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Chaitan Khosla - One of the best experts on this subject based on the ideXlab platform.

  • challenges and opportunities for engineering assembly line Polyketide Biosynthesis in escherichia coli
    Metabolic Engineering Communications, 2020
    Co-Authors: Kai P Yuet, Chaitan Khosla
    Abstract:

    Assembly-line Polyketide synthases generate natural products that have led to many live-saving drugs. The use of E. coli as a heterologous host for reconstituting these enormous and complex enzymatic machines has and will continue to be a critical strategy for understanding them. Here, we concisely summarize successful examples in exploiting E. coli for assembly-line Polyketide Biosynthesis as well as offer examples of new challenges in which this approach is primed to tackle.

  • From Active Sites to Machines: A Challenge for Enzyme Chemists.
    Israel journal of chemistry, 2018
    Co-Authors: Chaitan Khosla
    Abstract:

    As researchers who study enzyme chemistry embrace increasingly complex systems, especially biological machines, our attention is also shifting from steps involving covalent bond formation or cleavage to those that exclusively involve changes in non-covalent bonding. Assembly line Polyketide synthases are an example of this growing challenge. By now, the chemical reactions underpinning Polyketide Biosynthesis can be unequivocally mapped to well-defined active sites and are, for the most part, readily explicable in the language of physical organic chemistry. Yet, all of these insights merely serve as a backdrop to the real problem of explaining how the catalytic functions of dozens of active sites are synchronized in order to allow these remarkable machines to turn over with remarkable specificity. Notwithstanding the fact that the time-honored language of physical organic chemistry can teach us a lot, it is often insufficient to describe many of these events, and must therefore evolve.

  • A turnstile mechanism for the controlled growth of biosynthetic intermediates on assembly line Polyketide synthases
    ACS Central Science, 2016
    Co-Authors: Brian Lowry, Xiuyuan Li, Thomas Robbins, David E Cane, Chaitan Khosla
    Abstract:

    Vectorial Polyketide Biosynthesis on an assembly line Polyketide synthase is the most distinctive property of this family of biological machines, while providing the key conceptual tool for the bioinformatic decoding of new antibiotic pathways. We now show that the action of the entire assembly line is synchronized by a previously unrecognized turnstile mechanism that prevents the ketosynthase domain of each module from being acylated by a new Polyketide chain until the product of the prior catalytic cycle has been passed to the downstream module from the corresponding acyl carrier protein domain. The turnstile is closed by virtue of tight coupling to the signature decarboxylative condensation reaction catalyzed by the ketosynthase domain of each Polyketide synthase module. Reopening of the turnstile is coupled to the eventual chain translocation step that vacates the module. At the maximal rate of substrate turnover, one would expect the chain release step to initiate a cascade of chain translocation events that sequentially migrate back upstream, thereby repriming each module and setting up the assembly line for the next round of Polyketide chain elongation.

  • the biochemical basis for stereochemical control in Polyketide Biosynthesis
    Journal of the American Chemical Society, 2009
    Co-Authors: Chiara R Valenzano, Chaitan Khosla, Rachel J Lawson, Alice Y Chen, David E Cane
    Abstract:

    One of the most striking features of complex Polyketides is the presence of numerous methyl- and hydroxyl-bearing stereogenic centers. To investigate the biochemical basis for the control of Polyketide stereochemistry and to establish the timing and mechanism of the epimerization at methyl-bearing centers, a series of incubations was carried out using reconstituted components from a variety of modular Polyketide synthases. In all cases the stereochemistry of the product was directly correlated with the intrinsic stereospecificity of the ketoreductase domain, independent of the particular chain elongation domains that were used, thereby establishing that methyl group epimerization, when it does occur, takes place after ketosynthase-catalyzed chain elongation. The finding that there were only minor differences in the rates of product formation observed for parallel incubations using an epimerizing ketoreductase domain and the nonepimerizing ketoreductase domain supports the proposal that the epimerization is catalyzed by the ketoreductase domain itself.

