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David H Sherman - One of the best experts on this subject based on the ideXlab platform.
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Engineering the Substrate Specificity of a Modular Polyketide Synthase for Installation of Consecutive Non-Natural Extender Units
2019Co-Authors: Edward Kalkreuter, David H Sherman, Jared M. Crowetipton, Andrew N. Lowell, Gavin J. WilliamsAbstract:There is significant interest in diversifying the structures of polyketides to create new analogues of these bioactive molecules. This has traditionally been done by focusing on engineering the acyltransferase (AT) domains of polyketide synthases (PKSs) responsible for the incorporation of malonyl-CoA extender units. Non-natural extender units have been utilized by engineered PKSs previously; however, most of the work to date has been accomplished with ATs that are either naturally promiscuous and/or located in terminal modules lacking downstream bottlenecks. These limitations have prevented the engineering of ATs with low native promiscuity and the study of any potential gatekeeping effects by domains downstream of an engineered AT. In an effort to address this gap in PKS engineering knowledge, the substrate preferences of the final two modules of the Pikromycin PKS were compared for several non-natural extender units and through active site mutagenesis. This led to engineering of the methylmalonyl-CoA specificity of both modules and inversion of their selectivity to prefer consecutive non-natural derivatives. Analysis of the product distributions of these bimodular reactions revealed unexpected metabolites resulting from gatekeeping by the downstream ketoreductase and ketosynthase domains. Despite these new bottlenecks, AT engineering provided the first full-length polyketide products incorporating two non-natural extender units. Together, this combination of tandem AT engineering and the identification of previously poorly characterized bottlenecks provides a platform for future advancements in the field
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identification of a thioesterase bottleneck in the Pikromycin pathway through full module processing of unnatural pentaketides
Journal of the American Chemical Society, 2017Co-Authors: Douglas A Hansen, Aaron A Koch, David H ShermanAbstract:Polyketide biosynthetic pathways have been engineered to generate natural product analogs for over two decades. However, manipulation of modular type I polyketide synthases (PKSs) to make unnatural metabolites commonly results in attenuated yields or entirely inactive pathways, and the mechanistic basis for compromised production is rarely elucidated since rate-limiting or inactive domain(s) remain unidentified. Accordingly, we synthesized and assayed a series of modified Pikromycin (Pik) pentaketides that mimic early pathway engineering to probe the substrate tolerance of the PikAIII-TE module in vitro. Truncated pentaketides were processed with varying efficiencies to corresponding macrolactones, while pentaketides with epimerized chiral centers were poorly processed by PikAIII-TE and failed to generate 12-membered ring products. Isolation and identification of extended but prematurely offloaded shunt products suggested that the Pik thioesterase (TE) domain has limited substrate flexibility and function...
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a single active site mutation in the Pikromycin thioesterase generates a more effective macrocyclization catalyst
Journal of the American Chemical Society, 2017Co-Authors: Aaron A Koch, Douglas A Hansen, Vikram V Shende, Lawrence R Furan, K N Houk, Gonzalo Jimenezoses, David H ShermanAbstract:Macrolactonization of natural product analogs presents a significant challenge to both biosynthetic assembly and synthetic chemistry. In the preceding paper, we identified a thioesterase (TE) domain catalytic bottleneck processing unnatural substrates in the Pikromycin (Pik) system, preventing the formation of epimerized macrolactones. Here, we perform molecular dynamics simulations showing the epimerized hexaketide was accommodated within the Pik TE active site; however, intrinsic conformational preferences of the substrate resulted in predominately unproductive conformations, in agreement with the observed hydrolysis. Accordingly, we engineered the stereoselective Pik TE to yield a variant (TES148C) with improved reaction kinetics and gain-of-function processing of an unnatural, epimerized hexaketide. Quantum mechanical comparison of model TES148C and TEWT reaction coordinate diagrams revealed a change in mechanism from a stepwise addition–elimination (TEWT) to a lower energy concerted acyl substitution...
