr Gly Trp 195 200 205 Thr Glu Gln Glu Val Leu Ala Arg Val Gly Thr Trp Ile Thr Thr Leu 210 215 220 Gly Ala Lys Gly Cys Arg 225 230 18 416 PRT Streptomyces venezuelae 18 Val Arg Arg Thr Gln Gln Gly Thr Thr Ala Ser Pro Pro Val Leu Asp 1 5 10 15 Leu Gly Ala Leu Gly Gln Asp Phe Ala Ala Asp Pro Tyr Pro Thr Tyr 20 25 30 Ala Arg Leu Arg Ala Glu Gly Pro Ala His Arg Val Arg Thr Pro Glu 35 40 45 Gly Asp Glu Val Trp Leu Val Val Gly Tyr Asp Arg Ala Arg Ala Val 50 55 60 Leu Ala Asp Pro Arg Phe Ser Lys Asp Trp Arg Asn Ser Thr Thr Pro 65 70 75 80 Leu Thr Glu Ala Glu Ala Ala Leu Asn His Asn Met Leu Glu Ser Asp 85 90 95 Pro Pro Arg His Thr Arg Leu Arg Lys Leu Val Ala Arg Glu Phe Thr 100 105 110 Met Arg Arg Val Glu Leu Leu Arg Pro Arg Val Gln Glu Ile Val Asp 115 120 125 Gly Leu Val Asp Ala Met Leu Ala Ala Pro Asp Gly Arg Ala Asp Leu 130 135 140 Met Glu Ser Leu Ala Trp Pro Leu Pro Ile Thr Val Ile Ser Glu Leu 145 150 155 160 Leu Gly Val Pro Glu Pro Asp Arg Ala Ala Phe Arg Val Trp Thr Asp 165 170 175 Ala Phe Val Phe Pro Asp Asp Pro Ala Gln Ala Gln Thr Ala Met Ala 180 185 190 Glu Met Ser Gly Tyr Leu Ser Arg Leu Ile Asp Ser Lys Arg Gly Gln 195 200 205 Asp Gly Glu Asp Leu Leu Ser Ala Leu Val Arg Thr Ser Asp Glu Asp 210 215 220 Gly Ser Arg Leu Thr Ser Glu Glu Leu Leu Gly Met Ala His Ile Leu 225 230 235 240 Leu Val Ala Gly His Glu Thr Thr Val Asn Leu Ile Ala Asn Gly Met 245 250 255 Tyr Ala Leu Leu Ser His Pro Asp Gln Leu Ala Ala Leu Arg Ala Asp 260 265 270 Met Thr Leu Leu Asp Gly Ala Val Glu Glu Met Leu Arg Tyr Glu Gly 275 280 285 Pro Val Glu Ser Ala Thr Tyr Arg Phe Pro Val Glu Pro Val Asp Leu 290 295 300 Asp Gly Thr Val Ile Pro Ala Gly Asp Thr Val Leu Val Val Leu Ala 305 310 315 320 Asp Ala His Arg Thr Pro Glu Arg Phe Pro Asp Pro His Arg Phe Asp 325 330 335 Ile Arg Arg Asp Thr Ala Gly His Leu Ala Phe Gly His Gly Ile His 340 345 350 Phe Cys Ile Gly Ala Pro Leu Ala Arg Leu Glu Ala Arg Ile Ala Val 355 360 365 Arg Ala Leu Leu Glu Arg Cys Pro Asp Leu Ala Leu Asp Val Ser Pro 370 375 380 Gly Glu Leu Val Trp Tyr Pro Asn Pro Met Ile Arg Gly Leu Lys Ala 385 390 395 400 Leu Pro Ile Arg Trp Arg Arg Gly Arg Glu Ala Gly Arg Arg Thr Gly 405 410 415 19 38506 DNA Streptomyces venezuelae 19 gatcatgcgg agcactcctt ctctcgtgct cctaccggtg atgtgcgcgc cgaattgatt 60 cgtggagaga tgtcgacagt gtccaagagt gagtccgagg aattcgtgtc cgtgtcgaac 120 gacgccggtt ccgcgcacgg cacagcggaa cccgtcgccg tcgtcggcat ctcctgccgg 180 gtgcccggcg cccgggaccc gagagagttc tgggaactcc tggcggcagg cggccaggcc 240 gtcaccgacg tccccgcgga ccgctggaac gccggcgact tctacgaccc ggaccgctcc 300 gcccccggcc gctcgaacag ccggtggggc gggttcatcg aggacgtcga ccggttcgac 360 gccgccttct tcggcatctc gccccgcgag gccgcggaga tggacccgca gcagcggctc 420 gccctggagc tgggctggga ggccctggag cgcgccggga tcgacccgtc ctcgctcacc 480 ggcacccgca ccggcgtctt cgccggcgcc atctgggacg actacgccac cctgaagcac 540 cgccagggcg gcgccgcgat caccccgcac accgtcaccg gcctccaccg cggcatcatc 600 gcgaaccgac tctcgtacac gctcgggctc cgcggcccca gcatggtcgt cgactccggc 660 cagtcctcgt cgctcgtcgc cgtccacctc gcgtgcgaga gcctgcggcg cggcgagtcc 720 gagctcgccc tcgccggcgg cgtctcgctc aacctggtgc cggacagcat catcggggcg 780 agcaagttcg gcggcctctc ccccgacggc cgcgcctaca ccttcgacgc gcgcgccaac 840 ggctacgtac gcggcgaggg cggcggtttc gtcgtcctga agcgcctctc ccgggccgtc 900 gccgacggcg acccggtgct cgccgtgatc cggggcagcg ccgtcaacaa cggcggcgcc 960 gcccagggca tgacgacccc cgacgcgcag gcgcaggagg ccgtgctccg cgaggcccac 1020 gagcgggccg ggaccgcgcc ggccgacgtg cggtacgtcg agctgcacgg caccggcacc 1080 cccgtgggcg acccgatcga ggccgctgcg ctcggcgccg ccctcggcac cggccgcccg 1140 gccggacagc cgctcctggt cggctcggtc aagacgaaca tcggccacct ggagggcgcg 1200 gccggcatcg ccggcctcat caaggccgtc ctggcggtcc gcggtcgcgc gctgcccgcc 1260 agcctgaact acgagacccc gaacccggcg atcccgttcg aggaactgaa cctccgggtg 1320 aacacggagt acctgccgtg ggagccggag cacgacgggc agcggatggt cgtcggcgtg 1380 tcctcgttcg gcatgggcgg cacgaacgcg catgtcgtgc tcgaagaggc cccgggggtt 1440 gtcgagggtg cttcggtcgt ggagtcgacg gtcggcgggt cggcggtcgg cggcggtgtg 1500 gtgccgtggg tggtgtcggc gaagtccgct gccgcgctgg acgcgcagat cgagcggctt 1560 gccgcgttcg cctcgcggga tcgtacggat ggtgtcgacg cgggcgctgt cgatgcgggt 1620 gctgtcgatg cgggtgctgt cgctcgcgta ctggccggcg ggcgtgctca gttcgagcac 1680 cgggccgtcg tcgtcggcag cgggccggac gatctggcgg cagcgctggc cgcgcctgag 1740 ggtctggtcc ggggcgtggc ttccggtgtc gggcgagtgg cgttcgtgtt ccccgggcag 1800 ggcacgcagt gggccggcat gggtgccgaa ctgctggact cttccgcggt gttcgcggcg 1860 gccatggccg aatgcgaggc cgcactctcc ccgtacgtcg actggtcgct ggaggccgtc 1920 gtacggcagg cccccggtgc gcccacgctg gagcgggtcg atgtcgtgca gcctgtgacg 1980 ttcgccgtca tggtctcgct ggctcgcgtg tggcagcacc acggggtgac gccccaggcg 2040 gtcgtcggcc actcgcaggg cgagatcgcc gccgcgtacg tcgccggtgc cctgagcctg 2100 gacgacgccg ctcgtgtcgt gaccctgcgc agcaagtcca tcgccgccca cctcgccggc 2160 aagggcggca tgctgtccct cgcgctgagc gaggacgccg tcctggagcg actggccggg 2220 ttcgacgggc tgtccgtcgc cgctgtgaac gggcccaccg ccaccgtggt ctccggtgac 2280 cccgtacaga tcgaagagct tgctcgggcg tgtgaggccg atggggtccg tgcgcgggtc 2340 attcccgtcg actacgcgtc ccacagccgg caggtcgaga tcatcgagag cgagctcgcc 2400 gaggtcctcg ccgggctcag cccgcaggct ccgcgcgtgc cgttcttctc gacactcgaa 2460 ggcgcctgga tcaccgagcc cgtgctcgac ggcggctact ggtaccgcaa cctgcgccat 2520 cgtgtgggct tcgccccggc cgtcgagacc ctggccaccg acgagggctt cacccacttc 2580 gtcgaggtca gcgcccaccc cgtcctcacc atggccctcc ccgggaccgt caccggtctg 2640 gcgaccctgc gtcgcgacaa cggcggtcag gaccgcctcg tcgcctccct cgccgaagca 2700 tgggccaacg gactcgcggt cgactggagc ccgctcctcc cctccgcgac cggccaccac 2760 tccgacctcc ccacctacgc gttccagacc gagcgccact ggctgggcga gatcgaggcg 2820 ctcgccccgg cgggcgagcc ggcggtgcag cccgccgtcc tccgcacgga ggcggccgag 2880 ccggcggagc tcgaccggga cgagcagctg cgcgtgatcc tggacaaggt ccgggcgcag 2940 acggcccagg tgctggggta cgcgacaggc gggcagatcg aggtcgaccg gaccttccgt 3000 gaggccggtt gcacctccct gaccggcgtg gacctgcgca accggatcaa cgccgccttc 3060 ggcgtacgga tggcgccgtc catgatcttc gacttcccca cccccgaggc tctcgcggag 3120 cagctgctcc tcgtcgtgca cggggaggcg gcggcgaacc cggccggtgc ggagccggct 3180 ccggtggcgg cggccggtgc cgtcgacgag ccggtggcga tcgtcggcat ggcctgccgc 3240 ctgcccggtg gggtcgcctc gccggaggac ctgtggcggc tggtggccgg cggcggggac 3300 gcgatctcgg agttcccgca ggaccgcggc tgggacgtgg aggggctgta ccacccggat 3360 cccgagcacc ccggcacgtc gtacgtccgc cagggcggtt tcatcgagaa cgtcgccggc 3420 ttcgacgcgg ccttcttcgg gatctcgccg cgcgaggccc tcgccatgga cccgcagcag 3480 cggctcctcc tcgaaacctc ctgggaggcc gtcgaggacg ccgggatcga cccgacctcc 3540 ctgcggggac ggcaggtcgg cgtcttcact ggggcgatga cccacgagta cgggccgagc 3600 ctgcgggacg gcggggaagg cctcgacggc tacctgctga ccggcaacac ggccagcgtg 3660 atgtcgggcc gcgtctcgta cacactcggc cttgagggcc ccgccctgac ggtggacacg 3720 gcctgctcgt cgtcgctggt cgccctgcac ctcgccgtgc aggccctgcg caagggcgag 3780 gtcgacatgg cgctcgccgg cggcgtggcc gtgatgccca cgcccgggat gttcgtcgag 3840 ttcagccggc agcgcgggct ggccggggac ggccggtcga aggcgttcgc cgcgtcggcg 3900 gacggcacca gctggtccga gggcgtcggc gtcctcctcg tcgagcgcct gtcggacgcc 3960 cgccgcaacg gacaccaggt cctcgcggtc gtccgcggca gcgccgtgaa ccaggacggc 4020 gcgagcaacg gcctcacggc tccgaacggg ccctcgcagc agcgcgtcat ccggcgcgcg 4080 ctggcggacg cccggctgac gacctccgac gtggacgtcg tcgaggcaca cggcacgggc 4140 acgcgactcg gcgacccgat cgaggcgcag gccctgatcg ccacctacgg ccagggccgt 4200 gacgacgaac agccgctgcg cctcgggtcg ttgaagtcca acatcgggca cacccaggcc 4260 gcggccggcg tctccggtgt catcaagatg gtccaggcga tgcgccacgg actgctgccg 4320 aagacgctgc acgtcgacga gccctcggac cagatcgact ggtcggctgg cgccgtggaa 4380 ctcctcaccg aggccgtcga ctggccggag aagcaggacg gcgggctgcg ccgggccgcc 4440 gtctcctcct tcgggatcag cggcaccaat gcgcatgtgg tgctcgaaga ggccccggtg 4500 gttgtcgagg gtgcttcggt cgtcgagccg tcggttggcg ggtcggcggt cggcggcggt 4560 gtgacgcctt gggtggtgtc ggcgaagtcc gctgccgcgc tcgacgcgca gatcgagcgg 4620 cttgccgcat tcgcctcgcg ggatcgtacg gatgacgccg acgccggtgc tgtcgacgcg 4680 ggcgctgtcg ctcacgtact ggctgacggg cgtgctcagt tcgagcaccg ggccgtcgcg 4740 ctcggcgccg gggcggacga cctcgtacag gcgctggccg atccggacgg gctgatacgc 4800 ggaacggctt ccggtgtcgg gcgagtggcg ttcgtgttcc ccggtcaggg cacgcagtgg 4860 gctggcatgg gtgccgaact gctggactct tccgcggtgt tcgcggcggc catggccgag 4920 tgtgaggccg cgctgtcccc gtacgtcgac tggtcgctgg aggccgtcgt acggcaggcc 4980 cccggtgcgc ccacgctgga gcgggtcgat gtcgtgcagc ctgtgacgtt cgccgtcatg 5040 gtctcgctgg ctcgcgtgtg gcagcaccac ggtgtgacgc cccaggcggt cgtcggccac 5100 tcgcagggcg agatcgccgc cgcgtacgtc gccggagccc tgcccctgga cgacgccgcc 5160 cgcgtcgtca ccctgcgcag caagtccatc gccgcccacc tcgccggcaa gggcggcatg 5220 ctgtccctcg cgctgaacga ggacgccgtc ctggagcgac tgagtgactt cgacgggctg 5280 tccgtcgccg ccgtcaacgg gcccaccgcc actgtcgtgt cgggtgaccc cgtacagatc 5340 gaagagcttg ctcaggcgtg caaggcggac ggattccgcg cgcggatcat tcccgtcgac 5400 tacgcgtccc acagccggca ggtcgagatc atcgagagcg agctcgccca ggtcctcgcc 5460 ggtctcagcc cgcaggcccc gcgcgtgccg ttcttctcga cgctcgaagg cacctggatc 5520 accgagcccg tcctcgacgg cacctactgg taccgcaacc tccgtcaccg cgtcggcttc 5580 gcccccgcca tcgagaccct ggccgtcgac gagggcttca cgcacttcgt cgaggtcagc 5640 gcccaccccg tcctcaccat gaccctcccc gagaccgtca ccggcctcgg caccctccgt 5700 cgcgaacagg gaggccaaga gcgtctggtc acctcgctcg ccgaggcgtg ggtcaacggg 5760 cttcccgtgg catggacttc gctcctgccc gccacggcct cccgccccgg tctgcccacc 5820 tacgccttcc aggccgagcg ctactggctc gagaacactc ccgccgccct ggccaccggc 5880 gacgactggc gctaccgcat cgactggaag cgcctcccgg ccgccgaggg gtccgagcgc 5940 accggcctgt ccggccgctg gctcgccgtc acgccggagg accactccgc gcaggccgcc 6000 gccgtgctca ccgcgctggt cgacgccggg gcgaaggtcg aggtgctgac ggccggggcg 6060 gacgacgacc gtgaggccct cgccgcccgg ctcaccgcac tgacgaccgg tgacggcttc 6120 accggcgtgg tctcgctcct cgacggactc gtaccgcagg tcgcctgggt ccaggcgctc 6180 ggcgacgccg gaatcaaggc gcccctgtgg tccgtcaccc agggcgcggt ctccgtcgga 6240 cgtctcgaca cccccgccga ccccgaccgg gccatgctct ggggcctcgg ccgcgtcgtc 6300 gcccttgagc accccgaacg ctgggccggc ctcgtcgacc tccccgccca gcccgatgcc 6360 gccgccctcg cccacctcgt caccgcactc tccggcgcca ccggcgagga ccagatcgcc 6420 atccgcacca ccggactcca cgcccgccgc ctcgcccgcg cacccctcca cggacgtcgg 6480 cccacccgcg actggcagcc ccacggcacc gtcctcatca ccggcggcac cggagccctc 6540 ggcagccacg ccgcacgctg gatggcccac cacggagccg aacacctcct cctcgtcagc 6600 cgcagcggcg aacaagcccc cggagccacc caactcaccg ccgaactcac cgcatcgggc 6660 gcccgcgtca ccatcgccgc ctgcgacgtc gccgaccccc acgccatgcg caccctcctc 6720 gacgccatcc ccgccgagac gcccctcacc gccgtcgtcc acaccgccgg cgcgctcgac 6780 gacggcatcg tggacacgct gaccgccgag caggtccggc gggcccaccg tgcgaaggcc 6840 gtcggcgcct cggtgctcga cgagctgacc cgggacctcg acctcgacgc gttcgtgctc 6900 ttctcgtccg tgtcgagcac tctgggcatc cccggtcagg gcaactacgc cccgcacaac 6960 gcctacctcg acgccctcgc ggctcgccgc cgggccaccg gccggtccgc cgtctcggtg 7020 gcctggggac cgtgggacgg tggcggcatg gccgccggtg acggcgtggc cgagcggctg 7080 cgcaaccacg gcgtgcccgg catggacccg gaactcgccc tggccgcact ggagtccgcg 7140 ctcggccggg acgagaccgc gatcaccgtc gcggacatcg actgggaccg cttctacctc 7200 gcgtactcct ccggtcgccc gcagcccctc gtcgaggagc tgcccgaggt gcggcgcatc 7260 atcgacgcac gggacagcgc cacgtccgga cagggcggga gctccgccca gggcgccaac 7320 cccctggccg agcggctggc cgccgcggct cccggcgagc gtacggagat cctcctcggt 7380 ctcgtacggg cgcaggccgc cgccgtgctc cggatgcgtt cgccggagga cgtcgccgcc 7440 gaccgcgcct tcaaggacat cggcttcgac tcgctcgccg gtgtcgagct gcgcaacagg 7500 ctgacccggg cgaccgggct ccagctgccc gcgacgctcg tcttcgacca cccgacgccg 7560 