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PubMed2005

Combinatorial polyketide biosynthesis by de novo design and rearrangement of modular polyketide synthase genes.

Type I polyketide synthase (PKS) genes consist of modules approximately 3-6 kb long, which encode the structures of 2-carbon units in polyketide products. Alteration or replacement of individual PKS modules can lead to the biosynthesis of 'unnatural' natural products but existing techniques for this are time consuming. Here we describe a generic approach to the design of synthetic PKS genes where facile cassette assembly and interchange of modules and domains are facilitated by a repeated set of flanking restriction sites. To test the feasibility of this approach, we synthesized 14 modules from eight PKS clusters and associated them in 154 bimodular combinations spanning over 1.5-million bp of novel PKS gene sequences. Nearly half the combinations successfully mediated the biosynthesis of a polyketide in Escherichia coli, and all individual modules participated in productive bimodular combinations. This work provides a truly combinatorial approach for the production of polyketides.

Amino Acid Sequence↗
PubMed2006

Engineered biosynthesis of a novel amidated polyketide, using the malonamyl-specific initiation module from the oxytetracycline polyketide synthase.

Tetracyclines are aromatic polyketides biosynthesized by bacterial type II polyketide synthases (PKSs). Understanding the biochemistry of tetracycline PKSs is an important step toward the rational and combinatorial manipulation of tetracycline biosynthesis. To this end, we have sequenced the gene cluster of oxytetracycline (oxy and otc genes) PKS genes from Streptomyces rimosus. Sequence analysis revealed a total of 21 genes between the otrA and otrB resistance genes. We hypothesized that an amidotransferase, OxyD, synthesizes the malonamate starter unit that is a universal building block for tetracycline compounds. In vivo reconstitution using strain CH999 revealed that the minimal PKS and OxyD are necessary and sufficient for the biosynthesis of amidated polyketides. A novel alkaloid (WJ35, or compound 2) was synthesized as the major product when the oxy-encoded minimal PKS, the C-9 ketoreductase (OxyJ), and OxyD were coexpressed in CH999. WJ35 is an isoquinolone compound derived from an amidated decaketide backbone and cyclized with novel regioselectivity. The expression of OxyD with a heterologous minimal PKS did not afford similarly amidated polyketides, suggesting that the oxy-encoded minimal PKS possesses novel starter unit specificity.

Genetic Engineering↗
PubMed1994

Engineered biosynthesis of novel polyketides: evidence for temporal, but not regiospecific, control of cyclization of an aromatic polyketide precursor.

BACKGROUND: Aromatic polyketide synthases (PKSs) catalyze the formation and cyclization of polyketide chains of variable lengths, generating a family of compounds of proven medical significance. Initial control over the regiospecificity of cyclization is believed to be exercised by the minimal PKS, composed of the three essential components for polyketide biosynthesis, which catalyzes an intramolecular aldol condensation towards the middle of the chain. Subsequent cyclization reactions are either catalyzed by additional components of the PKS, or occur in the absence of specific catalysts. RESULTS: Structural and biosynthetic studies on SEK4b, a novel octaketide product of a minimal PKS, revealed an unusual cyclization pattern. The first cyclization (an aldol condensation) occurs at the methyl end of the unreduced polyketide backbone precursor. This is followed by hemiketal formation and lactonization. The overall structure of SEK4b is similar to that of SEK4, a previously-identified product of the same genetically-engineered strain, differing only in the positions of a methyl and a pyrone group around a common fused-ring system. The biosynthetic pathways of the two molecules are quite different, however. The yield of SEK4b relative to SEK4 is much higher in the absence of PKS components (aromatases and cyclases) acting later in the pathway. CONCLUSIONS: In this cyclization pathway, the regiospecificity of cyclization is not directly controlled by the minimal PKS. Instead, we propose that the enzyme influences cyclization by controlling the timing of chain release. Chain release and cyclization may be concurrent with synthesis. Other PKS subunits appear to stabilize the complex of the PKS with the nascent chain, preventing premature release.

Catalysis↗
PubMed2004

The crystal structure of the actIII actinorhodin polyketide reductase: proposed mechanism for ACP and polyketide binding.

We have determined the 2.5 angstroms crystal structure of an active, tetrameric Streptomyces coelicolor type II polyketide ketoreductase (actIII) with its bound cofactor, NADP+. This structure shows a Rossman dinucleotide binding fold characteristic of SDR enzymes. Of two subunits in the crystallographic asymmetric unit, one is closed around the active site. Formate is observed in the open subunit, indicating possible carbonyl binding sites of the polyketide intermediate. Unlike previous models we observe crystal contacts that may mimic the KR-ACP interactions that may drive active site opening. Based on these observations, we have constructed a model for ACP and polyketide binding. We propose that binding of ACP triggers a conformational change from the closed to the open, active form of the enzyme. The polyketide chain enters the active site and reduction occurs. The model also suggests a general mechanism for ACP recognition which is applicable to a range of protein families.