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Co-produced natural ketolides methymycin and Pikromycin inhibit bacterial growth by preventing synthesis of a limited number of proteins
Nucleic acids research, 2017Co-Authors: Mashal M. Almutairi, Douglas A Hansen, David H Sherman, Han-young Kang, Maxim S. Svetlov, Nelli F. Khabibullina, Dorota Klepacki, Nora Vázquez-laslop, Yury S. Polikanov, Alexander S. MankinAbstract:Antibiotics methymycin (MTM) and Pikromycin (PKM), co-produced by Streptomyces venezuelae, represent minimalist macrolide protein synthesis inhibitors. Unlike other macrolides, which carry several side chains, a single desosamine sugar is attached to the macrolactone ring of MTM and PKM. In addition, the macrolactone scaffold of MTM is smaller than in other macrolides. The unusual structure of MTM and PKM and their simultaneous secretion by S. venezuelae bring about the possibility that two compounds would bind to distinct ribosomal sites. However, by combining genetic, biochemical and crystallographic studies, we demonstrate that MTM and PKM inhibit translation by binding to overlapping sites in the ribosomal exit tunnel. Strikingly, while MTM and PKM readily arrest the growth of bacteria, ∼40% of cellular proteins continue to be synthesized even at saturating concentrations of the drugs. Gel electrophoretic analysis shows that compared to other ribosomal antibiotics, MTM and PKM prevent synthesis of a smaller number of cellular polypeptides illustrating a unique mode of action of these antibiotics.
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Identification of a Thioesterase Bottleneck in the Pikromycin Pathway through Full-Module Processing of Unnatural Pentaketides
2017Co-Authors: Douglas A. Hansen, Aaron A Koch, David H ShermanAbstract:Polyketide biosynthetic pathways have been engineered to generate natural product analogs for over two decades. However, manipulation of modular type I polyketide synthases (PKSs) to make unnatural metabolites commonly results in attenuated yields or entirely inactive pathways, and the mechanistic basis for compromised production is rarely elucidated since rate-limiting or inactive domain(s) remain unidentified. Accordingly, we synthesized and assayed a series of modified Pikromycin (Pik) pentaketides that mimic early pathway engineering to probe the substrate tolerance of the PikAIII-TE module in vitro. Truncated pentaketides were processed with varying efficiencies to corresponding macrolactones, while pentaketides with epimerized chiral centers were poorly processed by PikAIII-TE and failed to generate 12-membered ring products. Isolation and identification of extended but prematurely offloaded shunt products suggested that the Pik thioesterase (TE) domain has limited substrate flexibility and functions as a gatekeeper in the processing of unnatural substrates. Synthesis of an analogous hexaketide with an epimerized nucleophilic hydroxyl group allowed for direct evaluation of the substrate stereoselectivity of the excised TE domain. The epimerized hexaketide failed to undergo cyclization and was exclusively hydrolyzed, confirming the TE domain as a key catalytic bottleneck. In an accompanying paper, we engineer the standalone Pik thioesterase to yield a thioesterase (TES148C) and module (PikAIII-TES148C) that display gain-of-function processing of substrates with inverted hydroxyl groups
Kevin A Reynolds - One of the best experts on this subject based on the ideXlab platform.
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acyl coa subunit selectivity in the Pikromycin polyketide synthase pikaiv steady state kinetics and active site occupancy analysis by fticr ms
Chemistry & Biology, 2011Co-Authors: Shilah A Bonnett, Joseph A. Chemler, Christopher M. Rath, Kevin A Reynolds, Kristina Håkansson, Abdurrafay Shareef, Joanna R Joels, David H ShermanAbstract:Summary Polyketide natural products generated by type I modular polyketide synthases (PKSs) are vital components in our drug repertoire. To reprogram these biosynthetic assembly lines, we must first understand the steps that occur within the modular "black boxes." Herein, key steps of acyl-CoA extender unit selection are explored by in vitro biochemical analysis of the PikAIV PKS model system. Two complementary approaches are employed: a fluorescent-probe assay for steady-state kinetic analysis, and Fourier Transform Ion Cyclotron Resonance-mass spectrometry (FTICR-MS) to monitor active-site occupancy. Findings from five enzyme variants and four model substrates have enabled a model to be proposed involving catalysis based upon acyl-CoA substrate loading followed by differential rates of hydrolysis. These efforts suggest a strategy for future pathway engineering efforts using unnatural extender units with slow rates of hydrolytic off-loading from the acyltransferase domain.
