WO1999061599A2 - Recombinant narbonolide polyketide synthase - Google Patents
Recombinant narbonolide polyketide synthase Download PDFInfo
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- WO1999061599A2 WO1999061599A2 PCT/US1999/011814 US9911814W WO9961599A2 WO 1999061599 A2 WO1999061599 A2 WO 1999061599A2 US 9911814 W US9911814 W US 9911814W WO 9961599 A2 WO9961599 A2 WO 9961599A2
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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.
- FK506, FK520, narbomycin, picromycin, rapamycin, spinocyn, and tylosin are examples of such compounds.
- PKS 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.
- 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 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 Figure 1, compound 1), the 12-membered macrolides neomethymycin and methymycin (methymycin is shown in Figure 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.
- narbonolide Glycosylation of the C5 hydroxyl group of the polyketide precursor, narbonolide, is achieved through an endogenous desosaminyl transferase to produce narbomycin. In Streptomyces venezuelae, narbomycin is then converted to picromycin by the endogenously produced narbomycin hydroxylase. (See Figure 1)
- the macrolide product of the narbonolide PKS is further modified by hydroxylation and glycosylation.
- Figure 1 also shows the metabolic relationships of the compounds discussed above.
- Picromycin (Figure 1, compound 1) is of particular interest because of its close structural relationship to ketolide compounds (e.g. HMR 3004, Figure 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 hydroxy lated 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 (fhspicK gene), the desosamine biosynthesis and desosaminyl transferase enzymes, and the beta- glucosidase enzyme involved in picromycin biosynthesis in S.
- 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 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.
- Figure 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.
- Figure 2 shows a restriction site and function map of cosmid pKOS023-27.
- Figure 3 shows a restriction site and function map of cosmid pKOS023-26.
- Figure 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 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 t epicB thioesterase gene and two desosamine biosynthesis genes (picCII and picCIII).
- 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 acyltransf erase
- DH dehydratase
- ER is enoylreductase
- KR ketoreductase
- KS is ketosynthase
- TE is thioesterase.
- Figure 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.
- Figure 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, Figure 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. Patent No. 5,824,513, incorporated herein by reference. Recombinant methods for manipulating modular PKS genes are described in U.S.
- 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.
- 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.
- 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.
- 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.
- modules may contain additional enzymatic activities as well.
- 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 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.
- the narbonolide PKS is composed of a loading module, six extender modules, and two thioesterase domains one of which is on a separate protein.
- Figure 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. There is a second thioesterase domain (TEII) on a separate protein, designated PICB. The amino acid sequences of these proteins are shown below.
- PICAIII SEQ ID NO:3
- MANNEDKLRD YLKRVTAELQ
- QNTRRLREIE GRTHEPVAIV GMACRLPGGV ASPEDLWQLV
- 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.
- Figure 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.
- the invention includes DNA compounds of any sequence that encode the amino acid sequences of the polypeptides and proteins of the invention.
- 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.
- 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 Co A. 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 and 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 Co A, 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 pic AW 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 Figure 4, part B) of DNA flanking fhepicAl 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 desosaminylating 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.
- 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-deoxy glucose 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 fhe picCVI gene, also known as desVI, a homologue of eryCVI, which encodes a 3-amino dimethyltransf erase.
- 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 20 Aug 1998 under the Budapest Treaty and is available under the accession number ATCC 203141.
- Figure 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.
- ORF 13 also known as desIII, has no known ery gene homologue and encodes an NDP glucose synthase.
- the picCV gene also known as desll, a homologue of eryCV is required for desosamine biosynthesis.
- the picCIV gene also known as desl, 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 ORF 16, 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 hydroxy lates the C 12 of narbomycin and the CIO 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. Amino acid sequence of PICCI (desV) (SEQ ID NO:6)
- 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)
- 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)
- 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
- Contig 004 from cosmid pKOS023-26 contains 1693 nucleotides and the following ORFs: from nucleotide 1692 to 694 is ORF 15, which encodes a part of S-adenosylmethionine synthetase; and from nucleotide 692 to 1 is ORF 16, which encodes a part of a protein homologous to the M. tuberculosis cbhK gene. (SEQ ID NO:23)
- 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)
- 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 hydroxy lating 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 /?/ ⁇ r 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 apicK 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 C 12 position and YC-17 at either the CIO 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 pic K 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. To drive production of the RNA, 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, as 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 (tip), the beta- lactamase (bla), bacteriophage lambda PL, and T5 promoters. In addition, synthetic promoters, such as the tac promoter (U.S. Patent 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 tew gene promoters; an example of a Type I PKS gene cluster promoter is the spiramycin PKS gene promoter. 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. Patent No. 5,672,491, inco ⁇ orated 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. Gen. Genet.