Acyl Carrier Protein↗
PubMed1998

Engineering of a minimal modular polyketide synthase, and targeted alteration of the stereospecificity of polyketide chain extension.

BACKGROUND: Polyketides are a large and structurally diverse group of natural products that include antibiotics, antifungal agents and immunosuppressant compounds. Polyketides are biosynthesised in filamentous bacteria on modular polyketide synthases (PKSs) in which each cycle of chain extension requires a different 'module' of enzymatic activities. The recently proposed dimeric model for modular PKSs predicts that even a single-module PKS should be catalytically active in the absence of other PKS components. Researchers are also interested in manipulating the stereochemical outcome of polyketide chain extension using genetic engineering of domains within each module. RESULTS: We have constructed a minimal modular PKS from the erythromycin-producing PKS (DEBS) of Saccharopolyspora erythraea. The diketide synthase (DKS1-2) consists of a single chimaeric extension module, derived from the DEBS module 1 ketoacyl-ACP synthase (KS), sandwiched between a loading module and a chain-terminating thioesterase. When DKS1-2 was expressed in S. erythraea, the strain preferentially6 accumulated the diketide (2R, 3S)-2-methyl-3-hydroxy pentanoic acid. CONCLUSIONS: These results demonstrate that, as predicted, even a single-module PKS is catalytically active in the absence of other DEBS proteins. In its normal context, the ketosynthase domain KS1 is thought to generate a (2S)-2methyl-3-hydroxy intermediate by epimerising the initial product of carbon-carbon chain formation, the (2R)-2-methyl-3-ketoester. The observed formation of the alternative (2R)-methyl-3-hydroxy product catalysed by DKS1-2 provides strong support for this proposal, and indicates how targeted alteration of stereospecificity can be achieved on a modular PKS.

Dimerization↗
PubMed2003

Polyketide biosynthesis beyond the type I, II and III polyketide synthase paradigms.

Recent literature on polyketide biosynthesis suggests that polyketide synthases have much greater diversity in both mechanism and structure than the current type I, II and III paradigms. These examples serve as an inspiration for searching novel polyketide synthases to give new insights into polyketide biosynthesis and to provide new opportunities for combinatorial biosynthesis.

Biological Factors↗
PubMed1994

Engineered biosynthesis of novel polyketides: stereochemical course of two reactions catalyzed by a polyketide synthase.

A genetically engineered strain expressing the essential components of the tetracenomycin polyketide synthase (tcm PKS) along with the actinorhodin ketoreductase (act KR) was found to produce two new (diastereomeric) aromatic polyketides, designated RM20b and RM20c, in addition to RM20, whose structure was reported earlier [McDaniel, R., Ebert-Khosla, S., Hopwood, D. A., & Khosla, C. (1993) Science 262, 1546-1550]. Spectroscopic and in vivo isotopic labeling analysis of RM20b and RM20c revealed that their polyketide backbones were identical to that of RM20 with respect to chain length, regiospecificity of ketoreduction, and regiospecificity of the first intramolecular aldol condensation. This is consistent with earlier predictions that the essential components of the PKS--a bifunctional ketosynthase/acyltransferase, a chain length determining factor, and an acyl carrier protein--are responsible for controlling each of these features of the polyketide backbone [McDaniel, R., Ebert-Khosla, S., Hopwood, D. A., & Khosla, C. (1993) Science 262, 1546-1550; McDaniel, R., Ebert-Khosla, S., Hopwood, D. A., & Khosla, C. (1993) J. Am. Chem. Soc. 115, 11671-11675; Fu, H., Ebert-Khosla, S., Hopwood, D. A., & Khosla, C. (1994) J. Am. Chem. Soc. 116, 4166-4170]. In addition, however, RM20b and RM20c possess two unusual features. In both molecules the hydroxyls on sp3 C-9 and C-7 of the first six-membered ring, which arise as a result of ketoreduction and aldol condensation, respectively, are intact, rather than being lost via dehydration. Furthermore, the relative yield of RM20b (in which these hydroxyls are syn) is 7-fold greater than that of RM20c (in which they are anti).(ABSTRACT TRUNCATED AT 250 WORDS)

Carbon Isotopes↗
PubMed2000

Dissection of malonyl-coenzyme A decarboxylation from polyketide formation in the reaction mechanism of a plant polyketide synthase.