ctggccctcg tgtcgctgct ccgcagcgag ttcctcggtg acgaggagac ggcggacgcc 7620 cggcggtccg cggcgctgcc cgcgactgtc ggtgccggtg ccggcgccgg cgccggcacc 7680 gatgccgacg acgatccgat cgcgatcgtc gcgatgagct gccgctaccc cggtgacatc 7740 cgcagcccgg aggacctgtg gcggatgctg tccgagggcg gcgagggcat cacgccgttc 7800 cccaccgacc gcggctggga cctcgacggc ctgtacgacg ccgacccgga cgcgctcggc 7860 agggcgtacg tccgcgaggg cgggttcctg cacgacgcgg ccgagttcga cgcggagttc 7920 ttcggcgtct cgccgcgcga ggcgctggcc atggacccgc agcagcggat gctcctgacg 7980 acgtcctggg aggccttcga gcgggccggc atcgagccgg catcgctgcg cggcagcagc 8040 accggtgtct tcatcggcct ctcctaccag gactacgcgg cccgcgtccc gaacgccccg 8100 cgtggcgtgg agggttacct gctgaccggc agcacgccga gcgtcgcgtc gggccgtatc 8160 gcgtacacct tcggtctcga agggcccgcg acgaccgtcg acaccgcctg ctcgtcgtcg 8220 ctgaccgccc tgcacctggc ggtgcgggcg ctgcgcagcg gcgagtgcac gatggcgctc 8280 gccggtggcg tggcgatgat ggcgaccccg cacatgttcg tggagttcag ccgtcagcgg 8340 gcgctcgccc cggacggccg cagcaaggcc ttctcggcgg acgccgacgg gttcggcgcc 8400 gcggagggcg tcggcctgct gctcgtggag cggctctcgg acgcgcggcg caacggtcac 8460 ccggtgctcg ccgtggtccg cggtaccgcc gtcaaccagg acggcgccag caacgggctg 8520 accgcgccca acggaccctc gcagcagcgg gtgatccggc aggcgctcgc cgacgcccgg 8580 ctggcacccg gcgacatcga cgccgtcgag acgcacggca cgggaacctc gctgggcgac 8640 cccatcgagg cccagggcct ccaggccacg tacggcaagg agcggcccgc ggaacggccg 8700 ctcgccatcg gctccgtgaa gtccaacatc ggacacaccc aggccgcggc cggtgcggcg 8760 ggcatcatca agatggtcct cgcgatgcgc cacggcaccc tgccgaagac cctccacgcc 8820 gacgagccga gcccgcacgt cgactgggcg aacagcggcc tggccctcgt caccgagccg 8880 atcgactggc cggccggcac cggtccgcgc cgcgccgccg tctcctcctt cggcatcagc 8940 gggacgaacg cgcacgtcgt gctggagcag gcgccggatg ctgctggtga ggtgcttggg 9000 gccgatgagg tgcctgaggt gtctgagacg gtagcgatgg ctgggacggc tgggacctcc 9060 gaggtcgctg agggctctga ggcctccgag gcccccgcgg cccccggcag ccgtgaggcg 9120 tccctccccg ggcacctgcc ctgggtgctg tccgccaagg acgagcagtc gctgcgcggc 9180 caggccgccg ccctgcacgc gtggctgtcc gagcccgccg ccgacctgtc ggacgcggac 9240 ggaccggccc gcctgcggga cgtcgggtac acgctcgcca cgagccgtac cgccttcgcg 9300 caccgcgccg ccgtgaccgc cgccgaccgg gacgggttcc tggacgggct ggccacgctg 9360 gcccagggcg gcacctcggc ccacgtccac ctggacaccg cccgggacgg caccaccgcg 9420 ttcctcttca ccggccaggg cagtcagcgc cccggcgccg gccgtgagct gtacgaccgg 9480 caccccgtct tcgcccgggc gctcgacgag atctgcgccc acctcgacgg tcacctcgaa 9540 ctgcccctgc tcgacgtgat gttcgcggcc gagggcagcg cggaggccgc gctgctcgac 9600 gagacgcggt acacgcagtg cgcgctgttc gccctggagg tcgcgctctt ccggctcgtc 9660 gagagctggg gcatgcggcc ggccgcactg ctcggtcact cggtcggcga gatcgccgcc 9720 gcgcacgtcg ccggtgtgtt ctcgctcgcc gacgccgccc gcctggtcgc cgcgcgcggc 9780 cggctcatgc aggagctgcc cgccggtggc gcgatgctcg ccgtccaggc cgcggaggac 9840 gagatccgcg tgtggctgga gacggaggag cggtacgcgg gacgtctgga cgtcgccgcc 9900 gtcaacggcc ccgaggccgc cgtcctgtcc ggcgacgcgg acgcggcgcg ggaggcggag 9960 gcgtactggt ccgggctcgg ccgcaggacc cgcgcgctgc gggtcagcca cgccttccac 10020 tccgcgcaca tggacggcat gctcgacggg ttccgcgccg tcctggagac ggtggagttc 10080 cggcgcccct ccctgaccgt ggtctcgaac gtcaccggcc tggccgccgg cccggacgac 10140 ctgtgcgacc ccgagtactg ggtccggcac gtccgcggca ccgtccgctt cctcgacggc 10200 gtccgtgtcc tgcgcgacct cggcgtgcgg acctgcctgg agctgggccc cgacggggtc 10260 ctcaccgcca tggcggccga cggcctcgcg gacacccccg cggattccgc tgccggctcc 10320 cccgtcggct ctcccgccgg ctctcccgcc gactccgccg ccggcgcgct ccggccccgg 10380 ccgctgctcg tggcgctgct gcgccgcaag cggtcggaga ccgagaccgt cgcggacgcc 10440 ctcggcaggg cgcacgccca cggcaccgga cccgactggc acgcctggtt cgccggctcc 10500 ggggcgcacc gcgtggacct gcccacgtac tccttccggc gcgaccgcta ctggctggac 10560 gccccggcgg ccgacaccgc ggtggacacc gccggcctcg gtctcggcac cgccgaccac 10620 ccgctgctcg gcgccgtggt cagccttccg gaccgggacg gcctgctgct caccggccgc 10680 ctctccctgc gcacccaccc gtggctcgcg gaccacgccg tcctggggag cgtcctgctc 10740 cccggcgccg cgatggtcga actcgccgcg cacgctgcgg agtccgccgg tctgcgtgac 10800 gtgcgggagc tgaccctcct tgaaccgctg gtactgcccg agcacggtgg cgtcgagctg 10860 cgcgtgacgg tcggggcgcc ggccggagag cccggtggcg agtcggccgg ggacggcgca 10920 cggcccgtct ccctccactc gcggctcgcc gacgcgcccg ccggtaccgc ctggtcctgc 10980 cacgcgaccg gtctgctggc caccgaccgg cccgagcttc ccgtcgcgcc cgaccgtgcg 11040 gccatgtggc cgccgcaggg cgccgaggag gtgccgctcg acggtctcta cgagcggctc 11100 gacgggaacg gcctcgcctt cggtccgctg ttccaggggc tgaacgcggt gtggcggtac 11160 gagggtgagg tcttcgccga catcgcgctc cccgccacca cgaatgcgac cgcgcccgcg 11220 accgcgaacg gcggcgggag tgcggcggcg gccccctacg gcatccaccc cgccctgctc 11280 gacgcttcgc tgcacgccat cgcggtcggc ggtctcgtcg acgagcccga gctcgtccgc 11340 gtccccttcc actggagcgg tgtcaccgtg cacgcggccg gtgccgcggc ggcccgggtc 11400 cgtctcgcct ccgcggggac ggacgccgtc tcgctgtccc tgacggacgg cgagggacgc 11460 ccgctggtct ccgtggaacg gctcacgctg cgcccggtca ccgccgatca ggcggcggcg 11520 agccgcgtcg gcgggctgat gcaccgggtg gcctggcgtc cgtacgccct cgcctcgtcc 11580 ggcgaacagg acccgcacgc cacttcgtac gggccgaccg ccgtcctcgg caaggacgag 11640 ctgaaggtcg ccgccgccct ggagtccgcg ggcgtcgaag tcgggctcta ccccgacctg 11700 gccgcgctgt cccaggacgt ggcggccggc gccccggcgc cccgtaccgt ccttgcgccg 11760 ctgcccgcgg gtcccgccga cggcggcgcg gagggtgtac ggggcacggt ggcccggacg 11820 ctggagctgc tccaggcctg gctggccgac gagcacctcg cgggcacccg cctgctcctg 11880 gtcacccgcg gtgcggtgcg ggaccccgag gggtccggcg ccgacgatgg cggcgaggac 11940 ctgtcgcacg cggccgcctg gggtctcgta cggaccgcgc agaccgagaa ccccggccgc 12000 ttcggccttc tcgacctggc cgacgacgcc tcgtcgtacc ggaccctgcc gtcggtgctc 12060 tccgacgcgg gcctgcgcga cgaaccgcag ctcgccctgc acgacggcac catcaggctg 12120 gcccgcctgg cctccgtccg gcccgagacc ggcaccgccg caccggcgct cgccccggag 12180 ggcacggtcc tgctgaccgg cggcaccggc ggcctgggcg gactggtcgc ccggcacgtg 12240 gtgggcgagt ggggcgtacg acgcctgctg ctggtgagcc ggcggggcac ggacgccccg 12300 ggcgccgacg agctcgtgca cgagctggag gccctgggag ccgacgtctc ggtggccgcg 12360 tgcgacgtcg ccgaccgcga agccctcacc gccgtactcg acgccatccc cgccgaacac 12420 ccgctcaccg cggtcgtcca cacggcaggc gtcctctccg acggcaccct cccgtccatg 12480 acgacggagg acgtggaaca cgtactgcgg cccaaggtcg acgccgcgtt cctcctcgac 12540 gaactcacct cgacgcccgc atacgacctg gcagcgttcg tcatgttctc ctccgccgcc 12600 gccgtcttcg gtggcgcggg gcagggcgcc tacgccgccg ccaacgccac cctcgacgcc 12660 ctcgcctggc gccgccgggc agccggactc cccgccctct ccctcggctg gggcctctgg 12720 gccgagacca gcggcatgac cggcgagctc ggccaggcgg acctgcgccg gatgagccgc 12780 gcgggcatcg gcgggatcag cgacgccgag ggcatcgcgc tcctcgacgc cgccctccgc 12840 gacgaccgcc acccggtcct gctgcccctg cggctcgacg ccgccgggct gcgggacgcg 12900 gccgggaacg acccggccgg aatcccggcg ctcttccggg acgtcgtcgg cgccaggacc 12960 gtccgggccc ggccgtccgc ggcctccgcc tcgacgacag ccgggacggc cggcacgccg 13020 gggacggcgg acggcgcggc ggaaacggcg gcggtcacgc tcgccgaccg ggccgccacc 13080 gtggacgggc ccgcacggca gcgcctgctg ctcgagttcg tcgtcggcga ggtcgccgaa 13140 gtactcggcc acgcccgcgg tcaccggatc gacgccgaac ggggcttcct cgacctcggc 13200 ttcgactccc tgaccgccgt cgaactccgc aaccggctca actccgccgg tggcctcgcc 13260 ctcccggcga ccctggtctt cgaccaccca agcccggcgg cactcgcctc ccacctggac 13320 gccgagctgc cgcgcggcgc ctcggaccag gacggagccg ggaaccggaa cgggaacgag 13380 aacgggacga cggcgtcccg gagcaccgcc gagacggacg cgctgctggc acaactgacc 13440 cgcctggaag gcgccttggt gctgacgggc ctctcggacg cccccgggag cgaagaagtc 13500 ctggagcacc tgcggtccct gcgctcgatg gtcacgggcg agaccgggac cgggaccgcg 13560 tccggagccc cggacggcgc cgggtccggc gccgaggacc ggccctgggc ggccggggac 13620 ggagccgggg gcgggagtga ggacggcgcg ggagtgccgg acttcatgaa cgcctcggcc 13680 gaggaactct tcggcctcct cgaccaggac cccagcacgg actgatccct gccgcacggt 13740 cgcctcccgc cccggacccc gtcccgggca cctcgactcg aatcacttca tgcgcgcctc 13800 gggcgcctcc aggaactcaa ggggacagcg tgtccacggt gaacgaagag aagtacctcg 13860 actacctgcg tcgtgccacg gcggacctcc acgaggcccg tggccgcctc cgcgagctgg 13920 aggcgaaggc gggcgagccg gtggcgatcg tcggcatggc ctgccgcctg cccggcggcg 13980 tcgcctcgcc cgaggacctg tggcggctgg tggccggcgg cgaggacgcg atctcggagt 14040 tcccccagga ccgcggctgg gacgtggagg gcctgtacga cccgaacccg gaggccacgg 14100 gcaagagtta cgcccgcgag gccggattcc tgtacgaggc gggcgagttc gacgccgact 14160 tcttcgggat ctcgccgcgc gaggccctcg ccatggaccc gcagcagcgt ctcctcctgg 14220 aggcctcctg ggaggcgttc gagcacgccg ggatcccggc ggccaccgcg cgcggcacct 14280 cggtcggcgt cttcaccggc gtgatgtacc acgactacgc cacccgtctc accgatgtcc 14340 cggagggcat cgagggctac ctgggcaccg gcaactccgg cagtgtcgcc tcgggccgcg 14400 tcgcgtacac gcttggcctg gaggggccgg ccgtcacggt cgacaccgcc tgctcgtcct 14460 cgctggtcgc cctgcacctc gccgtgcagg ccctgcgcaa gggcgaggtc gacatggcgc 14520 tcgccggcgg cgtgacggtc atgtcgacgc ccagcacctt cgtcgagttc agccgtcagc 14580 gcgggctggc gccggacggc cggtcgaagt ccttctcgtc gacggccgac ggcaccagct 14640 ggtccgaggg cgtcggcgtc ctcctcgtcg agcgcctgtc cgacgcgcgt cgcaagggcc 14700 atcggatcct cgccgtggtc cggggcaccg ccgtcaacca ggacggcgcc agcagcggcc 14760 tcacggctcc gaacgggccg tcgcagcagc gcgtcatccg acgtgccctg gcggacgccc 14820 ggctcacgac ctccgacgtg gacgtcgtcg aggcccacgg cacgggtacg cgactcggcg 14880 acccgatcga ggcgcaggcc gtcatcgcca cgtacgggca gggccgtgac ggcgaacagc 14940 cgctgcgcct cgggtcgttg aagtccaaca tcggacacac ccaggccgcc gccggtgtct 15000 ccggcgtgat caagatggtc caggcgatgc gccacggcgt cctgccgaag acgctccacg 15060 tggagaagcc gacggaccag gtggactggt ccgcgggcgc ggtcgagctg ctcaccgagg 15120 ccatggactg gccggacaag ggcgacggcg gactgcgcag ggccgcggtc tcctccttcg 15180 gcgtcagcgg gacgaacgcg cacgtcgtgc tcgaagaggc cccggcggcc gaggagaccc 15240 ctgcctccga ggcgaccccg gccgtcgagc cgtcggtcgg cgccggcctg gtgccgtggc 15300 tggtgtcggc gaagactccg gccgcgctgg acgcccagat cggacgcctc gccgcgttcg 15360 cctcgcaggg ccgtacggac gccgccgatc cgggcgcggt cgctcgcgta ctggccggcg 15420 ggcgcgccga gttcgagcac cgggccgtcg tgctcggcac cggacaggac gatttcgcgc 15480 aggcgctgac cgctccggaa ggactgatac gcggcacgcc ctcggacgtg ggccgggtgg 15540 cgttcgtgtt ccccggtcag ggcacgcagt gggccgggat gggcgccgaa ctcctcgacg 15600 tgtcgaagga gttcgcggcg gccatggccg agtgcgagag cgcgctctcc cgctatgtcg 15660 actggtcgct ggaggccgtc gtccggcagg cgccgggcgc gcccacgctg gagcgggtcg 15720 acgtcgtcca gcccgtgacc ttcgctgtca tggtttcgct ggcgaaggtc tggcagcacc 15780 acggcgtgac gccgcaggcc gtcgtcggcc actcgcaggg cgagatcgcc gccgcgtacg 15840 tcgccggtgc cctcaccctc gacgacgccg cccgcgtcgt caccctgcgc agcaagtcca 15900 tcgccgccca cctcgccggc aagggcggca tgatctccct cgccctcagc gaggaagcca 15960 cccggcagcg catcgagaac ctccacggac tgtcgatcgc cgccgtcaac ggccccaccg 16020 ccaccgtggt ttcgggcgac cccacccaga tccaagagct cgctcaggcg tgtgaggccg 16080 acggggtccg cgcacggatc atccccgtcg actacgcctc ccacagcgcc cacgtcgaga 16140 ccatcgagag cgaactcgcc gaggtcctcg ccgggctcag cccgcggaca cctgaggtgc 16200 cgttcttctc gacactcgaa ggcgcctgga tcaccgagcc ggtgctcgac ggcacctact 16260 ggtaccgcaa cctccgccac cgcgtcggct tcgcccccgc cgtcgagacc ctcgccaccg 16320 acgaaggctt cacccacttc atcgaggtca gcgcccaccc cgtcctcacc atgaccctcc 16380 ccgagaccgt caccggcctc ggcaccctcc gccgcgaaca gggaggccag gagcgtctgg 16440 tcacctcact cgccgaagcc tggaccaacg gcctcaccat cgactgggcg cccgtcctcc 16500 ccaccgcaac cggccaccac cccgagctcc ccacctacgc cttccagcgc cgtcactact 16560 ggctccacga ctcccccgcc gtccagggct ccgtgcagga ctcctggcgc taccgcatcg 16620 actggaagcg cctcgcggtc gccgacgcgt ccgagcgcgc cgggctgtcc gggcgctggc 16680 tcgtcgtcgt ccccgaggac cgttccgccg aggccgcccc ggtgctcgcc gcgctgtccg 16740 gcgccggcgc cgaccccgta cagctggacg tgtccccgct gggcgaccgg cagcggctcg 16800 ccgcgacgct gggcgaggcc ctggcggcgg ccggtggagc cgtcgacggc gtcctctcgc 16860 