  • ketosynthases in the initiation and elongation modules of aromatic Polyketide synthases have orthogonal acyl carrier protein specificity
    Biochemistry, 2003
    Co-Authors: Yi Tang, Taek Soon Lee, Seiji Kobayashi, Chaitan Khosla
    Abstract:

    Many bacterial aromatic Polyketides are synthesized by type II Polyketide synthases (PKSs) which minimally consist of a ketosynthase-chain length factor (KS-CLF) heterodimer, an acyl carrier protein (ACP), and a malonyl-CoA:ACP transacylase (MAT). This minimal PKS initiates Polyketide Biosynthesis by decarboxylation of malonyl-ACP, which is catalyzed by the KS-CLF complex and leads to incorporation of an acetate starter unit. In non-acetate-primed PKSs, such as the frenolicin (fren) PKS and the R1128 PKS, decarboxylative priming is suppressed in favor of chain initiation with alternative acyl groups. Elucidation of these unusual priming pathways could lead to the engineered Biosynthesis of Polyketides containing novel starter units. Unique to some non-acetate-primed PKSs is a second catalytic module comprised of a dedicated homodimeric KS, an additional ACP, and a MAT. This initiation module is responsible for starter-unit selection and catalysis of the first chain elongation step. To elucidate the protei...

Yi Tang - One of the best experts on this subject based on the ideXlab platform.

  • coordinated and iterative enzyme catalysis in fungal Polyketide Biosynthesis
    ACS Catalysis, 2016
    Co-Authors: Leibniz Hang, Nicholas Liu, Yi Tang
    Abstract:

    Fungal Polyketides are natural products with great chemical diversity that exhibit a wide range of biological activity. This chemical diversity often stems from specialized enzymes encoded in the biosynthetic gene cluster responsible for natural product Biosynthesis. Fungal Polyketide synthases (PKS) are megasynthases that produce the carbon scaffold for the molecules. Subsequent downstream tailoring enzymes such as oxygenases will then further modify the organic framework. In fungi, many of these enzymes have been found to work iteratively—catalyzing multiple similar reactions on different sites of the substrate. This Perspective will analyze several examples of fungal Polyketides that are assembled from a scaffold-building iterative PKS and an accompanying, complexity-generating iterative tailoring oxygenase. In these examples, the PKS product is designed for downstream iterative oxygenations to elegantly generate additional complexity. Together, these iterative enzymes efficiently orchestrate the biosy...

  • biochemical and structural basis for controlling chemical modularity in fungal Polyketide Biosynthesis
    Journal of the American Chemical Society, 2015
    Co-Authors: Jaclyn M Winter, Duilio Cascio, David Dietrich, Michio Sato, Kenji Watanabe, Michael R. Sawaya, John C. Vederas, Yi Tang
    Abstract:

    Modular collaboration between iterative fungal Polyketide synthases (IPKSs) is an important mechanism for generating structural diversity of Polyketide natural products. Inter-PKS communication and substrate channeling are controlled in large by the starter unit acyl carrier protein transacylase (SAT) domain found in the accepting IPKS module. Here, we reconstituted the modular Biosynthesis of the benzaldehyde core of the chaetoviridin and chaetomugilin azaphilone natural products using the IPKSs CazF and CazM. Our studies revealed a critical role of CazM’s SAT domain in selectively transferring a highly reduced triketide product from CazF. In contrast, a more oxidized triketide that is also produced by CazF and required in later stages of Biosynthesis of the final product is not recognized by the SAT domain. The structural basis for the acyl unit selectivity was uncovered by the first X-ray structure of a fungal SAT domain, highlighted by a covalent hexanoyl thioester intermediate in the SAT active site....

  • Biochemical and Structural Basis for Controlling Chemical Modularity in Fungal Polyketide Biosynthesis
    2015
    Co-Authors: Jaclyn M. Winter, Duilio Cascio, David Dietrich, Michio Sato, Kenji Watanabe, Michael R. Sawaya, John C. Vederas, Yi Tang
    Abstract:

    Modular collaboration between iterative fungal Polyketide synthases (IPKSs) is an important mechanism for generating structural diversity of Polyketide natural products. Inter-PKS communication and substrate channeling are controlled in large by the starter unit acyl carrier protein transacylase (SAT) domain found in the accepting IPKS module. Here, we reconstituted the modular Biosynthesis of the benzaldehyde core of the chaetoviridin and chaetomugilin azaphilone natural products using the IPKSs CazF and CazM. Our studies revealed a critical role of CazM’s SAT domain in selectively transferring a highly reduced triketide product from CazF. In contrast, a more oxidized triketide that is also produced by CazF and required in later stages of Biosynthesis of the final product is not recognized by the SAT domain. The structural basis for the acyl unit selectivity was uncovered by the first X-ray structure of a fungal SAT domain, highlighted by a covalent hexanoyl thioester intermediate in the SAT active site. The crystal structure of SAT domain will enable protein engineering efforts aimed at mixing and matching different IPKS modules for the Biosynthesis of new compounds