- 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
- 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, plP, pll, and pBR.
- phage phiC31 and its derivative KC515 can be employed (see Hopwood et al, supra).
- plasmid pS ⁇ T152, plasmid pSAM, plasmids pSElOl and pSE211 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 pRMl and pRM5 vectors, as described in U.S. Patent No. 5,830,750 and U.S. patent application Serial Nos. 08/828,898, filed 31 Mar. 1997, and 09/181,833, filed 28 Oct. 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 melCl 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 dnrl, redD, and ptpA genes (see U.S. patent application Serial 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.
- 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.
- markers include 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 ( Figure 5, compound 4) and 10-deoxymethynolide ( Figure 5, compound 5), the respective 14 and 12-membered polyketide precursors of narbomycin and YC17.
- 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 (Pact/) 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 6-dEB-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. In one important embodiment, 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.
- 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.
- 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 CIO 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.
- 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 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, 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.
- 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°- 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.
- 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.
- 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.
- 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, 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 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 27 Jan. 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.
- 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.
- Streptomyces hygroscopicus analysis of the enzymatic domains in the modular polyketide synthase, Gene 169: 9-16.
- 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 hybrid PKS-encoding DNA compounds of the invention can be and often are hybrids of more than two PKS genes. Even where only two genes are used, there are often two or more modules in the hybrid gene in which all or part of the module is derived from a second (or third) PKS gene.
- the invention provides a hybrid narbonolide PKS that contains the naturally occurring loading module and thioesterase domain as well as extender modules one, two, four, and six of the narbonolide PKS and further contains hybrid or heterologous extender modules three and five.
- Hybrid or heterologous extender modules three and five contain AT domains specific for malonyl CoA and derived from, for example, the rapamycin PKS genes.
- a hybrid PKS or narbonolide derivative PKS of the invention one can employ a technique, described in PCT Pub. No. WO 98/27203, which is incorporated herein by reference, in which the large PKS gene cluster is divided into two or more, typically three, segments, and each segment is placed on a separate expression vector. In this manner, each of the segments of the gene can be altered, and various altered segments can be combined in a single host cell to provide a recombinant PKS gene of the invention.
- This technique makes more efficient the construction of large libraries of recombinant PKS genes, vectors for expressing those genes, and host cells comprising those vectors.
- hybrid PKS where alterations (including deletions, insertions and substitutions) are made directly using the narbonolide PKS as a substrate.
- the invention also provides libraries of PKS genes, PKS proteins, and ultimately, of polyketides. that are constructed by generating modifications in the narbonolide PKS so that the protein complexes produced have altered activities in one or more respects and thus produce polyketides other than the natural product of the PKS. Novel polyketides may thus be prepared, or polyketides in general prepared more readily, using this method.
- a modular PKS “derived from” the narbonolide or other naturally occurring PKS is a subset of the “hybrid” PKS family and includes a modular PKS (or its corresponding encoding gene(s)) that retains the scaffolding of the utilized portion of the naturally occurring gene. Not all modules need be included in the constructs.
- On the constant scaffold at least one enzymatic activity is mutated, deleted, replaced, or inserted so as to alter the activity of the resulting PKS relative to the original PKS. Alteration results when these activities are deleted or are replaced by a different version of the activity, or simply mutated in such a way that a polyketide other than the natural product results from these collective activities.
- the origin of the replacement activity may come from a corresponding activity in a different naturally occurring PKS or from a different region of the narbonolide PKS.
- Any or all of the narbonolide PKS genes may be included in the derivative or portions of any of these may be included, but the scaffolding of the PKS protein is retained in whatever derivative is constructed.
- the derivative preferably contains a thioesterase activity from the narbonolide or another PKS.
- a PKS "derived from” the narbonolide PKS includes a PKS that contains the scaffolding of all or a portion of the narbonolide PKS.
- the derived PKS also contains at least two extender modules that are functional, preferably three extender modules, and more preferably four or more extender modules, and most preferably six extender modules.
- the derived PKS also contains mutations, deletions, insertions, or replacements of one or more of the activities of the functional modules of the narbonolide PKS so that the nature of the resulting polyketide is altered. This definition applies both at the protein and DNA sequence levels.