tgctcgcgtg ggacgagagc gcgcaccccg gccaccccgc ccccttcacc cggggcaccg 16920 gcgccaccct caccctggtg caggcgctgg aggacgccgg cgtcgccgcc ccgctgtggt 16980 gcgtgaccca cggcgcggtg tccgtcggcc gggccgacca cgtcacctcc cccgcccagg 17040 ccatggtgtg gggcatgggc cgggtcgccg ccctggagca ccccgagcgg tggggcggcc 17100 tgatcgacct gccctcggac gccgaccggg cggccctgga ccgcatgacc acggtcctcg 17160 ccggcggtac gggtgaggac caggtcgcgg tacgcgcctc cgggctgctc gcccgccgcc 17220 tcgtccgcgc ctccctcccg gcgcacggca cggcttcgcc gtggtggcag gccgacggca 17280 cggtgctcgt caccggtgcc gaggagcctg cggccgccga ggccgcacgc cggctggccc 17340 gcgacggcgc cggacacctc ctcctccaca ccaccccctc cggcagcgaa ggcgccgaag 17400 gcacctccgg tgccgccgag gactccggcc tcgccgggct cgtcgccgaa ctcgcggacc 17460 tgggcgcgac ggccaccgtc gtgacctgcg acctcacgga cgcggaggcg gccgcccggc 17520 tgctcgccgg cgtctccgac gcgcacccgc tcagcgccgt cctccacctg ccgcccaccg 17580 tcgactccga gccgctcgcc gcgaccgacg cggacgcgct cgcccgtgtc gtgaccgcga 17640 aggccaccgc cgcgctccac ctggaccgcc tcctgcggga ggccgcggct gccggaggcc 17700 gtccgcccgt cctggtcctc ttctcctcgg tcgccgcgat ctggggcggc gccggtcagg 17760 gcgcgtacgc cgccggtacg gccttcctcg acgccctcgc cggtcagcac cgggccgacg 17820 gccccaccgt gacctcggtg gcctggagcc cctgggaggg cagccgcgtc accgagggtg 17880 cgaccgggga gcggctgcgc cgcctcggcc tgcgccccct cgcccccgcg acggcgctca 17940 ccgccctgga caccgcgctc ggccacggcg acaccgccgt cacgatcgcc gacgtcgact 18000 ggtcgagctt cgcccccggc ttcaccacgg cccggccggg caccctcctc gccgatctgc 18060 ccgaggcgcg ccgcgcgctc gacgagcagc agtcgacgac ggccgccgac gacaccgtcc 18120 tgagccgcga gctcggtgcg ctcaccggcg ccgaacagca gcgccgtatg caggagttgg 18180 tccgcgagca cctcgccgtg gtcctcaacc acccctcccc cgaggccgtc gacacggggc 18240 gggccttccg tgacctcgga ttcgactcgc tgacggcggt cgagctccgc aaccgcctca 18300 agaacgccac cggcctggcc ctcccggcca ctctggtctt cgactacccg accccccgga 18360 cgctggcgga gttcctcctc gcggagatcc tgggcgagca ggccggtgcc ggcgagcagc 18420 ttccggtgga cggcggggtc gacgacgagc ccgtcgcgat cgtcggcatg gcgtgccgcc 18480 tgccgggcgg tgtcgcctcg ccggaggacc tgtggcggct ggtggccggc ggcgaggacg 18540 cgatctccgg cttcccgcag gaccgcggct gggacgtgga ggggctgtac gacccggacc 18600 cggacgcgtc cgggcggacg tactgccgtg ccggtggctt cctcgacgag gcgggcgagt 18660 tcgacgccga cttcttcggg atctcgccgc gcgaggccct cgccatggac ccgcagcagc 18720 ggctcctcct ggagacctcc tgggaggccg tcgaggacgc cgggatcgac ccgacctccc 18780 ttcaggggca gcaggtcggc gtgttcgcgg gcaccaacgg cccccactac gagccgctgc 18840 tccgcaacac cgccgaggat cttgagggtt acgtcgggac gggcaacgcc gccagcatca 18900 tgtcgggccg tgtctcgtac accctcggcc tggagggccc ggccgtcacg gtcgacaccg 18960 cctgctcctc ctcgctggtc gccctgcacc tcgccgtgca ggccctgcgc aagggcgaat 19020 gcggactggc gctcgcgggc ggtgtgacgg tcatgtcgac gcccacgacg ttcgtggagt 19080 tcagccggca gcgcgggctc gcggaggacg gccggtcgaa ggcgttcgcc gcgtcggcgg 19140 acggcttcgg cccggcggag ggcgtcggca tgctcctcgt cgagcgcctg tcggacgccc 19200 gccgcaacgg acaccgtgtg ctggcggtcg tgcgcggcag cgcggtcaac caggacggcg 19260 cgagcaacgg cctgaccgcc ccgaacgggc cctcgcagca gcgcgtcatc cggcgcgcgc 19320 tcgcggacgc ccgactgacg accgccgacg tggacgtcgt cgaggcccac ggcacgggca 19380 cgcgactcgg cgacccgatc gaggcacagg ccctcatcgc cacctacggc caggggcgcg 19440 acaccgaaca gccgctgcgc ctggggtcgt tgaagtccaa catcggacac acccaggccg 19500 ccgccggtgt ctccggcatc atcaagatgg tccaggcgat gcgccacggc gtcctgccga 19560 agacgctcca cgtggaccgg ccgtcggacc agatcgactg gtcggcgggc acggtcgagc 19620 tgctcaccga ggccatggac tggccgagga agcaggaggg cgggctgcgc cgcgcggccg 19680 tctcctcctt cggcatcagc ggcacgaacg cgcacatcgt gctcgaagaa gccccggtcg 19740 acgaggacgc cccggcggac gagccgtcgg tcggcggtgt ggtgccgtgg ctcgtgtccg 19800 cgaagactcc ggccgcgctg gacgcccaga tcggacgcct cgccgcgttc gcctcgcagg 19860 gccgtacgga cgccgccgat ccgggcgcgg tcgctcgcgt actggccggc gggcgtgcgc 19920 agttcgagca ccgggccgtc gcgctcggca ccggacagga cgacctggcg gccgcactgg 19980 ccgcgcctga gggtctggtc cggggtgtgg cctccggtgt gggtcgagtg gcgttcgtgt 20040 tcccgggaca gggcacgcag tgggccggga tgggtgccga actcctcgac gtgtcgaagg 20100 agttcgcggc ggccatggcc gagtgcgagg ccgcgctcgc tccgtacgtg gactggtcgc 20160 tggaggccgt cgtccgacag gcccccggcg cgcccacgct ggagcgggtc gatgtcgtcc 20220 agcccgtgac gttcgccgtc atggtctcgc tggcgaaggt ctggcagcac cacggggtga 20280 ccccgcaagc cgtcgtcggc cactcgcagg gcgagatcgc cgccgcgtac gtcgccggtg 20340 ccctgagcct ggacgacgcc gctcgtgtcg tgaccctgcg cagcaagtcc atcggcgccc 20400 acctcgcggg ccagggcggc atgctgtccc tcgcgctgag cgaggcggcc gttgtggagc 20460 gactggccgg gttcgacggg ctgtccgtcg ccgccgtcaa cgggcctacc gccaccgtgg 20520 tttcgggcga cccgacccag atccaagagc tcgctcaggc gtgtgaggcc gacggggtcc 20580 gcgcacggat catccccgtc gactacgcct cccacagcgc ccacgtcgag accatcgaga 20640 gcgaactcgc cgacgtcctg gcggggttgt ccccccagac accccaggtc cccttcttct 20700 ccaccctcga aggcgcctgg atcaccgaac ccgccctcga cggcggctac tggtaccgca 20760 acctccgcca tcgtgtgggc ttcgccccgg ccgtcgaaac cctggccacc gacgaaggct 20820 tcacccactt cgtcgaggtc agcgcccacc ccgtcctcac catggccctg cccgagaccg 20880 tcaccggcct cggcaccctc cgccgtgaca acggcggaca gcaccgcctc accacctccc 20940 tcgccgaggc ctgggccaac ggcctcaccg tcgactgggc ctctctcctc cccaccacga 21000 ccacccaccc cgatctgccc acctacgcct tccagaccga gcgctactgg ccgcagcccg 21060 acctctccgc cgccggtgac atcacctccg ccggtctcgg ggcggccgag cacccgctgc 21120 tcggcgcggc cgtggcgctc gcggactccg acggctgcct gctcacgggg agcctctccc 21180 tccgtacgca cccctggctg gcggaccacg cggtggccgg caccgtgctg ctgccgggaa 21240 cggcgttcgt ggagctggcg ttccgagccg gggaccaggt cggttgcgat ctggtcgagg 21300 agctcaccct cgacgcgccg ctcgtgctgc cccgtcgtgg cgcggtccgt gtgcagctgt 21360 ccgtcggcgc gagcgacgag tccgggcgtc gtaccttcgg gctctacgcg cacccggagg 21420 acgcgccggg cgaggcggag tggacgcggc acgccaccgg tgtgctggcc gcccgtgcgg 21480 accgcaccgc ccccgtcgcc gacccggagg cctggccgcc gccgggcgcc gagccggtgg 21540 acgtggacgg tctgtacgag cgcttcgcgg cgaacggcta cggctacggc cccctcttcc 21600 agggcgtccg tggtgtctgg cggcgtggcg acgaggtgtt cgccgacgtg gccctgccgg 21660 ccgaggtcgc cggtgccgag ggcgcgcggt tcggccttca cccggcgctg ctcgacgccg 21720 ccgtgcaggc ggccggtgcg ggcggggcgt tcggcgcggg cacgcggctg ccgttcgcct 21780 ggagcgggat ctccctgtac gcggtcggcg ccaccgccct ccgcgtgcgg ctggcccccg 21840 ccggcccgga cacggtgtcc gtgagcgccg ccgactcctc cgggcagccg gtgttcgccg 21900 cggactccct cacggtgctg cccgtcgacc ccgcgcagct ggcggccttc agcgacccga 21960 ctctggacgc gctgcacctg ctggagtgga ccgcctggga cggtgccgcg caggccctgc 22020 ccggcgcggt cgtgctgggc ggcgacgccg acggtctcgc cgcggcgctg cgcgccggtg 22080 gcaccgaggt cctgtccttc ccggacctta cggacctggt ggaggccgtc gaccggggcg 22140 agaccccggc cccggcgacc gtcctggtgg cctgccccgc cgccggcccc ggtgggccgg 22200 agcatgtccg cgaggccctg cacgggtcgc tcgcgctgat gcaggcctgg ctggccgacg 22260 agcggttcac cgatgggcgc ctggtgctcg tgacccgcga cgcggtcgcc gcccgttccg 22320 gcgacggcct gcggtccacg ggacaggccg ccgtctgggg cctcggccgg tccgcgcaga 22380 cggagagccc gggccggttc gtcctgctcg acctcgccgg ggaagcccgg acggccgggg 22440 acgccaccgc cggggacggc ctgacgaccg gggacgccac cgtcggcggc acctctggag 22500 acgccgccct cggcagcgcc ctcgcgaccg ccctcggctc gggcgagccg cagctcgccc 22560 tccgggacgg ggcgctcctc gtaccccgcc tggcgcgggc cgccgcgccc gccgcggccg 22620 acggcctcgc cgcggccgac ggcctcgccg ctctgccgct gcccgccgct ccggccctct 22680 ggcgtctgga gcccggtacg gacggcagcc tggagagcct cacggcggcg cccggcgacg 22740 ccgagaccct cgccccggag ccgctcggcc cgggacaggt ccgcatcgcg atccgggcca 22800 ccggtctcaa cttccgcgac gtcctgatcg ccctcggcat gtaccccgat ccggcgctga 22860 tgggcaccga gggagccggc gtggtcaccg cgaccggccc cggcgtcacg cacctcgccc 22920 ccggcgaccg ggtcatgggc ctgctctccg gcgcgtacgc cccggtcgtc gtggcggacg 22980 cgcggaccgt cgcgcggatg cccgaggggt ggacgttcgc ccagggcgcc tccgtgccgg 23040 tggtgttcct gacggccgtc tacgccctgc gcgacctggc ggacgtcaag cccggcgagc 23100 gcctcctggt ccactccgcc gccggtggcg tgggcatggc cgccgtgcag ctcgcccggc 23160 actggggcgt ggaggtccac ggcacggcga gtcacgggaa gtgggacgcc ctgcgcgcgc 23220 tcggcctgga cgacgcgcac atcgcctcct cccgcaccct ggacttcgag tccgcgttcc 23280 gtgccgcttc cggcggggcg ggcatggacg tcgtactgaa ctcgctcgcc cgcgagttcg 23340 tcgacgcctc gctgcgcctg ctcgggccgg gcggccggtt cgtggagatg gggaagaccg 23400 acgtccgcga cgcggagcgg gtcgccgccg accaccccgg tgtcggctac cgcgccttcg 23460 acctgggcga ggccgggccg gagcggatcg gcgagatgct cgccgaggtc atcgccctct 23520 tcgaggacgg ggtgctccgg cacctgcccg tcacgacctg ggacgtgcgc cgggcccgcg 23580 acgccttccg gcacgtcagc caggcccgcc acacgggcaa ggtcgtcctc acgatgccgt 23640 cgggcctcga cccggagggt acggtcctgc tgaccggcgg caccggtgcg ctggggggca 23700 tcgtggcccg gcacgtggtg ggcgagtggg gcgtacgacg cctgctgctc gtgagccggc 23760 ggggcacgga cgccccgggc gccggcgagc tcgtgcacga gctggaggcc ctgggagccg 23820 acgtctcggt ggccgcgtgc gacgtcgccg accgcgaagc cctcaccgcc gtactcgact 23880 cgatccccgc cgaacacccg ctcaccgcgg tcgtccacac ggcaggcgtc ctctccgacg 23940 gcaccctccc ctcgatgaca gcggaggatg tggaacacgt actgcgtccc aaggtcgacg 24000 ccgcgttcct cctcgacgaa ctcacctcga cgcccggcta cgacctggca gcgttcgtca 24060 tgttctcctc cgccgccgcc gtcttcggtg gcgcggggca gggcgcctac gccgccgcca 24120 acgccaccct cgacgccctc gcctggcgcc gccggacagc cggactcccc gccctctccc 24180 tcggctgggg cctctgggcc gagaccagcg gcatgaccgg cggactcagc gacaccgacc 24240 gctcgcggct ggcccgttcc ggggcgacgc ccatggacag cgagctgacc ctgtccctcc 24300 tggacgcggc catgcgccgc gacgacccgg cgctcgtccc gatcgccctg gacgtcgccg 24360 cgctccgcgc ccagcagcgc gacggcatgc tggcgccgct gctcagcggg ctcacccgcg 24420 gatcgcgggt cggcggcgcg ccggtcaacc agcgcagggc agccgccgga ggcgcgggcg 24480 aggcggacac ggacctcggc gggcggctcg ccgcgatgac accggacgac cgggtcgcgc 24540 acctgcggga cctcgtccgt acgcacgtgg cgaccgtcct gggacacggc accccgagcc 24600 gggtggacct ggagcgggcc ttccgcgaca ccggtttcga ctcgctcacc gccgtcgaac 24660 tccgcaaccg tctcaacgcc gcgaccgggc tgcggctgcc ggccacgctg gtcttcgacc 24720 accccacccc gggggagctc gccgggcacc tgctcgacga actcgccacg gccgcgggcg 24780 ggtcctgggc ggaaggcacc gggtccggag acacggcctc ggcgaccgat cggcagacca 24840 cggcggccct cgccgaactc gaccggctgg aaggcgtgct cgcctccctc gcgcccgccg 24900 ccggcggccg tccggagctc gccgcccggc tcagggcgct ggccgcggcc ctgggggacg 24960 acggcgacga cgccaccgac ctggacgagg cgtccgacga cgacctcttc tccttcatcg 25020 acaaggagct gggcgactcc gacttctgac ctgcccgaca ccaccggcac caccggcacc 25080 accagccccc ctcacacacg gaacacggaa cggacaggcg agaacgggag ccatggcgaa 25140 caacgaagac aagctccgcg actacctcaa gcgcgtcacc gccgagctgc agcagaacac 25200 caggcgtctg cgcgagatcg agggacgcac gcacgagccg gtggcgatcg tgggcatggc 25260 ctgccgcctg ccgggcggtg tcgcctcgcc cgaggacctg tggcagctgg tggccgggga 25320 cggggacgcg atctcggagt tcccgcagga ccgcggctgg gacgtggagg ggctgtacga 25380 ccccgacccg gacgcgtccg gcaggacgta ctgccggtcc ggcggattcc tgcacgacgc 25440 cggcgagttc gacgccgact tcttcgggat ctcgccgcgc gaggccctcg ccatggaccc 25500 gcagcagcga ctgtccctca ccaccgcgtg ggaggcgatc gagagcgcgg gcatcgaccc 25560 gacggccctg aagggcagcg gcctcggcgt cttcgtcggc ggctggcaca ccggctacac 25620 ctcggggcag accaccgccg tgcagtcgcc cgagctggag ggccacctgg tcagcggcgc 25680 ggcgctgggc ttcctgtccg gccgtatcgc gtacgtcctc ggtacggacg gaccggccct 25740 gaccgtggac acggcctgct cgtcctcgct ggtcgccctg cacctcgccg tgcaggccct 25800 ccgcaagggc gagtgcgaca tggccctcgc cggtggtgtc acggtcatgc ccaacgcgga 25860 cctgttcgtg cagttcagcc ggcagcgcgg gctggccgcg gacggccggt cgaaggcgtt 25920 cgccacctcg gcggacggct tcggccccgc ggagggcgcc ggagtcctgc tggtggagcg 25980 cctgtcggac gcccgccgca acggacaccg gatcctcgcg gtcgtccgcg gcagcgcggt 26040 caaccaggac ggcgccagca acggcctcac ggctccgcac gggccctccc agcagcgcgt 26100 catccgacgg gccctggcgg acgcccggct