The Experts below are selected from a list of 32574 Experts worldwide ranked by ideXlab platform
Chaitan Khosla - One of the best experts on this subject based on the ideXlab platform.
-
Evolution of Polyketide synthases in bacteria.
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Christian P. Ridley, Chaitan KhoslaAbstract:The emergence of resistant strains of human pathogens to current antibiotics, along with the demonstrated ability of Polyketides as antimicrobial agents, provides strong motivation for understanding how Polyketide antibiotics have evolved and diversified in nature. Insights into how bacterial Polyketide synthases (PKSs) acquire new metabolic capabilities can guide future laboratory efforts in generating the next generation of Polyketide antibiotics. Here, we examine phylogenetic and structural evidence to glean answers to two general questions regarding PKS evolution. How did the exceptionally diverse chemistry of present-day PKSs evolve? And what are the take-home messages for the biosynthetic engineer?
-
Engineered biosynthesis of Polyketides in heterologous hosts
Chemical Engineering Science, 2004Co-Authors: Mathew A. Rude, Chaitan KhoslaAbstract:Polyketide natural products play an important role in the treatment of a wide range of human physiological disorders and in animal health and agriculture. The production of Polyketides in heterologous hosts offers many advantages over the use of natural producers. Heterologous production systems can facilitate analysis of the catalytic properties of Polyketide producing enzymes. These systems can also have an impact on the ability to make Polyketide compounds from organisms that are difficult or impossible to culture. A number of factors must be considered in choosing a heterologous host. The host must be able to express relatively large Polyketide proteins (300kDa or larger), post-translationally modify these proteins, and produce adequate supplies of intracellular building blocks such as malonyl-CoA and methylmalonyl-CoA. For example, Streptomyces coelicolor has been used extensively to produce a variety of Polyketides. Recent progress in increasing intracellular supplies of Polyketide building blocks combined with the development of stable high copy number vectors has led to substantial increases in S. coelicolor Polyketide titers. Escherichia coli has also emerged as an alternative expression host. This bacterium was extensively engineered to create a suitable host for Polyketide production. E. coli has been used to produce a variety of Polyketide compounds including 6-deoxyerythronolide B, yersiniabactin, and epothilone. With continued improvements to these and other systems, heterologous hosts promise to become a robust platform for large-scale Polyketide production.
-
Engineered Biosynthesis of Regioselectively Modified Aromatic Polyketides Using Bimodular Polyketide Synthases
PLOS Biology, 2004Co-Authors: Yi Tang, Chaitan KhoslaAbstract:Bacterial aromatic Polyketides such as tetracycline and doxorubicin are a medicinally important class of natural products produced as secondary metabolites by actinomyces bacteria. Their backbones are derived from malonyl-CoA units by Polyketide synthases (PKSs). The nascent Polyketide chain is synthesized by the minimal PKS, a module consisting of four dissociated enzymes. Although the biosynthesis of most aromatic Polyketide backbones is initiated through decarboxylation of a malonyl building block (which results in an acetate group), some Polyketides, such as the estrogen receptor antagonist R1128, are derived from nonacetate primers. Understanding the mechanism of nonacetate priming can lead to biosynthesis of novel Polyketides that have improved pharmacological properties. Recent biochemical analysis has shown that nonacetate priming is the result of stepwise activity of two dissociated PKS modules with orthogonal molecular recognition features. In these PKSs, an initiation module that synthesizes a starter unit is present in addition to the minimal PKS module. Here we describe a general method for the engineered biosynthesis of regioselectively modified aromatic Polyketides. When coexpressed with the R1128 initiation module, the actinorhodin minimal PKS produced novel hexaketides with propionyl and isobutyryl primer units. Analogous octaketides could be synthesized by combining the tetracenomycin minimal PKS with the R1128 initiation module. Tailoring enzymes such as ketoreductases and cyclases were able to process the unnatural Polyketides efficiently. Based upon these findings, hybrid PKSs were engineered to synthesize new anthraquinone antibiotics with predictable functional group modifications. Our results demonstrate that (i) bimodular aromatic PKSs present a general mechanism for priming aromatic Polyketide backbones with nonacetate precursors; (ii) the minimal PKS controls Polyketide chain length by counting the number of atoms incorporated into the backbone rather than the number of elongation cycles; and (iii) in contrast, auxiliary PKS enzymes such as ketoreductases, aromatases, and cyclases recognize specific functional groups in the backbone rather than overall chain length. Among the anthracyclines engineered in this study were compounds with (i) more superior activity than R1128 against the breast cancer cell line MCF-7 and (ii) inhibitory activity against glucose-6-phosphate translocase, an attractive target for the treatment of Type II diabetes.