- Particular preferred embodiments include those wherein a KS, AT, KR, DH, or ER has been deleted or replaced by a version of the activity from a different PKS or from another location within the same PKS. Also preferred are derivatives where at least one non- condensation cycle enzymatic activity (KR, DH, or ER) has been deleted or added or wherein any of these activities has been mutated so as to change the structure of the polyketide synthesized by the PKS.
- a PKS derived from the narbonolide PKS are functional PKS modules or their encoding genes wherein at least one portion, preferably two portions, of the narbonolide PKS activities have been inserted.
- exemplary is the use of the narbonolide AT for extender module 2 which accepts a malonyl CoA extender unit rather than methylmalonyl CoA to replace a methylmalonyl specific AT in a PKS.
- Other examples include insertion of portions of non-condensation cycle enzymatic activities or other regions of narbonolide synthase activity into a heterologous PKS.
- the derived from definition applies to the PKS at both the genetic and protein levels.
- the polyketide chain length is determined by the number of modules in the PKS.
- the nature of the carbon skeleton of the PKS is determined by the specificities of the acyl transferases that determine the nature of the extender units at each position, e.g., malonyl, methylmalonyl, ethylmalonyl, or other substituted malonyl.
- the loading module specificity also has an effect on the resulting carbon skeleton of the polyketide.
- the loading module may use a different starter unit, such as acetyl, butyryl, and the like.
- KS1 extender module 1
- diketides that are chemically synthesized analogs of extender module 1 diketide products
- extender module 2 extender module 2
- KS 1 activity was inactivated through mutation.
- the oxidation state at various positions of the polyketide will be determined by the dehydratase and reductase portions of the modules. This will determine the presence and location of ketone and alcohol moieties and C-C double bonds or C-C single bonds in the polyketide.
- 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
- 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.
- the compounds of the invention can occur as mixtures of stereoisomers, it may be beneficial in some instances to generate individual stereoisomers.
- 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/ ⁇ R corresponds to KR alone.
- 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.
- 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.
- 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, Hpofection, 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.
- desired targets such as receptors, signaling proteins, and the like.
- the supematants 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. 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, 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-l-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.
- Saccharopolyspora erythraea or Streptomyces venezuelae to make the conversion, preferably using mutants unable to synthesize macrolides.
- 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-l 8, in which the pikTE ORF was fused with the DEBS genes in place of the DEBS TE ORF (see Figure 5).
- the fusion junction was chosen between the AT and ACP to eliminate ketoreductase activity in DEBS extender module 6 (KR 6 ).
- 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 ery All genes were coexpressed with picATV 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 pic AN 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 picATV 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 fhe pic Al 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- 10,1 l-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 DEB S3 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,l 1- anhydroerythronolide B in Streptomyces lividans.
- the DEB S3 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,ll- anhydroerythronolide B and 5,6-dideoxy-4,5-anhydro-10-desmethyl-10,l l- 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,l 1- anhydroerythronolide B as well as 5,6-dideoxy-10-desmethyl-10,l l-anhydroerythronolide B in Streptomyces lividans, indicating that the rapamycin DH, KR, and ER domains functioned only inefficiently in this construct.
- the DEB S3 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,l 1- anhydroerythronolide B in Streptomyces lividans.
- the DEB S3 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.
- 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.
- 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. Patent 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. However, when 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.
- 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 hydroxy late 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 C 12 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 C 12 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.
- -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; with the proviso that: at least two of R'-R 6 are alkyl (1-4C).
- 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, com 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. Patent 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, inco ⁇ orated 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.
- Example 1 General Methodology Bacterial strains, plasmids, and culture conditions, streptomyces coelicolor CH999 described in WO 95/08548, published 30 March 1995, or S. lividans K4-114, described in Ziermann and Betlach, Jan. 99, Recombinant Polyketide Synthesis in Streptomyces: Engineering of Improved Host Strains, BioTechniques 26:106-110, inco ⁇ orated herein by reference, was used as an expression host. DNA manipulations were performed in Escherichia coli XLl-Blue, available from Stratagene. E. coli MCI 061 is also suitable for use as a host for plasmid manipulation. Plasmids were passaged through E.
- E. coli ET12567 (dam dcm hsdS Cmr) (MacNeil, 1988, J. Bacteriol. 170: 5607, inco ⁇ orated 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, inco ⁇ orated 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, inco ⁇ orated 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 actll- 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-bome 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 Manual (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.
- 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 2xSSC buffer + 0.1% SDS for 15 minutes, followed by two 15 minute washes with 2xSSC 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
- 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 Figure 4, parts B and C.