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functional dissection of a multimodular polypeptide of the Pikromycin polyketide synthase into monomodules by using a matched pair of heterologous docking domains
ChemBioChem, 2009Co-Authors: John Yan, David H Sherman, Shuchi Gupta, Kevin A ReynoldsAbstract:The pikromyin polyketide synthase (PKS) in Streptomyces venezulae is comprised of a loading module and six extension modules, which generate the corresponding 14-membered macrolactone product. PikAI is a multimodular component of this PKS and houses both the loading domain and the first two extension modules, joined by short intraprotein linkers. We have shown that PikAI can be separated into two proteins at either of these linkers, only when matched pairs of docking domains (DDs) from a heterologous modular phoslactomycin PKS are used in place of the intraprotein linker. In both cases the yields of Pikromycin produced by the S. venezuelae mutant were 50% of that of a S. venezuelae strain expressing the native trimodular PikAI. This observation provides the first demonstration that such separations do not dramatically impact the efficiency of the entire in vivo biosynthetic process. Expression of module 2 as a monomodular protein fused to a heterologous N-terminal docking domain was also observed to give almost a tenfold improvement in the in vivo generation of Pikromycin from a synthetic diketide intermediate. These results demonstrate the utility of DDs to manipulate biosynthetic processes catalyzed by modular PKSs and the quest to generate novel polyketide products.
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generation of novel Pikromycin antibiotic products through mutasynthesis
ChemBioChem, 2008Co-Authors: Shuchi Gupta, Beomseok Kim, Venkatraman Lakshmanan, Robert A Fecik, Kevin A ReynoldsAbstract:Mutasynthesis in Pikromycin PKS: The amenability of Pikromycin polyketide synthase to mutational biosynthesis has been demonstrated. A natural triketide and its analogues, activated as N-acetyl-cysteamine thioesters, were synthesized and fed to a pikAI-deleted strain; this led to the production of new antibiotics. A vinyl analogue was found to have better antibacterial activity than Pikromycin. The Pikromycin polyketide synthase (PKS) of S. venezuelae, which consists of one loading module and six extension modules, is responsible for the formation of the hexaketide narbonolide, a key intermediate ok? in the biosynthesis of the antibiotic Pikromycin. S. venezuelae strains in which PikAI, which houses the loading domain and first two modules of the PKS, is either absent or catalytically inactive, produce no Pikromycin product. When these strains are grown in the presence of a synthetically prepared triketide product, activated as the N-acetylcysteamine thioester, Pikromycin yields are restored to as much as 11 % of that seen in the wild-type strain. Feeding analogues of the triketide intermediate provides Pikromycin analogues bearing different alkyl substituents at C13 and C14. One of these analogues, Δ15,16-dehydroPikromycin, exhibits improved antimicrobial activity relative to Pikromycin.
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substrate recognition and channeling of monomodules from the Pikromycin polyketide synthase
Journal of the American Chemical Society, 2003Co-Authors: Brian J Beck, Robert A Fecik, Kevin A Reynolds, Courtney C Aldrich, David H ShermanAbstract:The unique ability of the Pikromycin (Pik) polyketide synthase to generate 12- and 14-membered ring macrolactones presents an opportunity to explore the fundamental processes underlying polyketide synthesis, specifically the mechanistic details of the chain extension process. We have overexpressed and purified PikAIII (module 5) and PikAIV (module 6) and assessed the ability of these proteins to generate tri- and tetraketide lactone products using N-acetylcysteamine-activated diketides and 14C-methylmalonyl-CoA as substrates. Comparison of the stereochemical specificities for PikAIII and PikAIV and the reported values for the DEBS modules reveals significant differences between these systems.