Chalcone synthase (CHS) catalyzes formation of the phenylpropanoid chalcone from one p-coumaroyl-CoA and three malonyl-coenzyme A (CoA) thioesters. The three-dimensional structure of CHS [Ferrer, J.-L., Jez, J. M., Bowman, M. E., Dixon, R. A., and Noel, J. P. (1999) Nat. Struct. Biol. 6, 775-784] suggests that four residues (Cys164, Phe215, His303, and Asn336) participate in the multiple decarboxylation and condensation reactions catalyzed by this enzyme. Here, we functionally characterize 16 point mutants of these residues for chalcone production, malonyl-CoA decarboxylation, and the ability to bind CoA and acetyl-CoA. Our results confirm Cys164's role as the active-site nucleophile in polyketide formation and elucidate the importance of His303 and Asn336 in the malonyl-CoA decarboxylation reaction. We suggest that Phe215 may help orient substrates at the active site during elongation of the polyketide intermediate. To better understand the structure-function relationships in some of these mutants, we also determined the crystal structures of the CHS C164A, H303Q, and N336A mutants refined to 1.69, 2.0, and 2.15 A resolution, respectively. The structure of the C164A mutant reveals that the proposed oxyanion hole formed by His303 and Asn336 remains undisturbed, allowing this mutant to catalyze malonyl-CoA decarboxylation without chalcone formation. The structures of the H303Q and N336A mutants support the importance of His303 and Asn336 in polarizing the thioester carbonyl of malonyl-CoA during the decarboxylation reaction. In addition, both of these residues may also participate in stabilizing the tetrahedral transition state during polyketide elongation. Conservation of the catalytic functions of the active-site residues may occur across a wide variety of condensing enzymes, including other polyketide and fatty acid synthases.

Acetyl Coenzyme A↗
PubMed2004

Identification of domains within megalomicin and erythromycin polyketide synthase modules responsible for differences in polyketide production levels in Escherichia coli.

The megalomicin and erythromycin polyketide synthases (PKSs) produce the same aglycon product, 6-deoxyerythronolide B (6-dEB). Both PKSs were examined in an Escherichia coli strain metabolically engineered to support complex polyketide biosynthesis. Production of 6-dEB in shake flask fermentations was undetectable by mass spectrometry in the strain expressing the megalomicin (Meg) PKS genes, whereas 31 mg/L 6-dEB was produced by the strain with the erythromycin (DEBS) PKS. The genes for each of the three subunits comprising the PKSs were expressed in different combinations from three compatible expression vectors (e.g., DEBS1, DEBS2, and MegA3) to identify two Meg PKS subunits, MegA1 and MegA3, which conferred lower 6-dEB titers than their DEBS counterparts. Comparison of protein expression levels and 6-dEB titers by engineered hybrid DEBS/Meg PKS genes further defined regions within modules 2 and 6 of MegA1 and MegA3, respectively, which limit protein expression and 6-dEB production in E. coli. Meg module 2 + TE (M2 + TE) and a hybrid DEBS M2/Meg M2 + TE protein were engineered and purified for in vitro comparisons with DEBS M2 + TE. The specific activity of the hybrid M2 + TE was approximately 16-fold lower than DEBS M2 + TE and only twice as high as the Meg M2 + TE enzyme in diketide elongation assays. Since the hybrid M2 worked comparably to DEBS M2 in vivo, this suggests that boosting subunit concentration could serve as a useful approach to overcome enzyme deficiencies in heterologous polyketide production.

Aminoglycosides↗
PubMed2005

Polyketide double bond biosynthesis. Mechanistic analysis of the dehydratase-containing module 2 of the picromycin/methymycin polyketide synthase.