cgcgccgggt gacgtggacg tcgtcgaggc 26160 gcacggcacg ggcacgcggc tcggcgaccc gatcgaggcg caggccctca tcgccaccta 26220 cggccaggag aagagcagcg aacagccgct gaggctgggc gcgttgaagt cgaacatcgg 26280 gcacacgcag gccgcggccg gtgtcgcagg tgtcatcaag atggtccagg cgatgcgcca 26340 cggactgctg ccgaagacgc tgcacgtcga cgagccctcg gaccagatcg actggtcggc 26400 gggcacggtg gaactcctca ccgaggccgt cgactggccg gagaagcagg acggcgggct 26460 gcgccgcgcg gctgtctcct ccttcggcat cagcgggacg aacgcgcacg tcgtcctgga 26520 ggaggccccg gcggtcgagg actccccggc cgtcgagccg ccggccggtg gcggtgtggt 26580 gccgtggccg gtgtccgcga agactccggc cgcgctggac gcccagatcg ggcagctcgc 26640 cgcgtacgcg gacggtcgta cggacgtgga tccggcggtg gccgcccgcg ccctggtcga 26700 cagccgtacg gcgatggagc accgcgcggt cgcggtcggc gacagccggg aggcactgcg 26760 ggacgccctg cggatgccgg aaggactggt acgcggcacg tcctcggacg tgggccgggt 26820 ggcgttcgtc ttccccggcc agggcacgca gtgggccggc atgggcgccg aactccttga 26880 cagctcaccg gagttcgctg cctcgatggc cgaatgcgag accgcgctct cccgctacgt 26940 cgactggtct cttgaagccg tcgtccgaca ggaacccggc gcacccacgc tcgaccgcgt 27000 cgacgtcgtc cagcccgtga ccttcgctgt catggtctcg ctggcgaagg tctggcagca 27060 ccacggcatc accccccagg ccgtcgtcgg ccactcgcag ggcgagatcg ccgccgcgta 27120 cgtcgccggt gcactcaccc tcgacgacgc cgcccgcgtc gtcaccctgc gcagcaagtc 27180 catcgccgcc cacctcgccg gcaagggcgg catgatctcc ctcgccctcg acgaggcggc 27240 cgtcctgaag cgactgagcg acttcgacgg actctccgtc gccgccgtca acggccccac 27300 cgccaccgtc gtctccggcg acccgaccca gatcgaggaa ctcgcccgca cctgcgaggc 27360 cgacggcgtc cgtgcgcgga tcatcccggt cgactacgcc tcccacagcc ggcaggtcga 27420 gatcatcgag aaggagctgg ccgaggtcct cgccggactc gccccgcagg ctccgcacgt 27480 gccgttcttc tccaccctcg aaggcacctg gatcaccgag ccggtgctcg acggcaccta 27540 ctggtaccgc aacctgcgcc atcgcgtggg cttcgccccc gccgtggaga ccttggcggt 27600 tgacggcttc acccacttca tcgaggtcag cgcccacccc gtcctcacca tgaccctccc 27660 cgagaccgtc accggcctcg gcaccctccg ccgcgaacag ggaggccagg agcgtctggt 27720 cacctcactc gccgaagcct gggccaacgg cctcaccatc gactgggcgc ccatcctccc 27780 caccgcaacc ggccaccacc ccgagctccc cacctacgcc ttccagaccg agcgcttctg 27840 gctgcagagc tccgcgccca ccagcgccgc cgacgactgg cgttaccgcg tcgagtggaa 27900 gccgctgacg gcctccggcc aggcggacct gtccgggcgg tggatcgtcg ccgtcgggag 27960 cgagccagaa gccgagctgc tgggcgcgct gaaggccgcg ggagcggagg tcgacgtact 28020 ggaagccggg gcggacgacg accgtgaggc cctcgccgcc cggctcaccg cactgacgac 28080 cggcgacggc ttcaccggcg tggtctcgct cctcgacgac ctcgtgccac aggtcgcctg 28140 ggtgcaggca ctcggcgacg ccggaatcaa ggcgcccctg tggtccgtca cccagggcgc 28200 ggtctccgtc ggacgtctcg acacccccgc cgaccccgac cgggccatgc tctggggcct 28260 cggccgcgtc gtcgcccttg agcaccccga acgctgggcc ggcctcgtcg acctccccgc 28320 ccagcccgat gccgccgccc tcgcccacct cgtcaccgca ctctccggcg ccaccggcga 28380 ggaccagatc gccatccgca ccaccggact ccacgcccgc cgcctcgccc gcgcacccct 28440 ccacggacgt cggcccaccc gcgactggca gccccacggc accgtcctca tcaccggcgg 28500 caccggagcc ctcggcagcc acgccgcacg ctggatggcc caccacggag ccgaacacct 28560 cctcctcgtc agccgcagcg gcgaacaagc ccccggagcc acccaactca ccgccgaact 28620 caccgcatcg ggcgcccgcg tcaccatcgc cgcctgcgac gtcgccgacc cccacgccat 28680 gcgcaccctc ctcgacgcca tccccgccga gacgcccctc accgccgtcg tccacaccgc 28740 cggcgcaccg ggcggcgatc cgctggacgt caccggcccg gaggacatcg cccgcatcct 28800 gggcgcgaag acgagcggcg ccgaggtcct cgacgacctg ctccgcggca ctccgctgga 28860 cgccttcgtc ctctactcct cgaacgccgg ggtctggggc agcggcagcc agggcgtcta 28920 cgcggcggcc aacgcccacc tcgacgcgct cgccgcccgg cgccgcgccc ggggcgagac 28980 ggcgacctcg gtcgcctggg gcctctgggc cggcgacggc atgggccggg gcgccgacga 29040 cgcgtactgg cagcgtcgcg gcatccgtcc gatgagcccc gaccgcgccc tggacgaact 29100 ggccaaggcc ctgagccacg acgagacctt cgtcgccgtg gccgatgtcg actgggagcg 29160 gttcgcgccc gcgttcacgg tgtcccgtcc cagccttctg ctcgacggcg tcccggaggc 29220 ccggcaggcg ctcgccgcac ccgtcggtgc cccggctccc ggcgacgccg ccgtggcgcc 29280 gaccgggcag tcgtcggcgc tggccgcgat caccgcgctc cccgagcccg agcgccggcc 29340 ggcgctcctc accctcgtcc gtacccacgc ggcggccgta ctcggccatt cctcccccga 29400 ccgggtggcc cccggccgtg ccttcaccga gctcggcttc gactcgctga cggccgtgca 29460 gctccgcaac cagctctcca cggtggtcgg caacaggctc cccgccacca cggtcttcga 29520 ccacccgacg cccgccgcac tcgccgcgca cctccacgag gcgtacctcg caccggccga 29580 gccggccccg acggactggg aggggcgggt gcgccgggcc ctggccgaac tgcccctcga 29640 ccggctgcgg gacgcggggg tcctcgacac cgtcctgcgc ctcaccggca tcgagcccga 29700 gccgggttcc ggcggttcgg acggcggcgc cgccgaccct ggtgcggagc cggaggcgtc 29760 gatcgacgac ctggacgccg aggccctgat ccggatggct ctcggccccc gtaacacctg 29820 acccgaccgc ggtcctgccc cacgcgccgc accccgcgca tcccgcgcac cacccgcccc 29880 cacacgccca caaccccatc cacgagcgga agaccacacc cagatgacga gttccaacga 29940 acagttggtg gacgctctgc gcgcctctct caaggagaac gaagaactcc ggaaagagag 30000 ccgtcgccgg gccgaccgtc ggcaggagcc catggcgatc gtcggcatga gctgccggtt 30060 cgcgggcgga atccggtccc ccgaggacct ctgggacgcc gtcgccgcgg gcaaggacct 30120 ggtctccgag gtaccggagg agcgcggctg ggacatcgac tccctctacg acccggtgcc 30180 cgggcgcaag ggcacgacgt acgtccgcaa cgccgcgttc ctcgacgacg ccgccggatt 30240 cgacgcggcc ttcttcggga tctcgccgcg cgaggccctc gccatggacc cgcagcagcg 30300 gcagctcctc gaagcctcct gggaggtctt cgagcgggcc ggcatcgacc ccgcgtcggt 30360 ccgcggcacc gacgtcggcg tgtacgtggg ctgtggctac caggactacg cgccggacat 30420 ccgggtcgcc cccgaaggca ccggcggtta cgtcgtcacc ggcaactcct ccgccgtggc 30480 ctccgggcgc atcgcgtact ccctcggcct ggagggaccc gccgtgaccg tggacacggc 30540 gtgctcctct tcgctcgtcg ccctgcacct cgccctgaag ggcctgcgga acggcgactg 30600 ctcgacggca ctcgtgggcg gcgtggccgt cctcgcgacg ccgggcgcgt tcatcgagtt 30660 cagcagccag caggccatgg ccgccgacgg ccggaccaag ggcttcgcct cggcggcgga 30720 cggcctcgcc tggggcgagg gcgtcgccgt actcctcctc gaacggctct ccgacgcgcg 30780 gcgcaagggc caccgggtcc tggccgtcgt gcgcggcagc gccatcaacc aggacggcgc 30840 gagcaacggc ctcacggctc cgcacgggcc ctcccagcag cgcctgatcc gccaggccct 30900 ggccgacgcg cggctcacgt cgagcgacgt ggacgtcgtg gagggccacg gcacggggac 30960 ccgtctcggc gacccgatcg aggcgcaggc gctgctcgcc acgtacgggc aggggcgcgc 31020 cccggggcag ccgctgcggc tggggacgct gaagtcgaac atcgggcaca cgcaggccgc 31080 ttcgggtgtc gccggtgtca tcaagatggt gcaggcgctg cgccacgggg tgctgccgaa 31140 gaccctgcac gtggacgagc cgacggacca ggtcgactgg tcggccggtt cggtcgagct 31200 gctcaccgag gccgtggact ggccggagcg gccgggccgg ctccgccggg cgggcgtctc 31260 cgcgttcggc gtgggcggga cgaacgcgca cgtcgtcctg gaggaggccc cggcggtcga 31320 ggagtcccct gccgtcgagc cgccggccgg tggcggcgtg gtgccgtggc cggtgtccgc 31380 gaagacctcg gccgcactgg acgcccagat cgggcagctc gccgcatacg cggaagaccg 31440 cacggacgtg gatccggcgg tggccgcccg cgccctggtc gacagccgta cggcgatgga 31500 gcaccgcgcg gtcgcggtcg gcgacagccg ggaggcactg cgggacgccc tgcggatgcc 31560 ggaaggactg gtacggggca cggtcaccga tccgggccgg gtggcgttcg tcttccccgg 31620 ccagggcacg cagtgggccg gcatgggcgc cgaactcctc gacagctcac ccgaattcgc 31680 cgccgccatg gccgaatgcg agaccgcact ctccccgtac gtcgactggt ctctcgaagc 31740 cgtcgtccga caggctccca gcgcaccgac actcgaccgc gtcgacgtcg tccagcccgt 31800 caccttcgcc gtcatggtct ccctcgccaa ggtctggcag caccacggca tcacccccga 31860 ggccgtcatc ggccactccc agggcgagat cgccgccgcg tacgtcgccg gtgccctcac 31920 cctcgacgac gccgctcgtg tcgtgaccct ccgcagcaag tccatcgccg cccacctcgc 31980 cggcaagggc ggcatgatct ccctcgccct cagcgaggaa gccacccggc agcgcatcga 32040 gaacctccac ggactgtcga tcgccgccgt caacgggcct accgccaccg tggtttcggg 32100 cgaccccacc cagatccaag aacttgctca ggcgtgtgag gccgacggca tccgcgcacg 32160 gatcatcccc gtcgactacg cctcccacag cgcccacgtc gagaccatcg agaacgaact 32220 cgccgacgtc ctggcggggt tgtcccccca gacaccccag gtccccttct tctccaccct 32280 cgaaggcacc tggatcaccg aacccgccct cgacggcggc tactggtacc gcaacctccg 32340 ccatcgtgtg ggcttcgccc cggccgtcga gaccctcgcc accgacgaag gcttcaccca 32400 cttcatcgag gtcagcgccc accccgtcct caccatgacc ctccccgaca aggtcaccgg 32460 cctggccacc ctccgacgcg aggacggcgg acagcaccgc ctcaccacct cccttgccga 32520 ggcctgggcc aacggcctcg ccctcgactg ggcctccctc ctgcccgcca cgggcgccct 32580 cagccccgcc gtccccgacc tcccgacgta cgccttccag caccgctcgt actggatcag 32640 ccccgcgggt cccggcgagg cgcccgcgca caccgcttcc gggcgcgagg ccgtcgccga 32700 gacggggctc gcgtggggcc cgggtgccga ggacctcgac gaggagggcc ggcgcagcgc 32760 cgtactcgcg atggtgatgc ggcaggcggc ctccgtgctc cggtgcgact cgcccgaaga 32820 ggtccccgtc gaccgcccgc tgcgggagat cggcttcgac tcgctgaccg ccgtcgactt 32880 ccgcaaccgc gtcaaccggc tgaccggtct ccagctgccg cccaccgtcg tgttcgagca 32940 cccgacgccc gtcgcgctcg ccgagcgcat cagcgacgag ctggccgagc ggaactgggc 33000 cgtcgccgag ccgtcggatc acgagcaggc ggaggaggag aaggccgccg ctccggcggg 33060 ggcccgctcc ggggccgaca ccggcgccgg cgccgggatg ttccgcgccc tgttccggca 33120 ggccgtggag gacgaccggt acggcgagtt cctcgacgtc ctcgccgaag cctccgcgtt 33180 ccgcccgcag ttcgcctcgc ccgaggcctg ctcggagcgg ctcgacccgg tgctgctcgc 33240 cggcggtccg acggaccggg cggaaggccg tgccgttctc gtcggctgca ccggcaccgc 33300 ggcgaacggc ggcccgcacg agttcctgcg gctcagcacc tccttccagg aggagcggga 33360 cttcctcgcc gtacctctcc ccggctacgg cacgggtacg ggcaccggca cggccctcct 33420 cccggccgat ctcgacaccg cgctcgacgc ccaggcccgg gcgatcctcc gggccgccgg 33480 ggacgccccg gtcgtcctgc tcgggcactc cggcggcgcc ctgctcgcgc acgagctggc 33540 cttccgcctg gagcgggcgc acggcgcgcc gccggccggg atcgtcctgg tcgaccccta 33600 tccgccgggc catcaggagc ccatcgaggt gtggagcagg cagctgggcg agggcctgtt 33660 cgcgggcgag ctggagccga tgtccgatgc gcggctgctg gccatgggcc ggtacgcgcg 33720 gttcctcgcc ggcccgcggc cgggccgcag cagcgcgccc gtgcttctgg tccgtgcctc 33780 cgaaccgctg ggcgactggc aggaggagcg gggcgactgg cgtgcccact gggaccttcc 33840 gcacaccgtc gcggacgtgc cgggcgacca cttcacgatg atgcgggacc acgcgccggc 33900 cgtcgccgag gccgtcctct cctggctcga cgccatcgag ggcatcgagg gggcgggcaa 33960 gtgaccgaca gacctctgaa cgtggacagc ggactgtgga tccggcgctt ccaccccgcg 34020 ccgaacagcg cggtgcggct ggtctgcctg ccgcacgccg gcggctccgc cagctacttc 34080 ttccgcttct cggaggagct gcacccctcc gtcgaggccc tgtcggtgca gtatccgggc 34140 cgccaggacc ggcgtgccga gccgtgtctg gagagcgtcg aggagctcgc cgagcatgtg 34200 gtcgcggcca ccgaaccctg gtggcaggag ggccggctgg ccttcttcgg gcacagcctc 34260 ggcgcctccg tcgccttcga gacggcccgc atcctggaac agcggcacgg ggtacggccc 34320 gagggcctgt acgtctccgg tcggcgcgcc ccgtcgctgg cgccggaccg gctcgtccac 34380 cagctggacg accgggcgtt cctggccgag atccggcggc tcagcggcac cgacgagcgg 34440 ttcctccagg acgacgagct gctgcggctg gtgctgcccg cgctgcgcag cgactacaag 34500 gcggcggaga cgtacctgca ccggccgtcc gccaagctca cctgcccggt gatggccctg 34560 gccggcgacc gtgacccgaa ggcgccgctg aacgaggtgg ccgagtggcg tcggcacacc 34620 agcgggccgt tctgcctccg ggcgtactcc ggcggccact tctacctcaa cgaccagtgg 34680 cacgagatct gcaacgacat ctccgaccac ctgctcgtca cccgcggcgc gcccgatgcc 34740 cgcgtcgtgc agcccccgac cagccttatc gaaggagcgg cgaagagatg gcagaaccca 34800 cggtgaccga cgacctgacg ggggccctca cgcagccccc gctgggccgc accgtccgcg 34860 cggtggccga ccgtgaactc ggcacccacc tcctggagac ccgcggcatc cactggatcc 34920 acgccgcgaa cggcgacccg tacgccaccg tgctgcgcgg ccaggcggac gacccgtatc 34980 ccgcgtacga gcgggtgcgt gcccgcggcg cgctctcctt cagcccgacg ggcagctggg 35040 tcaccgccga tcacgccctg gcggcgagca tcctctgctc gacggacttc ggggtctccg 35100 gcgccgacgg cgtcccggtg ccgcagcagg tcctctcgta cggggagggc tgtccgctgg 35160 agcgcgagca ggtgctgccg gcggccggtg acgtgccgga gggcgggcag cgtgccgtgg 35220 tcgaggggat ccaccgggag acgctggagg gtctcgcgcc ggacccgtcg gcgtcgtacg 35280 ccttcgagct gctgggcggt ttcgtccgcc cggcggtgac ggccgctgcc gccgccgtgc 35340 tgggtgttcc cgcggaccgg cgcgcggact tcgcggatct gctggagcgg ctccggccgc 35400 