Key Laboratory of Microbial Biochemistry and Metabolism Engineering of Zhejiang Province

Hangzhou, China

Key Laboratory of Microbial Biochemistry and Metabolism Engineering of Zhejiang Province

Hangzhou, China
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Mao X.-M.,Zhejiang University | Mao X.-M.,Key Laboratory of Microbial Biochemistry and Metabolism Engineering of Zhejiang Province | Mao X.-M.,University of California at Los Angeles | Ren N.-N.,Zhejiang University | And 11 more authors.
FEBS Letters | Year: 2014

In Streptomyces coelicolor, the ECF sigma factor SigT negatively regulates cell differentiation, and is degraded by ClpP protease in a dual positive feedback manner. Here we further report that the proteasome is required for degradation of SigT, but not for degradation of its anti-sigma factor RstA, and RstA can protect SigT from degradation independent of the proteasome. Meanwhile, deletion of the proteasome showed reduced production of secondary metabolites, and the fermentation medium from wild type could promote SigT degradation. Furthermore, overexpression of redD or actII-orf4 in the proteasome-deficiency mutant resulted in SigT degradation and over-production of both undecylprodigiosin and actinorhodin. Therefore the proteasome is required for SigT degradation by affecting the production of secondary metabolites during cell differentiation. © 2014 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.


Wang Y.-Y.,Zhejiang University | Zhang X.-S.,Zhejiang University | Ren N.-N.,Zhejiang University | Guo Y.-Y.,Zhejiang University | And 5 more authors.
Current Microbiology | Year: 2014

It is known that bacterial group II phosphopantetheinyl transferases (PPTases) usually phosphopantetheinylate acyl carrier proteins (ACPs) involved in the secondary metabolism. For example, a bacterial group II PPTase SchPPT has been known to phosphopantetheinylate only ACPs involved in secondary metabolism, such as scn ACP0-2 and scn ACP7. In this study, we found two bacterial group II PPTases, Hppt and Sppt, could phosphopantetheinylate not only scn ACP0-2 and scn ACP7, but also sch FAS ACP, an ACP involved in primary metabolism. Swapping of the N terminus and C terminus of PPTases showed that (i) both the hybrids Hppt-Sppt and Sppt-Hppt could phosphopantetheinylate sch FAS ACP but not scn ACP0-2; (ii) both the hybrids Sppt-SchPPT and SchPPT-Sppt lost abilities to phosphopantetheinylate sch FAS ACP and scn ACP0- 2. Hppt and Sppt represent group II PPTases which phosphopantetheinylate both ACPs involved in primary metabolism and ACPs involved in secondary metabolism. © 2014, Springer Science+Business Media New York.


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.


Jiang H.,Zhejiang University | Jiang H.,Key Laboratory of Microbial Biochemistry and Metabolism Engineering of Zhejiang Province | Wang Y.-Y.,Zhejiang University | Ran X.-X.,Zhejiang University | And 6 more authors.
Applied and Environmental Microbiology | Year: 2013

Phosphopantetheinyl transferases (PPTases) are essential to the activities of type I/II polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) through converting acyl carrier proteins (ACPs) in PKSs and peptidyl carrier proteins (PCPs) in NRPSs from inactive apo-forms into active holo-forms, leading to biosynthesis of polyketides and nonribosomal peptides. The industrial natamycin (NTM) producer, Streptomyces chattanoogensis L10, contains two PPTases (SchPPT and SchACPS) and five PKSs. Biochemical characterization of these two PPTases shows that SchPPT catalyzes the phosphopantetheinylation of ACPs in both type I PKSs and type II PKSs, SchACPS catalyzes the phosphopantetheinylation of ACPs in type II PKSs and fatty acid synthases (FASs), and the specificity of SchPPT is possibly controlled by its C terminus. Inactivation of SchPPT in S. chattanoogensis L10 abolished production of NTM but not the spore pigment, while overexpression of the SchPPT gene not only increased NTM production by about 40% but also accelerated productions of both NTM and the spore pigment. Thus, we elucidated a comprehensive phosphopantetheinylation network of PKSs and improved polyketide production by engineering the cognate PPTase in bacteria. © 2013, American Society for Microbiology.


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.


Mao X.-M.,Zhejiang University | Mao X.-M.,Key Laboratory of Microbial Biochemistry and Metabolism Engineering of Zhejiang Province | Sun N.,Zhejiang University | Sun N.,Key Laboratory of Microbial Biochemistry and Metabolism Engineering of Zhejiang Province | And 11 more authors.
Journal of Biological Chemistry | Year: 2013

Here we report that in Streptomyces coelicolor, the protein stability of an ECF σfactor SigT, which is involved in the negative regulation of cell differentiation, was completely dependent on its cognate anti-σ factor RstA. The degradation of RstA caused a ClpP/SsrA-dependent degradation of SigT during cell differentiation. This was consistent with the delayed morphological development or secondary metabolism in the ΔclpP background after rstA deletion or sigT overexpression. Meanwhile, SigT negatively regulated clpP/ssrA expression by directly binding to the clpP promoter (clpPp). The SigT-clpPp interaction could be disrupted by secondary metabolites, giving rise to the stabilized SigT protein and retarded morphological development in a non-antibiotic-producing mutant. Thus a novel regulatory mechanism was revealed that the protein degradation of the ECF σfactor was initiated by the degradation of its anti-σfactor, and was accelerated in a dual positive feedback manner, through regulation by secondary metabolites, to promote rapid and irreversible development of the secondary metabolism. This ingeniouscooperationofintracellularcomponentscanensureeconomical and exquisite control of the ECF σfactor protein level for the proper cell differentiation in Streptomyces. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.


Wang F.,Zhejiang University | Wang F.,Key Laboratory of Microbial Biochemistry and Metabolism Engineering of Zhejiang Province | Ren N.-N.,Zhejiang University | Ren N.-N.,Key Laboratory of Microbial Biochemistry and Metabolism Engineering of Zhejiang Province | And 8 more authors.
Gene | Year: 2014

Daptomycin, a novel cyclic lipopeptide antibiotic against Gram-positive bacteria, is produced by Streptomyces roseosporus. Though its biosynthetic mechanism, structural shuffling and fermentation optimization have been extensively studied, little is understood about its production regulation at the transcriptional levels. Here we reported that dptR2, encoding a DeoR-type regulator located close to the daptomycin biosynthesis gene cluster in S. roseosporus SW0702, is required for daptomycin production, but not for the expression of daptomycin gene cluster, suggesting that DptR2 was not a pathway-specific regulator. Furthermore, EMSA and qRT-PCR analysis suggested that DptR2 was positively auto-regulated by binding to its own promoter. Meanwhile, the binding sites on the dptR2 promoter were determined by a DNase I footprinting assay, and the essentiality of the inverted complementary sequences in the protected region for DptR2 binding was assessed. Our results for the first time reported the regulation of daptomycin production at the transcriptional level in S. roseosporus. © 2014 Elsevier B.V.


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.


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.

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