-
biosynthesis of Polyketides in heterologous hosts
Microbiology and Molecular Biology Reviews, 2001Co-Authors: Blaine A Pfeifer, Chaitan KhoslaAbstract:Polyketide natural products show great promise as medicinal agents. Typically the products of microbial secondary biosynthesis, Polyketides are synthesized by an evolutionarily related but architecturally diverse family of multifunctional enzymes called Polyketide synthases. A principal limitation for fundamental biochemical studies of these modular megasynthases, as well as for their applications in biotechnology, is the challenge associated with manipulating the natural microorganism that produces a Polyketide of interest. To ameliorate this limitation, over the past decade several genetically amenable microbes have been developed as heterologous hosts for Polyketide biosynthesis. Here we review the state of the art as well as the difficulties associated with heterologous Polyketide production. In particular, we focus on two model hosts, Streptomyces coelicolor and Escherichia coli. Future directions for this relatively new but growing technological opportunity are also discussed.
-
Generation of Polyketide libraries via combinatorial biosynthesis.
Trends in Biotechnology, 1996Co-Authors: Chaitan Khosla, Robert J. X. ZawadaAbstract:Polyketides are a family of structurally complex natural products that include a number of important pharmaceuticals. Motivated by the value of these natural products, there has been much research focused on developing guidelines for engineering Polyketide synthases (PKSs) to generate novel Polyketides. Recent studies have provided interesting insights into the enzymatic specificity of the Polyketide synthesis pathway, and have demonstrated that various PKSs can be genetically manipulated to synthesize 'unnatural' Polyketide natural products. In this article, we discuss the synthesis of Polyketides and Polyketide libraries by combinatorial biosynthesis.
Yi Tang - One of the best experts on this subject based on the ideXlab platform.
-
saccharomyces cerevisiae as a tool for mining studying and engineering fungal Polyketide synthases
Fungal Genetics and Biology, 2016Co-Authors: Carly M Bond, Yi TangAbstract:Small molecule secondary metabolites produced by organisms such as plants, bacteria, and fungi form a fascinating and important group of natural products, many of which have shown promise as medicines. Fungi in particular have been important sources of natural product Polyketide pharmaceuticals. While the structural complexity of these Polyketides makes them interesting and useful bioactive compounds, these same features also make them difficult and expensive to prepare and scale-up using synthetic methods. Currently, nearly all commercial Polyketides are prepared through fermentation or semi-synthesis. However, elucidation and engineering of Polyketide pathways in the native filamentous fungi hosts are often hampered due to a lack of established genetic tools and of understanding of the regulation of fungal secondary metabolisms. Saccharomyces cerevisiae has many advantages beneficial to the study and development of Polyketide pathways from filamentous fungi due to its extensive genetic toolbox and well-studied metabolism. This review highlights the benefits S. cerevisiae provides as a tool for mining, studying, and engineering fungal Polyketide synthases (PKSs), as well as notable insights this versatile tool has given us into the mechanisms and products of fungal PKSs.