tgtccgacag cctgctggcc ccgcagtccc tgcggacggt acgggcggcg gacggcgcgc 35460 tggccgagct cacggcgctg ctcgccgatt cggacgactc ccccggggcc ctgctgtcgg 35520 cgctcggggt caccgcagcc gtccagctca ccgggaacgc ggtgctcgcg ctcctcgcgc 35580 atcccgagca gtggcgggag ctgtgcgacc ggcccgggct cgcggcggcc gcggtggagg 35640 agaccctccg ctacgacccg ccggtgcagc tcgacgcccg ggtggtccgc ggggagacgg 35700 agctggcggg ccggcggctg ccggccgggg cgcatgtcgt cgtcctgacc gccgcgaccg 35760 gccgggaccc ggaggtcttc acggacccgg agcgcttcga cctcgcgcgc cccgacgccg 35820 ccgcgcacct cgcgctgcac cccgccggtc cgtacggccc ggtggcgtcc ctggtccggc 35880 ttcaggcgga ggtcgcgctg cggaccctgg ccgggcgttt ccccgggctg cggcaggcgg 35940 gggacgtgct ccgcccccgc cgcgcgcctg tcggccgcgg gccgctgagc gtcccggtca 36000 gcagctcctg agacaccggg gccccggtcc gcccggcccc ccttcggacg gaccggacgg 36060 ctcggaccac ggggacggct cagaccgtcc cgtgtgtccc cgtccggctc ccgtccgccc 36120 catcccgccc ctccaccggc aaggaaggac acgacgccat gcgcgtcctg ctgacctcgt 36180 tcgcacatca cacgcactac tacggcctgg tgcccctggc ctgggcgctg ctcgccgccg 36240 ggcacgaggt gcgggtcgcc agccagcccg cgctcacgga caccatcacc gggtccgggc 36300 tcgccgcggt gccggtcggc accgaccacc tcatccacga gtaccgggtg cggatggcgg 36360 gcgagccgcg cccgaaccat ccggcgatcg ccttcgacga ggcccgtccc gagccgctgg 36420 actgggacca cgccctcggc atcgaggcga tcctcgcccc gtacttctat ctgctcgcca 36480 acaacgactc gatggtcgac gacctcgtcg acttcgcccg gtcctggcag ccggacctgg 36540 tgctgtggga gccgacgacc tacgcgggcg ccgtcgccgc ccaggtcacc ggtgccgcgc 36600 acgcccgggt cctgtggggg cccgacgtga tgggcagcgc ccgccgcaag ttcgtcgcgc 36660 tgcgggaccg gcagccgccc gagcaccgcg aggaccccac cgcggagtgg ctgacgtgga 36720 cgctcgaccg gtacggcgcc tccttcgaag aggagctgct caccggccag ttcacgatcg 36780 acccgacccc gccgagcctg cgcctcgaca cgggcctgcc gaccgtcggg atgcgttatg 36840 ttccgtacaa cggcacgtcg gtcgtgccgg actggctgag tgagccgccc gcgcggcccc 36900 gggtctgcct gaccctcggc gtctccgcgc gtgaggtcct cggcggcgac ggcgtctcgc 36960 agggcgacat cctggaggcg ctcgccgacc tcgacatcga gctcgtcgcc acgctcgacg 37020 cgagtcagcg cgccgagatc cgcaactacc cgaagcacac ccggttcacg gacttcgtgc 37080 cgatgcacgc gctcctgccg agctgctcgg cgatcatcca ccacggcggg gcgggcacct 37140 acgcgaccgc cgtgatcaac gcggtgccgc aggtcatgct cgccgagctg tgggacgcgc 37200 cggtcaaggc gcgggccgtc gccgagcagg gggcggggtt cttcctgccg ccggccgagc 37260 tcacgccgca ggccgtgcgg gacgccgtcg tccgcatcct cgacgacccc tcggtcgcca 37320 ccgccgcgca ccggctgcgc gaggagacct tcggcgaccc caccccggcc gggatcgtcc 37380 ccgagctgga gcggctcgcc gcgcagcacc gccgcccgcc ggccgacgcc cggcactgag 37440 ccgcacccct cgccccaggc ctcacccctg tatctgcgcc gggggacgcc cccggcccac 37500 cctccgaaag accgaaagca ggagcaccgt gtacgaagtc gaccacgccg acgtctacga 37560 cctcttctac ctgggtcgcg gcaaggacta cgccgccgag gcctccgaca tcgccgacct 37620 ggtgcgctcc cgtacccccg aggcctcctc gctcctggac gtggcctgcg gtacgggcac 37680 gcatctggag cacttcacca aggagttcgg cgacaccgcc ggcctggagc tgtccgagga 37740 catgctcacc cacgcccgca agcggctgcc cgacgccacg ctccaccagg gcgacatgcg 37800 ggacttccgg ctcggccgga agttctccgc cgtggtcagc atgttcagct ccgtcggcta 37860 cctgaagacg accgaggaac tcggcgcggc cgtcgcctcg ttcgcggagc acctggagcc 37920 cggtggcgtc gtcgtcgtcg agccgtggtg gttcccggag accttcgccg acggctgggt 37980 cagcgccgac gtcgtccgcc gtgacgggcg caccgtggcc cgtgtctcgc actcggtgcg 38040 ggaggggaac gcgacgcgca tggaggtcca cttcaccgtg gccgacccgg gcaagggcgt 38100 gcggcacttc tccgacgtcc atctcatcac cctgttccac caggccgagt acgaggccgc 38160 gttcacggcc gccgggctgc gcgtcgagta cctggagggc ggcccgtcgg gccgtggcct 38220 cttcgtcggc gtccccgcct gagcaccgcc caagaccccc cggggcggga cgtcccgggt 38280 gcaccaagca aagagagaga aacgaaccgt gacaggtaag acccgaatac cgcgtgtccg 38340 ccgcggccgc accacgccca gggccttcac cctggccgtc gtcggcaccc tgctggcggg 38400 caccaccgtg gcggccgccg ctcccggcgc cgccgacacg gccaatgttc agtacacgag 38460 ccgggcggcg gagctcgtcg cccagatgac gctcgacgag aagatc 38506 20 2401 DNA Streptomyces venezuelae 20 cgtggcggcc gccgctcccg gcgccgccga cacggccaat gttcagtaca cgagccgggc 60 ggcggagctc gtcgcccaga tgacgctcga cgagaagatc agcttcgtcc actgggcgct 120 ggaccccgac cggcagaacg tcggctacct tcccggcgtg ccgcgtctgg gcatcccgga 180 gctgcgtgcc gccgacggcc cgaacggcat ccgcctggtg gggcagaccg ccaccgcgct 240 gcccgcgccg gtcgccctgg ccagcacctt cgacgacacc atggccgaca gctacggcaa 300 ggtcatgggc cgcgacggtc gcgcgctcaa ccaggacatg gtcctgggcc cgatgatgaa 360 caacatccgg gtgccgcacg gcggccggaa ctacgagacc ttcagcgagg accccctggt 420 ctcctcgcgc accgcggtcg cccagatcaa gggcatccag ggtgcgggtc tgatgaccac 480 ggccaagcac ttcgcggcca acaaccagga gaacaaccgc ttctccgtga acgccaatgt 540 cgacgagcag acgctccgcg agatcgagtt cccggcgttc gaggcgtcct ccaaggccgg 600 cgcgggctcc ttcatgtgtg cctacaacgg cctcaacggg aagccgtcct gcggcaacga 660 cgagctcctc aacaacgtgc tgcgcacgca gtggggcttc cagggctggg tgatgtccga 720 ctggctcgcc accccgggca ccgacgccat caccaagggc ctcgaccagg agatgggcgt 780 cgagctcccc ggcgacgtcc cgaagggcga gccctcgccg ccggccaagt tcttcggcga 840 ggcgctgaag acggccgtcc tgaacggcac ggtccccgag gcggccgtga cgcggtcggc 900 ggagcggatc gtcggccaga tggagaagtt cggtctgctc ctcgccactc cggcgccgcg 960 gcccgagcgc gacaaggcgg gtgcccaggc ggtgtcccgc aaggtcgccg agaacggcgc 1020 ggtgctcctg cgcaacgagg gccaggccct gccgctcgcc ggtgacgccg gcaagagcat 1080 cgcggtcatc ggcccgacgg ccgtcgaccc caaggtcacc ggcctgggca gcgcccacgt 1140 cgtcccggac tcggcggcgg cgccactcga caccatcaag gcccgcgcgg gtgcgggtgc 1200 gacggtgacg tacgagacgg gtgaggagac cttcgggacg cagatcccgg cggggaacct 1260 cagcccggcg ttcaaccagg gccaccagct cgagccgggc aaggcggggg cgctgtacga 1320 cggcacgctg accgtgcccg ccgacggcga gtaccgcatc gcggtccgtg ccaccggtgg 1380 ttacgccacg gtgcagctcg gcagccacac catcgaggcc ggtcaggtct acggcaaggt 1440 gagcagcccg ctcctcaagc tgaccaaggg cacgcacaag ctcacgatct cgggcttcgc 1500 gatgagtgcc accccgctct ccctggagct gggctgggtn acgccggcgg cggccgacgc 1560 gacgatcgcg aaggccgtgg agtcggcgcg gaaggcccgt acggcggtcg tcttcgccta 1620 cgacgacggc accgagggcg tcgaccgtcc gaacctgtcg ctgccgggta cgcaggacaa 1680 gctgatctcg gctgtcgcgg acgccaaccc gaacacgatc gtggtcctca acaccggttc 1740 gtcggtgctg atgccgtggc tgtccaagac ccgcgcggtc ctggacatgt ggtacccggg 1800 ccaggcgggc gccgaggcca ccgccgcgct gctctacggt gacgtcaacc cgagcggcaa 1860 gctcacgcag agcttcccgg ccgccgagaa ccagcacgcg gtcgccggcg acccgaccag 1920 ctacccgggc gtcgacaacc agcagacgta ccgcgagggc atccacgtcg ggtaccgctg 1980 gttcgacaag gagaacgtca agccgctgtt cccgttcggg cacggcctgt cgtacacctc 2040 gttcacgcag agcgccccga ccgtcgtgcg tacgtccacg ggtggtctga aggtcacggt 2100 cacggtccgc aacagcggga agcgcgccgg ccaggaggtc gtccaggcgt acctcggtgc 2160 cagcccgaac gtgacggctc cgcaggcgaa gaagaagctc gtgggctaca cgaaggtctc 2220 gctcgccgcg ggcgaggcga agacggtgac ggtgaacgtc gaccgccgtc agctgcagtt 2280 ctgggatgcc gccacggaca actggaagac gggaacgggc aaccgcctcc tgcagaccgg 2340 ttcgtcctcc gccgacctgc ggggcagcgc cacggtcaac gtctggtgac gtgacgccgt 2400 g 2401 21 5970 DNA Streptomyces venezuelae 21 ggcgagaagt aggcgcgggt gtgcacgcct tcggccttca ggacctccat gacgaggtcg 60 cggtggatgc cggtggtggc ctcgtcgatc tcgacgatca cgtactggtg gttgttgagg 120 ccgtggcggt cgtggtcggc gacgaggacg ccggggaggt ccgcgaggtg ctcgcggtag 180 scggcgtggt tgcgccggtt ccggtcgatg acctcgggaa acgcgtcgag ggaggtgagg 240 cccatggcgg cggcggcctc gctcatcttg gcgttggtcc cgccggcggg gctgccgccg 300 ggcaggtcga agccgaagtt gtggagggcg cggatccggg cggcgaggtc ggcgtcgtcg 360 gtgacgacgg cgccgccctc gaaggcgttg acggccttgg tggcgtggaa gctgaagacc 420 tcggcgtcgc cgaggctgcc ggcgggccgg ccgtcgaccg cgcagccgag ggcgtgcgcg 480 gcgtcgaagt acagccgcag gccgtgctcg tcggcgacct tccgcagctg gtcggcggcg 540 caggggcggc cccagaggtg gacgccgacg acggccgagg tgcggggtgt gaccgcggcg 600 gccacctggt ccgggtcgag gttgccggtg tccgggtcga tgtcggcgaa gaccggggtg 660 aggccgatcc agcgcagtgc gtgcggggtg gcggcgaacg tcatcgacgg catgatcact 720 tcgccggtga ggccggcggc gtgcgcgagg agctggagcc cggccgtggc gttgcaggtg 780 gccacggcat gccggacccc ggcgagcccg gcgacgcgct cctcgaactc gcggacgagc 840 gggccgccgt tggacagcca ctggctgtcg agggcccggt cgagccgctc gtacagcctg 900 gcgcggtcga tgcggttggg ccgccccacg aggagcggct ggtcgaaagc ggcggggccg 960 ccgaagaatg cgaggtcgga taaggcgctt ttcacggatg ttccctccgg gccaccgtca 1020 cgaaatgatt cgccgatccg ggaatcccga acgaggtcgc cgcgctccac cgtgacgtac 1080 gacgagatgg tcgattgtgg tggtcgattt cggggggact ctaatccgcg cggaacggga 1140 ccgacaagag cacgctatgc gctctcgatg tgcttcggat cacatccgcc tccggggtat 1200 tccatcggcg gcccgaatgt gatgatcctt gacaggatcc gggaatcagc cgagccgccg 1260 ggagggccgg ggcgcgctcc gcggaagagt acgtgtgaga agtcccgttc ctcttcccgt 1320 ttccgttccg cttccggccc ggtctggagt tctccgtgcg ccgtacccag cagggaacga 1380 ccgcttctcc cccggtactc gacctcgggg ccctggggca ggatttcgcg gccgatccgt 1440 atccgacgta cgcgagactg cgtgccgagg gtccggccca ccgggtgcgc acccccgagg 1500 gggacgaggt gtggctggtc gtcggctacg accgggcgcg ggcggtcctc gccgatcccc 1560 ggttcagcaa ggactggcgc aactccacga ctcccctgac cgaggccgag gccgcgctca 1620 accacaacat gctggagtcc gacccgccgc ggcacacccg gctgcgcaag ctggtggccc 1680 gtgagttcac catgcgccgg gtcgagttgc tgcggccccg ggtccaggag atcgtcgacg 1740 ggctcgtgga cgccatgctg gcggcgcccg acggccgcgc cgatctgatg gagtccctgg 1800 cctggccgct gccgatcacc gtgatctccg aactcctcgg cgtgcccgag ccggaccgcg 1860 ccgccttccg cgtctggacc gacgccttcg tcttcccgga cgatcccgcc caggcccaga 1920 ccgccatggc cgagatgagc ggctatctct cccggctcat cgactccaag cgcgggcagg 1980 acggcgagga cctgctcagc gcgctcgtgc ggaccagcga cgaggacggc tcccggctga 2040 cctccgagga gctgctcggt atggcccaca tcctgctcgt cgcggggcac gagaccacgg 2100 tcaatctgat cgccaacggc atgtacgcgc tgctctcgca ccccgaccag ctggccgccc 2160 tgcgggccga catgacgctc ttggacggcg cggtggagga gatgttgcgc tacgagggcc 2220 cggtggaatc cgcgacctac cgcttcccgg tcgagcccgt cgacctggac ggcacggtca 2280 tcccggccgg tgacacggtc ctcgtcgtcc tggccgacgc ccaccgcacc cccgagcgct 2340 tcccggaccc gcaccgcttc gacatccgcc gggacaccgc cggccatctc gccttcggcc 2400 acggcatcca cttctgcatc ggcgccccct tggcccggtt ggaggcccgg atcgccgtcc 2460 gcgcccttct cgaacgctgc ccggacctcg ccctggacgt ctcccccggc gaactcgtgt 2520 ggtatccgaa cccgatgatc cgcgggctca aggccctgcc gatccgctgg cggcgaggac 2580 gggaggcggg ccgccgtacc ggttgaaccc gcacgtcacc cattacgact ccttgtcacg 2640 gaagccccgg atcggtcccc cctcgccgta acaagacctg gttagagtga tggaggacga 2700 cgaagggttc ggcgcccgga cgagggggga cttccgcgat gaatctggtg gaacgcgacg 2760 gggagatagc ccatctcagg gccgttcttg acgcatccgc cgcaggtgac gggacgctct 2820 tactcgtctc cggaccggcc ggcagcggga agacggagct gctgcggtcg ctccgccggc 2880 tggccgccga gcgggagacc cccgtctggt cggtccgggc gctgccgggt gaccgcgaca 2940 tccccctggg cgtcctctgc cagttactcc gcagcgccga acaacacggt gccgacacct 3000 ccgccgtccg cgacctgctg gacgccgcct cgcggcgggc cggaacctca cctcccccgc 3060 cgacgcgccg ctccgcgtcg acgagacaca ccgcctgcac gactggctgc tctccgtctc 3120 ccgccggcac cccgttcctc gtcgccgtcg acgacctgac ccacgccgac accgcgtccc 3180 tgaggttcct cctgtactgc gccgcccacc acgaccaggg cggcatcggc ttcgtcatga 3240 ccgagcgggc ctcgcagcgc gccggatacc gggtgttccg cgccgagctg ctccgccagc 3300 cgcactgccg caacatgtgg ctctccgggc ttccccccag cggggtacgc cagttactcg 3360 cccactacta cggccccgag gccgccgagc ggcgggcccc cgcgtaccac gcgacgaccg 3420 gcgggaaccc gctgctcctg cgggcgctga cccaggaccg gcaggcctcc cacaccaccc 3480 tcggcgcggc cggcggcgac gagcccgtcc acggcgacgc cttcgcccag gccgtcctcg 3540 actgcctgca ccgcagcgcc gagggcacac tggagaccgc ccgctggctc gcggtcctcg 3600 aacagtccga cccgctcctg gtggagcggc tcacgggaac gaccgccgcc gccgtcgagc 3660 gccacatcca ggagctcgcc gccatcggcc tcctggacga ggacggcacc ctgggacagc 3720 ccgcgatccg cgaggccgcc ctccaggacc tgccggccgg cgagcgcacc gaactgcacc 3780 ggcgcgccgc ggagcagctg