Picromycin/methymycin synthase (PICS) is a modular polyketide synthase (PKS) that is responsible for the biosynthesis of both 10-deoxymethynolide (1) and narbonolide (2), the parent 12- and 14-membered aglycone precursors of the macrolide antibiotics methymycin and picromycin, respectively. PICS module 2 is a dehydratase (DH)-containing module that catalyzes the formation of the unsaturated triketide intermediate using malonyl-CoA as the chain extension substrate. Recombinant PICS module 2+TE, with the PICS thioesterase domain appended to the C-terminus to allow release of polyketide products, was expressed in Escherichia coli. Purified PICS module 2+TE converted malonyl-CoA and 4, the N-acetylcysteamine thioester of (2S,3R)-2-methyl-3-hydroxypentanoic acid, to a 1:2 mixture of the triketide acid (4S,5R)-4-methyl-5-hydroxy-2-heptenoic acid (5) and (3S,4S,5R)-3,5-dihydroxy-4-methyl-n-heptanoic acid-delta-lactone (10) with a combined kcat of 0.6 min(-1). The triketide lactone 10 is formed by thioesterase-catalyzed cyclization of the corresponding d-3-hydroxyacyl-SACP intermediate, a reaction which competes with dehydration catalyzed by the dehydratase domain. PICS module 2+TE showed a strong preference for the syn-diketide-SNAC 4, with a 20-fold greater kcat/K(m) than the anti-(2S,3S)-diketide-SNAC 14, and a 40-fold advantage over the syn-(2R,3S)-diketide-SNAC 13. PICS module 2(DH(0))+TE, with an inactivated DH domain, produced exclusively 10, while three PICS module 2(KR(0))+TE mutants, with inactivated KR domains, produced exclusively or predominantly the unreduced triketide ketolactone, (4S,5R)-3-oxo-4-methyl-5-hydroxy-n-heptanoic acid-delta-lactone (7). These studies establish for the first time the structure and stereochemistry of the intermediates of a polyketide chain elongation cycle catalyzed by a DH-containing module, while confirming the importance of key active site residues in both KR and DH domains.

Amino Acid Sequence↗
PubMed2006

Two polyketide synthase-encoding genes are required for biosynthesis of the polyketide virulence factor, T-toxin, by Cochliobolus heterostrophus.

Cochliobolus heterostrophus race T, causal agent of southern corn leaf blight, requires T-toxin (a family of C35 to C49 polyketides) for high virulence on T-cytoplasm maize. Production of T-toxin is controlled by two unlinked loci, Tox1A and Tox1B, carried on 1.2 Mb of DNA not found in race O, a mildly virulent form of the fungus that does not produce T-toxin, or in any other Cochliobolus spp. or closely related fungus. PKS1, a polyketide synthase (PKS)-encoding gene at Tox1A, and DEC1, a decarboxylase-encoding gene at Tox1B, are necessary for T-toxin production. Although there is evidence that additional genes are required for T-toxin production, efforts to clone them have been frustrated because the genes are located in highly repeated, A+T-rich DNA. To overcome this difficulty, ligation specificity-based expression analysis display (LEAD), a comparative amplified fragment length polymorphism/gel fractionation/capillary sequencing procedure, was applied to cDNAs from a near-isogenic pair of race T (Tox1+) and race O (Tox1-) strains. This led to discovery of PKS2, a second PKS-encoding gene that maps at Tox1A and is required for both T-toxin biosynthesis and high virulence to maize. Thus, the carbon chain of each T-toxin family member likely is assembled by action of two PKSs, which produce two polyketides, one of which may act as the starter unit for biosynthesis of the mature T-toxin molecule.

Ascomycota↗
PubMed1995

Polyketide synthesis in vitro on a modular polyketide synthase.

BACKGROUND: The 6-deoxyerythronolide B synthase (DEBS) of Saccharopolyspora erythraea, which synthesizes the aglycone core of the antibiotic erythromycin A, contains some 30 active sites distributed between three multienzyme polypeptides (designated DEBS1-3). This complexity has hitherto frustrated mechanistic analysis of such enzymes. We previously produced a mutant strain of S. erythraea in which the chain-terminating cyclase domain (TE) is fused to the carboxyl-terminus of DEBS1, the multienzyme that catalyzes the first two rounds of polyketide chain extension in S. erythraea. This mutant strain produces triketide lactone in vivo. We set out to purify the chimaeric enzyme and to determine its activity in vitro. RESULTS: The purified DEBS1-TE multienzyme catalyzes synthesis of triketide lactones in vitro. The synthase specifically uses the (2S)-isomer of methylmalonyl-CoA, as previously proposed, but has a more relaxed specificity for the starter unit than in vivo. CONCLUSIONS: We have obtained a purified polyketide synthase system, derived from DEBS, which retains catalytic activity. This approach opens the way for mechanistic and structural analyses of active multienzymes derived from any modular polyketide synthase.

Cell-Free System↗
PubMed1998

Engineering of modular polyketide synthases to produce novel polyketides.

Polyketides are important natural products produced by Actinomycetes and other organisms via the polymerization of coenzyme A-activated carboxylic acids. Modular polyketide synthases are large multifunctional enzymes that direct the biosynthetic process using a dedicated 'module' for each polymerization reaction, which specifies the unit to be polymerized, its oxidation state and stereochemistry. Over the past two years proof-of-principle has been demonstrated for technologies that modify or exchange modules to create hybrid enzymes that catalyze the biosynthesis of novel polyketides.