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iterative chain elongation by a Pikromycin monomodular polyketide synthase
Journal of the American Chemical Society, 2003Co-Authors: Brian J Beck, Robert A Fecik, Kevin A Reynolds, Courtney C Aldrich, David H ShermanAbstract:The unique ability of the Pikromycin polyketide synthase (Pik PKS) to generate 12- and 14-membered ring macrolactones presents an opportunity to explore the fundamental processes of polyketide synthesis, specifically, the mechanistic details of the chain extension process. We have overexpressed and purified PikAIII and PikAIV and demonstrated the ability of these proteins to generate triketide lactone products using 14C-methylmalonyl-CoA as the sole substrate. Monomodular PikAIII generates TKL (1) when reacted alone, and synthesizes TKL (2) upon reaction in combination with PikAIV. Product formation remains dependent on the enzymatic decarboxylation of methylmalonyl-CoA and transfer of the acyl chain within the enzyme rather than acylation by propionyl-CoA from spontaneous decarboxylation. We propose that synthesis of TKL (1) by PikAIII involves iterative assembly of the triketide chain within a PikAIII homodimer analogous to the nonmodular type I PKS systems.
David E Cane - One of the best experts on this subject based on the ideXlab platform.
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elucidation of the cryptic epimerase activity of redox inactive ketoreductase domains from modular polyketide synthases by tandem equilibrium isotope exchange
Journal of the American Chemical Society, 2014Co-Authors: Ashish Garg, Chaitan Khosla, Xinqiang Xie, Adrian T Keatingeclay, David E CaneAbstract:Many modular polyketide synthases harbor one or more redox-inactive domains of unknown function that are highly homologous to ketoreductase (KR) domains. A newly developed tandem equilibrium isotope exchange (EIX) assay has now established that such "KR(0)" domains catalyze the biosynthetically essential epimerization of transient (2R)-2-methyl-3-ketoacyl-ACP intermediates to the corresponding (2S)-2-methyl-3-ketoacyl-ACP diastereomers. Incubation of [2-(2)H]-(2R,3S)-2-methyl-3-hydroxypentanoyl-SACP ([2-(2)H]-3b) with the EryKR3(0) domain from module 3 of the 6-deoxyerythronolide B synthase, and the redox-active, nonepimerizing EryKR6 domain and NADP(+) resulted in time- and cofactor-dependent washout of deuterium from 3b, as a result of EryKR3(0)-catalyzed epimerization of transiently generated [2-(2)H]-2-methyl-3-ketopentanoyl-ACP (4). Similar results were obtained with redox-inactive PicKR3(0) from module 3 of the picromycin synthase. Four redox-inactive mutants of epimerase-active EryKR1 were engineered by mutagenesis of the NADPH binding site of this enzyme. Tandem EIX established that these EryKR1(0) mutants retained the intrinsic epimerase activity of the parent EryKR1 domain. These results establish the intrinsic epimerase activity of redox-inactive KR(0) domains, rule out any role for the NADPH cofactor in epimerization, and provide a general experimental basis for decoupling the epimerase and reductase activities of a large class of PKS domains.
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macrolactonization to 10 deoxymethynolide catalyzed by the recombinant thioesterase of the picromycin methymycin polyketide synthase
Bioorganic & Medicinal Chemistry Letters, 2006Co-Authors: Chaitan Khosla, David E CaneAbstract:The recombinant thioesterase (TE) domain of the picromycin/methymycin synthase (PICS) catalyzes the macrolactonization of 3, the N-acetylcysteamine thioester of seco-10-deoxymethynolide to generate 10-deoxymethynolide (1) with high efficiency. By contrast, 4, the 7-dihydro derivative of seco-thioester 3, undergoes exclusive hydrolysis by PICS TE to seco-acid 5. The recombinant TE domain of 6-deoxyerythronolide B synthase (DEBS TE) shows the same reaction specificity as PICS TE, but with significantly lower activity.
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polyketide double bond biosynthesis mechanistic analysis of the dehydratase containing module 2 of the picromycin methymycin polyketide synthase
Journal of the American Chemical Society, 2005Co-Authors: Toby J Zaleski, Chaitan Khosla, Chiara R Valenzano, David E CaneAbstract: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.