- the -4.5 kb Hindlll-Spel fragment from plasmid pKOS039-07 was ligated with the 2.5 kb Hindlll-Nhel fragment of integrating vector pS ⁇ T152, 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.
- the cosmids were digested with BamHl 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 2XSSC buffer with 0.1% SDS for 15 minutes, followed by two 15 minute washes with 2XSSC 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 Serial No. 09/181,833, filed 28 Oct. 1998, each of which is inco ⁇ orated 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. Patent No.
- pCK7'Kan' differs from pCK7 only in that it contains a kanamycin resistance conferring gene inserted at its Hindlll 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 x 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, inco ⁇ orated 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, inco ⁇ orated herein by reference) under control of the same promoter (P ⁇ cti) 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 Pad, a Shine-Dalgamo sequence, and restriction enzyme recognition sites for Ndel, Bglll, and Hindlll, 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 desl 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 Nsil and EcoRI to produce plasmid pKOS039-101.
- the -6 kb Sphl-Pstl restriction fragment of pKOS23-26 containing the desl, desll, desIII, desIV, and desV genes was inserted into plasmid pUC19 (Stratagene) to yield plasmid pKOS039-102.
- the -6 kb Sphl-EcoRI restriction fragment from plasmid pKOS039-102 was inserted into pKOS039-101 to produce plasmid pKOS039-103.
- the -6 kb Bglll-Pstl 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 Pacl-Pstl restriction fragment of pKOS39-100 and the -6.4 kb Nsil-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 fhepicK gene, the antibiotics methymycin, neomethymycin, and picromycin are produced.
- 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 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 N3918: 5'-AGGTCTAGAGCTCAGTGCCGGGCGTCGGCCGG-3' (SEQ ID NO:26), to give a 1.5 kb product.
- Plasmid pWHMl 104 described in Tang et al, 1996, Molec. Microbiol. 22(5): 801-813, inco ⁇ orated 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: N024-36B (forward): 5'-TTGCATGCATATGCGCCGTACCCAGCAGGGAACGACC (SEQ ID NO:27); and N024-37B (reverse):
- Plasmid pKOS023-61 was digested with restriction enzymes Spel 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/ ⁇ ET21c.
- 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- ⁇ phosphate, 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 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 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.
- 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 ⁇ M 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.
- narbomycin and formation of picromycin were determined by high performance liquid chromatography (HPLC, Beckman C-18 0.46x15 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).
- HPLC high performance liquid chromatography
- APCI atmospheric pressure chemical ionization
- Mass spectroscopic detection Perkin Elmer/Sciex API 100
- evaporative light scattering detection Alltech 500 ELSD
- the picK gene was amplified from cosmid pKOS023-26 using the primers: N3903: 5*-TCCTCTAGACGTTTCCGT-3' (SEQ ID NO:31); and N3904: 5'-TGAAGCTTGAATTCAACCGGT-3' (SEQ ID NO:32) to obtain an -1.3 kb product.
- the product was treated with restriction enzymes Xbal and Hindlll and ligated with the 7.6 kb Xba ⁇ -Hindlll restriction fragment of plasmid pWHMl 104 to provide plasmid pKOS039-01, placing fhepicK gene under the control of the ermE* promoter.
- the resulting plasmid was transformed into purified stocks of 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
- the 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 KSl domain is inactivated, i.e., the ketosynthase in extender module 1 is disabled.
- the inactive DEBS KSl domain and its construction are described in detail in PCT publication Nos. WO 97/02358 and WO 99/03986, each of which is inco ⁇ orated herein by reference.
- the primers used in the PCR were:
- N3905 5'-TTTATGCATCCCGCGGGTCCCGGCGAG-3' (SEQ ID NO:33); and N3906: 5'-TCAGAATTCTGTCGGTCACTTGCCCGC-3' (SEQ ID NO:34).
- the 1.6 kb PCR product was digested with Pstl and EcoRI and cloned into the corresponding sites of plasmid pKOSO 15-52 (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.
- Plasmid pJRJ2 is described in PCT publication Nos. WO 99/03986 and WO 97/02358, inco ⁇ orated herein by reference. Plasmids pKOS039-18 and pKOS039-19, respectively, were obtained. These two plasmids were transformed into 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.
- cells transformed with plasmid pKOS039-19 were provided (2S,3R)-2-methyl-3-hydroxyhexanoate NACS, 13-desethyl-l 3 -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.
- 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 ⁇ 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-6-phosphate, and 10 nmol 6-deoxyerythronolide B.
- the reaction was allowed to proceed for 90 minutes at 30°C.
- hemiketal formation in the above compound and compounds of similar structure.