-
expanding the structural diversity of Polyketides by exploring the cofactor tolerance of an inline methyltransferase domain
Organic Letters, 2013Co-Authors: Jaclyn M Winter, Grace Chiou, Ian R Bothwell, Wei Xu, Neil K Garg, Yi TangAbstract:A strategy for introducing structural diversity into Polyketides by exploiting the promiscuity of an in-line methyltransferase domain in a multidomain Polyketide synthase is reported. In vitro investigations using the highly-reducing fungal Polyketide synthase CazF revealed that its methyltransferase domain accepts the nonnatural cofactor propargylic Se-adenosyl-l-methionine and can transfer the propargyl moiety onto its growing Polyketide chain. This propargylated Polyketide product can then be further chain-extended and cyclized to form propargyl-α pyrone or be processed fully into the alkyne-containing 4′-propargyl-chaetoviridin A.
-
Engineered Polyketide biosynthesis and biocatalysis in Escherichia coli
Applied Microbiology and Biotechnology, 2010Co-Authors: Xue Gao, Peng Wang, Yi TangAbstract: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.
-
crystal structure and functional analysis of tetracenomycin aro cyc implications for cyclization specificity of aromatic Polyketides
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Brian D Ames, Tyler Paz Korman, T N Vu, P G R Smith, Yi Tang, Wenjun Zhang, Shiouchuan TsaiAbstract:Abstract Polyketides are a class of natural products with highly diverse chemical structures and pharmaceutical activities. Polyketide cyclization, promoted by the aromatase/cyclase (ARO/CYC), helps diversify aromatic Polyketides. How the ARO/CYC promotes highly specific cyclization is not well understood because of the lack of a first-ring ARO/CYC structure. The 1.9 A crystal structure of Tcm ARO/CYC reveals that the enzyme belongs to the Bet v1-like superfamily (or STAR domain family) with a helix–grip fold, and contains a highly conserved interior pocket. Docking, mutagenesis, and an in vivo assay show that the size, shape, and composition of the pocket are important to orient and specifically fold the Polyketide chain for C9-C14 first-ring and C7-C16 second-ring cyclizations. Two pocket residues, R69 and Y35, were found to be essential for promoting first- and second-ring cyclization specificity. Different pocket residue mutations affected the Polyketide product distribution. A mechanism is proposed based on the structure-mutation-docking results. These results strongly suggest that the regiospecific cyclizations of the first two rings and subsequent aromatizations take place in the interior pocket. The chemical insights gleaned from this work pave the foundation toward defining the molecular rules for the ARO/CYC cyclization specificity, whose rational control will be important for future endeavors in the engineered biosynthesis of novel anticancer and antibiotic aromatic Polyketides. aromatase cyclase natural product
-
enzymatic synthesis of aromatic Polyketides using pks4 from gibberella fujikuroi
Journal of the American Chemical Society, 2007Co-Authors: Suzanne M., Jixun Zhan, Kenji Watanabe, Xinkai Xie, Clay C. C. Wang, Wenjun Zhang, Kenji Watanabe, Clay C. C. Wang, Yi TangAbstract:Iterative fungal Polyketide synthases (PKSs) use a unique set of biochemical rules in the synthesis of complex Polyketides. These rules dictate Polyketide starter unit selection, chain length control, and post-PKS processing. We have demonstrated the E. coli expression and reconstitution of an iterative, unreduced fungal PKS. The Gibberella fujikuroi PKS4 was expressed at high levels, purified to homogeneity, and functionally characterized. In the presence of malonyl-CoA, PKS4 was able to synthesize the nonaketide 3,8,10,11-tetrahydroxy-1-methyl-12H-benzo[b]xanthen-12-one (2) as the predominant product. PKS4 selectively used octanoyl-CoA as the starter unit and synthesized two novel benzopyrone-containing Polyketides. Our work sets the stage for a comprehensive characterization of the intact PKS and its domains and offers significant opportunity toward the enzymatic synthesis of additional compounds.
Keqian Yang - One of the best experts on this subject based on the ideXlab platform.
