WO2000000620A2 - Dna encoding methymycin and pikromycin - Google Patents
Dna encoding methymycin and pikromycin Download PDFInfo
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- WO2000000620A2 WO2000000620A2 PCT/US1999/014398 US9914398W WO0000620A2 WO 2000000620 A2 WO2000000620 A2 WO 2000000620A2 US 9914398 W US9914398 W US 9914398W WO 0000620 A2 WO0000620 A2 WO 0000620A2
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
- C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
- C12P19/62—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin the hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/42—Hydroxy-carboxylic acids
Definitions
- PHAs Polyhydroxyalkanoates
- the first identified member of the PHAs thermoplastics was polyhydroxybutyrate (PHB), the polymeric ester of D(-)-3-hydroxybutyrate.
- the genes responsible for PHB synthesis in A. eutrophus have been cloned and sequenced. (Peoples et al., J. Biol. Chem. r 264, 15293 (1989); Peoples et al., J. Biol. Chem.. 264, 15298 (1989)).
- Three enzymes: ⁇ -ketothiolase (phbA), acetoacetyl-CoA reductase (phbB), and PHB synthase (phbC) are involved in the conversion of acetyl-CoA to PHB.
- PHB represents the archetypical form of a biodegradable thermoplastic
- its physical properties preclude significant use of the homopolymer form.
- Pure PHB is highly crystalline and, thus, very brittle.
- unique physical properties resulting form the structural characteristics of the R groups in a PHA copolymer may result in a polymer with more desirable characteristics. These characteristics include altered crystallinity, UV weathering resistance, glass to rubber transition temperature (T ), melting temperature of the crystalline phase, rigidity and durability (Holmes et al., EPO 00052 459; Anderson et al., Microbiol. Rev., 54, 450 (1990)).
- polyesters behave as thermoplastics, with melting temperatures of 50-180°C, which can be processed by conventional extension and molding equipment.
- Traditional strategies for producing random PHA copolymers involve feeding short- and long-chain fatty acid monomers to bacterial cultures.
- this technology is limited by the monomer units which can be incorporated into a polymer by the endogenous PHA synthase and the expense of manufacturing PHAs by existing fermentation methods (Haywood et al, FHMS Microbiol. T,ett. ; 51, 1 (1989); Poi et al., mt. J. Biol . Macrnmol.. 12, 106 (1990); Steinbuchel et al., In: Novel Biomaterials from Biological Sources. D.
- PHAs also occurs in some bacteria through the reduction and condensation pathway of fatty acids.
- This pathway employs a fatty acid synthase (FAS) which condenses malonate and acetate.
- FAS fatty acid synthase
- the resulting ⁇ -keto group undergoes three processing steps, ⁇ -keto reduction, dehydration, and enoyl reduction, to yield a fully saturated butyryl unit.
- FAS fatty acid synthase
- this pathway provides only a limited array of PHA monomers which vary in alkyl chain length but not in the degree of alkyl group branching, saturation, or functionalization along the acyl chain.
- polyketides such as erythromycin
- FPS polyketide synthase
- PHAs are biodegradable polymers that have the versatility to replace petrochemical-based thermoplastics, it is desirable that new, more economical methods be provided for the production of defined PHAs. Thus, what is needed are methods to produce recombinant PHA monomer synthases for the generation of PHA polymers.
- the invention provides an isolated and purified nucleic acid segment comprising a nucleic acid sequence comprising a sugar (desosamine) biosynthetic gene cluster, a biologically active variant or fragment thereof, wherein the nucleic acid sequence is not derived from the eryC gene cluster of Saccharopolyspora erythraea.
- the desosamine biosynthetic gene cluster from Streptomyces venezuelae was isolated, cloned and sequenced.
- the isolated nucleic acid segment comprising the gene cluster preferably includes a nucleic acid sequence comprising SEQ ID NO: 3, or a fragment or variant thereof.
- the cluster was found to encode nine polypeptides including Desl (e.g., SEQ ID NO:8 encoded by SEQ ID NO:7), DesII (e.g., SEQ ID NO: 10 encoded by SEQ ID NO:9), DesIII (e.g., SEQ ID NO: 12 encoded by SEQ ID NO:l 1), DesIV (e.g., SEQ ID NO: 14 encoded by SEQ ID NO: 13), DesV (e.g., SEQ ID NO: 16 encoded by SEQ ID NO: 15), DesVI (e.g., SEQ ID NO: 18 encoded by SEQ ID NO: 17), DesVII (e.g., SEQ ID NO:20 encoded by SEQ ID NO:19), DesVIII (e.g., SEQ ID NO:22 encoded by SEQ ID NO:21), and DesR (e.g., SEQ ID NO:24 encoded by SEQ ID NO:23) (see Figure 24).
- Desl e.g., SEQ ID NO:8
- nucleic acid segment of the invention encoding DesR is not derived from the eryB gene cluster of Saccharopolyspora erythraea or the oleD gene from Streptomyces antibioticus.
- the nucleic acid segment comprising the desosamine biosynthetic gene cluster hybridizes under moderate, or more preferably stringent, hybridization conditions to SEQ ID NO:3, or a fragment thereof. Moderate and stringent hybridization conditions are well known to the art, see, for example sections 9.47-9.51 of Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989).
- stringent conditions are those that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate (SSC); 0.1% sodium lauryl sulfate (SDS) at 50°C, or (2) employ a denaturing agent such as formamide during hybridization, e.g., 50% formamide with 0.1% bovine serum albumin/0.1%
- Another example is use of 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 ⁇ g/ml), 0.1% sodium dodecylsulfate (SDS), and 10% dextran sulfate at 42°C, with washes at 42°C in 0.2 x SSC and 0.1% SDS.
- the invention also provides a variant polypeptide having at least about 80%, more preferably at least about 90%, and even more preferably at least about 95%, but less than 100%, contiguous amino acid sequence identity to the polypeptide having an amino acid sequence comprising SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ED NO:24, or a fragment thereof.
- a preferred variant polypeptide, or a subunit or fragment of a polypeptide, of the invention includes a variant or subunit polypeptide having at least about 1%, more preferably at least about 10%, and even more preferably at least about 50%, the activity of the polypeptide having the amino acid sequence comprising SEQ ED NO:8, SEQ ED NO: 10, SEQ ID NO: 12, SEQ ED NO:14, SEQ ID NO: 16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, or SEQ ED NO:24.
- glycosyltransferase activity of a polypeptide of SEQ ID NO:20 can be compared to a variant of SEQ ID NO:20 having at least one amino acid substitution, insertion, or deletion relative to SEQ ID NO:20.
- a variant nucleic acid sequence of the invention has at least about 80%, more preferably at least about 90%, and even more preferably at least about 95%, but less than 100%, contiguous nucleic acid sequence identity to a nucleic acid sequence comprising SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:l 1, SEQ ED NO:13, SEQ ID NO:15, SEQ ID NO: 17, SEQ DD NO: 19, SEQ ID NO:21, SEQ ED NO:23, or a fragment thereof.
- an expression cassette comprising a nucleic acid sequence comprising a desosamine biosynthetic gene cluster, a biologically active variant or fragment thereof operably linked to a promoter functional in a host cell, as well as host cells comprising an expression cassette of the invention.
- the expression cassettes of the invention are useful to express individual genes within the cluster, e.g., the desR gene which encodes a glycosidase or the des VII gene which encodes a glycosyltransferase having relaxed substrate specificity for polyketides and deoxysugars, i.e., the glycosyltransferase processes sugar substrates other than TDP-desosamine.
- the desVII gene can be employed in combinatorial biology approaches to synthesize a library of macrolide compounds having various polyketide and deoxysugar structures.
- the expression of a glycosylase in a host cell which synthesizes a macrolide antibiotic may be useful in a method to reduce toxicity of, e.g., inactivate, the antibiotic.
- a host cell which produces the antibiotic is transformed with an expression cassette encoding the glycosyltransferase.
- the recombinant glycosyltransferase is expressed in an amount that reversibly inactivates the antibiotic.
- the antibiotic preferably the isolated antibiotic which is recovered from the host cell, is contacted with an appropriate native or recombinant glycosidase.
- the nucleic acid segment encoding desosamine in the expression cassette of the invention is not derived form the eryC gene cluster of Saccharopolyspora erythraea.
- Preferred host cells are prokaryotic cells, although eukaryotic host cells are also envisioned. These host cells are useful to express desosamine, analogs or derivatives thereof as well as individual polypeptides which can then be isolated from the host cell. Also provided is an expression cassette or host cell comprising antisense sequences from at least a portion of the desosamine biosynthetic gene cluster.
- Another embodiment of the invention is a recombinant host cell, e.g., a bacterial cell, in which at least a portion of a nucleic acid sequence encoding desosamine in the host chromosome is disrupted, e.g., deleted or interrupted (e.g., by an insertion) with heterologous sequences, or substituted with a variant nucleic acid sequence of the invention, so as to alter, preferably so as to result in a decrease or lack of, desosamine synthesis and/or so as to result in the synthesis of an analog or derivative of desosamine.
- a recombinant host cell e.g., a bacterial cell, in which at least a portion of a nucleic acid sequence encoding desosamine in the host chromosome is disrupted, e.g., deleted or interrupted (e.g., by an insertion) with heterologous sequences, or substituted with a variant nucleic acid sequence of the invention, so as
- the nucleic acid sequence which is disrupted is not derived from the eryC gene cluster of Saccharopolyspora erythraea.
- the recombinant host cell of the invention has at least one gene, i.e., desl, desll, desIII, desIV, desV, desVI, desVII, desVIII o ⁇ desR, which is disrupted.
- One embodiment of the invention includes a recombinant host cell in which the desVI gene, which encodes an N-methyltransferase, is disrupted, for example, by replacement with an antibiotic resistance gene.
- such a host cell produces an aglycone having an N-acetylated aminodeoxy sugar, 10-deoxy-methylonide, a compound of formula (7), a compound of formula (8), or a combination thereof.
- the deletion or disruption of the desVI gene may be useful in a method for preparing novel sugars.
- Another preferred embodiment of the invention is a recombinant bacterial host cell in which the desR gene, which encodes a glycosidase such as ⁇ -glucosidase, is disrupted.
- the host cell synthesizes C-2' ⁇ -glucosylated macrolide antibiotics, for example, a compound of formula (13), a compound of formula (14), or a combination thereof. Therefore, the invention further provides a compound of formula (8), (9), (13) or (14).
- each atom of the compounds of the invention having a chiral center may exist in and be isolated in optically active and racemic forms. Some compounds may exhibit polymo ⁇ hism.
- the present invention encompasses any racemic, optically active, polymo ⁇ hic or stereoisomeric form, or mixtures thereof, of a compound of the invention, which possess the useful properties described herein, it being well known in the art how to prepare optically active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase) and how to determine activity using the standard tests described herein, or using other similar tests which are well known in the art. Also provided is a method for directing the biosynthesis of specific glycosylation- modified polyketides by genetic manipulation of a polyketide-producing microorganism.
- the method comprises introducing into a polyketide-producing microorganism a DNA sequence encoding enzymes in desosamine biosynthesis, e.g., a DNA sequence comprising SEQ ID NO:3, a variant or fragment thereof, so as to yield a microorganism that produces specific glycosylation-modified polyketides.
- a DNA sequence encoding enzymes in desosamine biosynthesis
- an anti-sense DNA sequence of the invention may be employed.
- the glycosylation-modified polyketides are isolated from the microorganism. It is preferred that the DNA sequence is modified so as to result in the inactivation of at least one enzymatic activity in sugar biosynthesis or in the attachment of the sugar to a polyketide.
- nucleic acid segment comprising a nucleic acid sequence comprising a macrolide biosynthetic gene cluster (the "met/pik” or “pik” gene cluster) encoding polypeptides that synthesize methymycin, pikromycin, neomethymycin, narbomycin, or a combination thereof, or a biologically active variant or fragment thereof. It is preferred that the nucleic acid segment comprises SEQ ED NO:5, or a fragment or variant thereof, or hybridizes under moderate or more preferably stringent, conditions to SEQ ID NO: 5 or a fragment thereof.
- the isolated and purified nucleic acid segment is from Streptomyces sp., such as Streptomyces venezuelae (e.g., ATCC 15439, ATCC 15068, MCRL 0306, SC 2366 or 3629), Streptomyces narbonensis (e.g., ATCC 19790), Streptomyces eurocidicus, Streptomyces zaomyceticus (MCRL 0405), Streptomyces flavochromogens, Streptomyces sp.
- Streptomyces sp. such as Streptomyces venezuelae (e.g., ATCC 15439, ATCC 15068, MCRL 0306, SC 2366 or 3629), Streptomyces narbonensis (e.g., ATCC 19790), Streptomyces eurocidicus, Streptomyces zaomyceticus (MCRL 0405), Streptomyces flavochromogens,
- the cloned genes can be introduced into an expression system and genetically manipulated so as to yield novel macrolide antibiotics, e.g., ketolides, as well as monomers for polyhydroxyalkanoate (PHA) biopolymers.
- novel macrolide antibiotics e.g., ketolides, as well as monomers for polyhydroxyalkanoate (PHA) biopolymers.
- the nucleic acid sequence encodes PikRl (e.g., SEQ ID NO:27 encoded by SEQ ID NO:26), PikR2 (e.g., SEQ ID NO:29 encoded by SEQ ID NO:28), PikAI (e.g., SEQ ID NO:31 encoded by SEQ ID NO:30), PikAH (e.g., SEQ ID NO:33 encoded by SEQ ID NO:32), PikAIII (e.g., SEQ ID NO:35 encoded by SEQ ID NO:34), PikAIV (e.g., SEQ ID NO:37 encoded by SEQ ID NO:36), PikB (which is the desosamine gene cluster described above), PikC (e.g., SEQ ID NO:39 encoded by SEQ ID NO:38), and PikD (e.g., SEQ ID NO:41 encoded by SEQ ID NO:40), a variant or a fragment thereof, or hybridizes under moderate or preferably stringent conditions to
- the invention also provides a variant polypeptide having at least about 80%, more preferably at least about 90%, and even more preferably at least about 95%, but less than 100%, contiguous amino acid sequence identity to the polypeptide having an amino acid sequence comprising SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, or a fragment thereof.
- a preferred variant polypeptide, or a subunit or fragment of a polypeptide, of the invention includes a variant or subunit polypeptide having at least about 1%, more preferably at least about 10%, and even more preferably at least about 50%, the activity of the polypeptide having the amino acid sequence comprising SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, or SEQ ID NO:41.
- the activities of polypeptides of the macrolide biosynthetic pathway of the invention are described below.
- a variant nucleic acid sequence of the pik biosynthetic gene cluster of the invention has at least about 80%, more preferably at least about 90%, and even more preferably at least about 95%, but less than 100%, contiguous nucleic acid sequence identity to a nucleic acid sequence comprising SEQ ID NO:5, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, or a fragment thereof.
- the pikA gene encodes a polyketide synthase which synthesizes macrolactone 10- deoxymethonolide and narbolide,/?/&# encodes desosamine synthases which catalyze the formation and transfer of a deoxysugar moiety onto agly cones
- the pikC gene encodes a P450 hydoxylase which catalyzes the conversion of YC-17 and narbomycin into methymycin, neomethymycin, and pikromycin
- the pikRl, pikR2 (possibly one for a 12-membered ring and the other for a 14-membered ring) and desR genes which encode enzymes associated with bacterial self-protection.
- the isolated nucleic acid molecule of the invention encodes four active macrolide antibiotics two of which have a 12-membered ring while the other two have a 14-membered ring.
