Production of a polyketide natural product in nonpolyketide-producing prokaryotic and eukaryotic hosts
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The polyketides are a diverse group of natural products with great significance as human and veterinary pharmaceuticals. A significant barrier to the production of novel genetically engineered polyketides has been the lack of available heterologous expression systems for functional polyketide synthases (PKSs). Herein, we report the expression of an intact functional PKS in Escherichia coli and Saccharomyces cerevisiae . The fungal gene encoding 6-methylsalicylic acid synthase from Penicillium patulum was expressed in E. coli and S. cerevisiae and the polyketide 6-methylsalicylic acid (6-MSA) was produced. In both bacterial and yeast hosts, polyketide production required coexpression of 6-methylsalicylic acid synthase and a heterologous phosphopantetheinyl transferase that was required to convert the expressed apo-PKS to its holo form. Production of 6-MSA in E. coli was both temperature- and glycerol-dependent and levels of production were lower than those of P. patulum , the native host. In yeast, however, 6-MSA levels greater than 2-fold higher than the native host were observed. The heterologous expression systems described will facilitate the manipulation of PKS genes and consequent production of novel engineered polyketides and polyketide libraries.Keywords:
Polyketide synthase
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Acyl carrier protein
A unique highly reducing polyketide synthase (HR-PKS) with a reductase domain was identified in a betaenone biosynthetic gene cluster. Successful heterologous expression and characterization of the HR-PKS and trans-acting enoyl reductase (ER) provide insights into the core structure formation with a decalin scaffold and allow reconstitution of the betaenone biosynthetic machinery.
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Leinamycin (LNM) is biosynthesized by a hybrid nonribosomal peptide synthetase (NRPS)–acyltransferase (AT)-less type I polyketide synthase (PKS). Characterization of LnmI revealed ketosynthase (KS)–acyl carrier protein (ACP)–KS domains at the NRPS–PKS interface. Inactivation of the KS domain or ACP domain in vivo abolished LNM production, and the ACP domain can be phosphopantetheinylated in vitro. The LnmI KS–ACP–KS architecture represents a new mechanism for functional crosstalk between NRPS and AT-less type I PKS in the biosynthesis of hybrid peptide–polyketide natural products.
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The biosynthetic gene cluster of the fungal meroterpenoid chrodrimanin B (4) was discovered in Penicillium verruculosum TPU1311, and the complete biosynthetic pathway of 4 has been elucidated by heterologous reconstitution of its biosynthesis in Aspergillus oryzae, as well as by in vitro characterizations of selected enzymes. The present study has identified the polyketide synthase that produces 6-hydroxymellein (3) and also provided a biosynthetic platform of chrodrimanins for further bioengineering.
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Thioesterase
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Abstract The enzymes that comprise type II polyketide synthases (PKSs) are powerful biocatalysts that, once well-understood and strategically applied, could enable cost-effective and sustainable access to a range of pharmaceutically relevant molecules. Progress toward this goal hinges on gaining ample access to materials for in vitro characterizations and structural analysis of the components of these synthases. A central component of PKSs is the acyl carrier protein (ACP), which serves as a hub during the biosynthesis of type II polyketides. Herein, we share methods for accessing type II PKS ACPs via heterologous expression in E. coli . We also share how the installation of reactive and site-specific spectroscopic probes can be leveraged to study the conformational dynamics and interactions of type II PKS ACPs.
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Phenolic glycolipids (PGLs) are polyketide synthase-derived glycolipids unique to pathogenic mycobacteria. PGLs are found in several clinically relevant species, including various Mycobacterium tuberculosis strains, Mycobacterium leprae, and several nontuberculous mycobacterial pathogens, such as M. marinum. Multiple lines of investigation implicate PGLs in virulence, thus underscoring the relevance of a deep understanding of PGL biosynthesis. We report mutational and biochemical studies that interrogate the mechanism by which PGL biosynthetic intermediates (p-hydroxyphenylalkanoates) synthesized by the iterative polyketide synthase Pks15/1 are transferred to the noniterative polyketide synthase PpsA for acyl chain extension in M. marinum. Our findings support a model in which the transfer of the intermediates is dependent on a p-hydroxyphenylalkanoyl-AMP ligase (FadD29) acting as an intermediary between the iterative and the noniterative synthase systems. Our results also establish the p-hydroxyphenylalkanoate extension ability of PpsA, the first-acting enzyme of a multisubunit noniterative polyketide synthase system. Notably, this noniterative system is also loaded with fatty acids by a specific fatty acyl-AMP ligase (FadD26) for biosynthesis of phthiocerol dimycocerosates (PDIMs), which are nonglycosylated lipids structurally related to PGLs. To our knowledge, the partially overlapping PGL and PDIM biosynthetic pathways provide the first example of two distinct, pathway-dedicated acyl-AMP ligases loading the same type I polyketide synthase system with two alternate starter units to produce two structurally different families of metabolites. The studies reported here advance our understanding of the biosynthesis of an important group of mycobacterial glycolipids.