caccgggacg gcgccgacga ggacaccgtg gcccgccacc 3840 tgctggtcgg cggcgccccc gacgctccct gggcgctgcc cctgctcgaa cggggcgcgc 3900 agcaggccct gttcgacgac cgactcgacg acgccttccg gatcctcgag ttcgccgtgc 3960 ggtcgagcac cgacaacacc cagctggccc gcctcgcccc acacctggtc gcggcctcct 4020 ggcggatgaa cccgcacatg acgacccggg ccctcgcact cttcgaccgg ctcctgagcg 4080 gtgaactgcc gcccagccac ccggtcatgg ccctgatccg ctgcctcgtc tggtacggnc 4140 ggctgcccga ggccgccgac gcgctgtccc ggctgcggcc cagctccgac aacgatgcct 4200 tggagctgtc gctcacccgg atgtggctcg cggcgctgtg cccgccgctc ctggagtccc 4260 tgccggccac gccggagccg gagcggggtc ccgtccccgt acggctcgcg ccgcggacga 4320 ccgcgctcca ggcccaggcc ggcgtcttcc agcggggccc ggacaacgcc tcggtcgcgc 4380 aggccgaaca gatcctgcag ggctgccggc tgtcggagga gacgtacgag gccctggaga 4440 cggccctctt ggtcctcgtc cacgccgacc ggctcgaccg ggcgctgttc tggtcggacg 4500 ccctgctcgc cgaggccgtg gagcggcggt cgctcggctg ggaggcggtc ttcgccgcga 4560 cccgggcgat gatcgcgatc cgctgcggcg acctcccgac ggcgcgggag cgggccgagc 4620 tggcgctctc ccacgcggcg ccggagagct ggggcctcgc cgtgggcatg cccctctccg 4680 cgctgctgct cgcctgcacg gaggccggcg agtacgaaca ggcggagcgg gtcctgcggc 4740 agccggtgcc ggacgcgatg ttcgactcgc ggcacggcat ggagtacatg cacgcccggg 4800 gccgctactg gctggcganc ggccggctgc acgcggcgct gggcgagttc atgctctgcg 4860 gggagatcct gggcagctgg aacctcgacc agccctcgat cgtgccctgg cggacctccg 4920 ccgccgaggt gtacctgcgg ctcggcaacc gccagaaggc cagggcgctg gccgaggccc 4980 agctcgccct ggtgcggccc gggcgctccc gcacccgggg tctcaccctg cgggtcctgg 5040 cggcggcggt ggacggccag caggcggagc ggctgcacgc cgaggcggtc gacatgctgc 5100 acgacagcgg cgaccggctc gaacacgccc gcgcgctcgc cgggatgagc cgccaccagc 5160 aggcccaggg ggacaactac cgggcgagga tgacggcgcg gctcgccggc gacatggcgt 5220 gggcctgcgg cgcgtacccg ctggccgagg agatcgtgcc gggccgcggc ggccgccggg 5280 cgaaggcggt gagcacggag ctggaactgc cgggcggccc ggacgtcggc ctgctctcgg 5340 aggccgaacg ccgggtggcg gccctggcag cccgaggatt gacgaaccgc cagatagcgc 5400 gccggctctg cgtcaccgcg agcacggtcg aacagcacct gacgcgcgtc taccgcaaac 5460 tgaacgtgac ccgccgagca gacctcccga tcagcctcgc ccaggacaag tccgtcacgg 5520 cctgagccac ccccggtgtc cccgtgcgac gacccgccgc acgggccacc gggcccgccg 5580 ggacacgccg gtgcgacacg ggggcgcgcc aggtgccatg gggacctccg tgaccgcccg 5640 aggcgcccga ggcgcccggt gcggcacccg gagacgccag gaccgccggg accaccggag 5700 acgccaggga ccgctgggga caccgggacc tcagggaccg ccgggaccgc ccgagttgca 5760 cccggtgcgc ccggggacac cagaccgccg ggaccacccg agggtgcccg gtgtggcccc 5820 ggcggccggg gtgtccttca tcggtgggcc ttcatcggca ggaggaagcg accgtgagac 5880 ccgtcgtgcc gtcggcgatc agccgcctgt acgggcgtcg gactccctgg cggtcccgga 5940 cccgtcgtac gggctcgcgg gacccggtgc 5970 22 3292 DNA Streptomyces venezuelae 22 accccccaaa ggggtggtga cactccccct gcgcagcccc tagcgccccc ctaactcgcc 60 acgccgaccg ttatcaccgg cgccctgctg ctagtttccg agaatgaagg gaatagtcct 120 ggccggcggg agcggaactc ggctgcatcc ggcgacctcg gtcatttcga agcagattct 180 tccggtctac aacaaaccga tgatctacta tccgctgtcg gttctcatgc tcggcggtat 240 tcgcgagatt caaatcatct cgacccccca gcacatcgaa ctcttccagt cgcttctcgg 300 aaacggcagg cacctgggaa tagaactcga ctatgcggtc cagaaagagc ccgcaggaat 360 cgcggacgca cttctcgtcg gagccgagca catcggcgac gacacctgcg ccctgatcct 420 gggcgacaac atcttccacg ggcccggcct ctacacgctc ctgcgggaca gcatcgcgcg 480 cctcgacggc tgcgtgctct tcggctaccc ggtcaaggac cccgagcggt acggcgtcgc 540 cgaggtggac gcgacgggcc ggctgaccga cctcgtcgag aagcccgtca agccgcgctc 600 caacctcgcc gtcaccggcc tctacctcta cgacaacgac gtcgtcgaca tcgccaagaa 660 catccggccc tcgccgcgcg gcgagctgga gatcaccgac gtcaaccgcg tctacctgga 720 gcggggccgg gccgaactcg tcaacctggg ccgcggcttc gcctggctgg acaccggcac 780 ccacgactcg ctcctgcggg ccgcccagta cgtccaggtc ctggaggagc ggcagggcgt 840 ctggatcgcg ggccttgagg agatcgcctt ccgcatgggc ttcatcgacg ccgaggcctg 900 tcacggcctg ggagaaggcc tctcccgcac cgagtacggc agctatctga tggagatcgc 960 cggccgcgag ggagccccgt gagggcacct cgcggccgac gcgttcccac gaccgacagc 1020 gccaccgaca gtgcgaccca caccgcgacc cgcaccgcca ccgacagtgc gacccacacc 1080 gcgacctaca gcgcgaccga aaggaagacg gcagtgcggc ttctggtgac cggaggtgcg 1140 ggcttcatcg gctcgcactt cgtgcggcag ctcctcgccg gggcgtaccc cgacgtgccc 1200 gccgatgagg tgatcgtcct ggacagcctc acctacgcgg gcaaccgcgc caacctcgcc 1260 ccggtggacg cggacccgcg actgcgcttc gtccacggcg acatccgcga cgccggcctc 1320 ctcgcccggg aactgcgcgg cgtggacgcc atcgtccact tcgcggccga gagccacgtg 1380 gaccgctcca tcgcgggcgc gtccgtgttc accgagacca acgtgcaggg cacgcagacg 1440 ctgctccagt gcgccgtcga cgccggcgtc ggccgggtcg tgcacgtctc caccgacgag 1500 gtgtacgggt cgatcgactc cggctcctgg accgagagca gcccgctgga gcccaactcg 1560 ccctacgcgg cgtccaaggc cggctccgac ctcgttgccc gcgcctacca ccggacgtac 1620 ggcctcgacg tacggatcac ccgctgctgc aacaactacg ggccgtacca gcaccccgag 1680 aagctcatcc ccctcttcgt gacgaacctc ctcgacggcg ggacgctccc gctgtacggc 1740 gacggcgcga acgtccgcga gtgggtgcac accgacgacc actgccgggg catcgcgctc 1800 gtcctcgcgg gcggccgggc cggcgagatc taccacatcg gcggcggcct ggagctgacc 1860 aaccgcgaac tcaccggcat cctcctggac tcgctcggcg ccgactggtc ctcggtccgg 1920 aaggtcgccg accgcaaggg ccacgacctg cgctactccc tcgacggcgg caagatcgag 1980 cgcgagctcg gctaccgccc gcaggtctcc ttcgcggacg gcctcgcgcg gaccgtccgc 2040 tggtaccggg agaaccgcgg ctggtgggag ccgctcaagg cgaccgcccc gcagctgccc 2100 gccaccgccg tggaggtgtc cgcgtgagca gccgcgccga gaccccccgc gtccccttcc 2160 tcgacctcaa ggccgcctac gaggagctcc gcgcggagac cgacgccgcg atcgcccgcg 2220 tcctcgactc ggggcgctac ctcctcggac ccgaactcga aggattcgag gcggagttcg 2280 ccgcgtactg cgagacggac cacgccgtcg gcgtgaacag cgggatggac gccctccagc 2340 tcgccctccg cggcctcggc atcggacccg gggacgaggt gatcgtcccc tcgcacacgt 2400 acatcgccag ctggctcgcg gtgtccgcca ccggcgcgac ccccgtgccc gtcgagccgc 2460 acgaggacca ccccaccctg gacccgctgc tcgtcgagaa ggcgatcacc ccccgcaccc 2520 gggcgctcct ccccgtccac ctctacgggc accccgccga catggacgcc ctccgcgagc 2580 tcgcggaccg gcacggcctg cacatcgtcg aggacgccgc gcaggcccac ggcgcccgct 2640 accggggccg gcggatcggc gccgggtcgt cggtggccgc gttcagcttc tacccgggca 2700 agaacctcgg ctgcttcggc gacggcggcg ccgtcgtcac cggcgacccc gagctcgccg 2760 aacggctccg gatgctccgc aactacggct cgcggcagaa gtacagccac gagacgaagg 2820 gcaccaactc ccgcctggac gagatgcagg ccgccgtgct gcggatccgg ctcgnccacc 2880 tggacagctg gaacggccgc aggtcggcgc tggccgcgga gtacctctcc gggctcgccg 2940 gactgcccgg catcggcctg ccggtgaccg cgcccgacac cgacccggtc tggcacctct 3000 tcaccgtgcg caccgagcgc cgcgacgagc tgcgcagcca cctcgacgcc cgcggcatcg 3060 acaccctcac gcactacccg gtacccgtgc acctctcgcc cgcctacgcg ggcgaggcac 3120 cgccggaagg ctcgctcccg cgggccgaga gcttcgcgcg gcaggtcctc agcctgccga 3180 tcggcccgca cctggagcgc ccgcaggcgc tgcgggtgat cgacgccgtg cgcgaatggg 3240 ccgagcgggt cgaccaggcc tagtcaggtg gtccggtaga cccagcaggc cg 3292 23 1693 DNA Streptomyces venezuelae 23 atgcggcacc ccttggcgcc gagcgtggtg atccaggtgc cgacccgggc gagcacctcc 60 tgctcggtcc agcccgtctt gctgagcagc agcgcccgct cgtaggcgtt cgtgaacagc 120 agctcggctc cgtcgacgag ctcccggacg ctgtcgccct ccagccgggc gagctgctgc 180 gaggggtccg cggcccggcg gaggcccagc tcgcggcaga cccgcgtgtg ccgcaccatc 240 gcctcggggt cgtccgcgcc gacgaggacg aggtcgatcc cgccgggccg gccggccgtc 300 tcgcccaggt cgatgtcgcg cgcctcggcc atcgcgcccg cgtagaacga ggcgagctga 360 ttgccgtcct cgtcggtggt gcacatgaag cgggcggtgt gctgacggtc cgacacccgc 420 acggagtcgg tgtcgacgcc cgcggcgcgg agcagctgcc cgtacccgtc gaagtccttg 480 ccgacggcgc cgacgaggac ggggcggcga ccgagcaggc cgaggccgta cgcgatgttg 540 gcggcgacgc cgccgtgccg gatgtccagg gtgtcgacga ggaacgacag ggacacgtgg 600 gcgagctggt ccggcaggat ctgctcggcg aagcggcccg ggaaggtcat caggtggtcg 660 gtggcgatcg acccggtgac ggctatacgc atgtcagagc cccgcggcct tcttcagggc 720 gtccacgcgg tcggtgcgct cccaggtgaa gtccggcagc tcgcggccga agtggccgta 780 ggcggcggtc tgggagtaga tcgggcggag caggtcgagg tcgcggatga tcgcggccgg 840 gcggaggtcg aagacctcgc cgatggcgtt ctcgatcttc tcggtctcga tcttgtgggt 900 gccgaaggtc tcgacgaaga ggccgacggg ctcggccttg ccgatcgcgt acgcgacctg 960 gacctcgcag cgcgaggcga gaccggcggc gacgacgttc ttcgccaccc agcgcatcgc 1020 gtacgcggcg gagcggtcga ccttcgacgg gtccttgccg gagaaggcgc cgccaccgtg 1080 gcgggccatg ccgccgtagg tgtcgatgat gatcttgcgg ccggtgaggc cggcgtcgcc 1140 catcgggccg ccgatctcga agcgaccggt cgggttcacg agcaggcggt agccgtcggt 1200 gtcgagcttg atgccgtcct cgacgagctg cgcaagcacg tgctcgacga cgaacttccg 1260 cacgtcgggg gcgagcagcg actccaggtc gatgtccgag gcgtgctgcg aggagacgac 1320 gaccgtgtcg agacggaccg ccctgtcgcc gtcgtactcg atggtgacct gggtcttgcc 1380 gtcgggacgc aggtacggga tggtcccgtt cttgcggacc tcggtcaggc ggcgcgagag 1440 acggtgcgcg aggtggatcg gcagcggcat cagctcgggc gtctcgtccg aggcatagcc 1500 gaacatcagg ccctggtcac cggcgccctg cttgtcgagc tcgtccccct cgtcccgctg 1560 ggaggcaccc tcgacccgct tctcgtacgc ggtgtcgaca ccctgggcga tgtccgggga 1620 ctgcgacccg atggacaccg acacgccgca ggaggcgccg tcgaagccct tcttcgagga 1680 gtcgtacccg atc 1693 24 1565 DNA Streptomyces venezuelae 24 ccccgctcgc ggccccccag acatccacgc ccacgattgg acgctcccga tgaccgcccc 60 cgccctctcc gccaccgccc cggccgaacg ctgcgcgcac cccggagccg atctgggggc 120 ggcggtccac gccgtcggcc agaccctcgc cgccggcggc ctcgtgccgc ccgacgaggc 180 cggaacgacc gcccgccacc tcgtccggct cgccgtgcgc tacggcaaca gccccttcac 240 cccgctggag gaggcccgcc acgacctggg cgtcgaccgg gacgccttcc ggcgcctcct 300 cgccctgttc gggcaggtcc cggagctccg caccgcggtc gagaccggcc ccgccggggc 360 gtactggaag aacaccctgc tcccgctcga acagcgcggc gtcttcgacg cggcgctcgc 420 caggaagccc gtcttcccgt acagcgtcgg cctctacccc ggcccgacct gcatgttccg 480 ctgccacttc tgcgtccgtg tgaccggcgc ccgctacgac ccgtccgccc tcgacgccgg 540 caacgccatg ttccggtcgg tcatcgacga gatacccgcg ggcaacccct cggcgatgta 600 cttctccggc ggcctggagc cgctcaccaa ccccggcctc gggagcctgg ccgcgcacgc 660 caccgaccac ggcctgcggc ccaccgtcta cacgaactcc ttcgcgctca ccgagcgcac 720 cctggagcgc cagcccggcc tctggggcct gcacgccatc cgcacctcgc tctacggcct 780 caacgacgag gagtacgagc agaccaccgg caagaaggcc gccttccgcc gcgtccgcga 840 gaacctgcgc cgcttccagc agctgcgcgc cgagcgcgag tcgccgatca acctcggctt 900 cgcctacatc gtgctcccgg gccgtgcctc ccgcctgctc gacctggtcg acttcatcgc 960 cgacctcaac gacgccgggc agggcaggac gatcgacttc gtcaacattc gcgaggacta 1020 cagcggccgt gacgacggca agctgccgca ggaggagcgg gccgagctcc aggaggccct 1080 caacgccttc gaggagcggg tccgcgagcg cacccccgga ctccacatcg actacggcta 1140 cgccctgaac agcctgcgca ccggggccga cgccgaactg ctgcggatca agcccgccac 1200 catgcggccc accgcgcacc cgcaggtcgc ggtgcaggtc gatctcctcg gcgacgtgta 1260 cctgtaccgc gaggccggct tccccgacct ggacggcgcg acccgctaca tcgcgggccg 1320 cgtgaccccc gacacctccc tcaccgaggt cgtcagggac ttcgtcgagc gcggcggcga 1380 ggtggcggcc gtcgacggcg acgagtactt catggacggc ttcgatcagg tcgtcaccgc 1440 ccgcctgaac cagctggagc gcgacgccgc ggacggctgg gaggaggccc gcggcttcct 1500 gcgctgaccc gcacccgccc cgatcccccc gatccccccc ccacgatccc cccacctgag 1560 ggccc 1565 25 31 DNA Streptomyces venezuelae 25 ccctgcagcg gcaaggaagg acacgacgcc a 31 26 32 DNA Streptomyces venezuelae 26 aggtctagag ctcagtgccg ggcgtcggcc gg 32 27 37 DNA Streptomyces venezuelae 27 ttgcatgcat atgcgccgta cccagcaggg aacgacc 37 28 38 DNA Streptomyces venezuelae 28 ttgaattctc aactagtacg gcggcccgcc tcccgtcc 38 29 29 DNA Streptomyces venezuelae 29 ctagtatgca tcatcatcat catcattaa 29 30 29 DNA Streptomyces venezuelae 30 aattttaatg atgatgatga tgatgcata 29 31 18 DNA Streptomyces venezuelae 31 tcctctagac gtttccgt 18 32 21 DNA Streptomyces venezuelae 32 tgaagcttga attcaaccgg t 21 33 27 DNA Streptomyces venezuelae 33 tttatgcatc ccgcgggtcc cggcgag 27 34 27 DNA Streptomyces venezuelae 34 tcagaattct gtcggtcact tgcccgc 27