-
Efficient production of Polyketide products in Streptomyces hosts - A review
Wei sheng wu xue bao = Acta microbiologica Sinica, 2016Co-Authors: Yongpeng Yao, Weishan Wang, Keqian YangAbstract:Polyketides represent an important class of structurally and functionally diverse secondary metabolites with high economic value. Among bacteria, Streptomycetes are the main producers of Polyketides. To enhance Polyketide production in Streptomyces hosts, rational metabolic engineering approaches have been applied, such as overexpressing rate-limiting enzymes, or transcriptional activator, increasing the supply of precursor, removing feedback inhibition by end products and heterologous expression of Polyketide biosynthetic gene clusters. In this review, we discuss examples of successful metabolic engineering strategies used to improve Polyketide production in Streptomycetes. Meanwhile, we also address future prospective, emerging synthetic biology strategies to dynamically adjust the metabolic fluxes of pathways related to Polyketide synthesis.
-
Localization of the ActIII actinorhodin Polyketide ketoreductase to the cell wall
FEMS Microbiology Letters, 2008Co-Authors: Zhijun Wang, Keqiang Fan, Shenglan Wang, Cui-juan Jia, Hui Han, Eswar Ramalingam, Keqian YangAbstract:Structurally diverse Polyketides provide a rich reservoir of bioactive molecules. Actinorhodin, a model aromatic Polyketide, is synthesized by minimal type II Polyketide synthase and tailoring enzymes. The ActIII actinorhodin ketoreductase is a key tailoring enzyme in actinorhodin biosynthesis. With purified antibodies against actinorhodin Polyketide synthase α subunit (KSα) and ketoreductase, we conducted systematic localization experiments of the two proteins in Streptomyces coelicolor subproteomes. The results support the membrane location of KSα and cell-wall location of ketoreductase. Considering previous evidence that some other tailoring enzymes of actinorhodin biosynthesis may be located outside the cytoplasm, a picture is emerging of an extensive role for extracellular biochemistry in the synthesis of type II Polyketide antibiotic.
-
functional analyses of oxygenases in jadomycin biosynthesis and identification of jadh as a bifunctional oxygenase dehydrase
Journal of Biological Chemistry, 2005Co-Authors: Yihua Chen, Chenchen Wang, Lisa Greenwell, Uwe Rix, Dirk Hoffmeister, Leo C. Vining, Jürgen Rohr, Keqian YangAbstract:A novel angucycline metabolite, 2,3-dehydro-UWM6, was identified in a jadH mutant of Streptomyces venezuelae ISP5230. Both UWM6 and 2,3-dehydro-UWM6 could be converted to jadomycin A or B by a ketosynthase alpha (jadA) mutant of S. venezuelae. These angucycline intermediates were also converted to jadomycin A by transformant of the heterologous host Streptomyces lividans expressing the jadFGH oxygenases in vivo and by its cell-free extracts in vitro; thus the three gene products JadFGH are implicated in catalysis of the post-Polyketide synthase biosynthetic reactions converting UWM6 to jadomycin aglycone. Genetic and biochemical analyses indicate that JadH possesses dehydrase activity, not previously associated with Polyketide-modifying oxygenase. Since the formation of aromatic Polyketides often requires multiple dehydration steps, bifunctionality of oxygenases modifying aromatic Polyketides may be a general phenomenon.
-
Functional Analyses of Oxygenases in Jadomycin Biosynthesis and Identification of JadH as a Bifunctional Oxygenase/Dehydrase
The Journal of biological chemistry, 2005Co-Authors: Yihua Chen, Chenchen Wang, Lisa Greenwell, Uwe Rix, Dirk Hoffmeister, Leo C. Vining, Jürgen Rohr, Keqian YangAbstract:A novel angucycline metabolite, 2,3-dehydro-UWM6, was identified in a jadH mutant of Streptomyces venezuelae ISP5230. Both UWM6 and 2,3-dehydro-UWM6 could be converted to jadomycin A or B by a ketosynthase alpha (jadA) mutant of S. venezuelae. These angucycline intermediates were also converted to jadomycin A by transformant of the heterologous host Streptomyces lividans expressing the jadFGH oxygenases in vivo and by its cell-free extracts in vitro; thus the three gene products JadFGH are implicated in catalysis of the post-Polyketide synthase biosynthetic reactions converting UWM6 to jadomycin aglycone. Genetic and biochemical analyses indicate that JadH possesses dehydrase activity, not previously associated with Polyketide-modifying oxygenase. Since the formation of aromatic Polyketides often requires multiple dehydration steps, bifunctionality of oxygenases modifying aromatic Polyketides may be a general phenomenon.