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expression and kinetic analysis of the substrate specificity of modules 5 and 6 of the picromycin methymycin polyketide synthase
Journal of the American Chemical Society, 2003Co-Authors: Yifeng Yin, Chaitan Khosla, David E CaneAbstract:Picromycin synthase (PICS) is a multifunctional, modular polyketide synthase (PKS) that catalyzes the conversion of methylmalonyl-CoA to narbonolide and 10-deoxymethynolide, the macrolide aglycone precursors of the antibiotics picromycin and methymycin, respectively. PICS modules 5 and 6 were each expressed in Escherichia coli with a thioesterase domain at the C-terminus to allow release of polyketide products. The substrate specificity of PICS modules 5+TE and 6+TE was investigated using N-acetylcysteamine thioesters of 2-methyl-3-hydroxy-pentanoic acid as diketide analogues of the natural polyketide chain elongation substrates. PICS module 5+TE could catalyze the chain elongation of only the syn diketide (2S,3R)-4, while PICS module 6+TE processed both syn diastereomers, (2S,3R)-4 and (2R,3S)-5, with a 2.5:1 preference in kcat/Km for 5 but did not turn over either of the two anti diketides. The observed substrate specificity patterns are in contrast to the 15−100:1 preference for 4 over 5 previously est...
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insights into channel architecture and substrate specificity from crystal structures of two macrocycle forming thioesterases of modular polyketide synthases
Biochemistry, 2002Co-Authors: Shiouchuan Tsai, Chaitan Khosla, David E Cane, Robert M StroudAbstract:Modular polyketide synthases (PKSs) synthesize the polyketide cores of pharmacologically important natural products such as erythromycin and picromycin. Understanding PKSs at high resolution could present new opportunities for chemoenzymatic synthesis of complex molecules. The crystal structures of macrocycle-forming thioesterase (TE) domains from the picromycin synthase (PICS) and 6-deoxyerythronolide B synthase (DEBS) were determined to 1.8-3.0 A with an R(crys) of 19.2-24.4%, including three structures of PICS TE (crystallized at pH 7.6, 8.0, and 8.4) and a second crystal form of DEBS TE. As predicted by the previous work on DEBS TE [Tsai, S. C., et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 14808-14813], PICS TE contains an open substrate channel and a hydrophobic dimer interface. Notwithstanding their similarity, the dimer interfaces and substrate channels of DEBS TE and PICS TE reveal key differences. The structural basis for the divergent substrate specificities of DEBS TE and PICS TE is analyzed. The size of the substrate channel increases with increasing pH, presumably due to electrostatic repulsion in the channel at elevated pH. Together, these structures support previous predictions that macrocycle-forming thioesterases from PKSs share the same protein fold, an open substrate channel, a similar catalytic mechanism, and a hydrophobic dimer interface. They also provide a basis for the design of enzymes capable of catalyzing regioselective macrocyclization of natural or synthetic substrates. A series of high-resolution snapshots of a protein channel at different pHs is presented alongside analysis of channel residues, which could help in the redesign of the protein channel architecture.
Yeo Joon Yoon - One of the best experts on this subject based on the ideXlab platform.
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Biosynthesis of glycosylated derivatives of tylosin in Streptomyces venezuelae.
Journal of microbiology and biotechnology, 2011Co-Authors: Ah Reum Han, Sung Ryeol Park, Je Won Park, Eun Yeol Lee, Dong-myung Kim, Byung-gee Kim, Yeo Joon YoonAbstract:Streptomyces venezuelae YJ028, bearing a deletion of the entire biosynthetic gene cluster encoding the Pikromycin polyketide synthases and desosamine biosynthetic enzymes, was used as a bioconversion system for combinatorial biosynthesis of glycosylated derivatives of tylosin. Two engineered deoxysugar biosynthetic pathways for the biosynthesis of TDP-3-O-demethyl-D-chalcose or TDP-Lrhamnose in conjunction with the glycosyltransferaseauxiliary protein pair DesVII/DesVIII were expressed in a S. venezuelae YJ028 mutant strain. Supplementation of each mutant strain capable of producing TDP-3-O-demethyl- D-chalcose or TDP-L-rhamnose with tylosin aglycone tylactone resulted in the production of the 3-O-demethyl- D-chalcose, D-quinovose, or L-rhamnose-glycosylated tylactone.