- 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.
- MIC minimum inhibitory concentrations
Abstract
Description
Claims
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
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EP99925954A EP1082439A2 (en) | 1998-05-28 | 1999-05-27 | Recombinant narbonolide polyketide synthase |
AU42137/99A AU762399C (en) | 1998-05-28 | 1999-05-27 | Recombinant narbonolide polyketide synthase |
JP2000550984A JP2002516090A (en) | 1998-05-28 | 1999-05-27 | Recombinant narvonolide polyketide synthase |
CA002328427A CA2328427A1 (en) | 1998-05-28 | 1999-05-27 | Recombinant narbonolide polyketide synthase |
NZ509006A NZ509006A (en) | 1998-05-28 | 1999-05-27 | Recombinant narbonolide polyketide synthase |
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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 | |
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AU (1) | AU762399C (en) |
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US6265202B1 (en) | 1998-06-26 | 2001-07-24 | Regents Of The University Of Minnesota | DNA encoding methymycin and pikromycin |
WO2002059322A2 (en) * | 2000-10-17 | 2002-08-01 | Cubist Pharmaceuticlas, Inc. | Compositions and methods relating to the daptomycin biosynthetic gene cluster |
US6524841B1 (en) | 1999-10-08 | 2003-02-25 | Kosan Biosciences, Inc. | Recombinant megalomicin biosynthetic genes and uses thereof |
WO2003078411A1 (en) | 2002-03-12 | 2003-09-25 | Bristol-Myers Squibb Company | C3-cyano epothilone derivatives |
US6838265B2 (en) | 2000-05-02 | 2005-01-04 | Kosan Biosciences, Inc. | Overproduction hosts for biosynthesis of polyketides |
US7033818B2 (en) | 1999-10-08 | 2006-04-25 | Kosan Biosciences, Inc. | Recombinant polyketide synthase genes |
EP1754700A2 (en) | 1999-01-27 | 2007-02-21 | Kosan Biosciences, Inc. | Synthesis of oligoketides |
WO2012103516A1 (en) | 2011-01-28 | 2012-08-02 | Amyris, Inc. | Gel-encapsulated microcolony screening |
WO2012158466A1 (en) | 2011-05-13 | 2012-11-22 | Amyris, Inc. | Methods and compositions for detecting microbial production of water-immiscible compounds |
WO2014025941A1 (en) | 2012-08-07 | 2014-02-13 | Jiang Hanxiao | Methods for stabilizing production of acetyl-coenzyme a derived compounds |
WO2014144135A2 (en) | 2013-03-15 | 2014-09-18 | Amyris, Inc. | Use of phosphoketolase and phosphotransacetylase for production of acetyl-coenzyme a derived compounds |
WO2015020649A1 (en) | 2013-08-07 | 2015-02-12 | Amyris, Inc. | Methods for stabilizing production of acetyl-coenzyme a derived compounds |
WO2016210350A1 (en) | 2015-06-25 | 2016-12-29 | Amyris, Inc. | Maltose dependent degrons, maltose-responsive promoters, stabilization constructs, and their use in production of non-catabolic compounds |
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- 1999-05-27 JP JP2000550984A patent/JP2002516090A/en not_active Withdrawn
- 1999-05-27 EP EP99925954A patent/EP1082439A2/en not_active Withdrawn
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BROWN M J B ET AL: "A MUTANT GENERATED BY EXPRESSION OF AN ENGINEERED DEBS1 PROTEIN FROM THE ERYTHROMYCIN-PRODUCING POLYKETIDE SYNTHASE (PKS) IN STREPTOMYCES COELICOLOR PRODUCES THE TRIKETIDE AS A LACTONE, BUT THE MAJOR PRODUCT IS THE NOR-ANALOGUE DERIVED FROM ACETATE AS STARTER ACID" JOURNAL OF THE CHEMICAL SOCIETY, CHEMICAL COMMUNICATIONS,GB,CHEMICAL SOCIETY. LETCHWORTH, no. 15, 1995, page 1517-1518 XP002044729 ISSN: 0022-4936 * |
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Also Published As
Publication number | Publication date |
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AU4213799A (en) | 1999-12-13 |
AU762399C (en) | 2004-02-05 |
WO1999061599A3 (en) | 2000-01-27 |
CA2328427A1 (en) | 1999-12-02 |
AU762399B2 (en) | 2003-06-26 |
EP1082439A2 (en) | 2001-03-14 |
NZ509006A (en) | 2003-09-26 |
JP2002516090A (en) | 2002-06-04 |
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