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Wang Y.-Y.,Zhejiang University | Bai L.F.,Zhejiang University | Ran X.-X.,Zhejiang University | Jiang X.-H.,Zhejiang University | And 7 more authors.
Protein and Peptide Letters | Year: 2015

Acyltransferases (ATs) play an essential role in the polyketide biosynthesis through transferring acyl units into acyl carrier proteins (ACPs) via a self-acylation reaction and a transacylation reaction. Here we used AT10FkbA of FK506 biosynthetic polyketide synthase (PKS) from Streptomyces tsukubaensis YN06 as a model to study the specificity of ATs for acyl units. Our results show that AT10FkbA can form both malonyl-O-AT10FkbA and methylmalonyl-O-AT10FkbA in the self-acylation reaction, however, only malonyl-O-AT10FkbA but not methylmalonyl-O-AT10FkbA can transfer the acyl unit into ACPs in the transacylation reaction. Unlike some ATs that are known to control the acyl specificity in self-acylation reactions, AT10FkbA controls the acyl specificity in transacylation reactions. © 2015 Bentham Science Publishers.

Wang Y.-Y.,Zhejiang University | Ran X.-X.,Zhejiang University | Chen W.-B.,Zhejiang University | Liu S.-P.,Zhejiang University | And 7 more authors.
FEBS Letters | Year: 2014

The known functions of type II thioesterases (TEIIs) in type I polyketide synthases (PKSs) include selecting of starter acyl units, removal of aberrant extender acyl units, releasing of final products, and dehydration of polyketide intermediates. In this study, we characterized two TEIIs (ScnI and PKSIaTEII) from Streptomyces chattanoogensis L10. Deletion of scnI in S. chattanoogensis L10 decreased the natamycin production by about 43%. Both ScnI and PKSIaTEII could remove acyl units from the acyl carrier proteins (ACPs) involved in the natamycin biosynthesis. Our results show that the TEII could play important roles in both the initiation step and the elongation steps of a polyketide biosynthesis; the intracellular TEIIs involved in different biosynthetic pathways could complement each other. © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Jiang H.,Zhejiang University | Jiang H.,Key Laboratory of Microbial Biochemistry and Metabolism Engineering of Zhejiang Province | Wang Y.-Y.,Zhejiang University | Guo Y.-Y.,Zhejiang University | And 7 more authors.
FEBS Journal | Year: 2015

Acyltransferase (AT) domains of polyketide synthases (PKSs) usually use coenzyme A (CoA) as an acyl donor to transfer common acyl units to acyl carrier protein (ACP) domains, initiating incorporation of acyl units into polyketides. Two clinical immunosuppressive agents, FK506 and FK520, are biosynthesized by the same PKSs in several Streptomyces strains. In this study, characterization of AT4FkbB (the AT domain of the fourth module of FK506 PKS) in transacylation reactions showed that AT4FkbB recognizes both an ACP domain (ACPTcsA) and CoA as acyl donors for transfer of a unique allylmalonyl (AM) unit to an acyl acceptor ACP domain (ACP4FkbB), resulting in FK506 production. In addition, AT4FkbB uses CoA as an acyl donor to transfer an unusual ethylmalonyl (EM) unit to ACP4FkbB, resulting in FK520 production, and transfers AM units to non-native ACP acceptors. Characterization of AT4FkbB in self-acylation reactions suggests that AT4FkbB controls acyl unit specificity in transacylation reactions but not in self-acylation reactions. Generally, AT domains of PKSs only recognize one acyl donor; however, we report here that AT4FkbB recognizes two acyl donors for the transfer of different acyl units. © 2015 FEBS.

Liu S.-P.,Zhejiang University | Yu P.,Zhejiang University | Yuan P.-H.,Zhejiang University | Zhou Z.-X.,Zhejiang University | And 5 more authors.
Applied Microbiology and Biotechnology | Year: 2015

The roles of many sigma factors are unclear in regulatory mechanism of secondary metabolism in Streptomyces. Here, we report the regulation network of a group 3 sigma factor, WhiGch, from a natamycin industrial strain Streptomyces chattanoogensis L10. WhiGch regulates the growth and morphological differentiation of S. chattanoogensis L10. The whiGch deletion mutant decreased natamycin production by about 30 % and delayed natamycin production more than 24 h by delaying the growth. Overexpression of the whiGch gene increased natamycin production in large scale production medium by about 26 %. WhiGch upregulated the transcription of natamycin biosynthetic gene cluster and inhibited the expression of migrastatin and jadomycin analog biosynthetic polyketide synthase genes. WhiGch positively regulated natamycin biosynthetic gene cluster by directly binding to the promoters of scnC and scnD, which were involved in natamycin biosynthesis, and these binding sites adjacent to translation start codon were determined. Thus, this paper further elucidates the high natamycin yield mechanisms of industrial strains and demonstrates that a valuable improvement in the yield of the target metabolites can be achieved through manipulating the transcription regulators. © 2015, Springer-Verlag Berlin Heidelberg.

Wang Y.-Y.,Zhejiang University | Li Y.-D.,Zhejiang GongShang University | Liu J.-B.,Zhejiang University | Ran X.-X.,Zhejiang University | And 7 more authors.
PLoS ONE | Year: 2014

Phosphopantetheinyl transferases (PPTases), which play an essential role in both primary and secondary metabolism, are magnesium binding enzymes. In this study, we characterized the magnesium binding residues of all known group II PPTases by biochemical and evolutionary analysis. Our results suggested that group II PPTases could be classified into two subgroups, two-magnesium-binding- residue-PPTases containing the triad Asp-Xxx-Glu and three-magnesium-binding- residue-PPTases containing the triad Asp-Glu-Glu. Mutations of two three-magnesium-binding-residue-PPTases and one two-magnesium-binding-residue- PPTase indicate that the first and the third residues in the triads are essential to activities; the second residues in the triads are non-essential. Although variations of the second residues in the triad Asp-Xxx-Glu exist throughout the whole phylogenetic tree, the second residues are conserved in animals, plants, algae, and most prokaryotes, respectively. Evolutionary analysis suggests that: the animal group II PPTases may originate from one common ancestor; the plant two-magnesium-binding-residue-PPTases may originate from one common ancestor; the plant three-magnesium-binding-residue-PPTases may derive from horizontal gene transfer from prokaryotes. © 2014 Wang et al.

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