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Biosynthesis of plant-specific flavones and flavonols in Streptomyces venezuelae.
Journal of microbiology and biotechnology, 2010Co-Authors: Sung Ryeol Park, Jihye Paik, Je Won Park, Mi Sun Ahn, Yeo Joon YoonAbstract:Recently, recombinant Streptomyces venezuelae has been established as a heterologous host for microbial production of flavanones and stilbenes, a class of plant-specific polyketides. In the present work, we expanded the applicability of the S. venezuelae system to the production of more diverse plant polyketides including flavones and flavonols. A plasmid with the synthetic codon-optimized flavone synthase I gene from Petroselium crispum was introduced to S. venezuelae DHS2001 bearing a deletion of the native Pikromycin polyketide synthase gene, and the resulting strain generated flavones from exogenously fed flavanones. In addition, a recombinant S. venezuelae mutant expressing a codon-optimized flavanone 3beta-hydroxylase gene from Citrus siensis and a flavonol synthase gene from Citrus unshius also successfully produced flavonols.
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genetic reconstruction of the biosynthetic route to tylosin attached deoxysugarby heterologous expression in streptomyces venezuelae
한국생물공학회 학술대회, 2009Co-Authors: Eun Ae Choi, Sung Ryeol Park, Eunji Kim, Ah Reum Han, Mi Sun Ahn, So Ra Lee, Yeo Joon YoonAbstract:Tylosin is a 16-membered macrolide antibiotics produced by Streptomyces fradiae, composed of a branched lactone ring, tylactone and three deoxysugars, mycinose, mycaminose and mycarose. Of these different deoxysugars, mycinose is attached to C-23 position of tylactone. Inthis study, the mycinose biosynthetic pathway was identified through the heterologous expression of several gene sets in an engineered strain of Streptomyces venezuelae. A plasmid harboring TDPdeoxyallose biosynthetic gene set (tylD-tylJ-tylN-desIII-desIV) wasconstructed and introduced into the S. venezuelae YJ028 bearing a deletion of native Pikromycin polyketide synthase and desosamine gene cluster. As a result, deoxyallosyl-OMT and deoxyallosyl-DMT were produced from 5-O-mycaminosyl tylonolide (OMT) and demycinosyl tylosin (DMT), respectively. In addition, expression of TDP-deoxyallose biosynthetic genes plus three different sets of methyltransferase genes tylE and tylF (tylE, tylF and tylE-tylF) in theS. venezuelae YJ028 supplemented with OMT and DMT, led to the production of 2'''-or/and 3'''-methyldeoxylallosyl OMT and 2'''-or/and 3'''-methyldeoxylallosyl DMT, respectively. These results describe more precise mycinose biosynthetic pathway and biosynthetic routesto tylosin of S. fradiae.
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Engineering of plant-specific phenylpropanoids biosynthesis in Streptomyces venezuelae
Journal of Biotechnology, 2009Co-Authors: Sung Ryeol Park, Jin A Yoon, Jihye Paik, Je Won Park, Won Seok Jung, Yeon-hee Ban, Eunji Kim, Young Ji Yoo, Ah Reum Han, Yeo Joon YoonAbstract:Abstract Phenylpropanoids, including flavonoids and stilbenes, are plant secondary metabolites with potential pharmacological and nutraceutical properties. To expand the applicability of Streptomyces venezuelae as a heterologous host to plant polyketide production, flavonoid and stilbene biosynthetic genes were expressed in an engineered strain of S. venezuelae DHS2001 bearing a deletion of native Pikromycin polyketide synthase gene. A plasmid expressing the 4-coumarate/cinnamate:coenzyme A ligase from Streptomyces coelicolor (ScCCL) and the chalcone synthase from Arabidopsis thaliana (atCHS) under the control of a single ermE* promoter was constructed and introduced into S. venezuelae DHS2001. The resulting strain produced racemic naringenin and pinocembrin from 4-coumaric acid and cinnamic acid, respectively. Placement of an additional ermE* promoter upstream of the codon-optimized atCHS (atCHSop) gene significantly increased the yield of both flavanones. Expression of codon-optimized chalcone isomerase gene from Medicago sativa, together with ScCCL and atCHSop genes led to production of (2S)-flavanones, but the yield was reduced. On the other hand, a recombinant strain harboring the ScCCL and codon-optimized stilbene synthase gene from Arachis hypogaea generated stilbenes such as resveratrol and pinosylvin. This is the first report on the heterologous expression of plant phenylpropanoid biosynthetic pathways in Streptomyces genus.