  • Engineered Polyketide Biosynthesis and biocatalysis in Escherichia coli
    Applied Microbiology and Biotechnology, 2010
    Co-Authors: Xue Gao, Peng Wang, Yi Tang
    Abstract:

    Polyketides are important bioactive natural products biosynthesized by bacteria, fungi, and plants. The enzymes that synthesize Polyketides are collectively referred to as Polyketide synthases (PKSs). Because many of the natural hosts that produce Polyketides are difficult to culture or manipulate, establishing a universal heterologous host that is genetically tractable has become an important goal toward the engineered Biosynthesis of Polyketides and analogues. Here, we summarize the recent progresses in engineering Escherichia coli as a heterologous host for reconstituting PKSs of different types. Our increased understanding of PKS enzymology and structural biology, combined with new tools in protein engineering, metabolic engineering, and synthetic biology, has firmly established E. coli as a powerful host for producing Polyketides.

  • ketosynthases in the initiation and elongation modules of aromatic Polyketide synthases have orthogonal acyl carrier protein specificity
    Biochemistry, 2003
    Co-Authors: Yi Tang, Taek Soon Lee, Seiji Kobayashi, Chaitan Khosla
    Abstract:

    Many bacterial aromatic Polyketides are synthesized by type II Polyketide synthases (PKSs) which minimally consist of a ketosynthase-chain length factor (KS-CLF) heterodimer, an acyl carrier protein (ACP), and a malonyl-CoA:ACP transacylase (MAT). This minimal PKS initiates Polyketide Biosynthesis by decarboxylation of malonyl-ACP, which is catalyzed by the KS-CLF complex and leads to incorporation of an acetate starter unit. In non-acetate-primed PKSs, such as the frenolicin (fren) PKS and the R1128 PKS, decarboxylative priming is suppressed in favor of chain initiation with alternative acyl groups. Elucidation of these unusual priming pathways could lead to the engineered Biosynthesis of Polyketides containing novel starter units. Unique to some non-acetate-primed PKSs is a second catalytic module comprised of a dedicated homodimeric KS, an additional ACP, and a MAT. This initiation module is responsible for starter-unit selection and catalysis of the first chain elongation step. To elucidate the protei...

Daniel V. Santi - One of the best experts on this subject based on the ideXlab platform.

David A Hopwood - One of the best experts on this subject based on the ideXlab platform.

Gong-li Tang - One of the best experts on this subject based on the ideXlab platform.

  • discovery of 16 demethylrifamycins by removing the predominant Polyketide Biosynthesis pathway in micromonospora sp strain tp a0468
    Applied and Environmental Microbiology, 2018
    Co-Authors: Guangcai Luo, Qiang Zhou, Huizhan Zhang, Gong-li Tang
    Abstract:

    A number of strategies have been developed to mine novel natural products based on biosynthetic gene clusters and there have been dozens of successful cases facilitated by the development of genomic sequencing. During our study on Biosynthesis of the antitumor Polyketide kosinostatin (KST), we found that the genome of Micromonospora sp. strain TP-A0468, the producer of KST, contains other potential Polyketide gene clusters, with no encoded products detected. Deletion of kst cluster led to abolishment of KST and the enrichment of several new compounds, which were isolated and characterized as 16-demethylrifamycins (referred to here as compounds 3 to 6). Transcriptional analysis demonstrated that the expression of the essential genes related to the Biosynthesis of compounds 3 to 6 was comparable to the level in the wild-type and in the kst cluster deletion strain. This indicates that the accumulation of these compounds was due to the redirection of metabolic flux rather than transcriptional activation. Genetic disruption, chemical complementation, and bioinformatic analysis revealed that the production of compounds 3 to 6 was accomplished by cross talk between the two distantly placed Polyketide gene clusters pks3 and M-rif This finding not only enriches the analogue pool and the biosynthetic diversity of rifamycins but also provides an auxiliary strategy for natural product discovery through genome mining in Polyketide-producing microorganisms.IMPORTANCE Natural products are essential in the development of novel clinically used drugs. Discovering new natural products and modifying known compounds are still the two main ways to generate new candidates. Here, we have discovered several rifamycins with varied skeleton structures by redirecting the metabolic flux from the predominant Polyketide biosynthetic pathway to the rifamycin pathway in the marine actinomycetes species Micromonospora sp. strain TP-A0468. Rifamycins are indispensable chemotherapeutics in the treatment of various diseases such as tuberculosis, leprosy, and AIDS-related mycobacterial infections. This study exemplifies a useful method for the discovery of cryptic natural products in genome-sequenced microbes. Moreover, the 16-demethylrifamycins and their genetically manipulable producer provide a new opportunity in the construction of novel rifamycin derivates to aid in the defense against the ever-growing drug resistance of Mycobacterium tuberculosis.