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Polyketide synthase
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Escherichia
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The polyketides are a diverse group of natural products with great significance as human and veterinary pharmaceuticals. A significant barrier to the production of novel genetically engineered polyketides has been the lack of available heterologous expression systems for functional polyketide synthases (PKSs). Herein, we report the expression of an intact functional PKS in Escherichia coli and Saccharomyces cerevisiae . The fungal gene encoding 6-methylsalicylic acid synthase from Penicillium patulum was expressed in E. coli and S. cerevisiae and the polyketide 6-methylsalicylic acid (6-MSA) was produced. In both bacterial and yeast hosts, polyketide production required coexpression of 6-methylsalicylic acid synthase and a heterologous phosphopantetheinyl transferase that was required to convert the expressed apo-PKS to its holo form. Production of 6-MSA in E. coli was both temperature- and glycerol-dependent and levels of production were lower than those of P. patulum , the native host. In yeast, however, 6-MSA levels greater than 2-fold higher than the native host were observed. The heterologous expression systems described will facilitate the manipulation of PKS genes and consequent production of novel engineered polyketides and polyketide libraries.
Polyketide synthase
Heterologous expression
Heterologous
Acyl carrier protein
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Bacterial aromatic polyketides are important therapeutic compounds including front line antibiotics and anticancer drugs. It is one of the last remaining major classes of natural products of which the biosynthesis has not been reconstituted in the genetically superior host Escherichia coli. Here, we demonstrate the engineered biosynthesis of bacterial aromatic polyketides in E. coli by using a dissected and reassembled fungal polyketide synthase (PKS). The minimal PKS of the megasynthase PKS4 from Gibberella fujikuroi was extracted by using two approaches. The first approach yielded a stand-alone Ketosynthase (KS)_malonyl-CoA:ACP transferase (MAT) didomain and an acyl-carrier protein (ACP) domain, whereas the second approach yielded a compact PKS (PKS_WJ) that consists of KS, MAT, and ACP on a single polypeptide. Both minimal PKSs produced nonfungal polyketides cyclized via different regioselectivity, whereas the fungal-specific C2-C7 cyclization mode was not observed. The kinetic properties of the two minimal PKSs were characterized to confirm both PKSs can synthesize polyketides with similar efficiency as the parent PKS4 megasynthase. Both minimal PKSs interacted effectively with exogenous polyketide cyclases as demonstrated by the synthesis of predominantly PK8 3 or NonaSEK4 6 in the presence of a C9-C14 or a C7-C12 cyclase, respectively. When PKS_WJ and downstream tailoring enzymes were expressed in E. coli, the expected nonaketide anthraquinone SEK26 was recovered in good titer. High-cell density fermentation was performed to demonstrate the scale-up potential of the in vivo platform for the biosynthesis of bacterial polyketides. Using engineered fungal PKSs can therefore be a general approach toward the heterologous biosynthesis of bacterial aromatic polyketides in E. coli.
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Journal Article Construction and performance of heterologous polyketide‐producing K‐12‐ and B‐derived Escherichia coli Get access J. Wu, J. Wu State Key Laboratory of Bioreactor Engineering, National Engineering Research Center for Biotechnology, East China University of Science & Technology, Shanghai, China Department of Chemical & Biological Engineering, Science & Technology Center, Tufts University, Medford, MA, USA Search for other works by this author on: Oxford Academic Google Scholar B.A. Boghigian, B.A. Boghigian Department of Chemical & Biological Engineering, Science & Technology Center, Tufts University, Medford, MA, USA Search for other works by this author on: Oxford Academic Google Scholar M. Myint, M. Myint Department of Chemical & Biological Engineering, Science & Technology Center, Tufts University, Medford, MA, USA Search for other works by this author on: Oxford Academic Google Scholar H. Zhang, H. Zhang Department of Chemical & Biological Engineering, Science & Technology Center, Tufts University, Medford, MA, USA Search for other works by this author on: Oxford Academic Google Scholar S. Zhang, S. Zhang State Key Laboratory of Bioreactor Engineering, National Engineering Research Center for Biotechnology, East China University of Science & Technology, Shanghai, China Search for other works by this author on: Oxford Academic Google Scholar B.A. Pfeifer B.A. Pfeifer Department of Chemical & Biological Engineering, Science & Technology Center, Tufts University, Medford, MA, USA Blaine A. Pfeifer, Department of Chemical & Biological Engineering, Science & Technology Center, Tufts University, 4 Colby Street, Medford, MA 02155, USA. E‐mail: blaine.pfeifer@tufts.edu Search for other works by this author on: Oxford Academic Google Scholar Letters in Applied Microbiology, Volume 51, Issue 2, 1 August 2010, Pages 196–204, https://doi.org/10.1111/j.1472-765X.2010.02880.x Published: 01 August 2010 Article history Received: 24 March 2010 Revision received: 26 May 2010 Accepted: 27 May 2010 Published: 01 August 2010
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