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Heterologous production of epothilones B and D in Streptomyces venezuelae
Applied Microbiology and Biotechnology, 2008Co-Authors: Sung Ryeol Park, Jae Kyung Sohng, Je Won Park, Won Seok Jung, Yeon-hee Ban, Eunji Kim, Ah Reum Han, Sang Jun Sim, Yeo Joon YoonAbstract:Epothilones, produced from the myxobacterium Sorangium cellulosum , are potential anticancer agents that stabilize microtubules in a similar manner to paclitaxel. The entire epothilone biosynthetic gene cluster was heterologously expressed in an engineered strain of Streptomyces venezuelae bearing a deletion of Pikromycin polyketide synthase gene cluster. The resulting strains produced approximately 0.1 μg/l of epothilone B as a sole product after 4 days cultivation. Deletion of an epoF encoding the cytochrome P450 epoxidase gave rise to a mutant that selectively produces 0.4 μg/l of epothilone D. To increase the production level of epothilones B and D, an additional copy of the positive regulatory gene pikD was introduced into the chromosome of both S. venezuleae mutant strains. The resulting strains showed enhanced production of corresponding compounds (approximately 2-fold). However, deletion of putative transport genes, orf3 and orf14 in the epothilone D producing S. venezuelae mutant strain, led to an approximately 3-fold reduction in epothilone D production. These results introduce S. venezuelae as an alternative heterologous host for the production of these valuable anticancer agents and demonstrate the possibility of engineering this strain as a generic heterologous host for the production of polyketides and hybrid polyketide-nonribosomal peptides.
Chaitan Khosla - One of the best experts on this subject based on the ideXlab platform.
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elucidation of the cryptic epimerase activity of redox inactive ketoreductase domains from modular polyketide synthases by tandem equilibrium isotope exchange
Journal of the American Chemical Society, 2014Co-Authors: Ashish Garg, Chaitan Khosla, Xinqiang Xie, Adrian T Keatingeclay, David E CaneAbstract:Many modular polyketide synthases harbor one or more redox-inactive domains of unknown function that are highly homologous to ketoreductase (KR) domains. A newly developed tandem equilibrium isotope exchange (EIX) assay has now established that such "KR(0)" domains catalyze the biosynthetically essential epimerization of transient (2R)-2-methyl-3-ketoacyl-ACP intermediates to the corresponding (2S)-2-methyl-3-ketoacyl-ACP diastereomers. Incubation of [2-(2)H]-(2R,3S)-2-methyl-3-hydroxypentanoyl-SACP ([2-(2)H]-3b) with the EryKR3(0) domain from module 3 of the 6-deoxyerythronolide B synthase, and the redox-active, nonepimerizing EryKR6 domain and NADP(+) resulted in time- and cofactor-dependent washout of deuterium from 3b, as a result of EryKR3(0)-catalyzed epimerization of transiently generated [2-(2)H]-2-methyl-3-ketopentanoyl-ACP (4). Similar results were obtained with redox-inactive PicKR3(0) from module 3 of the picromycin synthase. Four redox-inactive mutants of epimerase-active EryKR1 were engineered by mutagenesis of the NADPH binding site of this enzyme. Tandem EIX established that these EryKR1(0) mutants retained the intrinsic epimerase activity of the parent EryKR1 domain. These results establish the intrinsic epimerase activity of redox-inactive KR(0) domains, rule out any role for the NADPH cofactor in epimerization, and provide a general experimental basis for decoupling the epimerase and reductase activities of a large class of PKS domains.
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macrolactonization to 10 deoxymethynolide catalyzed by the recombinant thioesterase of the picromycin methymycin polyketide synthase
Bioorganic & Medicinal Chemistry Letters, 2006Co-Authors: Chaitan Khosla, David E CaneAbstract:The recombinant thioesterase (TE) domain of the picromycin/methymycin synthase (PICS) catalyzes the macrolactonization of 3, the N-acetylcysteamine thioester of seco-10-deoxymethynolide to generate 10-deoxymethynolide (1) with high efficiency. By contrast, 4, the 7-dihydro derivative of seco-thioester 3, undergoes exclusive hydrolysis by PICS TE to seco-acid 5. The recombinant TE domain of 6-deoxyerythronolide B synthase (DEBS TE) shows the same reaction specificity as PICS TE, but with significantly lower activity.
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polyketide double bond biosynthesis mechanistic analysis of the dehydratase containing module 2 of the picromycin methymycin polyketide synthase
Journal of the American Chemical Society, 2005Co-Authors: Toby J Zaleski, Chaitan Khosla, Chiara R Valenzano, David E CaneAbstract: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.
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expression and kinetic analysis of the substrate specificity of modules 5 and 6 of the picromycin methymycin polyketide synthase
Journal of the American Chemical Society, 2003Co-Authors: Yifeng Yin, Chaitan Khosla, David E CaneAbstract:Picromycin synthase (PICS) is a multifunctional, modular polyketide synthase (PKS) that catalyzes the conversion of methylmalonyl-CoA to narbonolide and 10-deoxymethynolide, the macrolide aglycone precursors of the antibiotics picromycin and methymycin, respectively. PICS modules 5 and 6 were each expressed in Escherichia coli with a thioesterase domain at the C-terminus to allow release of polyketide products. The substrate specificity of PICS modules 5+TE and 6+TE was investigated using N-acetylcysteamine thioesters of 2-methyl-3-hydroxy-pentanoic acid as diketide analogues of the natural polyketide chain elongation substrates. PICS module 5+TE could catalyze the chain elongation of only the syn diketide (2S,3R)-4, while PICS module 6+TE processed both syn diastereomers, (2S,3R)-4 and (2R,3S)-5, with a 2.5:1 preference in kcat/Km for 5 but did not turn over either of the two anti diketides. The observed substrate specificity patterns are in contrast to the 15−100:1 preference for 4 over 5 previously est...
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insights into channel architecture and substrate specificity from crystal structures of two macrocycle forming thioesterases of modular polyketide synthases
Biochemistry, 2002Co-Authors: Shiouchuan Tsai, Chaitan Khosla, David E Cane, Robert M StroudAbstract:Modular polyketide synthases (PKSs) synthesize the polyketide cores of pharmacologically important natural products such as erythromycin and picromycin. Understanding PKSs at high resolution could present new opportunities for chemoenzymatic synthesis of complex molecules. The crystal structures of macrocycle-forming thioesterase (TE) domains from the picromycin synthase (PICS) and 6-deoxyerythronolide B synthase (DEBS) were determined to 1.8-3.0 A with an R(crys) of 19.2-24.4%, including three structures of PICS TE (crystallized at pH 7.6, 8.0, and 8.4) and a second crystal form of DEBS TE. As predicted by the previous work on DEBS TE [Tsai, S. C., et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 14808-14813], PICS TE contains an open substrate channel and a hydrophobic dimer interface. Notwithstanding their similarity, the dimer interfaces and substrate channels of DEBS TE and PICS TE reveal key differences. The structural basis for the divergent substrate specificities of DEBS TE and PICS TE is analyzed. The size of the substrate channel increases with increasing pH, presumably due to electrostatic repulsion in the channel at elevated pH. Together, these structures support previous predictions that macrocycle-forming thioesterases from PKSs share the same protein fold, an open substrate channel, a similar catalytic mechanism, and a hydrophobic dimer interface. They also provide a basis for the design of enzymes capable of catalyzing regioselective macrocyclization of natural or synthetic substrates. A series of high-resolution snapshots of a protein channel at different pHs is presented alongside analysis of channel residues, which could help in the redesign of the protein channel architecture.