  • new insights into bacterial type ii Polyketide Biosynthesis
    F1000Research, 2017
    Co-Authors: Zhuan Zhang, Haixue Pan, Gong-li Tang
    Abstract:

    Bacterial aromatic Polyketides, exemplified by anthracyclines, angucyclines, tetracyclines, and pentangular polyphenols, are a large family of natural products with diverse structures and biological activities and are usually biosynthesized by type II Polyketide synthases (PKSs). Since the starting point of Biosynthesis and combinatorial Biosynthesis in 1984-1985, there has been a continuous effort to investigate the biosynthetic logic of aromatic Polyketides owing to the urgent need of developing promising therapeutic candidates from these compounds. Recently, significant advances in the structural and mechanistic identification of enzymes involved in aromatic Polyketide Biosynthesis have been made on the basis of novel genetic, biochemical, and chemical technologies. This review highlights the progress in bacterial type II PKSs in the past three years (2013-2016). Moreover, novel compounds discovered or created by genome mining and biosynthetic engineering are also included.

  • characterization of the azinomycin b biosynthetic gene cluster revealing a different iterative type i Polyketide synthase for naphthoate Biosynthesis
    Chemistry & Biology, 2008
    Co-Authors: Qunfei Zhao, Wei Ding, Mancheng Tang, Qianjin Kang, Wei Deng, Qi Zhang, Jie Fang, Gong-li Tang
    Abstract:

    Azinomycin B is a complex natural product containing densely assembled functionalities with potent antitumor activity. Cloning and sequence analysis of the azi gene cluster revealed an iterative type I Polyketide synthase (PKS) gene, five nonribosomal peptide synthetases (NRPSs) genes and numerous genes encoding the Biosynthesis of unusual building blocks and tailoring steps for azinomycin B production. Characterization of AziB as a 5-methyl-naphthoic acid (NPA) synthase showed a distinct selective reduction pattern in aromatic Polyketide Biosynthesis governed by bacterial iterative type I PKSs. Heterologous expression established the PKS-post modification route from 5-methyl-NPA to reach the first building block 3-methoxy-5-methyl-NPA. This proposed azinomycin B biosynthetic pathway sets the stage to investigate the enzymatic mechanisms for building structurally unique and pharmaceutically important groups, including the unprecedented azabicyclic ring system and highly active epoxide moiety.

  • Type I Polyketide synthase requiring a discrete acyltransferase for Polyketide Biosynthesis
    Proceedings of the National Academy of Sciences of the United States of America, 2003
    Co-Authors: Yi-qiang Cheng, Gong-li Tang
    Abstract:

    Type I Polyketide synthases (PKSs) are multifunctional enzymes that are organized into modules, each of which minimally contains a β-ketoacyl synthase, an acyltransferase (AT), and an acyl carrier protein. Here we report that the leinamycin (LNM) biosynthetic gene cluster from Streptomyces atroolivaceus S-140 consists of two PKS genes, lnmI and lnmJ, that encode six PKS modules, none of which contain the cognate AT domain. The only AT activity identified within the lnm gene cluster is a discrete AT protein encoded by lnmG. Inactivation of lnmG, lnmI, or lnmJ in vivo abolished LNM Biosynthesis. Biochemical characterization of LnmG in vitro showed that it efficiently and specifically loaded malonyl CoA to all six PKS modules. These findings unveiled a previously unknown PKS architecture that is characterized by a discrete, iteratively acting AT protein that loads the extender units in trans to “AT-less” multifunctional type I PKS proteins for Polyketide Biosynthesis. This PKS structure provides opportunities for PKS engineering as exemplified by overexpressing lnmG to improve LNM production.