- the genetic mechanism underlying the alternative termination of polyketide synthesis may be useful to prepare novel compounds, e.g., antibiotics, and PHA monomers.
- the invention further provides isolated and purified nucleic acid segments, e.g., in the form of an expression cassette, for each of the individual genes in the macrolide biosynthetic gene cluster.
- the invention provides an isolated and purified pikA V gene that encodes a thioesterase II.
- the thioesterase may be useful to enhance the structural diversity of antibiotics and in PHA production, as the thioesterase modulates chain release and cyclization.
- a thioesterase II gene having acyl- ACP coenzyme A transferase activity e.g., a mutant pik TEII, bacterial, fungal or plant medium-chain-length thioesterase, an animal fatty acid thioesterase or a thioesterase from a polyketide synthase
- a recombinant monomer synthase see Figure 36
- a recombinant monomer synthase see Figure 36
- a recombinant monomer synthase see Figure 36
- a recombinant monomer synthase see Figure 36
- a fusion of a portion of PKS gene cluster with a PHA synthase may result in the transfer of an acyl chain from the PHA to the polymerase.
- ap ⁇ kC gene that encodes a hydroxylase which is active at two positions on a 12-membered ring or at one position on a 14-membered ring. Such a gene may be particularly useful to prepare novel compounds through bioconversion or biotransformation.
- the invention also provides an expression cassette comprising a nucleic acid segment comprising a macrolide biosynthetic gene cluster encoding polypeptides that synthesize methymycin, pikromycin, neomethymycin, narbomycin, or a combination thereof, or a biologically active variant or fragment thereof, operably linked to a promoter functional in a host cell.
- a host cell comprising the nucleic acid segment encoding methymycin, pikromycin, neomethymycin, narbomycin, or a combination thereof, or a biologically active variant or fragment thereof.
- the invention provides isolated and purified polypeptides of the invention, preferably obtained from host cells having the nucleic acid molecules of the invention.
- expression cassettes and host cells comprising antisense sequences of at least a portion of the macrolide biosynthetic gene cluster of the invention are envisioned.
- Yet another embodiment of the invention is a recombinant host cell, e.g., a bacterial cell, in which a portion of the macrolide biosynthetic gene cluster of the invention is disrupted or replaced with a heterologous sequence or a variant nucleic acid segment of the invention, so as to alter, preferably so as to result in a decrease or lack of methymycin, pikromycin, neomethymycin, narbomycin, or a combination thereof, and/or so as to result in the synthesis of novel macrolides.
- a recombinant host cell e.g., a bacterial cell, in which a portion of the macrolide biosynthetic gene cluster of the invention is disrupted or replaced with a heterologous sequence or a variant nucleic acid segment of the invention, so as to alter, preferably so as to result in a decrease or lack of methymycin, pikromycin, neomethymycin, narbomycin, or a
- the invention provides a recombinant host cell in which a pikAI gene, a pikAII gene, a pikAIII gene (12-membered rings), a piklV gene (14- membered rings), apikB gene cluster, a pikAV gene, ap ⁇ kC gene, apikD gene, a pikRl gene, ap ⁇ kR2 gene, or a combination thereof, is disrupted or replaced.
- a preferred embodiment of the invention is a host cell wherein the p ⁇ kB (e.g., the desVI and desV genes), pikAl , pikAV ⁇ pikC gene, is disrupted.
- the invention also provides a method for combinatorial biosynthesis.
- the method comprises expressing in a host cell an expression cassette comprising a DNA fragment of a biosynthetic gene cluster, e.g., a polyketide synthase gene wherein the expression cassette is present on a plasmid, wherein the genome of the host cell comprises a portion of the gene which is different than the portion of the gene present on the plasmid.
- the DNA fragment and the portion of the gene which is one the host chromosome together comprise the entire gene. Synchronized expression of genes from the plasmid and the chromosome thus creates a combinatorial pathway that produces a product.
- the DNA fragment and the portion of the gene cluster on the host chromosome are linked to the native promoter, e.g., pik genes are linked to VpikA.
- the nucleic acid segment comprising the macrolide biosynthetic gene cluster of the invention encodes a polyketide synthase, modules of that synthase are useful in methods to prepare recombinant polyhydroxyalkanoate monomer synthases and polymers in addition to macrolide antibiotics and derivatives thereof.
- the invention provides an isolated and purified DNA molecule comprising a first DNA segment encoding a first module and a second DNA segment encoding a second module, wherein the DNA segments together encode a recombinant polyhydroxyalkanoate monomer synthase, and wherein at least one DNA segment is derived from the pikA gene cluster of Streptomyces venezuel ⁇ e.
- no more than one DNA segment is derived from the eryh gene cluster of Saccharopolyspora erythraea.
- the 3' most DNA segment of the isolated DNA molecule of the invention encodes a thioesterase II.
- an expression cassette comprising a nucleic acid molecule encoding the polyhydroxyalkanoate monomer synthase operably linked to a promoter functional in a host cell.
- Yet another embodiment of the invention is a method of providing a polyhydroxyalkanoate monomer.
- the method comprises introducing into a host cell a DNA molecule comprising a DNA segment encoding a recombinant polyhydroxyalkanoate monomer synthase operably linked to a promoter functional in the host cell.
- the DNA molecule comprises a plurality of DNA segments, e.g., a first module and a second module, wherein at least one DNA segment is derived from the pikA gene cluster of Streptomyces venezuelae.
- the DNA encoding the recombinant polyhydroxyalkanoate monomer synthase is then expressed in the host cell so as to generate a polyhydroxyalkanoate monomer.
- a second DNA molecule may be introduced into the host cell.
- the second DNA molecule comprises a DNA segment encoding a polyhydroxyalkanoate synthase operably linked to a promoter functional in the host cell.
- the two DNA molecules are expressed in the host cell so as to generate a polyhydroxyalkanoate polymer.
- Another embodiment of the invention is an isolated and purified DNA molecule comprising a first DNA segment encoding a fatty acid synthase and a second DNA segment encoding a module from the pikA gene cluster of Streptomyces venezuelae.
- a DNA molecule can be employed in a method of providing a polyhydroxyalkanoate monomer.
- a DNA molecule comprising a first DNA segment encoding a fatty acid synthase and a second DNA segment encoding a polyketide synthase is introduced into a host cell.
- the first DNA segment is 5' to the second DNA segment and the first DNA segment is operably linked to a promoter functional in the host cell.
- the first DNA segment is linked to the second DNA segment so that the linked DNA segments express a fusion protein.
- the DNA molecule is expressed in the host cell so as to generate a polyhydroxyalkanoate monomer.
- the method comprises introducing an expression cassette comprising a DNA molecule encoding a polyhydroxyalkanoate synthase operably linked to a promoter functional in a host cell.
- the DNA molecule comprises a first DNA segment encoding a first module and a second DNA segment encoding a second module wherein the DNA segments together encode a polyhydroxyalkanoate monomer synthase.
- At least one DNA segment is derived from the pikA gene cluster of Streptomyces venezuelae.
- the DNA molecule is expressed in the host cell.
- the DNA molecule further comprises a DNA segment encoding a polyhydroxyalkanoate synthase.
- a second, separate DNA molecule encoding a polyhydroxyalkanoate synthase is introduced into the host cell.
- a further embodiment of the invention is an isolated and purified DNA molecule comprising a DNA segment which encodes a Streptomyces venezuelae polyketide synthase, e.g., a polyhydroxyalkanoate monomer synthase, a biologically active variant or subunit (fragment) thereof.
- the DNA segment encodes a polypeptide having an amino acid sequence comprising SEQ ID NO:2.
- the DNA segment comprises SEQ ID NO: 1.
- the DNA molecules of the invention are double stranded or single stranded.
- a preferred embodiment of the invention is a DNA molecule that has at least about 70%, more preferably at least about 80%, and even more preferably at least about 90%, but less than 100%, contiguous sequence identity to the DNA segment comprising SEQ ID NO:l, e.g., a "variant" DNA molecule.
- a variant DNA molecule of the invention can be prepared by methods well known to the art, including oligonucleotide-mediated mutagenesis. See Adelman et al., DNA, 2, 183 (1983) and Sambrook et al., Molecular Cloning: A Laboratory Manual (1989).
- the invention also provides an isolated, purified polyhydroxyalkanoate monomer synthase, e.g., a polypeptide having an amino acid sequence comprising SEQ ID NO:2, a biologically active subunit, or a biologically active variant thereof.
- a variant polypeptide having at least about 80%, more preferably at least about 90%, and even more preferably at least about 95%, but less than 100%, contiguous amino acid sequence identity to the polypeptide having an amino acid sequence comprising SEQ ID NO:2.
- a preferred variant polypeptide, or a subunit of a polypeptide, of the invention includes a variant or subunit polypeptide having at least about 10%, more preferably at least about 50%, and even more preferably at least about 90%, the activity of the polypeptide having the amino acid sequence comprising SEQ ID NO:2.
- a variant polypeptide of the invention has one or more conservative amino acid substitutions relative to the polypeptide having the amino acid sequence comprising SEQ ID NO:2.
- conservative substitutions include aspartic-glutamic as acidic amino acids; lysine/arginine/histidine as basic amino acids; leucine/isoleucine, methionine/valine, alanine/valine as hydrophobic amino acids; serine/glycine/alanine/threonine as hydrophilic amino acids.
- the biological activity of a polypeptide of the invention can be measured by methods well known to the art, including but not limited to, methods described hereinbelow.
- modules encoded by the nucleic acid segments of the invention may be employed in the methods described hereinabove to prepare polyhydroxyalkanoates of varied chain length or having various side chain substitutions and/or to prepare glycosylated biopolymers.
- the compounds produced by the recombinant host cells of the invention are useful as biopolymers, e.g., in packaging or biomedical applications, to engineer PHA monomer synthases, or to prepare biologically active agents, such as those useful to prepare a medicament for the treatment of a pathological condition or a symptom in a mammal, e.g., a human.
- the agents include pharmaceuticals such as chemotherapeutic agents, immunosuppressants, agents to treat asthma, chronic obstructive pulmonary disease as well as other diseases involving respiratory inflammation, cholesterol-lowering agents, or macrolide- based antibiotics which are active against a variety of organisms, e.g., bacteria, including multi-drug-resistant pneumococci and other respiratory pathogens, as well as viral and parasitic pathogens; or as crop protection agents (e.g., fungicides or insecticides) via expression of polyketides in plants.
- pharmaceuticals such as chemotherapeutic agents, immunosuppressants, agents to treat asthma, chronic obstructive pulmonary disease as well as other diseases involving respiratory inflammation, cholesterol-lowering agents, or macrolide- based antibiotics which are active against a variety of organisms, e.g., bacteria, including multi-drug-resistant pneumococci and other respiratory pathogens, as well as viral and parasitic pathogens; or as crop protection agents (e.g.,
- Methods employing these compounds e.g., to treat a mammal, bird or fish in need of such therapy, such as a patient having a bacterial, viral or parasitic infection, cancer, respiratory disease, or in need of immunosuppression, e.g., during cell, tissue or organ transplantation, are also envisioned.
- FIG. 3 Comparison of the natural and recombinant pathways for PHB synthesis.
- the three enzymatic steps of PHB synthesis in bacteria involving 3-ketothiolase, acetoacetyl- CoA reductase, and PHB synthase are shown on the left.
- the two enzymatic steps involved in PHB synthesis in the pathway in Sfll cells containing a rat fatty acid synthase with an inactivated dehydrase domain (ratFAS206) are shown on the right.
- Figure 4 Schematic diagram of the molecular organization of the tyl polyketide synthase (PKS) gene cluster.
- Open arrows correspond to individual open reading frames (ORFs) and numbers above an ORF denote a multifunctional module or synthase unit (SU).
- AT acyltransferase
- ACP acyl carrier protein
- KS ⁇ -ketoacyl synthase
- KR ketoreductase
- DH dehydrase
- ER enoyl reductase
- TE thioesterase
- MM methylmalonylCoA
- M malonyl CoA
- EM ethylmalonyl CoA
- Module 7 in tyl is also known as Module F.
- Figure 5 Schematic diagram of the molecular organization of the met PKS gene cluster.
- Figure 6 Strategy for producing a recombinant PHA monomer synthase by domain replacement.
- FIG. 7 (A) 10% SDS-PAGE gel showing samples from various stages of the purification of PHA synthase; lane 1, molecular weight markers; lane 2, total protein of uninfected insect cells; lane 3, total protein or insect cells expressing a rat FAS (200 kDa; Joshi et al., Biochem. I.. 226, 143 (1993)); lane 4, total protein of insect cells expressing PHA synthase; lane 5, soluble protein from sample in lane 4; lane 6, pooled hydroxylapatite (HA) fractions containing PHA synthase.
- B Western analysis of an identical gel using rabbit- ⁇ - PHA synthase antibody as probe.
- Bands designated with arrows are: a, intact PHB synthase with N-terminal alanine at residue 7 and serine at residue 10 (A7/S10); b, 44 kDa fragment of PHB synthase with N-terminal alanine at residue 181 and asparagine at residue 185 (Al 81/Nl 85); c, PHB synthase fragment of approximately 30 kDa apparently blocked based on resistance to Edman degradation; d, 22 kDa fragment with N-terminal glycine at residue 187 (G187). Band d apparently does not react with rabbit- ⁇ -PHB synthase antibody (B, lane 6). The band of similar size in B, lane 4 was not further identified.
- Figure 8 N-terminal analysis of PHA synthase purified from insect cells, (a) The expected N-terminal 25 amino acid sequence of A. eutrophus PHA synthase. (b&c) The two N-terminal sequences determined for the A. eutrophus PHA synthase produced in insect cells. The bolded sequences are the actual N-termini determined.
- FIG. 9 Spectrophotometric scans of substrate, 3-hydroxybutyrate CoA (HBCoA) and product, CoA.
- the wavelength at which the direct spectrophotometric assays were carried out (232 nm) is denoted by the arrow; substrate, HBCoA (•) and product, CoA (o).
- FIG. 10 Velocity of the hydrolysis of HBCoA as a function of substrate concentration. Assays were carried out in 40 or 200 ⁇ l assay volumes with enzyme concentration remaining constant at 0.95 mg/ml (3.8 ⁇ g/40 ⁇ l assay). Velocities were calculated from the linear portions of the assay curves subsequent to the characteristic lag period. The substrate concentration at half-optimal velocity, the apparent K,,, value, was estimated to be 2.5 mM from this data.
- FIG 14. pH activity curve for soluble PHA synthase produced using the baculovirus system. Reactions were carried out in the presence of 200 mM P,. Buffers of pH ⁇ 10 were prepared with potassium phosphate, while buffers of pH > 10 were prepared with the appropriate proportion of Na 3 PO 4 .
- Figure 15. Assays of the hydrolysis of HBCoA with varying amounts of PHA synthase. Assays were carried out in 40 ⁇ l assay volumes with the concentration of HBCoA remaining constant at 8 ⁇ M. Initial A 232 values, originally between 0.62 and 0.77, were normalized to 0.70.
- Enzyme amounts used in these assays were, from the uppermost curve, 0.38, 0.76, 1.14, 1.52, 1.90, 2.28, 2.66, 3.02, 3.42, 7.6, and 15.2 ⁇ g, respectively.
- Figure 16 SDS/PAGE analysis of proteins synthesized at various time points during infection o S ⁇ l cells. Approximately 0.5 mg of total cellular protein from various samples was fractionated on a 10% polyacrylamide gel.