Stephen Mann - One of the best experts on this subject based on the ideXlab platform.
-
stabilization and enhanced reactivity of actinorhodin Polyketide synthase minimal complex in polymer nucleotide coacervate droplets
Chemical Communications, 2012Co-Authors: John Crosby, Tom Treadwell, Michelle Hammerton, Konstantinos Vasilakis, Matthew P. Crump, David S. Williams, Stephen MannAbstract:Compartmentalization of the minimal complex of actinorhodin Polyketide synthase in coacervate liquid droplets produces enhanced yields of shunt Polyketides under conditions of low and high ionic strength.
-
Stabilization and enhanced reactivity of actinorhodin Polyketide synthase minimal complex in polymer–nucleotide coacervate droplets
Chemical Communications, 2012Co-Authors: John Crosby, Tom Treadwell, Michelle Hammerton, Konstantinos Vasilakis, Matthew P. Crump, David S. Williams, Stephen MannAbstract:Compartmentalization of the minimal complex of actinorhodin Polyketide synthase in coacervate liquid droplets produces enhanced yields of shunt Polyketides under conditions of low and high ionic strength.
John Crosby - One of the best experts on this subject based on the ideXlab platform.
-
The Determinants of Activity and Specificity in Actinorhodin Type II Polyketide Ketoreductase
Chemistry & Biology, 2013Co-Authors: Pouya Javidpour, John Crosby, Matthew P. Crump, Michael D Burkart, Joel Bruegger, Supawadee Srithahan, Tyler P. Korman, Shiouchuan TsaiAbstract:Summary In the actinorhodin type II Polyketide synthase, the first Polyketide modification is a regiospecific C9-carbonyl reduction, catalyzed by the ketoreductase ( act KR). Our previous studies identified the act KR 94-PGG-96 motif as a determinant of stereospecificity. The molecular basis for reduction regiospecificity is, however, not well understood. In this study, we examined the activities of 20 act KR mutants through a combination of kinetic studies, PKS reconstitution, and structural analyses. Residues have been identified that are necessary for substrate interaction, and these observations have suggested a structural model for this reaction. Polyketides dock at the KR surface and are steered into the enzyme pocket where C7–C12 cyclization is mediated by the KR before C9-ketoreduction can occur. These molecular features can potentially serve as engineering targets for the biosynthesis of novel, reduced Polyketides.
-
stabilization and enhanced reactivity of actinorhodin Polyketide synthase minimal complex in polymer nucleotide coacervate droplets
Chemical Communications, 2012Co-Authors: John Crosby, Tom Treadwell, Michelle Hammerton, Konstantinos Vasilakis, Matthew P. Crump, David S. Williams, Stephen MannAbstract:Compartmentalization of the minimal complex of actinorhodin Polyketide synthase in coacervate liquid droplets produces enhanced yields of shunt Polyketides under conditions of low and high ionic strength.
-
Stabilization and enhanced reactivity of actinorhodin Polyketide synthase minimal complex in polymer–nucleotide coacervate droplets
Chemical Communications, 2012Co-Authors: John Crosby, Tom Treadwell, Michelle Hammerton, Konstantinos Vasilakis, Matthew P. Crump, David S. Williams, Stephen MannAbstract:Compartmentalization of the minimal complex of actinorhodin Polyketide synthase in coacervate liquid droplets produces enhanced yields of shunt Polyketides under conditions of low and high ionic strength.