Claims (27)

1. A recombinant DNA compound that comprises a coding sequence for a domain of a narbonolide PKS.
2. The recombinant DNA compound of claim 1, wherein said domain is selected from the group consisting of a thioesterase domain, a KSQ domain, an AT domain, a KS domain, an ACP domain, a KR domain, a DH domain, and an ER domain.
3. The recombinant DNA compound of claim 2 that comprises the coding sequence for a loading module, thioesterase domain, and all six extender modules of the narbonolide PKS.
4. The recombinant DNA compound of claim 2 that comprises a hybrid PKS.
5. The recombinant DNA compound of claim 4 wherein said hybrid PKS comprises at least a portion of a narbonolide PKS gene and at least a portion of a second PKS gene for a macrolide aglycone other than narbonolide.
6. The recombinant DNA compound of claim 5 wherein said second PKS gene is a DEBS gene.
7. The recombinant DNA compound of claim 6 wherein said hybrid PKS is composed of a loading module and extender modules 1 through 6 of DEBS excluding a KR domain of extender module 6 of DEBS and an ACP of extender module 6 and a thioesterase domain of the narbonolide PKS.
8. A recombinant DNA compound that comprises a coding sequence for a desosamine biosynthetic gene or a desosaminyl transferase gene or a beta-glucosidase gene of Streptomyces venezuelae.
9. A recombinant DNA compound that comprises a coding sequence for a picK hydroxylase gene of Streptomyces venezuelae.
10. The DNA compound of any of claims 1-9 that further comprises a promoter operably linked to said coding sequence.
11. The recombinant DNA compound of claim 10, wherein said promoter is a promoter derived from a cell other than a Streptomyces venezuelae cell.
12. The recombinant DNA compound of claim 11 that is a recombinant DNA expression vector.
13. The expression vector of claim 12 that expresses a PKS in Streptomyces host cells.
14. A recombinant host cell, which in its untransformed state does not produce 10-deoxymethynolide or narbonolide, that comprises a recombinant DNA expression vector of claim 12 that encodes a narbonolide PKS and said cell produces 10-deoxymethynolide or narbonolide.
15. The recombinant host cell of claim 14 that further comprises a picB gene.
16. The recombinant host cell of claim 14 that further comprises desosamine biosynthetic genes and a gene for desosaminyl transferase and produces YC17 or narbomycin.
17. The recombinant host cell of claim 16 that further comprises a picK gene and produces methymycin, neomethymycin, or picromycin.
18. The recombinant host cell of any of claim 17 that is Streptomyces coelicolor or Streptomyces lividans.
19. A recombinant host cell other than a Streptomyces venezuelae cell that expresses the picK hydroxylase gene of S. venezuelae.
20. A recombinant host cell other than a Streptomyces venezuelae host cell that expresses a desosamine biosynthetic gene or desosaminyl transferase gene of S. venezuelae.
21. A method for increasing the yield of a desosaminylated polyketide in a cell, which method comprises transforming the cell with a recombinant expression vector that encodes a functional beta-glucosidase gene.
22. A hybrid PKS which comprises at least one domain of a narbonolide PKS.
23. The hybrid PKS of claim 22 wherein said hybrid PKS comprises at least a portion of a narbonolide PKS gene and at least a portion of a second PKS gene for a macrolide aglycone other than narbonolide.
24. The hybrid PKS of claim 23 wherein said second PKS gene is a DEBS gene.
25. The hybrid PKS of claim 24 wherein said hybrid PKS is composed of a loading module and extender modules 1 through 6 of DEBS excluding a KR domain of extender module 6 of DEBS and an ACP of extender module 6 and a thioesterase domain of the narbonolide PKS.
26. A method to produce a polyketide which comprises providing starter, extender and/or intermediate ketide units to the hybrid PKS of claim 22.
27. A polyketide produced by the method of claim 26.
US10/160,539 1998-05-28 2002-05-29 Recombinant narbonolide polyketide synthase Abandoned US20030162262A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US10/160,539 US20030162262A1 (en) 1998-05-28 2002-05-29 Recombinant narbonolide polyketide synthase