- Samples include: uninfected cells, lanes 1-4, days 0, 1, 2, 3, respectively; infection with BacPAK6::phbC alone, lanes 5-8, days, 0, 1, 2, 3, respectively, infection with baculo viral clone containing ratFAS206 alone, lanes 9-12, days 0, 1, 2, 3, respectively; and ratFAS206 and BacPAK ⁇ infected cells, lanes 13-16, days 0, 1, 2, 3, respectively.
- A mobility of FAS
- B mobility of PHA synthase.
- M mobility of PHA synthase.
- FIG. 17 Gas chromatographic evidence for PHB accumulation in 5/21 cells. Gas chromatograms from various samples are superimposed. PHB standard (Sigma) is chromatogram #7 showing a propylhydroxybutyrate elution time of 10.043 minutes (s, arrow). The gas chromatograms of extracts of the uninfected (#1); singly infected with ratFAS206 (#2, day 3); and singly infected with PHA synthase (#3, day 3) are shown at the bottom of the figure. Gas chromatograms of extracts of dual-infected cells at day 1 (#4), 2 (#5), and 3 (#6) are also shown exhibiting a peak eluting at 10.096 minutes (x, arrow). The peak of dual-infected, day 3 extract (#6) was used for mass spectrometry (MS) analysis.
- MS mass spectrometry
- FIG. 18 Gas chromatography-mass spectrometry analysis of PHB. The characteristic fragmentation of propylhydroxybutyrate at m/z of 43, 60, 87, and 131 is shown.
- FIG. 1 Map of the vep (Streptomyces venezuelae polyene encoding) gene cluster.
- Figure 20 Plasmid map of pDHS502.
- Figure 21 Plasmid map of pDHS505.
- FIG. 23 Nucleotide sequence (SEQ ID NO:l) and corresponding amino acid sequence (SEQ ID NO:22) of vep ORFI.
- Figure 24 Schematic diagram of the desosamine biosynthetic pathway and the enzymatic activity associated with each of the desosamine biosynthetic polypeptides.
- Figure 25 Schematic of the conversion of the inactive (diglycosylated) form of methymycin and pikromycin to the active form of methymycin and pikromycin.
- FIG. 26 Schematic diagram of the desosamine biosynthetic pathway.
- Figure 27 Pathway for the synthesis of a compound of formula 7 and 8 in desVT mutants of Streptomyces .
- FIG. 28 Structure and biosynthesis of methymycin, pikromycin, and related compounds in Streptomyces venezuelae ATCC 15439.
- Polyketide synthase components PikAI, PikAII, PikAIII, PikAIV, and PikAV are represented by solid bars. Each circle represents an enzymatic domain in the Pik PKS system.
- KS ⁇ -ketoacyl- ACP synthase
- AT acyltransferase
- ACP acyl carrier protein
- KR ⁇ -ketoacyl- ACP reductase
- DH ⁇ -hydroxyl-thioester dehydratase
- ER enoyl reductase
- KS Q a KS-like domain
- KR with a cross nonfunctional KR
- TE thioesterase domain
- TEII type II thioesterase.
- Des represents all eight enzymes for desosamine biosynthesis and transfer and PikC is the cytochrome P450 monooxygenase responsible for hydroxylation at R, , R 2 , and R 3 positions (Xu et al, 1998).
- FIG. 29 Organization of the pik cluster in 5. venezuelae. Each arrow represents an open reading frame (ORF). The direction of transcription and relative sizes of the ORFs deduced from nucleotide sequence are indicated.
- the cluster is composed of four genetic loci: pikA, pikB (des), pikC, and pikR. Cosmid clones are denoted as overlapping lines.
- Figure 30 Conversion of YC-17 and narbomycin by PikC P450 hydroxylase.
- FIG. 31 Nucleotide sequence (SEQ ID NO:5) and inferred amino acid sequence (SEQ ID NO:6) of the pik gene cluster.
- Figure 32 Nucleotide sequence (SEQ ID NO:3) and inferred amino acid sequence (SEQ ID NO:4) of the desosamine gene cluster.
- FIG. 33 5. venezuelae AX916 construct useful to prepare a polyketide having a shorter chain length compared to wild-type pikA.
- pik module 2 is fused to pik module 5, and module 3 and 4 are deleted, so as to encode a three module PKS which produces two macrolides, a triketide and a tetraketide.
- FIG. 34 Recombinant PKS having a wild-type thioesterase II.
- FIG. 35 pAX703 construct, an expression and complementation vector.
- the PikTEII gene can be replaced with an EcoRI-NsiI fragment.
- the phaCl gene can be replaced with a Pacl-Dral fragment.
- mTEII is a mutant pikTEII, an acyl- ACP CoA transferase; phaCl is a PHA polymerase 1 from P. olivarus which may have racemase activity.
- AX916, a PHA polymer is produced.
- FIG. 37 Strategy for C5 polymer production.
- a PHA polymerase gene phaCl is directly fused to pik module 2, so as to result in a fusion that transfers an acyl chain from the PKS protein directly to the polymerase by the prosthetic group on the ACP domain of the PKS.
- Figure 39 Exemplary and preferred amino acid substitutions.
- Figure 40 Plasmid complementation of 5. venezuelae AX912. The relevant genotype
- PikAIV Proteins PikAIII and PikAIV are stacked one on top of the other according to their order in polyketide biosynthesis (PikAI and PikAII are not shown).
- a sphere represents an enzymatic domain in the PKSs with its diameter proportional to the size of the domain.
- Each PKS module/protein was first dimerized (each peptide chain is shown as either red or blue) and then twisted 180 degrees to form a half helix following the model for erythromycin PKS (Staunton et al., 1999). Two sets of independent active sites are thus formed along two grooves of the helix that lead to the production of two polyketides in each biosynthetic cycle.
- venezuelae under culture conditions for pikromycin production B) Wild type 5. venezuelae under culture conditions for methymycin production. C) 5. venezuelae AX912 (pDHS704) under culture conditions for methymycin production. D) 5. venezuelae AX912 (pDHS704) under culture conditions for pikromycin production. E) 5. venezuelae AX912 (pDHS708) under culture conditions for pikromycin production. F) 5. venezuelae AX912 (pDHS708) under culture conditions for methymycin production. Gene products expressed from the plasmid construct used for complementation are underlined.
- FIG. 42 Pathway for desosamine biosynthesis.
- FIG 43 Schematic of pathway leading to methymycin/neomethymycin analogs 18 and 19.
- Figure 44 Macrolide having D-quinovose.
- Figure 45 Products produced by desl mutant.
- FIG. 46 Pik sequences from Streptomyces spp.
- a "linker region” is an amino acid sequence present in a multifunctional protein which is less well conserved in an amino acid sequence than an amino acid sequence with catalytic activity.
- an "extender unit” catalytic or enzymatic domain is an acyl transferase in a module that catalyzes chain elongation by adding 2-4 carbon units to an acyl chain and is located carboxy-terminal to another acyl transferase.
- an extender unit with methylmalonylCoA specificity adds acyl groups to a methylmalonylCoA molecule.
- a "polyhydroxyalkanoate” or “PHA” polymer includes, but is not limited to, linked units of related, preferably heterologous, hydroxyalkanoates such as 3- hydroxybutyrate, 3-hydroxyvalerate, 3-hydroxycaproate, 3-hydroxyheptanoate, 3- hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxyundecanoate, and 3-hydroxydodecanoate, and their 4-hydroxy and 5-hydroxy counte ⁇ arts.
- a "Type I polyketide synthase” is a single polypeptide with a single set of iteratively used active sites. This is in contrast to a Type II polyketide synthase which employs active sites on a series of polypeptides.
- a "recombinant" nucleic acid or protein molecule is a molecule where the nucleic acid molecule which encodes the protein has been modified in vitro, so that its sequence is not naturally occurring, or cooesponds to naturally occurring sequences that are not positioned as they would be positioned in a genome which has not been modified.
- a "recombinant" host cell of the invention has a genome that has been manipulated in vitro so as to alter, e.g., decrease or disrupt, or, alternatively, increase, the function or activity of at least one gene in the macrolide or desosamine biosynthetic gene cluster of the invention.
- a “multifunctional protein” is one where two or more enzymatic activities are present on a single polypeptide.
- a “module” is one of a series of repeated units in a multifunctional protein, such as a Type I polyketide synthase or a fatty acid synthase.
- a "premature termination product” is a product which is produced by a recombinant multifunctional protein which is different than the product produced by the non- recombinant multifunctional protein.
- the product produced by the recombinant multifunctional protein has fewer acyl groups.
- a DNA that is "derived from" a gene cluster is a DNA that has been isolated and purified in vitro from genomic DNA, or synthetically prepared on the basis of the sequence of genomic DNA.
- the "pik” or “pik/mef gene cluster includes sequences encoding a polyketide synthase (pikA), desosamine biosynthetic enzymes (pikB, also referred to as des), a cytochrome P450 (pikC), regulatory factors (pikD) and enzymes for cellular self-resistance (pikR).
- pikA polyketide synthase
- pikB desosamine biosynthetic enzymes
- pikC cytochrome P450
- pikD regulatory factors
- enzymes for cellular self-resistance cellular self-resistance
- the DNA may encode more than one recombinant Type I polyketide synthase and/or fatty acid synthase.
- an isolated DNA molecule encoding a polyhydroxyalkanoate monomer synthase is RNA or DNA containing greater than 7, preferably 15, and more preferably 20 or more sequential nucleotide bases that encode a biologically active polypeptide, fragment, or variant thereof, that is complementary to the non-coding, or complementary to the coding strand, of a polyhydroxyalkanoate monomer synthase RNA, or hybridizes to the RNA or DNA encoding the polyhydroxyalkanoate monomer synthase and remains stably bound under stringent conditions, as defined by methods well known to the art, e.g., in Sambrook et al., supra.
- an "antibiotic” as used herein is a substance produced by a microorganism which, either naturally or with limited chemical modification, will inhibit the growth of or kill another microorganism or eukaryotic cell.
- an "antibiotic biosynthetic gene” is a nucleic acid, e.g., DNA, segment or sequence that encodes an enzymatic activity which is necessary for an enzymatic reaction in the process of converting primary metabolites into antibiotics.
- An "antibiotic biosynthetic pathway” includes the entire set of antibiotic biosynthetic genes necessary for the process of converting primary metabolites into antibiotics. These genes can be isolated by methods well known to the art, e.g., see U.S. Patent No. 4,935,340.
- Antibiotic-producing organisms include any organism, including, but not limited to, Actinoplanes, Actinomadura, Bacillus, Cephalosporium, Micromonospora, Penicillium, Nocardia, and Streptomyces, which either produces an antibiotic or contains genes which, if expressed, would produce an antibiotic.
- An antibiotic resistance-conferring gene is a DNA segment that encodes an enzymatic or other activity which confers resistance to an antibiotic.
- polyketide refers to a large and diverse class of natural products, including but not limited to antibiotic, antifungal, anticancer, and anti-helminthic compounds.
- Antibiotics include, but are not limited to anthracyclines and macrolides of different types (polyenes and avermectins as well as classical macrolides such as erythromycins). Macrolides are produced by, for example, 5. erytheus, 5. antibioticus, 5. venezuelae, 5. fradiae and 5. narbonensis.
- glycos refers to any polyketide that contains one or more sugar residues.
- glycosylation-modified polyketide refers to a polyketide having a changed glycosylation pattern or configuration relative to that particular polyketide's unmodified or native state.
- polyketide-producing microorganism includes any microorganism that can produce a polyketide naturally or after being suitably engineered (i.e., genetically).
- actinomycetes that naturally produce polyketides include but are not limited to Micromonospora rosaria, Micromonospora megalomicea, Saccharopolyspora erythraea, Streptomyces antibioticus, , Streptomyces albereticuli, Streptomyces ambofaciens, Streptomyces avermitilis, Streptomyces fradiae, Streptomyces griseus, Streptomyces hydroscopicus, Streptomyces tsukulubaensis, Streptomyces mycarofasciens, Streptomyces platenesis, Streptomyces violaceoniger, Streptomyces violaceoniger, Streptomyces thermotolerans, Streptomyces rimosus
- sugar biosynthesis genes refers to nucleic acid sequences from organisms such as Streptomyces venezuelae that encode sugar biosynthesis enzymes and is intended to include sequences of DNA from other polyketide-producing microorganisms which are identical or analogous to those obtained from Streptomyces venezuelae.
- sugar biosynthesis enzymes refers to polypeptides which are involved in the biosynthesis and/or attachment of polyketide-associated sugars and their derivatives and intermediates.
- polyketide-associated sugar refers to a sugar that is known to attach to polyketides or that can be attached to polyketides by the processes described herein.
- sugar derivative refers to a sugar which is naturally associated with a polyketide but which is altered relative to the unmodified or native state, including but not limited to, N-3- ⁇ -desdimethyl D-desosamine.
- sugar intermediate refers to an intermediate compound produced in a sugar biosynthesis pathway.
- derivative means that a particular compound produced by a host cell of the invention or prepared in vitro using polypeptides encoded by the nucleic acid molecules of the invention, is modified so that it comprises other moieties, e.g., peptide or polypeptide molecules, such as antibodies or fragments thereof, nucleic acid molecules, sugars, lipids, fats, a detectable signal molecule such as a radioisotope, e.g., gamma emitters, small chemicals, metals, salts, synthetic polymers, e.g., polylactide and polyglycolide, surfactants and glycosaminoglycans, which are covalently or non-covalently attached or linked to the compound.
- moieties e.g., peptide or polypeptide molecules, such as antibodies or fragments thereof, nucleic acid molecules, sugars, lipids, fats, a detectable signal molecule such as
- a "recombinant" host cell of the invention has a genome that has been manipulated in vitro so as to alter, e.g., decrease or disrupt, or alternatively, increase, the function or activity of at least one gene, e.g., in the pik biosynthetic gene cluster, of the invention.
- the term "derivative" means that a particular compound produced by a host cell of the invention or prepared in vitro using polypeptides encoded by the nucleic acid molecules of the invention, is modified so that it comprises other moieties, e.g., peptide or polypeptide molecules, such as antibodies or fragments thereof, nucleic acid molecules, sugars, lipids, fats, a detectable signal molecule such as a radioisotope, e.g., gamma emitters, small chemicals, metals, salts, synthetic polymers, e.g., polylactide and polyglycolide, surfactants and glycosaminoglycans, which are covalently or non-covalently attached or linked to the compound.
- moieties e.g., peptide or polypeptide molecules, such as antibodies or fragments thereof, nucleic acid molecules, sugars, lipids, fats, a detectable signal molecule such as a radioisotope, e.g.,
- each atom of the compounds of the invention having a chiral center may exist in and be isolated in optically active and racemic forms. Some compounds may exhibit polymo ⁇ hism.
- the present invention encompasses any racemic, optically active, polymo ⁇ hic or stereoisomeric form, or mixtures thereof, of a compound of the invention, which possess the useful properties described herein, it being well known in the art how to prepare optically active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase) and how to determine activity using the standard tests described herein, or using other similar tests which are well known in the art.
- sequence homology or “sequence identity” means the proportion of base matches between two nucleic acid sequences or the proportion amino acid matches between two amino acid sequences.
- sequence homology is expressed as a percentage, e.g., 50%, the percentage denotes the proportion of matches over the length of sequence that is compared to some other sequence. Gaps (in either of the two sequences) are permitted to maximize matching; gap lengths of 15 bases or less are usually used, 6 bases or less are preferred with 2 bases or less more preferred.
- the sequence homology between the target nucleic acid and the oligonucleotide sequence is generally not less than 17 target base matches out of 20 possible oligonucleotide base pair matches (85%); preferably not less than 9 matches out of 10 possible base pair matches (90%), and more preferably not less than 19 matches out of 20 possible base pair matches (95%).
- Two amino acid sequences are homologous if there is a partial or complete identity between their sequences. For example, 85% homology means that 85% of the amino acids are identical when the two sequences are aligned for maximum matching. Gaps (in either of the two sequences being matched) are allowed in maximizing matching; gap lengths of 5 or less are preferred with 2 or less being more preferred.
- two protein sequences are homologous, as this term is used herein, if they have an alignment score of at more than 5 (in standard deviation units) using the program ALIGN with the mutation data matrix and a gap penalty of 6 or greater. See Dayhoff, M. O., in Atlas of Protein Sequence and Structure, 1972, volume 5, National Biomedical Research Foundation, pp. 101-110, and Supplement 2 to this volume, pp. 1-10.
- the two sequences or parts thereof are more preferably homologous if their amino acids are greater than or equal to 50% identical when optimally aligned using the ALIGN program.
- reference sequence is a defined sequence used as a basis for a sequence comparison; a reference sequence may be a subset of a larger sequence, for example, as a segment of a full-length cDNA or gene sequence given in a sequence listing, or may comprise a complete cDNA or gene sequence. Generally, a reference sequence is at least 20 nucleotides in length, frequently at least 25 nucleotides in length, and often at least 50 nucleotides in length.
- two polynucleotides may each (1) comprise a sequence (i.e., a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) may further comprise a sequence that is divergent between the two polynucleotides
- sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity.
- a “comparison window”, as used herein, refers to a conceptual segment of at least 20 contiguous nucleotides and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
- Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2: 482, by the homology alignment algorithm of Needleman and Wunsch (1970) I. Mol. Biol. 48: 443, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad.
- sequence identity means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison.
- percentage of sequence identity means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison.
- percentage of sequence identity is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
- the identical nucleic acid base e.g., A, T, C, G, U, or I
- substantially identical denote a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 85 percent sequence identity, preferably at least 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 nucleotide positions, frequently over a window of at least 20-50 nucleotides, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the window of comparison.
- substantially identical means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least about 80 percent sequence identity, preferably at least about 90 percent sequence identity, more preferably at least about 95 percent sequence identity, and most preferably at least about 99 percent sequence identity.
- nucleic acid molecule which encodes the entire pathway for methymycin, pikromycin, neomethymycin, narbomycin, or a combination thereof, which includes sugar biosynthetic genes that are linked thereto.
- the nucleic acid molecule is DNA isolated from Streptomyces spp.
- the present invention further includes isolated and purified nucleic acid sequences which hybridize under standard or stringent conditions to the nucleic acid molecules of the invention. It is also understood that the invention encompasses isolated and purified polypeptides which may be encoded by the nucleic acid molecules of the invention.
- the invention described herein can be used for the production of a diverse range of novel compounds including polyketides, e.g., antibiotics, and biodegradable PHA polymers through genetic redesign of DNA encoding a FAS or a PKS such as that found in Streptomyces spp.
- polyketides e.g., antibiotics
- biodegradable PHA polymers through genetic redesign of DNA encoding a FAS or a PKS such as that found in Streptomyces spp.
- the isolation and characterization of this gene cluster allows for the selective production of antibiotics, the ove ⁇ roduction or under production of particular compounds, e.g., ove ⁇ roduction of certain antibiotics, and the production of novel compounds.
- combinational biosynthetic-based modification of compounds may be accomplished by selective activation or disruption of specific genes within the cluster or inco ⁇ oration of the genes into biased biosynthetic libraries which are assayed for a wide range of biological activities, to derive greater chemical diversity.
- a further example includes the introduction of biosynthetic gene(s) into a particular host cell so as to result in the production of a novel compound due to the activity of the biosynthetic gene(s) on other metabolites, intermediates or components of the host cells.
- PHA synthases can be tested for their ability to polymerize monomers produced by the recombinant PKS or PHA monomer synthase into a biodegradable polymer.
- the invention also provides a method by which various PHA synthases can be tested for their specificity with respect to different monomer substrates.
- the potential uses and applications of PHAs produced by PHA monomer synthases and PHA synthases include both medical and industrial applications. Medical applications of PHAs include surgical pins, sutures, staples, swabs, wound dressings, blood vessel replacements, bone replacements and plates, stimulation of bone growth by piezoelectric properties, and biodegradable carrier for long-term dosage of pharmaceuticals.
- Industrial applications of PHAs include disposable items such as baby diapers, packaging containers, bottles, wrappings, bags, and films, and biodegradable carriers for long-term dosage of herbicides, fungicides, insecticides, or fertilizers.
- the biosynthesis of fatty acids de novo from malonyl-CoA is catalyzed by FAS.
- the rat FAS is a homodimer with a subunit structure consisting of 2505 amino acid residues having a molecular weight of 272,340 Da. Each subunit consists of seven catalytic activities in separate physical domains (Amy et al., Proc. Natl. Acad. Sci. US ⁇ , &6, 3114 (1989)).
- ketoacyl synthase KS
- malonyl/acetyltransferase M/AT
- ER enoyl reductase
- KR ketoreductase
- ACP acyl carrier protein
- TE thioesterase
- the seventh catalytic activity, dehydrase (DH), was identified as physically residing between AT and ER by an amino acid comparison of FAS with the amino acid sequences encoded by the three open reading frames of the eryA polyketide synthase (PKS) gene cluster of Saccharopolyspora erythraea.
- PKS eryA polyketide synthase
- the three polypeptides that comprise this PKS are constructed from "modules" which resemble animal FAS, both in terms of their amino acid sequence and in the ordering of the constituent domains (Donadio et al., Gene, Hi, 51 (1992); Benh et al., Eur. I. Biochem., 204, 39 (1992)).
- the FAS DH- employed in this embodiment of the invention is preferably a eukaryotic FAS DH- and, more preferably, a mammalian FAS DH- .
- the most preferred embodiment of the invention is a FAS where the active site in the DH has been inactivated by mutation.
- Joshi et al. J. Biol. Chem. r 26S, 22508 (1993) changed the His 878 residue in the rat FAS to an alanine residue by site-directed mutagenesis.
- ratFAS206 produced 3-hydroxybutyrylCoA as a premature termination product from acetyl-CoA, malonyl-CoA and NADPH.
- a FAS DH- effectively replaces the ⁇ -ketothiolase and acetoacetyl- CoA reductase activities of the natural pathway by producing D(-)-3-hydroxybutyrate as a premature termination product, rather than the usual 16-carbon product, palmitic acid.
- This premature termination product can then be inco ⁇ orated into PHB by a PHB synthase (See Example 2).
- Another embodiment of the invention employs a recombinant Streptomyces spp. PKS to produce a variety of ⁇ -hydroxyCoA esters that can serve as monomers for a PHA synthase.
- a DNA encoding a Type I PKS is the eryA gene cluster, which governs the synthesis of erythromycin aglycone deoxyerythronolide B (DEB).
- the gene cluster encodes six repeated units, termed modules or synthase units (SUs). Each module or SU, which comprises a series of putative FAS-like activities, is responsible for one of the six elongation cycles required for DEB formation.
- SUs synthase units
- Two other Type I PKS are encoded by the tyl (tylosin) ( Figure 4) and met (methymycin) ( Figure 5) gene clusters.
- the macrolide multifunctional synthases encoded by tyl and met provide a greater degree of metabolic diversity than that found in the eryA gene cluster.
- the PKSs encoded by the eryA gene cluster only catalyze chain elongation with methylmalonylCoA, as opposed to tyl and met PKSs, which catalyze chain elongation with malonylCoA, methylmalonylCoA and ethylmalonylCoA.
- the tyl PKS includes two malonylCoA extender units and one ethylmalonylCoA extender unit, and the wet PKS includes one malonylCoA extender unit.
- a preferred embodiment of the invention includes, but is not limited to, replacing catalytic activities encoded in met PKS open reading frame 1 (ORFl) to provide a DNA encoding a protein that possesses the required keto group processing capacity and short-chain acylCoA ester starter and extender unit specificity necessary to provide a saturated ⁇ -hydroxyhexanoylCoA or unsaturated ⁇ - hydroxyhexenoylCoA monomer.
- Linker regions amino acid sequences of related modules, preferably those encoded by more than one gene cluster, are compared. Linker regions are amino acid sequences which are less well conserved than amino acid sequences with catalytic activity. Witkowski et al., Eur. J. Biochem., 198, 571 (1991).
- a DNA encoding a module F containing KS, MT, KR, ACP, and TE catalytic activities, is introduced at the 3' end of a DNA encoding a first module ( Figure 6).
- Module F introduces the final (R)-3 -hydroxyl acyl group at the final step of PHA monomer synthesis, as a result of the presence of a TE domain.
- DNA encoding a module F is not present in the eryA PKS gene cluster (Donadio et al., supra, 1991).
- a DNA encoding a recombinant monomer synthase is inserted into an expression vector.
- the expression vector employed varies depending on the host cell to be transformed with the expression vector. That is, vectors are employed with transcription, translation and/or post-translational signals, such as targeting signals, necessary for efficient expression of the genes in various host cells into which the vectors are introduced. Such vectors are constructed and transformed into host cells by methods well known in the art. See Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor (1 89)
- Preferred host cells for the vectors of the invention include insect, bacterial, and plant cells.
- Preferred insect cells include Spodoptera frugiperda cells such as Sf2 ⁇ , and Trichoplusia ni cells.
- Preferred bacterial cells include Escherichia coli, Streptomyces and Pseudomonas.
- Preferred plant cells include monocot and dicot cells, such as maize, rice, wheat, tobacco, legumes, carrot, squash, canola, soybean, potato, and the like.
- the appropriate subcellular compartment in which to locate the enzyme in eukaryotic cells must be considered when constructing eukaryotic expression vectors. Two factors are important: the site of production of the acetyl-CoA substrate, and the available space for storage of the PHA polymer. To direct the enzyme to a particular subcellular location, targeting sequences may be added to the sequences encoding the recombinant molecules.
- the baculovirus system is particularly amenable to the introduction of DNA encoding a recombinant FAS or a PKS monomer synthase because an increasing variety of transfer plasmids are becoming available which can accommodate a large insert, and the virus can be propagated to high titers.
- insect cells are adapted readily to suspension culture, facilitating relatively large-scale recombinant protein production.
- recombinant proteins tend to be produced exclusively as soluble proteins in insect cells, thus, obviating the need for refolding, a task that might be particularly daunting in the case of a large multifunctional protein.
- the Sf2 ⁇ /baculo virus system has routinely expressed milligram quantities of catalytically active recombinant fatty acid synthase.
- the baculovirus/insect cell system provides the ability to construct and analyze different synthase proteins for the ability to polymerize monomers into unique biodegradable polymers.
- a further embodiment of the invention is the introduction of at least one DNA encoding a PHA synthase and a DNA encoding a PHA monomer synthase into a host cell.
- Such synthases include, but are not limited to, A.
- DNA(s) encoding more than one PHA synthase may be necessary to produce a particular PHA polymer due to the specificities exhibited by different PHA synthases. As multifunctional proteins are altered to produce unusual monomeric structures, synthase specificity may be problematic for particular substrates. Although the A.
- PHB synthase utilizes only C4 and C5 compounds as substrates, it appears to be a good prototype synthase for initial studies since it is known to be capable of producing copolymers of 3-hydroxybutyrate and 4-hydroxybutyrate (Kunioka et al., Macromolecules, 22, 694 (1989)) as well as copolymers of 3-hydroxyvalerate, 3-hydroxybutyrate, and 5- hydroxyvalerate (Doi et al., Macromolecules, 12, 2860 (1986)).
- Other synthases especially those of Pseudomonas aeruginosa (Tirnm et al., Eur. I.
- a DNA encoding a FAS and a PHA synthase can be introduced into a single expression vector, obviating the need to introduce the genes into a host cell individually.
- a further embodiment of the invention is the generation of a DNA encoding a recombinant multifunctional protein, which comprises a FAS, of either eukaryotic or prokaryotic origin, and a PKS module F.
- Module F will carry out the final chain extension to include two additional carbons and the reduction of the ⁇ -keto group, which results in a (R)- 3-hydroxy acyl CoA moiety.
- DNA encoding the FAS TE is replaced with a DNA encoding a linker region which is normally found in the ACP-KS interdomain region of bimodular ORFs.
- DNA encoding a module F is then inserted 3 ' to the DNA encoding the linker region.
- Different linker regions such as those described below which vary in length and amino acid composition, can be tested to determine which linker most efficiently mediates or allows the required transfer of the nascent saturated fatty acid intermediate to module F for the final chain elongation and keto reduction steps.
- the resulting DNA encoding the protein can then be tested for expression of long-chain ⁇ -hydroxy fatty acids in insect cells, such as Sf2 ⁇ cells, or Streptomyces, or Pseudomonas.
- the expected 3-hydroxy C-18 fatty acid can serve as a potential substrate for PHA synthases which are able to accept long-chain alkyl groups.
- a preferred embodiment of the invention is a FAS that has a chain length specificity between 4-22 carbons.
- linker regions examples include, but are not limited to, the ACP-KS linker regions encoded by the tyl ORFl (ACP,- KS 2 ; ACP 2 -KS 3 ), and ORF3 (ACP 5 -KS 6 ), and eryA ORFl (ACP,-KS,; ACP 2 -KS 2 ), ORF2 (ACP 3 -KS 4 ) and ORF3 (ACP 5 -KS 6 ).
- This approach can also be used to produce shorter chain fatty acid groups by limiting the ability of the FAS unit to generate long-chain fatty acids. Mutagenesis of DNA encoding various FAS catalytic activities, starting with the KS, may result in the synthesis of short- chain (R)-3-hydroxy fatty acids.
- PHA polymers are then recovered from the biomass. Large-scale solvent extraction can be used, but is expensive. An alternative method involving heat shock with subsequent enzymatic and detergent digestive processes is also available (Byron, Trends Biotechnical, 5_, 246 (1987); Holmes, In: Developments in Crystalline Polymers, D. C. Bassett (ed.), pp. 1-65 (1988)). PHB and other PHAs are readily extracted from microorganisms by chlorinated hydrocarbons. Refluxing with chloroform has been extensively used; the resulting solution is filtered to remove debris and concentrated, and the polymer is precipitated with methanol or ethanol, leaving low-molecular-weight lipids in solution.
- the present invention also contemplates nucleic acid sequences which hybridize under stringent hybridization conditions to the nucleic acid sequences set forth herein.
- Stringent hybridization conditions are well known in the art and define a degree of sequence identity greater than about 80 to about 90%.
- nucleic acid sequences encoding variant polypeptides Figure 38
- nucleic acid sequences having conservative (silent) nucleotide substitutions Figure 37
- variant polypeptides encoded by the nucleic acid sequences of the invention are biologically active.
- the present invention also contemplates naturally occurring allelic variations and mutations of the nucleic acid sequences described herein.
- DNA and RNA molecules that can code for the same polypeptides as those encoded by the exempUfied biosynthetic genes and fragments thereof.
- the present invention contemplates those other DNA and RNA molecules which, on expression, encode the polypeptides of, for example, portions of SEQ ID NO:4 or SEQ ID NO:6. Having identified the amino acid residue sequence encoded by a sugar biosynthetic or macrolide biosynthetic gene, and with knowledge of all triplet codons for each particular amino acid residue, it is possible to describe all such encoding RNA and DNA sequences. DNA and RNA molecules other than those specifically disclosed herein and, which molecules are characterized simply by a change in a codon for a particular amino acid, are within the scope of this invention.
- a TCT codon for serine exists at nucleotide positions 1735-1737.
- serine can be encoded by a TCA codon (see, e.g., nucleotide positions 1738-1740) and a TCC codon (see, e.g., nucleotide positions 1874-1876).
- TCA codon see, e.g., nucleotide positions 1738-1740
- TCC codon see, e.g., nucleotide positions 1874-1876.
- substitution of the latter codons for serine with the TCT codon for serine or vice versa does not substantially alter the DNA sequence of SEQ ID NO:6 and results in production of the same polypeptide.
- substitutions of the recited codons with other equivalent codons can be made in a like manner without departing from the scope of the present invention.
- a nucleic acid molecule, segment or sequence of the present invention can also be an RNA molecule, segment or sequence.
- An RNA molecule contemplated by the present invention corresponds to, is complementary to or hybridizes under stringent conditions to any of the DNA sequences set forth herein.
- Exemplary and preferred RNA molecules are mRNA molecules that encode sugar biosynthetic or macrolide biosynthetic enzymes of this invention. Mutations can be made to the native nucleic acid sequences of the invention and such mutants used in place of the native sequence, so long as the mutants are able to function with other sequences to collectively catalyze the synthesis of an identifiable polyketide or macrolide.
- Such mutations can be made to the native sequences using conventional techniques such as by preparing synthetic oligonucleotides including the mutations and inserting the mutated sequence into the gene using restriction endonuclease digestion. (See, e.g., Kunkel, T. A. Proc. Natl. Acad. Sci. USA (1985) 82:448; Geisselsoder et al. BioTechniques (1987) 5:786.)
- the mutations can be effected using a mismatched primer (generally 10-20 nucleotides in length) which hybridizes to the native nucleotide sequence (generally cDNA corresponding to the RNA 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. Zoller and Smith, Methods Enzymol., (1983) 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. Selection 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., Proc. Natl. Acad. Sci. USA (1982) 79:6409. PCR mutagenesis will also find use for effecting the desired mutations.
- Random mutagenesis of the nucleotide sequence can be accomplished by several different techniques known in the art, such as by altering sequences within restriction endonuclease sites, inserting an oligonucleotide linker randomly into a plasmid, by irradiation with X-rays or ultraviolet light, by inco ⁇ orating 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, hydroxylamine, agents which damage or remove bases thereby preventing normal base-pairing such as hydrazine or formic acid, analogues of nucleotide precursors such as nitrosoguanidine, 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.
- the gene sequences can be inserted into one or more expression vectors, using methods known to those of skill in the art.
- Expression vectors may include control sequences operably linked to the desired genes.
- Suitable expression systems for use with the present invention include systems which function in eukaryotic and prokaryotic host cells.
- Prokaryotic systems are preferred, and in particular, systems compatible with Streptomyces spp. are of particular interest.
- Control elements for use in such systems include promoters, optionally containing operator sequences, and ribosome binding sites.
- Particularly useful promoters include control sequences derived from the gene clusters of the invention.
- other bacterial promoters such as those derived from sugar metabolizing enzymes, such as galactose, lactose (lac) and maltose, will also find use in the expression cassettes encoding desosamine.
- Preferred promoters are Streptomyces promoters, including but not limited to the ermE*, pikA and tip A promoters.
- promoter sequences derived from biosynthetic enzymes such as tryptophan (tip), the ⁇ -lactamase (bla) promoter system, bacteriophage lambda PL, and T5.
- synthetic promoters such as the tac promoter (U.S. Pat. No. 4,551,433), which do not occur in nature, also function in bacterial host cells.
- Other regulatory sequences may also be desirable which allow for regulation of expression of the genes relative to the growth of the host cell. Regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound.
- Other types of regulatory elements may also be present in the vector, for example, enhancer sequences.
- Selectable markers can also be included in the recombinant expression vectors.
- a variety of markers are known which are useful in selecting for transformed cell lines and generally comprise a gene whose expression confers a selectable phenotype on transformed cells when the cells are grown in an appropriate selective medium.
- markers include, for example, genes which confer antibiotic resistance or sensitivity to the plasmid.
- polyketides are naturally colored and this characteristic provides a built-in marker for selecting cells successfully transformed by the present constructs.
- the various subunits of interest 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 subunits can include flanking restriction sites to allow for the easy deletion and insertion of other subunits 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.
- sequences generated by random mutagenesis the choice of vector depends on the pool of mutant sequences, i.e., donor or recipient, with which they are to be employed.
- any transducible cloning vector can be used as a cloning vector for the donor pool of mutants. It is preferred, however, that phagemids, cosmids, or similar cloning vectors be used for cloning the donor pool of mutant encoding nucleotide sequences into the host cell.
- Phagemids and cosmids are advantageous vectors due to the ability to insert and stably propagate therein larger fragments of DNA than in Ml 3 phage and ⁇ phage, respectively.
- Phagemids which will find use in this method generally include hybrids between plasmids and filamentous phage cloning vehicles.
- Cosmids which will find use in this method generally include ⁇ phage-based vectors into which cos sites have been inserted.
- Recipient pool cloning vectors can be any suitable plasmid.
- the cloning vectors into which pools of mutants are inserted may be identical or may be constructed to harbor and express different genetic markers (see, e.g., Sambrook et al., supra). The utility of employing such vectors having different marker genes may be exploited to facilitate a determination of successful transduction.
- the cloning vector employed may be an E. coli/Streptomyces shuttle vector (see, for example, U.S. Patent Nos. 4,416,994, 4,343,906, 4,477,571, 4,362,816, and 4,340,674), a cosmid, a plasmid, an artificial bacterial chromosome (see, e.g., Zhang and Wing, Plant Mol. Biol., 25, 115 (1997); Schalkwyk et al., Curr. Op. Biotech.. 6, 37 91995); and Monaco and Lavin, Trends in Biotech., 12, 280 (1994), or a phagemid, and the host cell may be a bacterial cell such as E.
- coli Penicillium patulum, and Streptomyces spp. such as 5. lividans, S. venezuelae, or 5. lavendulae, or a eukaryotic cell such as fungi, yeast or a plant cell, e.g., monocot and dicot cells, preferably cells that are regenerable.
- recombinant polypeptides having a particular activity may be prepared via
- phagemids upon infection of the host cell which contains a phagemid, single- stranded phagemid DNA is produced, packaged and extruded from the cell in the form of a transducing phage in a manner similar to other phage vectors.
- clonal amplification of mutant encoding nucleotide sequences carried by phagemids is accomplished by propagating the phagemids in a suitable host cell.
- the cloned donor pool of mutants is infected with a helper phage to obtain a mixture of phage particles containing either the helper phage genome or phagemids mutant alleles of the wild-type encoding nucleotide sequence.
- Infection, or transfection, of host cells with helper phage is generally accomplished by methods well known in the art (see., e.g., Sambrook et al., supra; and Russell et al. (1986) Gene 45:333-338).
- the helper phage may be any phage which can be used in combination with the cloning phage to produce an infective transducing phage.
- the helper phage will necessarily be a ⁇ phage.
- the clomng vector is a phagemid and the helper phage is a filamentous phage, and preferably phage Ml 3.
- the transducing phage can be separated from helper phage based on size difference (Barnes et al. (1983) Methods Enzymol. 101:98-122), or other similarly effective technique.
- Recipient cells which may be employed in the method disclosed and claimed herein may be, for example, E. coli, or other bacterial expression systems which are not recombination deficient.
- a recombination deficient cell is a cell in which recombinatorial events is greatly reduced, such as rec ⁇ mutants of E. coli (see, Clark et al. (1965) Proc. Natl. Acad. Sci. USA 53:451 -459)
- transductants can now be selected for the desired expressed protein property or characteristic and, if necessary or desirable, amplified.
- transductants may be selected by way of their expression of both donor and recipient plasmid markers.
- the recombinants generated by the above-described methods can then be subjected to selection or screening by any appropriate method, for example, enzymatic or other biological activity.
- the above cycle of amplification, infection, transduction, and recombination may be repeated any number of times using additional donor pools cloned on phagemids.
- the phagemids into which each pool of mutants is cloned may be constructed to express a different marker gene.
- Each cycle could increase the number of distinct mutants by up to a factor of 10 6 .
- the probability of occurrence of an inter-allelic recombination event in any individual cell is f (a parameter that is actually a function of the distance between the recombining mutations)
- the transduced culture from two pools of 10 6 allelic mutants will express up to 10 12 distinct mutants in a population of 10 I2 /f cells.
- Plasmid pFAS206 and a recombinant baculoviral clone encoding FAS206 were generous gifts of A. Joshi and S. Smith.
- Plasmid pAet41 Peoples et al, I. Biol. Chem., 264, 15298 (1989)
- Baculovirus transfer vector, pBacPAK9, and linearized baculoviral DNA were obtained from Clontech Inc. (Palo Alto, CA).
- R-(-)-3 HBCoA was prepared by the mixed anhydride method described by Haywood et al., FEMS Microbiol. Lett., 51, 1 (1989). 60 mg (0.58 nmol) of R-(-)-3 hydroxybutyric acid was freeze dried and added to a solution of 72 mg of pyridine in 10 ml diethyl ether at 0°C. Ethylchloroformate (100 mg) was added, and the mixture was allowed to stand at 4°C for 60 minutes. Insoluble pyridine hydrochloride was removed by centrifugation.
- the phbC gene (approximately 1.8 kb) was excised from pAet41 (Peoples et al., J. Biol. Chem., 264, 15293 (1989)) by digestion with BstB and Stul, purified as described by Williams et al. (Gene, 102, 445 (1991)), and ligated to pBacPAK9 digested with BstBI and Stul. This resulted in pBP-phbC, the baculovirus transfer vector used in formation of recombinant baculovirus particles carrying phbC. Large-scale expression of PHA synthase. A I L culture of J. ni cells
- Insect cell maintenance and recombinant baculovirus formation 5/21 cells were maintained at 26-28°C in Grace's insect cell medium supplemented with 10% FBS, 1.0% pluronic F68, and 1.0% antibiotic/antimycotic (GIBCO-BRL). Cells were typically maintained in suspension at 0.2-2.0 107ml in 60 ml total culture volume in 100 ml spinner flasks at 55-65 ⁇ m. Cell viability during the culture period was typically 95-100%. The procedures for use of the transfer vector and baculovirus were essentially those described by the manufacturer (Clontech, Inc.). Purified pBP-phbC and linearized baculovirus DNA were used for cotransfection of Sf2l cells using the liposome-mediated method (Feigner et al.,
- Recombinant viral clones were purified from plaque assay plates containing 1.5% Seaplaque GTG after 5-7 days at 28°C. Recombinant viral clone stocks were then amplified in T25-flask cultures (4 ml, 3 x 107ml on day 0) for 4 days; infected cells were determined by their mo ⁇ hology and size and then screened by SDS/PAGE using 10% polyacrylamide gels (Laemmli, Nature, 222, 680 (1970)) for production of PHA synthase.
- This mixture was homogenized with three series of 10 strokes each in two Thomas homogenizers while partially submerged in an ice bath and then sonicated for 2 minutes in a Branson Sonifier 250 at 30% cycle, 30% power while on ice. All subsequent procedures were carried out at 4°C.
- the lysate was immediately centrifuged at 100000 g in a Beckman 50.2Ti rotor for 80 minutes, and the resulting supernatant (10.5 ml, 47 mg) was immediately filtered through a 0.45 mm Uniflow filter (Schleicher and Schuell Inc., Keene, N.H.) to remove any remaining insoluble matter. Aliquots of the soluble fraction (1.5 ml, 7 mg) were loaded onto a 5 ml BioRad Econo-Pac HTP column that had been equilibrated with Buffer A (+ protease inhibitor mix) attached to a BioRad Econo-system, and the column was washed with 30 ml Buffer A. All chromatographic steps were carried out at a flow rate of 0.8 ml/minute. PHA synthase was eluted form the HA column with a 32 x 32 ml linear gradient from 10 to 300 mM KPi.
- Fraction collection tubes were prepared by addition of 30 ml of 100 mM EDTA to provide a metalloprotease inhibitor at 1 mM immediately after HA chromatography. PHA synthase was eluted in a broad peak between 110-180 mM KPi. Fractions (3 ml) containing significant PHA synthase activity were pooled and stored at 0°C until the entire soluble fraction had been run through the chromatographic process. Pooled fractions then were concentrated at 4°C by use of a Centriprep-30 concentrator (Amicon) to 3.8 mg/ml. Aliquots (0.5 ml) were either flash frozen and stored in liquid N 2 or glycerol was added to a final concentration of 50% and samples (1.9 mg/ml) were stored at -20°C.
- Membranes were then washed four times for 10 minutes with PBS-Tween followed by the addition of horseradish peroxidase-conjugated goat-anti-rabbit antibody (Boehringer-Mannheim) diluted 10,000X in fresh blocking solution and incubated at 25°C for 1 hour. Membranes were washed finally in three changes (10 minutes) of PBS, and the immobilized peroxidase label was detected using the chemiluminescent LumiGLO substrate kit (Kirkegaard and Perry, Gaithersburg, MD) and X-ray film.
- horseradish peroxidase-conjugated goat-anti-rabbit antibody Boehringer-Mannheim
- Double-infection protocol Four 100 ml spinner flasks were each inoculated with 8
- HBCoA was monitored spectrophotometrically. Assays were performed at 25°C in a Hewlett Packard 8452A diode array spectrophotometer equipped with a water-jacketed cell holder. Two-piece Starna Spectrosil spectrophotometer cells with pathlengths of 0.1 and 0.01 cm were employed to avoid errors arising from the compression of the absorbance scale at higher values. Absorbance was monitored at 232 nm, and E 232 nm of 4.5 103 M "1 cm “1 was used in calculations. One unit (U) of enzyme is the amount required to hydrolyze 1 mmol of substrate minute "1 .
- Buffer (0.15 M KPi, pH 7.2) and substrate were equilibrated to 25°C and then combined in an Eppendorf tube also at 25°C. Enzyme was added and mixed once in the pipet tip used to transfer the entire mixture to the spectrophotometer cell.
- the two-piece cell was immediately assembled, placed in the spectrophotometer with the cell holder (type CH) adapted for the standard 10 mm pathlength cell holder of the spectrophotometer. Manipulations of sample, from mixing to initiation of monitoring, took only 10-15 seconds. Absorbance was continually monitored for up to 10 minutes. Calibration of reactions was against a solution of buffer and enzyme (no substrate) which led to absorbance values that represented substrate only.
- PHB assay PHB was assayed from 5jt21 cell samples according to the propano lysis method of Riis et al., J. Chromo., 445, 285 (1988). Cell pellets were thawed on ice, resuspended in 1 ml cold ddH 2 O and transferred to 5 ml screwtop test tubes with teflon seals. Two ml of ddH 2 O were added, the cells were washed and centrifuged and then 3 ml of acetone were added and the cells washed and centrifuged. The samples were then desiccated by placing them in a 94°C oven for 12 hours.
- Transferase (AT) activity was assayed, using malonyl-CoA as donor and pantetheine as acceptor, by determining spectrophotometrically the free CoA released in a coupled ATP citrate-lyase-malate dehydrogenase reaction (see, Rangen et al., J. Biol. Chem., 266, 19180 (1991).
- Ketoreductase (KR) was assayed spectrophotometrically at 340 nm: assay systems contained 0.1 M potassium phosphate buffer (pH 7), 0.15 mM NADPH, enzyme and either 10 mM trans- 1-decalone or 0.1 mM acetoacetyl-CoA substrate.