Journal Article↗
PubMed2003

Enhancing the modularity of the modular polyketide synthases: transacylation in modular polyketide synthases catalyzed by malonyl-CoA:ACP transacylase.

Selective incorporation of extender units in modular polyketide synthases is primarily controlled by acyl transferase (AT) domains. The AT domains catalyze transacylation of the extender unit from acyl-CoA to the phosphopantetheine arm of an acyl carrier protein (ACP) domain in the same module. New methods that can modulate the extender unit specificity of individual modules with minimal structural or kinetic perturbations in the engineered module are desirable for the efficient biosynthesis of novel natural product analogues. We have demonstrated that transacylation of malonyl groups onto an AT-null form of a mutant modular polyketide synthase by malonyl-CoA:ACP transacylase is an effective strategy for the engineered biosynthesis of site specifically modified polyketides. Using this strategy, 6-deoxyerythronolide B synthase was engineered to exclusively produce 2-desmethyl-6-deoxyerythronolide B. The productivity of the modified system was comparable to that of the wild-type synthase in vitro and in vivo.

Acyl Carrier Protein↗
PubMed2002

Enzymatic formation of an unnatural C(6)-C(5) aromatic polyketide by plant type III polyketide synthases.

[reaction: see text] Substrate specificities of plant polyketide synthases (PKSs) were investigated using analogues of malonyl-CoA, the extension unit of the polyketide chain elongation reactions. When incubated with methylmalonyl-CoA and 4-coumaroyl-CoA, plant PKSs (chalcone synthase from Scutellaria baicalensis, stilbene synthase from Arachis hypogaea, and benzalacetone synthase from Rheum palmatum) afforded an unnatural C(6)-C(5) aromatic polyketide, 1-(4-hydroxyphenyl)pent-1-en-3-one, formed by one-step decarboxylative condensation of the two substrates. In contrast, succinyl-CoA was not accepted as a substrate by the enzymes.

Butanones↗
PubMed2006

Structural and mechanistic insights into polyketide macrolactonization from polyketide-based affinity labels.

Polyketides are a diverse class of natural products having important clinical properties, including antibiotic, immunosuppressive and anticancer activities. They are biosynthesized by polyketide synthases (PKSs), which are modular, multienzyme complexes that sequentially condense simple carboxylic acid derivatives. The final reaction in many PKSs involves thioesterase-catalyzed cyclization of linear chain elongation intermediates. As the substrate in PKSs is presented by a tethered acyl carrier protein, introduction of substrate by diffusion is problematic, and no substrate-bound type I PKS domain structure has been reported so far. We describe the chemical synthesis of polyketide-based affinity labels that covalently modify the active site serine of excised pikromycin thioesterase from Streptomyces venezuelae. Crystal structures reported here of the affinity label-pikromycin thioesterase adducts provide important mechanistic insights. These results suggest that affinity labels can be valuable tools for understanding the mechanisms of individual steps within multifunctional PKSs and for directing rational engineering of PKS domains for combinatorial biosynthesis.

Binding Sites↗
PubMed1999

Analysis of two chromosomal regions adjacent to genes for a type II polyketide synthase involved in the biosynthesis of the antitumor polyketide mithramycin in Streptomyces argillaceus.

Mithramycin is an aromatic antitumour polyketide synthesized by Streptomyces argillaceus. Two chromosomal regions located upstream and downstream of the locus for the mithramycin type II polyketide synthase were cloned and sequenced. Analysis of the sequence revealed the presence of eight genes encoding three oxygenases (mtmOI, mtmOII and mtmOIII), three reductases (mtmTI, mtmTII and mtmTIII), a cyclase (mtm Y) and an acyl CoA ligase (mtmL). The three oxygenase genes were each inactivated by gene replacement. Inactivation of one of them (mtmOII) generated a non-producing mutant, while inactivation of the other two (mtmOl and mtmOIII) did not affect the biosynthesis of mithramycin. The mtmOII gene may code for an oxygenase responsible for the introduction of oxygen atoms at early steps in the biosynthesis of mithramycin leading to 4-demethylpremithramycinone. One of the reductases may be responsible for reductive cleavage of an intermediate from an enzyme and another for the reduction of a keto group in the side-chain of the mithramycin aglycon moiety. A hypothetical biosynthetic pathway showing in particular the involvement of oxygenase MtmOII and of various other gene products in mithramycin biosynthesis is proposed.

Amino Acid Sequence↗