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
US8708098P 1998-05-28 1998-05-28
US09/141,908 US6503741B1 (en) 1998-05-28 1998-08-28 Polyketide synthase genes from Streptomyces venezuelae
US10088098P 1998-09-22 1998-09-22
US11913999P 1999-02-08 1999-02-08
US13499099P 1999-05-20 1999-05-20
US09/320,878 US6117659A (en) 1997-04-30 1999-05-27 Recombinant narbonolide polyketide synthase
US09/657,440 US6509455B1 (en) 1997-04-30 2000-09-07 Recombinant narbonolide polyketide synthase
US10/160,539 US20030162262A1 (en) 1998-05-28 2002-05-29 Recombinant narbonolide polyketide synthase

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US09/657,440 Continuation US6509455B1 (en) 1997-04-30 2000-09-07 Recombinant narbonolide polyketide synthase

Publications (1)

Publication Number Publication Date
US20030162262A1 true US20030162262A1 (en) 2003-08-28

Family

ID=27568329

Family Applications (3)

Application Number Title Priority Date Filing Date
US09/320,878 Expired - Fee Related US6117659A (en) 1997-04-30 1999-05-27 Recombinant narbonolide polyketide synthase
US09/657,440 Expired - Fee Related US6509455B1 (en) 1997-04-30 2000-09-07 Recombinant narbonolide polyketide synthase
US10/160,539 Abandoned US20030162262A1 (en) 1998-05-28 2002-05-29 Recombinant narbonolide polyketide synthase

Family Applications Before (2)

Application Number Title Priority Date Filing Date
US09/320,878 Expired - Fee Related US6117659A (en) 1997-04-30 1999-05-27 Recombinant narbonolide polyketide synthase
US09/657,440 Expired - Fee Related US6509455B1 (en) 1997-04-30 2000-09-07 Recombinant narbonolide polyketide synthase

Country Status (1)

Country Link
US (3) US6117659A (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210163964A1 (en) * 2018-04-06 2021-06-03 University Of Florida Research Foundation, Incorporated Engineered streptomyces albus strains

Families Citing this family (42)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6495348B1 (en) * 1993-10-07 2002-12-17 Regents Of The University Of Minnesota Mitomycin biosynthetic gene cluster
US5958672A (en) 1995-07-18 1999-09-28 Diversa Corporation Protein activity screening of clones having DNA from uncultivated microorganisms
US6204388B1 (en) 1996-12-03 2001-03-20 Sloan-Kettering Institute For Cancer Research Synthesis of epothilones, intermediates thereto and analogues thereof
EP0977563B1 (en) * 1996-12-03 2005-10-12 Sloan-Kettering Institute For Cancer Research Synthesis of epothilones, intermediates thereto, analogues and uses thereof
US6902913B2 (en) * 1997-04-30 2005-06-07 Kosan Biosciences, Inc. Recombinant narbonolide polyketide synthase
US6265202B1 (en) * 1998-06-26 2001-07-24 Regents Of The University Of Minnesota DNA encoding methymycin and pikromycin
EP1229934B1 (en) 1999-10-01 2014-03-05 Immunogen, Inc. Compositions and methods for treating cancer using immunoconjugates and chemotherapeutic agents
US7033818B2 (en) * 1999-10-08 2006-04-25 Kosan Biosciences, Inc. Recombinant polyketide synthase genes
US6524841B1 (en) 1999-10-08 2003-02-25 Kosan Biosciences, Inc. Recombinant megalomicin biosynthetic genes and uses thereof
US20030194784A1 (en) * 2001-04-17 2003-10-16 Sherman David H. DNA encoding methymycin and pikromycin
AR034703A1 (en) * 2001-05-16 2004-03-17 Syngenta Participations Ag METHODS AND COMPOSITIONS TO PREPARE EMAMECTINE.
US20040161839A1 (en) * 2001-10-05 2004-08-19 Hung-Wen Liu Method to alter sugar moieties
AU2003248694A1 (en) * 2002-06-13 2003-12-31 Kosan Biosciences, Inc. Methods and cells for improved production of polyketides
AU2003258978A1 (en) 2002-06-28 2004-01-19 Kosan Biosciences, Inc. Recombinant genes for polyketide modifying enzymes
WO2004024744A2 (en) * 2002-07-31 2004-03-25 The Board Of Trustees Of The Leland Stanford Junior University Production of glycosylated macrolides in e. coli
EP1767535B1 (en) 2002-08-23 2009-12-02 Sloan-Kettering Institute For Cancer Research Synthesis of epothilones, intermediates thereto, analogues and uses thereof
US7649006B2 (en) 2002-08-23 2010-01-19 Sloan-Kettering Institute For Cancer Research Synthesis of epothilones, intermediates thereto and analogues thereof
AU2003291534A1 (en) * 2002-11-12 2004-06-03 Kosan Biosciences, Inc. Method for producing polyketides
WO2004053065A2 (en) * 2002-12-06 2004-06-24 Kosan Biosciences, Inc. Disorazole polyketide synthase encoding polynucleotides
US7288396B2 (en) * 2003-09-11 2007-10-30 Kosan Biosciences Incorporated Biosynthetic gene cluster for leptomycins
WO2012036241A1 (en) * 2010-09-16 2012-03-22 独立行政法人海洋研究開発機構 Novel extracellularly secreted nuclease
KR101972303B1 (en) 2011-06-10 2019-04-25 메르사나 테라퓨틱스, 인코포레이티드 Protein-Polymer-Drug Conjugates
AU2013359506B2 (en) 2012-12-10 2018-05-24 Mersana Therapeutics, Inc. Protein-polymer-drug conjugates
US9872918B2 (en) 2012-12-12 2018-01-23 Mersana Therapeutics, Inc. Hydroxyl-polymer-drug-protein conjugates
MA38656A1 (en) 2013-06-11 2018-05-31 Bayer Pharma AG Combinations for the treatment of cancer comprising an inhibitor of the mps-1 kinase and a mitosis inhibitor
CN105813655B (en) 2013-10-11 2022-03-15 阿萨纳生物科技有限责任公司 Protein-polymer-drug conjugates
KR102087850B1 (en) 2013-10-11 2020-03-12 메르사나 테라퓨틱스, 인코포레이티드 Protein-Polymer-Drug Conjugates
WO2015156739A1 (en) * 2014-04-07 2015-10-15 Agency For Science, Technology And Research Biosynthetic polypeptide and methods of use
US11617799B2 (en) 2016-06-27 2023-04-04 Tagworks Pharmaceuticals B.V. Cleavable tetrazine used in bio-orthogonal drug activation
US11135307B2 (en) 2016-11-23 2021-10-05 Mersana Therapeutics, Inc. Peptide-containing linkers for antibody-drug conjugates
CN111315414A (en) 2017-06-22 2020-06-19 梅尔莎纳医疗公司 Methods of producing drug-loaded polymer scaffolds and protein-polymer-drug conjugates
US20210308207A1 (en) 2018-05-04 2021-10-07 Tagworks Pharmaceuticals B.V. Compounds comprising a linker for increasing transcyclooctene stability
AU2019262520A1 (en) 2018-05-04 2021-01-14 Tagworks Pharmaceuticals B.V. Tetrazines for high click conjugation yield in vivo and high click release yield
CA3117050A1 (en) 2018-10-29 2020-05-07 Mersana Therapeutics, Inc. Cysteine engineered antibody-drug conjugates with peptide-containing linkers
EP3983363B1 (en) 2019-06-17 2024-04-10 Tagworks Pharmaceuticals B.V. Compounds for fast and efficient click release
IL289094A (en) 2019-06-17 2022-02-01 Tagworks Pharmaceuticals B V Tetrazines for high click release speed and yield
WO2023031445A2 (en) 2021-09-06 2023-03-09 Veraxa Biotech Gmbh Novel aminoacyl-trna synthetase variants for genetic code expansion in eukaryotes
WO2023094525A1 (en) 2021-11-25 2023-06-01 Veraxa Biotech Gmbh Improved antibody-payload conjugates (apcs) prepared by site-specific conjugation utilizing genetic code expansion
EP4186529A1 (en) 2021-11-25 2023-05-31 Veraxa Biotech GmbH Improved antibody-payload conjugates (apcs) prepared by site-specific conjugation utilizing genetic code expansion
WO2023104941A1 (en) 2021-12-08 2023-06-15 European Molecular Biology Laboratory Hydrophilic tetrazine-functionalized payloads for preparation of targeting conjugates
EP4314031B1 (en) 2022-02-15 2024-03-13 Tagworks Pharmaceuticals B.V. Masked il12 protein
WO2024013724A1 (en) 2022-07-15 2024-01-18 Pheon Therapeutics Ltd Antibody-drug conjugates

Citations (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4551433A (en) * 1981-05-18 1985-11-05 Genentech, Inc. Microbial hybrid promoters
US4874748A (en) * 1986-03-24 1989-10-17 Abbott Laboratories Cloning vectors for streptomyces and use thereof in macrolide antibiotic production
US5063155A (en) * 1988-03-28 1991-11-05 Eli Lilly And Company Method for producing 2"'-o-demethyltylosin
US5098837A (en) * 1988-06-07 1992-03-24 Eli Lilly And Company Macrolide biosynthetic genes for use in streptomyces and other organisms
US5149639A (en) * 1986-03-24 1992-09-22 Abbott Laboratories Biologically pure cultures of streptomyces and use thereof in macrolide antibiotic production
US5168052A (en) * 1988-03-28 1992-12-01 Eli Lilly And Company Method for producing 20-deoxotylosin
US5252474A (en) * 1989-03-31 1993-10-12 Merck & Co., Inc. Cloning genes from Streptomyces avermitilis for avermectin biosynthesis and the methods for their use
US5514544A (en) * 1991-07-26 1996-05-07 Eli Lilly And Company Activator gene for macrolide biosynthesis
US5672491A (en) * 1993-09-20 1997-09-30 The Leland Stanford Junior University Recombinant production of novel polyketides
US5712146A (en) * 1993-09-20 1998-01-27 The Leland Stanford Junior University Recombinant combinatorial genetic library for the production of novel polyketides
US5712496A (en) * 1992-01-17 1998-01-27 Seiko Instruments, Inc. MOS Poly-Si thin film transistor with a flattened channel interface and method of producing same
US5824513A (en) * 1991-01-17 1998-10-20 Abbott Laboratories Recombinant DNA method for producing erythromycin analogs
US5998194A (en) * 1995-12-21 1999-12-07 Abbott Laboratories Polyketide-associated sugar biosynthesis genes
US6200813B1 (en) * 1991-01-17 2001-03-13 Abbott Laboratories Polyketide derivatives and recombinant methods for making same
US6265202B1 (en) * 1998-06-26 2001-07-24 Regents Of The University Of Minnesota DNA encoding methymycin and pikromycin

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IL81892A (en) * 1986-03-21 1995-10-31 Lilly Co Eli Recombinant DNA molecule comprising a sequence encoding macrocin o-methyltransferase
WO1993013663A1 (en) * 1992-01-17 1993-07-22 Abbott Laboratories Method of directing biosynthesis of specific polyketides
AU678058B2 (en) * 1993-09-20 1997-05-15 John Innes Centre Recombinant production of novel polyketides
NZ313383A (en) * 1995-07-06 1999-08-30 Univ Leland Stanford Junior Cell-free synthesis of polyketides using pks (polyketide synthase)
DE69636195T2 (en) * 1995-10-13 2007-03-29 President And Fellows Of Harvard College, Cambridge PHOSPHOPANTETHENYL TRANSFERASES AND ITS USES
EP0870053A4 (en) * 1995-12-19 2002-09-11 Univ Minnesota Metabolic engineering of polyhydroxyalkanoate monomer synthases
CA2197524A1 (en) * 1996-02-22 1997-08-22 Bradley Stuart Dehoff Polyketide synthase genes
CA2197160C (en) * 1996-02-22 2007-05-01 Stanley Gene Burgett Platenolide synthase gene
DE69739688D1 (en) * 1996-07-05 2010-01-21 Biotica Tech Ltd Hybrid polyketide synthase I gene
US5712354A (en) * 1996-07-10 1998-01-27 Mobil Oil Corporation Bridged metallocene compounds
US6033883A (en) * 1996-12-18 2000-03-07 Kosan Biosciences, Inc. Production of polyketides in bacteria and yeast

Patent Citations (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4551433A (en) * 1981-05-18 1985-11-05 Genentech, Inc. Microbial hybrid promoters
US4874748A (en) * 1986-03-24 1989-10-17 Abbott Laboratories Cloning vectors for streptomyces and use thereof in macrolide antibiotic production
US5149639A (en) * 1986-03-24 1992-09-22 Abbott Laboratories Biologically pure cultures of streptomyces and use thereof in macrolide antibiotic production
US5063155A (en) * 1988-03-28 1991-11-05 Eli Lilly And Company Method for producing 2"'-o-demethyltylosin
US5168052A (en) * 1988-03-28 1992-12-01 Eli Lilly And Company Method for producing 20-deoxotylosin
US5098837A (en) * 1988-06-07 1992-03-24 Eli Lilly And Company Macrolide biosynthetic genes for use in streptomyces and other organisms
US5252474A (en) * 1989-03-31 1993-10-12 Merck & Co., Inc. Cloning genes from Streptomyces avermitilis for avermectin biosynthesis and the methods for their use
US5824513A (en) * 1991-01-17 1998-10-20 Abbott Laboratories Recombinant DNA method for producing erythromycin analogs
US6200813B1 (en) * 1991-01-17 2001-03-13 Abbott Laboratories Polyketide derivatives and recombinant methods for making same
US5514544A (en) * 1991-07-26 1996-05-07 Eli Lilly And Company Activator gene for macrolide biosynthesis
US5712496A (en) * 1992-01-17 1998-01-27 Seiko Instruments, Inc. MOS Poly-Si thin film transistor with a flattened channel interface and method of producing same
US5672491A (en) * 1993-09-20 1997-09-30 The Leland Stanford Junior University Recombinant production of novel polyketides
US5712146A (en) * 1993-09-20 1998-01-27 The Leland Stanford Junior University Recombinant combinatorial genetic library for the production of novel polyketides
US5998194A (en) * 1995-12-21 1999-12-07 Abbott Laboratories Polyketide-associated sugar biosynthesis genes
US6265202B1 (en) * 1998-06-26 2001-07-24 Regents Of The University Of Minnesota DNA encoding methymycin and pikromycin

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210163964A1 (en) * 2018-04-06 2021-06-03 University Of Florida Research Foundation, Incorporated Engineered streptomyces albus strains

Also Published As

Publication number Publication date
US6509455B1 (en) 2003-01-21
US6117659A (en) 2000-09-12

Similar Documents

Publication Publication Date Title
US6509455B1 (en) Recombinant narbonolide polyketide synthase
US6902913B2 (en) Recombinant narbonolide polyketide synthase
AU762399C (en) Recombinant narbonolide polyketide synthase
Rowe et al. Engineering a polyketide with a longer chain by insertion of an extra module into the erythromycin-producing polyketide synthase
US6251636B1 (en) Recombinant oleandolide polyketide synthase
Tang et al. Formation of functional heterologous complexes using subunits from the picromycin, erythromycin and oleandomycin polyketide synthases
EP0979286B1 (en) Combinatorial polyketide libraries produced using a modular pks gene cluster as scaffold
US20030077707A1 (en) Combinatorial polyketide libraries produced using a modular PKS gene cluster as scaffold
US20040224394A1 (en) Recombinant Streptomyces hygroscopicus host cells that produce 17-desmethylrapamycin
US5691140A (en) Bidirectional in vitro transcription vectors utilizing a single RNA polymerase for both directions
AU784995B2 (en) Novel genes encoding a nystatin polyketide synthase and their manipulation and utility
US6303767B1 (en) Nucleic acids encoding narbonolide polyketide synthase enzymes from streptomyces narbonensis
US6399789B1 (en) Multi-plasmid method for preparing large libraries of polyketides and non-ribosomal peptides
US6503741B1 (en) Polyketide synthase genes from Streptomyces venezuelae
US20060269528A1 (en) Production detection and use of transformant cells
US6828126B2 (en) Methods for introducing hydroxyl or epoxide groups into polyketides using OleP
Boom Recent developments in the molecular genetics of the erythromycin-producing organism Saccharopolyspora erythraea
CA2369633A1 (en) A multi-plasmid method for preparing large libraries of polyketides and non-ribosomal peptides
US20040072165A1 (en) Method for producing dna encoding polypeptides that are composed of several section, and for producing polypeptides by expressing the dna thus obtained
US20030148469A1 (en) Combinatorial polyketide libraries produced using a modular PKS gene cluster as scaffold
US7285405B2 (en) Biosynthetic gene cluster for jerangolids
US20040209322A1 (en) Combinatorial polyketide libraries produced using a modular PKS gene cluster as scaffold
AU5780501A (en) Combinatorial polyketide libraries produced using a modular PKS gene cluster as scaffold

Legal Events

Date Code Title Description
STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION