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Chaitan Khosla - One of the best experts on this subject based on the ideXlab platform.
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evolution and diversity of assembly line Polyketide Synthases
Chemical Reviews, 2019Co-Authors: Aleksandra Nivina, Kai P Yuet, Jake Hsu, Chaitan KhoslaAbstract:Assembly-line Polyketide Synthases (PKSs) are among the most complex protein machineries known in nature, responsible for the biosynthesis of numerous compounds used in the clinic. Their present-day diversity is the result of an evolutionary path that has involved the emergence of a multimodular architecture and further diversification of assembly-line PKSs. In this review, we provide an overview of previous studies that investigated PKS evolution and propose a model that challenges the currently prevailing view that gene duplication has played a major role in the emergence of multimodularity. We also analyze the ensemble of orphan PKS clusters sequenced so far to evaluate how large the entire diversity of assembly-line PKS clusters and their chemical products could be. Finally, we examine the existing techniques to access the natural PKS diversity in natural and heterologous hosts and describe approaches to further expand this diversity through engineering.
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engineering of chimeric Polyketide Synthases using synzip docking domains
ACS Chemical Biology, 2019Co-Authors: Chaitan Khosla, Maja Klaus, Aleksandra Nivina, Alicia D Dsouza, Martin GriningerAbstract:Engineering of assembly line Polyketide Synthases (PKSs) to produce novel bioactive compounds has been a goal for over 20 years. The apparent modularity of PKSs has inspired many engineering attempts in which entire modules or single domains were exchanged. In recent years, it has become evident that certain domain–domain interactions are evolutionarily optimized and, if disrupted, cause a decrease of the overall turnover rate of the chimeric PKS. In this study, we compared different types of chimeric PKSs in order to define the least invasive interface and to expand the toolbox for PKS engineering. We generated bimodular chimeric PKSs in which entire modules were exchanged, while either retaining a covalent linker between heterologous modules or introducing a noncovalent docking domain, or SYNZIP domain, mediated interface. These chimeric systems exhibited non-native domain–domain interactions during intermodular Polyketide chain translocation. They were compared to otherwise equivalent bimodular PKSs in...
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Engineering of Chimeric Polyketide Synthases Using SYNZIP Docking Domains
2019Co-Authors: Maja Klaus, Chaitan Khosla, Aleksandra Nivina, Alicia D. D’souza, Martin GriningerAbstract:Engineering of assembly line Polyketide Synthases (PKSs) to produce novel bioactive compounds has been a goal for over 20 years. The apparent modularity of PKSs has inspired many engineering attempts in which entire modules or single domains were exchanged. In recent years, it has become evident that certain domain–domain interactions are evolutionarily optimized and, if disrupted, cause a decrease of the overall turnover rate of the chimeric PKS. In this study, we compared different types of chimeric PKSs in order to define the least invasive interface and to expand the toolbox for PKS engineering. We generated bimodular chimeric PKSs in which entire modules were exchanged, while either retaining a covalent linker between heterologous modules or introducing a noncovalent docking domain, or SYNZIP domain, mediated interface. These chimeric systems exhibited non-native domain–domain interactions during intermodular Polyketide chain translocation. They were compared to otherwise equivalent bimodular PKSs in which a noncovalent interface was introduced between the condensing and processing parts of a module, resulting in non-native domain interactions during the extender unit acylation and Polyketide chain elongation steps of their catalytic cycles. We show that the natural PKS docking domains can be efficiently substituted with SYNZIP domains and that the newly introduced noncovalent interface between the condensing and processing parts of a module can be harnessed for PKS engineering. Additionally, we established SYNZIP domains as a new tool for engineering PKSs by efficiently bridging non-native interfaces without perturbing PKS activity
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engineering of chimeric Polyketide Synthases using synzip docking domains
bioRxiv, 2018Co-Authors: Maja Klaus, Chaitan Khosla, Aleksandra Nivina, Alicia D Dsouza, Martin GriningerAbstract:Engineering of assembly line Polyketide Synthases (PKSs) to produce novel bioactive compounds has been a goal for over twenty years. The apparent modularity of PKSs has inspired many engineering attempts in which entire modules or single domains were exchanged. In recent years, it has become evident that certain domain-domain interactions are evolutionarily optimized, and if disrupted, cause a decrease of the overall turnover rate of the chimeric PKS. In this study, we compared different types of chimeric PKSs in order to define the least invasive interface and to expand the toolbox for PKS engineering. We generated bimodular chimeric PKSs in which entire modules were exchanged, while either retaining a covalent linker between heterologous modules or introducing a non-covalent docking domain- or SYNZIP domain-mediated interface. These chimeric systems exhibited non-native domain-domain interactions during intermodular Polyketide chain translocation. They were compared to otherwise equivalent bimodular PKSs in which a non-covalent interface was introduced between the condensing and processing parts of a module, resulting in non-native domain interactions during the extender unit acylation and Polyketide chain elongation steps of their catalytic cycles. We show that the natural PKS docking domains can be efficiently substituted with SYNZIP domains and that the newly introduced non-covalent interface between the condensing and processing parts of a module can be harnessed for PKS engineering. Additionally, we established SYNZIP domains as a new tool for engineering PKSs by efficiently bridging non-native interfaces without perturbing PKS activity.
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elucidation of the stereospecificity of c methyltransferases from trans at Polyketide Synthases
Journal of the American Chemical Society, 2017Co-Authors: Xinqiang Xie, Chaitan Khosla, David E. CaneAbstract:S-Adenosyl methionine (SAM)-dependent C-methyltransferases are responsible for the C2-methylation of 3-ketoacyl-acyl carrier protein (ACP) intermediates to give the corresponding 2-methy-3-ketoacyl-ACP products during bacterial Polyketide biosynthesis mediated by trans-AT Polyketide Synthases that lack integrated acyl transferase (AT) domains. A coupled ketoreductase (KR) assay was used to assign the stereochemistry of the C-methyltransferase-catalyzed reaction. Samples of chemoenzymatically generated 3-ketopentanoyl-ACP (9) were incubated with SAM and BonMT2 from module 2 of the bongkrekic acid Polyketide synthase. The resulting 2-methyl-3-ketopentanoyl-ACP (10) was incubated separately with five (2R)- or (2S)-methyl specific KR domains. Analysis of the derived 2-methyl-3-hydroxypentanoate methyl esters (8) by chiral GC-MS established that the BonMT2-catalyzed methylation generated exclusively (2R)-2-methyl-3-ketopentanoyl-ACP ((2R)-10). Identical results were also obtained with three additional C-methyl...
David E. Cane - One of the best experts on this subject based on the ideXlab platform.
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elucidation of the stereospecificity of c methyltransferases from trans at Polyketide Synthases
Journal of the American Chemical Society, 2017Co-Authors: Xinqiang Xie, Chaitan Khosla, David E. CaneAbstract:S-Adenosyl methionine (SAM)-dependent C-methyltransferases are responsible for the C2-methylation of 3-ketoacyl-acyl carrier protein (ACP) intermediates to give the corresponding 2-methy-3-ketoacyl-ACP products during bacterial Polyketide biosynthesis mediated by trans-AT Polyketide Synthases that lack integrated acyl transferase (AT) domains. A coupled ketoreductase (KR) assay was used to assign the stereochemistry of the C-methyltransferase-catalyzed reaction. Samples of chemoenzymatically generated 3-ketopentanoyl-ACP (9) were incubated with SAM and BonMT2 from module 2 of the bongkrekic acid Polyketide synthase. The resulting 2-methyl-3-ketopentanoyl-ACP (10) was incubated separately with five (2R)- or (2S)-methyl specific KR domains. Analysis of the derived 2-methyl-3-hydroxypentanoate methyl esters (8) by chiral GC-MS established that the BonMT2-catalyzed methylation generated exclusively (2R)-2-methyl-3-ketopentanoyl-ACP ((2R)-10). Identical results were also obtained with three additional C-methyl...
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Elucidation of the Stereospecificity of C‑Methyltransferases from trans-AT Polyketide Synthases
2017Co-Authors: Xinqiang Xie, Chaitan Khosla, David E. CaneAbstract:S-Adenosyl methionine (SAM)-dependent C-methyltransferases are responsible for the C2-methylation of 3-ketoacyl-acyl carrier protein (ACP) intermediates to give the corresponding 2-methy-3-ketoacyl-ACP products during bacterial Polyketide biosynthesis mediated by trans-AT Polyketide Synthases that lack integrated acyl transferase (AT) domains. A coupled ketoreductase (KR) assay was used to assign the stereochemistry of the C-methyltransferase-catalyzed reaction. Samples of chemoenzymatically generated 3-ketopentanoyl-ACP (9) were incubated with SAM and BonMT2 from module 2 of the bongkrekic acid Polyketide synthase. The resulting 2-methyl-3-ketopentanoyl-ACP (10) was incubated separately with five (2R)- or (2S)-methyl specific KR domains. Analysis of the derived 2-methyl-3-hydroxypentanoate methyl esters (8) by chiral GC-MS established that the BonMT2-catalyzed methylation generated exclusively (2R)-2-methyl-3-ketopentanoyl-ACP ((2R)-10). Identical results were also obtained with three additional C-methyltransferasesBaeMT9, DifMT1, and MupMT1from the bacillaene, difficidin, and mupirocin trans-AT Polyketide synthase
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structure and mechanism of assembly line Polyketide Synthases
Current Opinion in Structural Biology, 2016Co-Authors: Thomas Robbins, David E. Cane, Yuchen Liu, Chaitan KhoslaAbstract:Assembly line Polyketide Synthases (PKSs) are remarkable biosynthetic machines with considerable potential for structure-based engineering. Several types of protein-protein interactions, both within and between PKS modules, play important roles in the catalytic cycle of a multimodular PKS. Additionally, vectorial biosynthesis is enabled by the energetic coupling of Polyketide chain elongation to the channeling of intermediates between successive modules. A combination of high-resolution analysis of smaller PKS components and lower resolution characterization of intact modules and bimodules has yielded insights into the structure and organization of a prototypical assembly line PKS. This review discusses our understanding of key structure-function relationships in this family of megaSynthases, along with a recap of key unanswered questions in the field.
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assembly line Polyketide Synthases mechanistic insights and unsolved problems
Biochemistry, 2014Co-Authors: Chaitan Khosla, David E. Cane, Daniel Herschlag, Christopher T WalshAbstract:Two hallmarks of assembly line Polyketide Synthases have motivated an interest in these unusual multienzyme systems, their stereospecificity and their capacity for directional biosynthesis. In this review, we summarize the state of knowledge regarding the mechanistic origins of these two remarkable features, using the 6-deoxyerythronolide B synthase as a prototype. Of the 10 stereocenters in 6-deoxyerythronolide B, the stereochemistry of nine carbon atoms is directly set by ketoreductase domains, which catalyze epimerization and/or diastereospecific reduction reactions. The 10th stereocenter is established by the sequential action of three enzymatic domains. Thus, the problem has been reduced to a challenge in mainstream enzymology, where fundamental gaps remain in our understanding of the structural basis for this exquisite stereochemical control by relatively well-defined active sites. In contrast, testable mechanistic hypotheses for the phenomenon of vectorial biosynthesis are only just beginning to emerge. Starting from an elegant theoretical framework for understanding coupled vectorial processes in biology [Jencks, W. P. (1980) Adv. Enzymol. Relat. Areas Mol. Biol. 51, 75–106], we present a simple model that can explain assembly line Polyketide biosynthesis as a coupled vectorial process. Our model, which highlights the important role of domain–domain interactions, not only is consistent with recent observations but also is amenable to further experimental verification and refinement. Ultimately, a definitive view of the coordinated motions within and between Polyketide synthase modules will require a combination of structural, kinetic, spectroscopic, and computational tools and could be one of the most exciting frontiers in 21st Century enzymology.
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Stereochemistry of Reductions Catalyzed by Methyl-Epimerizing Ketoreductase Domains of Polyketide Synthases
2013Co-Authors: Young-ok You, Chaitan Khosla, David E. CaneAbstract:Ketoreductase (KR) domains from modular Polyketide Synthases (PKSs) catalyze the reduction of 2-methyl-3-ketoacyl acyl carrier protein (ACP) substrates and in certain cases epimerization of the 2-methyl group as well. The structural and mechanistic basis of epimerization is poorly understood, and only a small number of such KRs been studied. In this work, we studied three recombinant KR domains with putative epimerase activity: NysKR1 from module 1 of the nystatin PKS, whose stereospecificity can be predicted from both the protein sequence and the product structure; RifKR7 from module 7 of the rifamycin PKS, whose stereospecificity cannot be predicted from the protein sequence; and RifKR10 from module 10 of the rifamycin PKS, whose specificity is unclear from both the sequence and the structure. Each KR was individually incubated with NADPH and (2R)- or (2RS)-2-methyl-3-ketopentanoyl-ACP generated enzymatically in situ or via chemoenzymatic synthesis, respectively. Chiral GC–MS analysis revealed that each KR stereospecifically produced the corresponding (2S,3S)-2-methyl-3-hydroxypentanoyl-ACP in which the 2-methyl substituent had undergone KR-catalyzed epimerization. Thus, our results have led to the identification of a prototypical set of KR domains that generate (2S,3S)-2-methyl-3-hydroxyacyl products in the course of Polyketide biosynthesis
Adrian T. Keatinge-clay - One of the best experts on this subject based on the ideXlab platform.
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An in vitro platform for engineering and harnessing modular Polyketide Synthases
Nature Communications, 2020Co-Authors: Takeshi Miyazawa, Melissa Hirsch, Zhicheng Zhang, Adrian T. Keatinge-clayAbstract:A robust platform to study modular Polyketide Synthases (PKSs) in vitro is still unavailable. Here, the authors report the reconstitution of the venemycin PKS, engineer hybrid venemycin/pikromycin PKSs, and obtain much improved yields through employing the updated module boundaries. To harness the synthetic power of modular Polyketide Synthases (PKSs), many aspects of their biochemistry must be elucidated. A robust platform to study these megadalton assembly lines has not yet been described. Here, we in vitro reconstitute the venemycin PKS, a short assembly line that generates an aromatic product. Incubating its polypeptides, VemG and VemH, with 3,5-dihydroxybenzoic acid, ATP, malonate, coenzyme A, and the malonyl-CoA ligase MatB, venemycin production can be monitored by HPLC and NMR. Multi-milligram quantities of venemycin are isolable from dialysis-based reactors without chromatography, and the enzymes can be recycled. Assembly line engineering is performed using pikromycin modules, with Synthases designed using the updated module boundaries outperforming those using the traditional module boundaries by over an order of magnitude. Using combinations of VemG, VemH, and their engineered derivatives, as well as the alternate starter unit 3-hydroxybenzoic acid, a combinatorial library of six Polyketide products is readily accessed.
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Portability and Structure of the Four-Helix Bundle Docking Domains of trans-Acyltransferase Modular Polyketide Synthases.
ACS chemical biology, 2016Co-Authors: Jia Zeng, Drew T. Wagner, Zhicheng Zhang, Luisa Moretto, Janci D. Addison, Adrian T. Keatinge-clayAbstract:The polypeptides of multimodular Polyketide Synthases self-assemble into biosynthetic factories. While the docking domains that mediate the assembly of cis-acyltransferase Polyketide synthase polypeptides are well-studied, those of the more recently discovered trans-acyltransferase Polyketide Synthases have just started to be described. Located at the C- and N-termini of many polypeptides, these 25-residue, two-helix, pseudosymmetric motifs noncovalently connect domains both between and within modules. Domains expressed with their natural, cognate docking motifs formed complexes stable to size-exclusion chromatography with 1–10 μM dissociation constants as measured by isothermal titration calorimetry. Deletion and swapping experiments demonstrate portability of the docking motifs. A 1.72 A-resolution structure of the N-terminal portion of the macrolactin synthase polypeptide MlnE shows an uncomplexed N-terminal docking motif to be preorganized in the conformation it assumes within the docking domain complex.
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Methyltransferases excised from trans-AT Polyketide Synthases operate on N-acetylcysteamine-bound substrates
The Journal of antibiotics, 2016Co-Authors: D. Cole Stevens, Drew T. Wagner, Hannah R. Manion, Bradley K Alexander, Adrian T. Keatinge-clayAbstract:Methyltransferases excised from trans -AT Polyketide Synthases operate on N -acetylcysteamine-bound substrates
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The structures of type I Polyketide Synthases
Natural product reports, 2012Co-Authors: Adrian T. Keatinge-clayAbstract:Covering: up to 2012 With the recent structural characterization of each of the component enzymes of type I Polyketide Synthases, scientists are coming tantalizingly close to elucidating the overall architectures and mechanisms of these enormous molecular factories. This review highlights not only what has been revealed about the structures and activities of each of the domains but also the mysteries that remain to be solved.
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Divergence of multimodular Polyketide Synthases revealed by a didomain structure
Nature Chemical Biology, 2012Co-Authors: Jianting Zheng, Borries Demeler, Mark A White, Adrian T. Keatinge-clayAbstract:The first crystal structure and in vitro biochemical characterization of an enoylreductase domain from a multimodular Polyketide synthase indicates substantial architectural deviations from the mammalian fatty acid synthase and identifies an active site residue that controls catalytic activity. The enoylreductase (ER) is the final common enzyme from modular Polyketide Synthases (PKSs) to be structurally characterized. The 3.0 Å–resolution structure of the didomain comprising the ketoreductase (KR) and ER from the second module of the spinosyn PKS reveals that ER shares an ∼600-Å^2 interface with KR distinct from that of the related mammalian fatty acid synthase (FAS). In contrast to the ER domains of the mammalian FAS, the ER domains of the second module of the spinosyn PKS do not make contact across the two-fold axis of the synthase. This monomeric organization may have been necessary in the evolution of multimodular PKSs to enable acyl carrier proteins to access each of their cognate enzymes. The isolated ER domain showed activity toward a substrate analog, enabling us to determine the contributions of its active site residues.
Sueharu Horinouchi - One of the best experts on this subject based on the ideXlab platform.
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Enzymatic synthesis of bis-5-alkylresorcinols by resorcinol-producing type III Polyketide Synthases
The Journal of Antibiotics, 2009Co-Authors: Akimasa Miyanaga, Sueharu HorinouchiAbstract:No enzyme systems responsible for the biosynthesis of structurally and biosynthetically intriguing bis-5-alkylresorcinols produced by plants have been identified. Herein, we show that bacterial, fungal and plant alkylresorcinol-producing type III Polyketide Synthases (PKSs), such as ArsB in the Gram-negative bacterium Azotobacter vinelandii , ORAS in the fungus Neurospora crassa and ARAS2 in the rice plant Oryza sativa , can synthesize bis-5-alkylresorcinol from alkanedioic acid N -acetylcysteamine dithioester as a starter substrate and from malonyl-CoA as an extender substrate by two-step conversion. Plants presumably use a type III PKS for the biosynthesis of bis-5-alkylresorcinols.
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curcuminoid biosynthesis by two type iii Polyketide Synthases in the herb curcuma longa
Journal of Biological Chemistry, 2009Co-Authors: Yohei Katsuyama, Nobutaka Funa, Tomoko Kita, Sueharu HorinouchiAbstract:Curcuminoids found in the rhizome of turmeric, Curcuma longa, possess various biological activities. Despite much attention regarding the biosynthesis of curcuminoids because of their pharmaceutically important properties and biosynthetically intriguing structures, no enzyme systems have been elucidated. Here we propose a pathway for curcuminoid biosynthesis in the herb C. longa, which includes two novel type III Polyketide Synthases. One of the type III Polyketide Synthases, named diketide-CoA synthase (DCS), catalyzed the formation of feruloyldiketide-CoA by condensing feruloyl-CoA and malonyl-CoA. The other, named curcumin synthase (CURS), catalyzed the in vitro formation of curcuminoids from cinnamoyldiketide-N-acetylcysteamine (a mimic of the CoA ester) and feruloyl-CoA. Co-incubation of DCS and CURS in the presence of feruloyl-CoA and malonyl-CoA yielded curcumin at high efficiency, although CURS itself possessed low activity for the synthesis of curcumin from feruloyl-CoA and malonyl-CoA. These findings thus revealed the curcumin biosynthetic route in turmeric, in which DCS synthesizes feruloyldiketide-CoA, and CURS then converts the diketide-CoA esters into a curcuminoid scaffold.
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direct transfer of starter substrates from type i fatty acid synthase to type iii Polyketide Synthases in phenolic lipid synthesis
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Akimasa Miyanaga, Nobutaka Funa, Takayoshi Awakawa, Sueharu HorinouchiAbstract:Alkylresorcinols and alkylpyrones, which have a polar aromatic ring and a hydrophobic alkyl chain, are phenolic lipids found in plants, fungi, and bacteria. In the Gram-negative bacterium Azotobacter vinelandii, phenolic lipids in the membrane of dormant cysts are essential for encystment. The aromatic moieties of the phenolic lipids in A. vinelandii are synthesized by two type III Polyketide Synthases (PKSs), ArsB and ArsC, which are encoded by the ars operon. However, details of the synthesis of hydrophobic acyl chains, which might serve as starter substrates for the type III Polyketide Synthases (PKSs), were unknown. Here, we show that two type I fatty acid Synthases (FASs), ArsA and ArsD, which are members of the ars operon, are responsible for the biosynthesis of C22–C26 fatty acids from malonyl-CoA. In vivo and in vitro reconstitution of phenolic lipid synthesis systems with the Ars enzymes suggested that the C22–C26 fatty acids produced by ArsA and ArsD remained attached to the ACP domain of ArsA and were transferred hand-to-hand to the active-site cysteine residues of ArsB and ArsC. The type III PKSs then used the fatty acids as starter substrates and carried out two or three extensions with malonyl-CoA to yield the phenolic lipids. The phenolic lipids in A. vinelandii were thus found to be synthesized solely from malonyl-CoA by the four members of the ars operon. This is the first demonstration that a type I FAS interacts directly with a type III PKS through substrate transfer.
Satoshi Yuzawa - One of the best experts on this subject based on the ideXlab platform.
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short chain ketone production by engineered Polyketide Synthases in streptomyces albus
Nature Communications, 2018Co-Authors: Satoshi Yuzawa, Mona Mirsiaghi, Renee Jocic, Tatsuya Fujii, Fabrice Masson, Veronica T Benites, Edward E K Baidoo, Eric R Sundstrom, Deepti TanjoreAbstract:Microbial production of fuels and commodity chemicals has been performed primarily using natural or slightly modified enzymes, which inherently limits the types of molecules that can be produced. Type I modular Polyketide Synthases (PKSs) are multi-domain enzymes that can produce unique and diverse molecular structures by combining particular types of catalytic domains in a specific order. This catalytic mechanism offers a wealth of engineering opportunities. Here we report engineered microbes that produce various short-chain (C5-C7) ketones using hybrid PKSs. Introduction of the genes into the chromosome of Streptomyces albus enables it to produce >1 g · l-1 of C6 and C7 ethyl ketones and several hundred mg · l-1 of C5 and C6 methyl ketones from plant biomass hydrolysates. Engine tests indicate these short-chain ketones can be added to gasoline as oxygenates to increase the octane of gasoline. Together, it demonstrates the efficient and renewable microbial production of biogasolines by hybrid enzymes.
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Synthetic biology of Polyketide Synthases
Journal of Industrial Microbiology & Biotechnology, 2018Co-Authors: Satoshi Yuzawa, Tyler W. H. Backman, Jay D. Keasling, Leonard KatzAbstract:Complex reduced Polyketides represent the largest class of natural products that have applications in medicine, agriculture, and animal health. This structurally diverse class of compounds shares a common methodology of biosynthesis employing modular enzyme systems called Polyketide Synthases (PKSs). The modules are composed of enzymatic domains that share sequence and functional similarity across all known PKSs. We have used the nomenclature of synthetic biology to classify the enzymatic domains and modules as parts and devices, respectively, and have generated detailed lists of both. In addition, we describe the chassis (hosts) that are used to assemble, express, and engineer the parts and devices to produce Polyketides. We describe a recently developed software tool to design PKS system and provide an example of its use. Finally, we provide perspectives of what needs to be accomplished to fully realize the potential that synthetic biology approaches bring to this class of molecules.
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commodity chemicals from engineered modular type i Polyketide Synthases
Methods in Enzymology, 2018Co-Authors: Satoshi Yuzawa, Leonard Katz, Amin Zargar, Bo Pang, Jay D. KeaslingAbstract:Abstract Reduced Polyketides are a subclass of natural products that have a variety of medical, veterinary, and agricultural applications and are well known for their structural diversity. Although these compounds do not resemble each other, they are all made by a class of enzymes known as modular Polyketide Synthases (PKSs). The commonality of PKS domains/modules that compose PKSs and the understanding of the relationship between the sequence of the PKS and the structure of the compound it produces render modular PKSs as excellent targets for engineering to produce novel compounds with predicted structures. Here, we describe experimental protocols and considerations for modular PKS engineering and two case studies to produce commodity chemicals by engineered PKSs.
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Bio-based production of fuels and industrial chemicals by repurposing antibiotic-producing type I modular Polyketide Synthases: opportunities and challenges
The Journal of Antibiotics, 2017Co-Authors: Satoshi Yuzawa, Jay D. Keasling, Leonard KatzAbstract:Complex Polyketides comprise a large number of natural products that have broad application in medicine and agriculture. They are produced in bacteria and fungi from large enzyme complexes named type I modular Polyketide Synthases (PKSs) that are composed of multifunctional polypeptides containing discrete enzymatic domains organized into modules. The modular nature of PKSs has enabled a multitude of efforts to engineer the PKS genes to produce novel Polyketides of predicted structure. We have repurposed PKSs to produce a number of short-chain mono- and di-carboxylic acids and ketones that could have applications as fuels or industrial chemicals.
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role of a conserved arginine residue in linkers between the ketosynthase and acyltransferase domains of multimodular Polyketide Synthases
Biochemistry, 2012Co-Authors: Satoshi Yuzawa, David E. Cane, Shiven Kapur, Chaitan KhoslaAbstract:The role of interdomain linkers in modular Polyketide Synthases is poorly understood. Analysis of the 6-deoxyerythronolide B synthase (DEBS) has yielded a model in which chain elongation is governed by interactions between the acyl carrier protein domain and the ketosynthase domain plus an adjacent linker. Alanine scanning mutagenesis of the conserved residues of this linker in DEBS module 3 led to the identification of the R513A mutant with a markedly reduced rate of chain elongation. Limited proteolysis supported a structural role for this Arg. Our findings highlight the importance of domain–linker interactions in assembly line Polyketide biosynthesis.