- Dehydrase (DH) activity was assayed spectrophotometrically at 270 nm using S-DL- ⁇ -hydyroxybutyryl N-acetylcysteamine as substrate (Kumar et al., J. Biol. Chem., 245, 4732 (1970)).
- Enoyl reductase (ER) activity was assayed spectrophotometrically at 340 nm essentially as described by Strom et al. (J. Biol. Chem., 254, 8159 (1979)); the assay system contained 0.1 M potassium phosphate buffer (pH 7), 0.15 mM NADPH, 0.375 nM crotonoyl- CoA, 20 ⁇ M CoA and enzyme.
- Thioesterase (TE) activity was assessed radiochemically by extracting and assaying the [ 14 C]palmitic acid formed from [l- 14 C]palmitoyl-CoA during a 3 minute incubation Smith, Meth. Enzymol., 21£, 181 ( 1981 ); the assay was in a final volume of 0.1 ml, 25 mM potassium phosphate buffer (pH 8), 20 ⁇ M [l- 14 C]palmitoyl-CoA (20 nCi) and enzyme.
- PHA synthase from A. eutrophus can be overexpressed in E. coli, in the absence of 3-ketothiolase and acetoacetyl-CoA reductase (Gerngross et al., supra) and can be expressed in plants (See Poirier et al., Biotech, 12, 142 (1995) for a review). Isolation of the soluble form of PHA synthase provides opportunities to examine the mechanistic details of the priming and initiation reactions.
- the baculovirus system has been successful for the expression of a number of prokaryotic genes as soluble proteins, and insect cells, unlike bacterial expression systems, carry out a wide array of post- translational modifications, the baculovirus expression system appeared ideal for the expression of large quantities of soluble PHA synthase, a protein that must be modified by phosphopantetheine in order to be catalytically active (Gerngross et al., supra).
- PHA synthase Purification of PHA synthase.
- the purification procedure employed for PHA synthase is a modification of Gerngross et al. (supra) involving the elimination of the second liquid chromatographic step and inclusion of a protease-inhibitor cocktail in all buffers. All steps were carried out on ice or at 4°C except where noted. Frozen cells were thawed on ice in 10 ml of Buffer A (10 mM KPi, pH 7.2, 05% glycerol, and 0.05% Hecameg) and then immediately homogenized prior to centrifugation and HA chromatography.
- Buffer A 10 mM KPi, pH 7.2, 05% glycerol, and 0.05% Hecameg
- N-terminal sequencing of the 64 kDa protein confirmed its identity as PHA synthase ( Figure 8). Two prominent N-termini, at amino acid residue 7 (alanine) and residue 10 (serine) were obtained in a 3:2 ratio. This heterogeneous N-terminus presumably is the result of aminopeptidase activity.
- Western analysis using a rabbit-anti-PHA synthase antibody corroborated the results of the sequencing and indicated the presence of at least three bands that resulted from proteolysis of PHA synthase (Figure 7B, lanes 4-6). The antibody was specific for PHA synthase since neither T. ni nor baculoviral proteins showed reactivity (Figure 7B, lanes 2 and 3).
- N-terminal protein sequencing ( Figure 8) showed directly that the 44 kDa (band b) and 32 kDa (band d) proteins were derived from PHA synthase (fragments beginning at Al 81/Nl 85 and at G387, respectively).
- the 35-40 kDa (band c) protein gave low sequencing yields and may contain a blocked N-terminus. Inspection of Figure 7B suggests that most degradation occurs following cell disruption since the total protein sample of this gel (lane 4) was prepared by boiling intact cells directly in SDS sample buffer while the HA sample (lane 6) went through the purification procedure described above.
- HBCoA and CoA at 232 nm occurs at a trough between two well-separated peaks.
- Assays were carried out at pH 7.2 for comparative analysis with previous studies (Gerngross et al., supra).
- Substrate (R-(-)3-HBCoA) substrate for these studies was prepared using the mixed anhydride method (Haywood et al., supra), and its concentration was determined by measuring A 260 .
- the short pathlength cells (0.1 cm and 0.01 cm) allowed use of relatively high reaction concentrations while conserving substrate and enzyme.
- Assay results showed an initial lag period of 60 seconds prior to the linear decrease in A 232 , and velocities were determined from the slope of these linear regions of the assay curves.
- the pH activity curve for A. eutrophus PHA synthase purified from T. ni cells is shown in Figure 14.
- the curve shows a broad activity maximum centered around pH 8.5. This result agrees well with prior work on the A. eutrophus PHB synthase although it is significantly different than results obtained for the PHB synthase from Z. ramigera for which the optimum was determined to be pH 7.0.
- PHA synthase purified from insect cells appears to be relatively stable. Examination of activity following storage, in liquid N 2 and at -20°C in the presence of 50% glycerol showed that approximately 50% of synthase activity remained after 7 weeks when stored in liquid N 2 and approximately 75% of synthase activity remained after 7 weeks when stored at - 20°C in the presence of 50% glycerol.
- PHA synthase from eutrophus in a baculovirus expression system results in the synthase constituting approximately 50% of total protein 60 hours post- infection; however, approximately 50-75% of the synthase is observed in the membrane- associated fraction.
- This elevated level of expression allowed purification of the soluble PHA synthase using a single chromatographic step on HA.
- the purity of this preparation is estimated to be approximately 90% (intact PHA synthase and 3 proteolysis products).
- the initial specific activity of 12 U/mg was approximately 20-fold higher than the most successful previous efforts at overexpression of A. eutrophus PHA synthase.
- the synthase reported here was isolated from a 250 ml culture with 70% recovery which represents an improvement of 500-fold (1000 U / 64 U ⁇ 8 L / 0.25 L) when compared to an 8 L E. coli culture with 40% recovery.
- This high expression level should provide sufficient PHA synthase for extensive structural, functional, and mechanistic studies.
- the baculovirus expression system is an attractive option for isolation of other PHA synthases from various sources.
- PHA synthase produced in the baculovirus system was of sufficient potency to allow direct spectrophotometric analysis of the hydrolysis of the thioester bond of HBCoA at 232 nm. These assays revealed a lag period of approximately 60 seconds, the length of which was variable and inversely related to enzyme concentration. Such a lag period presumably reflects a slow step in the reaction, perhaps correlating to dimerization of the enzyme, the priming, and/or initiation steps in formation of PHB. Size exclusion chromatographic examination of the PHB synthase native MW indicated two forms of the synthase.
- the high level of expression obtained using the baculoviral system should allow convenient analysis for substrate-specificity and structure-function studies of PHA synthases from relatively crude insect cell extracts.
- ni cells which had been infected with a baculovirus vector encoding rat FAS DH° and/or a baculovirus vector encoding PHA synthase were analyzed for the presence of granules. Infected cells were fixed in paraformaldehyde and incubated with anti-PHA synthase antibodies (Williams et al., Protein Exp. Purif, 2, 203 (1996)). Granules were observed only in doubly infected cells (Williams et al., App. Environ. Micro., 62, 2540 (1996)).
- Figure 18 shows mass spectroscopy data cooesponding to the material obtained from peak 10.1 in the gas chromatograph compared to a propylhydroxybutyrate standard.
- the results show that PHB synthesis is occurring only in 5/21 cells co-expressing the rat FAS DH mutant cDNA and thephbC gene from A. eutrophus. Integration of the peak in the gas chromatograph corresponding to propylhydroxybutyrate revealed that approximately 1 mg of PHB was isolated from 1 liter culture of 5/21 cells (approximately 600 mg dry cell weight of 5/21 cells).
- the ratFAS206 protein effectively replaces the ⁇ - ketothiolase and acetoacetyl-CoA reductase functions, resulting in the production of PHB by a novel pathway.
- the approach described here provides a new strategy to combine metabolic pathways that are normally engaged in primary anabolic functions for production of polyesters.
- the premature termination of the normal fatty acid biosynthetic pathway to provide suitably modified acylCoA monomers for use in PHA synthesis can be applied to both prokaryotic and eukaryotic expression since the formation of polymer will not be dependent on specialized feedstocks.
- novel bipolymer formation can occur.
- the entire PKS cluster form Streptomyces venezuelae was cloned using a heterologous hybridization strategy.
- a 1.2 kb DNA fragment that hybridized strongly to a DNA encoding an eryA PKS ⁇ -ketoacyl synthase domain was cloned and used to generate a plasmid for gene disruption. This method generated a mutant strain blocked in the synthesis of the antibiotic.
- a 5. venezuelae genomic DNA library was generated and used to clone a cosmid containing the complete methymycin aglycone PKS DNA. Fine-mapping analysis was performed to identify the order and sequence of catalytic domains along the multifunctional PKS ( Figure 19).
- DNA sequence analysis of the vep ORFl showed that the order of catalytic domains is KS Q /AT/ACP/KS/AT/KR ACP/KS/AT/DH/KR/ACP.
- the complete DNA sequence, and corresponding amino acid sequence, of the vep ORFl is shown in Figure 23 (SEQ ID NO:l and SEQ ID NO:2, respectively).
- the sequence data indicated that the PKS gene cluster encodes a polyene of twelve carbons.
- the vep gene cluster contains 5 polyketide synthase modules, with a loading module at its 5' end and an ending domain at its 3' end.
- Each of the sequenced modules includes a keto- ACP (KS), an acyltransferase (AT), a dehydratase (DH), a keto-reductase (KR), and an acyl carrier protein domain.
- the six acyltransferase domains in the cluster are responsible for the inco ⁇ oration of six acetyl-CoA moieties into the product.
- the loading module contains a KS Q , an AT and an ACP domain.
- KS Q refers to a domain that is homologous to a KS domain except that the active site cysteine (C) is replaced by glutamine (Q). There is no counte ⁇ art to the KS Q domain in the PKS clusters which have been previously characterized.
- the ending domain is an enzyme which is responsible for the attachment of the nascent polyketide chain onto another molecule.
- the amino acid sequence of ED resembles an enzyme, HetM, which is involved in Anabaena heterocyst formation.
- HetM an enzyme which is involved in Anabaena heterocyst formation.
- the homology between vep and HetM suggests that the polypeptide encoded by the vep gene cluster may synthesize a polyene-containing composition which is present in the spore coat or cell wall of its natural host, 5. venezuelae.
- a DNA encoding a TE is added to the 3' end of an ORFl of a Type I PKS, preferably the met ORF I ( Figure 6) as recently described by Cortes et al. (Science, 268, 1487 (1995)) in the erythromycin system.
- DNA encoding a linker region separating a normal ACP-TE region in a PKS for example, the one found in met PKS ORF5 ( Figure 5) will be inco ⁇ orated into the DNA.
- the resulting vector can be introduced into a host cell and the TE activity, rate of release of the CoA product, and identity of the fatty acid chain determined.
- the acyl chain that is most likely to be released is the CoA ester, specifically the 3- hydroxy-4-methyl heptenoylCoA ester, since the fully elongated chain is presumably released in this form prior to macrolide cyclization. If the CoA form of the acyl chain is not observed, then a gene encoding a CoA ligase will be cloned and co-expressed in the host cell to catalyze formation of the desired intermediate.
- DNA encoding the extender unit AT in met module 1 is replaced to change the specificity from methylmalonylCoA to malonylCoA ( Figures 4-6). This change eliminates methyl group branching in the ⁇ -hydroxy acyl chain. While comparison of known AT amino acid sequences shows high overall amino acid sequence conservation, distinct regions are readily apparent where significant deletions or insertions have occuoed. For example, comparison of malonyl and methylmalonyl amino acid sequences reveals a 37 amino acid deletion in the central region of the malonyltransferase.
- the met ORFl DNA encoding the 37 amino acid sequence of MMT will be deleted, and the resulting gene will be tested in a host cell for production of the desmethyl species, 3-hydroxyheptenoylCoA.
- the DNA encoding the entire MMT can be replaced with a DNA encoding an intact MT to affect the desired chain construction.
- DNA encoding DH/ER will be introduced into DNA encoding met ORFl module 1. This modification results in a multifunctional protein that generates a methylene group at C-3 of the acyl chain ( Figure 6).
- the DNA encoding DH/ER will be PCR amplified from the available eryA or tyl PKS sequences, including the DNA encoding the required linker regions, employing a primer pair to conserved sequences 5' and 3' of the DNA encoding DH/ER.
- the PCR fragment will then be cloned into the met ORFl.
- the result is a DNA encoding a multifunctional protein (MT* DH/ER*TE*). This protein possesses the full complement of keto group processing steps and results in the production of heptenoylCoA.
- the DNA encoding dehydrase in met module 2 is then inactivated, using site-directed mutagenesis in a scheme similar to that used to generate the rat FAS DH- described above (Joshi et al, J. Biol. Chem., 268, 22508 (1993)). This preserves the required (R)-3-hydroxy group which serves as the substrate for PHA synthases and results in (R)-3- hydroxyheptanoylCoA species.
- the final domain replacement will involve the DNA encoding the starter unit acyltransferase in met module 1 ( Figure 5), to change the specificity from propionyl CoA to acetyl CoA. This shortens the (R)-3-hydroxy acyl chain from heptanoyl to hexanoyl.
- the DNA encoding the catalytic domain will need to be generated based on a FAS or 6- methylsalicylic acid synthase model (Beck et al., Eur. J. Biochem., 122, 487 (1990)) or by using site-directed mutagenesis to alter the specificity of the resident met PKS propionyltransferase sequence.
- Example 5 Preparation of a Vector Encoding a Recombinant Monomer Synthase that Synthesizes 3- hydroxyl-4-hexenoic Acid
- a precursor for polyhydroxyhexenoate the DNA segment encoding the loading and the first module of the vep gene cluster was linked to the DNA segment encoding module 7 of the tyl gene cluster so as to yield a recombinant DNA molecule encoding a fusion polypeptide which has no amino acid differences relative to the corresponding amino acid sequence of the parent modules.
- the fusion polypeptide catalyzes the synthesis of 3-hydroxyl-4-hexenoic acid.
- the recombinant DNA molecule was introduced into SCP2, a Streptomyces vector, under the control of the act promoter (pDHS502, Figure 20).
- the DNA segment encoding the polyhydroxyalkanoate polymerase is linked to the DNA segment encoding the recombinant PKS synthase so as to yield a fusion polypeptide which synthesizes polyhydroxyhexenoate in Streptomyces.
- Polyhydroxyhexenoate a biodegradable thermoplastic
- Streptomyces or as a major product in any other organism.
- unsaturated double bond in the side chain of polyhydroxyhexenoate may result in a polymer which has superior physical properties as a biodegradable thermoplastic over the known polyhydroxyalkanoates.
- a DNA library was constructed by partially digesting the genomic DNA of 5. venezuelae (ATCC 15439) with Sau A I into 35- 40 kb fragments which were ligated into the cosmid vector pNJl (Tuan et al., 1990). The recombinant DNA was packaged into bacteriophage ⁇ which was used to transfect E. coli DH5 ⁇ . The resulting cosmid library was screened for desired clones using the tylAl and tylA2 genes from the tylosin biosynthetic cluster as probes (Baltz et al., 1988; Merson-Davies et al., 1994).
- ORFs open reading frames downstream of the PKS genes
- Figure 24 Based on sequence similarities to other sugar biosynthetic genes, especially those derived form the erythromycin cluster (Gaisser et al., 1997; Summers et al., 1997), eight of these nine ORFs are believed to be involved in the biosynthesis of TDP-D- desosamine.
- the ery cluster lacks homologs of the tylAl and tylA2 genes that are responsible for the first two steps in desosamine pathway.
- erythromycin biosynthetic machinery may rely on a general cellular pool of TDP-4-keto-6- deoxy-D-glucose for mycarose and desosamine formation.
- Depicted in Figure 24 is a biosynthetic pathway for TDP-D-desosamine.
- the double crossover mutants in which chromosomal desR had been replaced with the disrupted gene were selected according to their thiostrepton-resistant and apramycin-sensitive characteristics. Southern blot hybridization analysis was used to confirm the gene replacement.
- the desired mutant was first grown at 29°C in seed medium for 48 hours, and then inoculated and grown in vegetative medium for another 48 hours (Cane et al., 1993). After the fermentation broth was centrifuged at 10,000 g to remove cellular debris and mycelia, the supernatant was adjusted to pH 9.5 with concentrated KOH, and extracted with an equivolume of chloroform (four times). The organic layer was dried over sodium sulfate and evaporated to dryness.
- amber oil-like crude products were first subjected to flash chromatography on silica gel using a gradient of 0-40% methanol in chloroform, followed by HPLC purification on a C 18 column eluted isocratically with 45% acetonitrile in 57 mM ammonium acetate (pH 6.7).
- methymycin a compound of formula (1)
- neomethymycin a compound of formula (2)
- two new products were isolated.
- the yield of a compound of formula (13) and a compound of formula (14) was each in the range of 5-10 mg/L of fermentation broth.
- a compound of formula (1) and a compound of formula (2) remained to be the major products.
- glycosyltransferases have been cloned in a few cases, such as mgtA of 5. lividans and oleD of 5. antibioticus, the whereabouts of macrolide ⁇ -glycosidase genes remain obscure.
- eryBI sequence which is part of the erythromycin biosynthetic cluster, is highly homologous to desR (55% identity) (Gaisser et al., 1997).
- desR a macrolide ⁇ -glucosidase gene
- the accumulation of deactivated compounds of formula (13) and (14) after desR disruption provides direct molecular evidence indicating that a similar self-defense mechanism via glycosylation/deglycosylation may also be operative in 5.
- venezuelae because a significant amount of methymycin and neomethymycin also exist in the fermentation broth of the mutant strain, glucosylation of desosamine may not be the primary self-resistance mechanism in 5.
- venezuelae an rRNA methyltransferase gene found upstream from the PKS genes in this cluster may confer the primary self- resistance protection.
- the entire desosamine biosynthetic gene cluster from the methymycin and neomethymycin producing strain Streptomyces venezuelae was cloned, sequenced, and mapped. Eight of the nine mapped genes were assigned to the biosynthesis of TDP-D-desosamine based on sequence similarities to those derived from the erythromycin cluster. The remaining gene, designated desR, showed strong sequence homology to ⁇ -glucosidases.
- the encoded protein (DesR)
- tsr thiosfrepton resistance
- two new products were isolated from the fermentation of the mutant strain. These two new compounds, which are biologically inactive, were found to be C-2' ⁇ -glucosylated methymycin and neomethymycin. Since the translated desR gene has a leader sequence characteristic of secretory proteins, the DesR protein may be an extracellular ⁇ -glucosidase capable of removing the added glucose from the modified antibiotics to activate them.
- the desR gene can be used as a probe to identify homologs in other antibiotic biosynthetic pathways. Deletion of the corresponding macrolide glycosidase gene in other antibiotic biosynthetic pathways may lead to the accumulation of the glycosylated products which may be used as prodrugs with reduced cytotoxicity. Glycosylation also holds promise as a tool to regulate and/or minimize the potential toxicity associated with new macrolide antibiotics produced by genetically engineered microorganisms.
- macrolide glycosidases which can be used for the activation of newly formed antibiotics that have been deliberately deactivated by engineered glycosyltransferases, may be useful in the development of novel antibiotics using the combinatorial biosynthetic approach (Hopwood et al., 1990; Katz et al., 1993; Hutchinson et al., 1995; Carreras et al., 1997; Kramer et al., 1996; Khosla et al., 1996; Jacobsen et al., 1997; Marsden et al., 1998).
- Example 7 Deletion of the desVI Gene of the Desosamine Biosynthetic Gene Cluster
- the emergence of pathogenic bacteria resistant to many commonly used antibiotics poses a serious threat to human health and has been the impetus of the present resurgent search for new antimicrobial agents (Box et al., 1997; Davies, 1996; Service, 1995). Since the first report on using genetic engineering techniques to create "hybrid" polyketides (Hopwood et al., 1995), the potential of manipulating the genes governing the biosynthesis of secondary metabolites to create new bioactive compounds, especially macrolide antibiotics, has received much attention (Kramer et al., 1996; Khosla et al., 1996).
- This class of clinically important drugs consists of two essential structural components: a polyketide aglycone and the appended deoxy sugars (Omura, 1984).
- the aglycone is synthesized via sequential condensations of acyl thioesters catalyzed by a highly organized multi-enzyme complex, polyketide synthase (PKS) (Hopwood et al., 1990; Katz, 1993; Hutchinson et al., 1995; Carreras et al., 1997).
- PKS polyketide synthase
- the des VI gene which has been predicted to encode the N-methyltransferase, was chosen as a target (Gaisser et al., 1997; Summers et al., 1997).
- the deduced desVI product is most closely related to that of eryCVI from the erythromycin producing strain Saccharopolyspora erythraea (70% identity), and also strongly resembles the predicted products ofrdmD from the rhodomycin cluster of Streptomyces purpurascens (Niemi et al., 1995), srmX from the spiromycin cluster of Streptomyces ambofaciens (Geistlich et al., 1992), and tylMl from the tylosin cluster of Streptomyces fradiae (Gandecha et al., 1997).
- All of these enzymes contain the consensus sequence LLDV(I)ACGTG (SEQ ID NO:25) (Gaisser et al., 1997; Summers et al., 1997), near their N-terminus, which is part of the S-adenosylmethionine binding site (Ingrosso et al., 1989; Haydock et al., 1991).
- des VI should have little polar effect (Lin et al., 1984) on the expression of other desosamine biosynthetic genes because the ORF (desR) lying immediately downstream from des VI is not directly involved in desosamine formation, and those lying further downstream are transcribed in the opposite direction.
- a plasmid pBL3001 in which des VI was replaced by the thiosfrepton gene (tsr) (Bibb et al., 1985), was constructed and introduced into wild type 5. venezuelae by conjugal transfer using E. coli SI 7-1 (Bierman et al., 1992). Two identical double crossover mutants, KdesVI- 21 and KdesVI-22 with phenotypes of thiosfrepton resistance (Thio R ) and apamycin sensitivity (Apm s ) were obtained. Southern blot hybridization using tsr or a 1.1 kb Hindi fragment from the desVII region further confirmed that the des VI gene was indeed replaced by tsr on the chromosome of these mutants.
- the KdesVI-21 mutant was first grown at 29°C in seed medium (100 mL) for 48 hours, and then inoculated and grown in vegetative medium (3 L) for another 48 hours (Cane et al., 1993).
- the fermentation broth was centrifuged to remove the cellular debris and mycelia, and the supernatant was adjusted to pH 9.5 with concentrated KOH, followed by extraction with chloroform.
- No methymycin or neomethymycin was found; instead, the 10-deoxy-methynolide (6) (350 mg) (Lambalot et al., 1992) and two new macrolides containing an N-acetylated amino sugar, a compound of formula (7) (20 mg) and a compound of formula (8) (15 mg), were isolated.
- Their structures were determined by spectral analyses and high-resolution MS.
- venezuelae deletion mutant strain resulted in the accumulation of a methymycin/neomethymycin analogue carrying an N- acetylated aminodeoxy sugar. Isolation and characterization of these derivatives not only provide the first direct evidence confirming the identity of des VI as the N-methyltransferase gene, but also demonstrate the feasibility of preparing novel sugars by the gene deletion approach. Most significantly, the results also revealed that the glycosyltransferase of methymycin/neomethymycin exhibits a relaxed specificity towards its sugar substrates.
- E. coli DH5 ⁇ was used as a cloning host.
- E. coli LE392 was the host for a cosmid library derived from 5. venezuelae genomic D ⁇ A.
- LB medium was used in E. coli propagation.
- Streptomyces venezuelae ATCC 15439 was obtained as a freeze- dried pellet from ATCC.
- Media for vegetative growth and antibiotic production were used as described (Lambalot et al., 1992). Briefly, SGGP liquid medium was for propagation of 5. venezuelae mycelia.
- Sporulation agar (SPA) was used for production of 5. venezuelae spores. Methymycin production was conducted in either SCM or vegetative medium and pikromycin production was performed in Suzuki glucose-peptone medium.
- Plasmid vectors for gene disruption were either pGM160 (Muth et al., 1989) or pKCl 139 (Bierman et al., 1992). Plasmid, cosmid, and genomic D ⁇ A preparation, restriction digestion, fragment isolation, and cloning were performed using standard procedures (Sambrook et al., 1989; Hopwood et al, 1985). The cosmid library was made according to instructions from the Packagene ⁇ -packaging system (Promega).
- a replicative plasmid-mediated homologous recombination approach was developed to conduct gene disruption in 5.
- venezuelae Plasmids for insertional inactivation were constructed by cloning a kanamycin resistance marker into target genes, and plasmid for gene deletion/replacement was constructed by replacing the target gene with a kanamycin or thiosfrepton resistance gene in the plasmid.
- Disruption plasmids were introduced into 5. venezuelae by either PEG-mediated protoplast transformation (Hopwood et al., 1985) or RK2-mediated conjugation (Bierman et al., 1992).
- Methymycin, pikromycin, and related compounds were extracted following published procedures (Cane et al., 1993). Thin layer chromatography (TLC) was routinely used to detect methymycin, neomethymycin, narbomycin and pikromycin. Further purification was conducted using flash column chromatography and HPLC, and the purified compounds were analyzed by ⁇ , 13 C NMR spectroscopy and MS spectrometry. Results Cloning and Identification of the pik Cluster. Heterologous hybridization was used to identify genes for methymycin, neomethymycin, narbomycin and pikromycin biosynthesis in 5. venezuelae.
- Nucleotide Sequence of the pik Cluster The nucleotide sequence of the pik cluster was completely determined and shown to contain 18 open reading frames (ORFs) that span approximately 60 kb. Central to the cluster are four large ORFs,pikAI, p ⁇ kAII, pikAIII, and pikAIV, encoding a multifunctional PKS ( Figure 28). Analysis of the six modules comprising the pik PKS indicated that it would specify production of narbonolide, the 14-membered ring aglycone precursor of narbomycin and pikromycin ( Figure 28).
- pikA may produce the 12-membered ring macrolactone 10- deoxymethynolide as well.
- the domain organization of PikAI - Ai ⁇ (module L-5) is consistent with the predicted biosynthesis of 10-deoxymethynolide except for the absence of a TE function at the C-terminus of Pik module 5 (PikAi ⁇ ).
- the lack of a TE domain in PikAi ⁇ may be compensated by the type II TE (encoded by pikA V) immediately downstream of pikAIV.
- Desl, desll, desIII, desIV, desV, des VI, and des VIII are responsible for the biosynthesis of the deoxysugar
- the eighth gene, desVII encodes a glycosyltransferase that apparently catalyzes transfer of desosamine onto the alternate (12- and 14-membered ring) polyketide aglycones.
- the existence of only one set of desosamine genes indicates that Des HI can accept both 10-deoxymethynolide and narbonolide as substrates (Jacobsen et al., 1997).
- the largest ORF in the des locus, desR encodes a ⁇ - glycosidase that is involved in a drug inactivation-reactivation cycle for bacterial self- protection.
- a gene (pikC) encoding a cytochrome P450 hydroxylase similar to eryF (Andersen et al., 1992), and eryK (Stassi et al., 1993), PikC, and a gene (pikD) encoding a putative regulator protein, PikD ( Figure 28).
- PikC is the only P450 hydroxylase identified in the entire pik cluster, suggesting that the enzyme can accept both 12- and 14-membered ring macrolide substrates and, more remarkably, it is active on both C-10 and C- 12 of the YC- 17 (12-membered ring intermediate) to produce methymycin and neomethymycin (Figure 30).
- PikD is a putative regulatory protein similar to ORFH in the rapamycin gene cluster (Schwecke et al., 1995).
- the combined functionality coded by the eighteen genes in the pik cluster predicts biosynthesis of methymycin, neomethymycin, narbomycin and pikromycin (Table 2). Flanking the pik cluster locus are genes presumably involved in primary metabolism and genes that may be involved in both primary and secondary metabolism.
- An S-adenosyl- methionine synthase gene is located downstream of pikD that may help to provide the methyl group in desosamine synthesis.
- a threonine dehydratase gene was identified upstream of pikRl that may provide precursors for polyketide biosynthesis. It is not apparent that any of these genes are dedicated to antibiotic biosynthesis and they are not directly linked to the pik cluster. Table 2. Deduced function of ORFs in the pik cluster
- PikAI the first putative enzyme involved in the biosynthesis of 10-deoxymethynolide and narbonolide was inactivated by insertional mutagenesis.
- the resulting mutant, AX903 produced neither methymycin or neomethymycin, nor narbomycin or pikromycin, indicating that pikA encodes a PKS required for both 12- and 14-membered ring macrolactone formation.
- mutant LZ3001 in which mutation in an enzyme downstream of pikA V accumulated 10-deoxymethynolide and narbonolide.
- mutant AX905 failed to accumulate these intermediates suggested that the polyketide chains were not efficiently released from this PKS protein in the absence of Pik TEII. Therefore, Pik TEII plays a crucial role in polyketide chain release and cyclization, and it presumably provides the mechanism for alternative termination in pik polyketide biosynthesis.
- PikC is the sole enzyme catalyzing hydroxylation of both YC- 17 (at C- 10 and C- 12) and narbomycin (at C- 12).
- the relaxed substrate specificity of PikC and its regional specificity at C-10 and C-12 provide another layer of metabolite diversity in the ?t " :-encoded biosynthetic system. Discussion
- pikA evolved in a line analogous to eryA and oleA since each of these PKSs specify the synthesis of 14-membered ring macrolactones.
- pik may have acquired the capacity to generate methymycin when a mutation in the primordial pikAIII-pikAIV linker region caused splitting of Pik module 5 and 6 into two separate gene products.
- This notion is raised by two features of the nucleotide sequence.
- the intergenic region between pikAIII and pikAIV which is 105 bp, may be the remanent of an intramodular linker peptide of 35 amino acids.
- the potential for independently regulated expression of pikAIV is implied by the presence of a 100 nucleotide region at the 5' end of the gene that is relatively AT-rich (62% as comparing 74% G+C content in coding region).
- the mutation in an original ORF encoding the bimodular multifunctional protein (PikAIII-PikATV) occurred, so too may have evolved a mechanism for regulated synthesis of the new gene product (PikAIV).
- Pik TEII in alternative termination of polyketide chain elongation intermediates provides a unique aspect of diversity generation in natural product biosynthesis.
- Engineered polyketides of different chain length are typically generated by moving the TE catalytic domain to alternate positions in a modular PKS (Cortes et al., 1995). Repositioning of the TE domain necessarily abolishes production of the original full-length polyketide so only one macrolide is produced each time.
- the independent Pik TEII polypeptide presumably has the flexibility to catalyze termination at different stages of polyketide assembly, therefore enabling the system to produce multiple products of variant chain length.
- the enzymes involved in post-polyketide assembly of 10-deoxymethynolide and narbonolide are particularly interesting, especially the glycosyltransferase, DesVII, and P450 hydroxylase, PikC. Both have the remarkable ability to accept substrates with significant structural variability. Moreover, disruption of des VI demonstrated that DesVII also tolerates variations in deoxysugar structure (Example 6). Likewise, PikC has recently been shown to convert YC-17 to methymycin/neomethymycin and narbomycin to pikromycin in vitro. Targeted gene disruption of ORFl abolished both pikromycin and methymycin production, indicating that the single cluster is responsible for biosynthesis of both antibiotics.
- TE2 in contrast to the position- fixed TE1 domain, has the capacity to release polyketide chain at different points during the assembly process, thereby producing polyketides of different chain length.
- the pik cluster represents the least complex yet most versatile modular PKS system so far investigated. This simplicity provides the basis for a compelling expression system in which novel active ketoside products are engineered and produced with considerable facility for discovery of a diverse range of new biologically active compounds. Summary
- PKSs represent one of the most amenable systems for combinatorial technologies because of their inherent genetic organization and ability to produce polyketide metabolites, a large group of natural products generated by bacteria (primarily actinomycetes and myxobacteria) and fungi with diverse structures and biological activities.
- Complex polyketides are produced by multifunctional PKSs involving a mechanism similar to long-chain fatty acid synthesis in animals (Hopwood et al., 1990).
- Streptomyces venezuelae ATCC 15439 is notable in its ability to produce two distinct groups of macrolide antibiotics.
- Methymycin and neomethymycin are derived from the 12-membered ring macrolactone 10-deoxymethynolide, while narbomycin and pikromycin are derived from the 14-membered ring macrolactone, narbonolide.
- the cloning and characterization of the biosynthetic gene cluster for these antibiotics reveals the key role of a type II thioesterase in forming a metabolic branch through which polyketides of different chain length are generated by the pikromycin multifunctional polyketide synthase (PKS).
- PKS pikromycin multifunctional polyketide synthase
- pikA a set of genes for desosamine (des) biosynthesis and macrolide ring hydroxylation.
- the glycosyl transferase encoded by des VIII
- the/>/£C-encoded P450 hydroxylase provides yet another layer of structural variability by introducing regiochemical diversity into the macrolide ring systems.
- PKSs e.g., modules of pikA or a FAS
- PHA monomers One strategy to exploit modular PKSs, e.g., modules of pikA or a FAS, to provide PHA monomers is to harvest polyketide intermediates as CoA derivatives using a TEII which is converted to an acyl-CoA transferase (mTEII).
- mTEII acyl-CoA transferase
- PikTEII is a small enzyme (281 amino acids) encoded by pikAV in 5. venezuelae. The primary function of the wild-type enzyme is to catalyze the release of a polyketide chain at the fifth module in the pikA pathway as 10- deoxymethonolide.
- the enzyme most likely binds to the fifth module (PikAIII) ACP (ACP 5 ) and releases the acyl chain attached to it.
- This relationship, TEII and its cognate ACP 5 can be exploited to produce a polyketide having different chain lengths by moving Pik ACP 5 to a different position in the cluster. For example, by moving ACP 5 into the second module in place of ACP 2 , a triketide instead of hexoketide may be produced by the cluster. Further, moving K ⁇ together with ACP 5 into the second module, and replacing the DH, KR, and ACP domains, a 3-hydroxyl triketide is produced that is structurally suitable as PHA monomer.
- a mutant TEII catalyzes the release of the triketide as CoA form.
- the triketide-CoA 3,5-dihydroxyl-4-methyl-heptonyl-CoA, is a subsfrate for PHA polymerase, e.g., PhaCl from P. olivarus, which, in turn, can inco ⁇ orate the monomer into a polymer.
- a second strategy includes the harvesting of a polyketide intermediate as a CoA derivative using a TEI which has been converted to an acyl-CoA transferase (mTE).
- mTE acyl-CoA transferase
- the second strategy for 3-hydroxyacyl-CoA monomer production is to exploit the TE domain (TEI) within the PKS module. It has been demonstrated that the TE domain can release polyketide intermediates attached to the ACP domain within the same module. Moving the TEI to a different position in a PKS cluster results in the production of a polyketide having a different chain length.
- a mutant TEI (i.e., one which is an acyl-CoA transferase) releases the polyketide intermediate to acyl-CoA, which then is polymerized by PHA synthetase.
- a mutant TE domain in the pikA gene cluster is moved into pik module 1, fusing it immediately downstream of ACPI .
- the recombinant enzyme produces 2- (S)-methyl-3(R)-hydroxylveleratyl-CoA, which is a suitable substrate for PHA polymerase PhaCl. Therefore, the coexpression of the polymerase with the recombinant PKS produces a polymer.
- a third strategy is to directly collect polyketide intermediates as substrates for PHA synthesis by fusing a PHA polymerase with a polyketide synthase.
- the first two strategies produce 3-hydroxylacyl-CoA as a substrate for PHA synthesis by employing a mutant PKS enzyme (TEI or TEU).
- TEI or TEU mutant PKS enzyme
- the third strategy fuses a PHA polymerase downstream of an ACP in a PKS protein.
- the PHA synthetase then serves as a domain within the chimeric multifunctional enzyme in place of a TE domain.
- the PKS portion of the protein catalyzes the synthesis of a 3-hydroxylacyl-ACP intermediate and then the PHA synthetase domain accepts it as substrate and adds the 3-hydroxylacyl monomer to the growing polyhydroxyalkanoate chain.
- the process regenerates ACP function so that the reaction can go on repeatedly to synthesize a PHA of multiple units.
- a phaCl gene is fused directly downstream of pik ACPI so as to produce a chimeric enzyme that catalyzes the synthesis of a polymer.
- the strategies described above can produce PHAs of complex structure, and having superior properties.
- the structure can be easily fine-tuned by modifying the PKS gene, thus resulting in PHAs having desired properties or functions.
- Streptomyces venezuelae ATCC 15439 produces two groups of macrolide antibiotics: the 12-membered ring macrolides methymycin and neomethymycin, and the 14- membered ring macrolides pikromycin and narbomycin ( Figure 28).
- Methymycin and neomethymycin are derived from the 12-membered ring macrolactone 10-deoxymethynolide and are produced in SCM medium (Lambalot et al., 1992), whereas pikromycin and narbomycin are derived from the 14-membered ring macrolactone narbonolide and are produced in PGM medium (Xue et al., 1998). Genetic Manipulation of 5. venezuelae. Mutant AX910 and AX912 were created by targeted gene replacement. The mutation plasmid pDHS910 was created by ligating two DNA fragments flanking the TE domain so that the TE domain was deleted and a hexa- histidine sequence was introduced at its position. The primer pairs that were used to amplified the flanking DNA in polymerase chain reaction (PCR) are 5'- CCCGAATTCGCCGCCGCCATGGCCGAA - 3' (SEQ ID NO:42) and 5' -
- Plasmid pDHS910 was introduced into 5.
- venezuelae AX905 (Xue et al., 1998) which has a kanamycin resistance marker at the position of pikA V.
- mutant AX910 (12 colonies) was isolated by screening for a kanamycin sensitive phenotype.
- Mutation plasmid pDHS912 was generated by replacing a BamUI- BgHI fragment (the DNA fragment corresponding to the pikAV gene immediately downstream of the TE domain) in pDHS910 with a kanamycin resistance gene (Denis et al., 1992). Thus, the TE domain as well as the TEII gene pikAV were disrupted in the mutant AX912. Plasmid pDHS912 was transferred into wild type 5. venezuelae and mutant AX912 (12 colonies) was selected according to the procedures of Xue et al. (1998).
- the pikA promoter, PpikA was isolated as an EcoRV-EcoRI fragment between pikAI and pikRl in the pik cluster (Xue et al., 1998).
- a DNA fragment encoding PikAV was first PCR- amplified and placed downstream of the EcoRI site in such a way that PikAV was translationally coupled to the leader sequence of pikAI in pikA to give plasmid pDHS702.
- plasmids pDHS704, pDHS705, pDHS706, pDHS707, and pDHS708 were constructed by cloning various lengths of the pikAIV-pikA V region into pDHS702 replacing pikA V
- the various lengths of pikAIV were PCR-amplified from cosmid pLZ51 (Xue et al., 1998) by the following primer pairs: prepared with primers 5' -
- pDHS705 (EcoRl-BamHl) and pDHS706 (EcoW-Bglll) was isolated directly from restriction digestion of cosmid pLZ51 (Xue et al., 1998) and ligated into EcoRI- BgH treated pDHS702.
- PikAV is an independent thioesterase (TEII) that is distinct from the thioesterase domain (TE) located at the C-terminus of PikAIV.
- TEII thioesterase domain
- the modular organization of PikA indicates that PikAI-PikAIII produces a hexaketide that cyclizes into 10-deoxymethynolide, and that PikAI-PikATV produces a heptaketide that cyclizes into narbonolide ( Figure 28). Termination of polyketide assembly at the heptaketide stage is likely catalyzed by the C- teoninal TE domain in PikAIV, which is analogous to chain termination in the erythromycin pathway.
- AX910 mutant strain would at least produce the 12- membered ring macrolides methymycin and neomethymycin because the sixth condensation cycle catalyzed by PikAIV is not required for 10-deoxymethynolide formation.
- mutant AX910 produced trace amounts of pikromycin, however, methymycin and neomethymycin were completely absent from the fermentation broth. Since the AX910 mutant contains an inframe deletion of the ⁇ z - ⁇ /F-encoded TE domain, the potential for a downstream polar effect (on the pikA F-encoded TEII enzyme) was avoided. This result suggested that PikAIV, or at least the TE domain within PikAIV, is involved directly in the production of the 12- as well as 14-membered ring macrolactones.
- PikATV Probing the expression of PikATV.
- SCM medium methymycin
- PGM medium pikromycin
- the PikAIV protein was first tagged by a hexa-histidine sequence replacing the TE domain at its C-terminus. Expression of PikAIV was then probed with anti- 6xHis antibody in a Western blot that revealed a single protein band under conditions for either methymycin or pikromycin production in the mutant strains (AX910 and AX912).
- the protein detected from cell extracts obtained under culture conditions for methymycin production was approximately 25 kDa lower in molecular weight compared to the protein detected under conditions for pikromycin production (PGM medium).
- the molecular weight of the protein detected under pikromycin culture conditions is 110 kDa, which is consistent with the predicted TE-truncated (6xHis-tag replaced) form of PikAIV. Therefore, the protein detected under conditions for methymycin production must be an N-terminal truncated form of PikAIV (Figure 41). Indeed, two potential alternative translation start sites have been located in the pikAIV sequence, with either predicted to generate the truncated form of PikAIV.
- pikAIV creates a protein product that contains only half of the Pik module 6 KS (KS 6 ) domain ( Figure 41). This result immediately pointed to a mechanism for alternative termination in the PikA system. Since the KS 6 domain is responsible for the condensation of the final extender unit, a PKS that is unable to catalyze this reaction could only produce the 12-membered ring macrolactone.
- TE domain in PikAIV is critical for 10— deoxymethynolide formation.
- all of the plasmid constructs that contain the TE domain including, pDHS704 (TE alone), pDHS705 (ACP 6 -TE), pDHS706 (ACP 6 -TE: :TEII), pDHS708 (AT 6 -ACP 6 -TE), and pDHS707 (KS 6 -AT 6 -ACP 6 -TE), complemented mutant AX912 to give 10-deoxymethynolide.
- TE domain directly with PikAIII ( Figure 42C) and/or formation of a wild type-like PKS complex ( Figure 42B) by the TE domain expressed from the plasmid interacting with the rest of PikAIV (expressed from the corresponding AX912 chromosomal allele) through noncovalent interactions.
- the TE domain alone did not complement AX912 (TE-TEII double mutant) to give narbonolide production ( Figure 41).
- TEII TEII
- venezuelae expresses a full length PikAIV module, which interacts with PikAIII and elongates the growing polyketide chain on ACP 5 by adding a methylmalonate unit (the activity of KS 6 ) to ultimately produce the 14-membered ring macrolactone, narbonolide ( Figure 42A).
- the truncated form of PikAIV that lacks KS 6 is expressed under culture conditions for methymycin production. The molecular space left unoccupied by KS 6 truncation is then presumably filled by the TE domain that would be aligned to interact directly with ACP 5 to release the 12-membered ring macrolactone ( Figure 42B).
- pikA to produce the rare 12-membered ring macrolactone may be the result of endogenous genetic selection to overcome antibiotic resistance within the ecological milieu of the antibiotic producing microorganism.
- the pikA system provides a natural example of a branched metabolic pathway with the capacity to generate multiple macrolactone systems that may be readily exploited for combinatorial biosynthetic creation of novel natural products.
- Example 11 A mutant of 5. venezuelae (KdesV-41) was constructed that had the desV gene disrupted (Zhao et al., J. Am. Chem. Son , 120, 12159 (1998)). Since desV encodes the 3- aminotransferase that catalyzes the conversion of the 3-keto sugar 17 ( Figure 42) to the corresponding amino sugar 4, deletion of this gene should prevent C-3 transamination, resulting in the accumulation of 17. It was expected that if the glycosyltransferase (DesVII) of this pathway is capable of recognizing and processing the keto sugar intermediate 17, the macrolide product(s) produced by the KdesV-41 mutant should have an attached 3-keto sugar.
- the two products isolated were the methymycin/neomethymycin analogues 18 and 19, each carrying a 4,6-dideoxyhexose (Figure 43). While this result demonstrated a relaxed specificity for the glycosyltransferase toward its sugar substrate, it also indicated the existence of a pathway-independent reductase in 5. venezuelae that can stereospecifically reduce the C-3 keto group of the sugar metabolite.
- KdesI-80 was selected and grown at 29°C in seed medium (100 mL) for 48 hours and then inoculated and grown in vegetative medium (5 L) for another 48 hours (Cane et al., 1993). The fermentation broth was centrifuged to remove cellular debris and mycelia, and the supernatant was adjusted to pH 9.5 with concentrated potassium hydroxide solution.
- cytochrome P450 hydroxylase which catalyzes the hydroxylation of 10-deoxy-methynolide at either its C- 10 or C- 12 position (Xue et al., Chem. Biol., 5, 661 (1998)), is sensitive to structural variations in the appended sugar. It could be argued that the presence of the 4-OH group in the sugar moiety is somehow responsible for decreasing or preventing hydroxylation of the macrolide.
- the results demonstrate the feasibility of combining pathway-dependent genetic manipulations and pathway-independent enzymatic reactions to engineer a sugar of designed structure. It is conceivable that the pathway-independent enzymes could also be used in concert with the natural biosynthetic machinery to generate further structural diversity, which can provide an array of random compounds.
- Hopwood, D. A. Malpartida, F., Kieser, H. M., Ikeda, H., Duncan, J., Fujii, I., Rudd, B. A., Floss, H. G., Omura, S. Nature, 214:642-644 (1985).
Abstract
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JP2000557373A JP2002536959A (en) | 1998-06-26 | 1999-06-25 | Genes encoding methymycin and picromycin |
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EP99930718A EP1090125A2 (en) | 1998-06-26 | 1999-06-25 | Dna encoding methymycin and pikromycin |
AU47199/99A AU4719999A (en) | 1998-06-26 | 1999-06-25 | Dna encoding methymycin and pikromycin |
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EP1090125A2 (en) | 2001-04-11 |
AU4719999A (en) | 2000-01-17 |
JP2002536959A (en) | 2002-11-05 |
US20020164742A1 (en) | 2002-11-07 |
US20030087405A1 (en) | 2003-05-08 |
WO2000000620A3 (en) | 2000-04-13 |
US6265202B1 (en) | 2001-07-24 |
CA2332129A1 (en) | 2000-01-06 |
US20020110897A1 (en) | 2002-08-15 |
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