Study On The Branched-chain Amino Acid Dehydrogenase Gene Of Streptomyces Roseosporus

Daptomycin has been the blockbuster drug for infection therapy caused by gram-positive pathogens especially the drug-resistant bacterica. Daptomycin (Cubicin) has been approved for clinic therapy for skin infection caused by gram-positive pathogens and endocarditis caused by Staphylococcus aureus (MRSA). Daptomycin is a membership of secondary metabolite A21978C, a lipopeptide complex mix produced by S.roseosporus. The A21978C complex shares a 13 amino acid chain bone consisting of a ten amino acid ring with three exocyclic residues. Daptomycin is isolated as a minor component of A21978C with a n-decanoyl tail linked to the terminal amino group of L-Trp,Which can be distinguished from 11-,12-or 13-carbon N-terminal branched-chain fatty acid tails of other three major memberships of A21978C. The daptomycin biosynthesis can be enriched by supplementing precursor decanoic acid during S.roseosporus fermentation.Branched chain fatty acids consist the major fatty acid in streptomyces. The acyl-CoA and its derivatives are thought to be the starter units of branched chain fatty acids, which play an important role in A21978C biosynthesis as precursor. Regulation metabolic pathways of fatty acid has an important significance for directional biosynthetic of A21978C.Branched-chain-keto acid dehydrogenase(BCDH) is a multisubunit enzyme complex that responsible for branched-chain amino acid catabolism. BCDH catalyze a-keto, which is derived from the transamination of branched-chain amino acid mediated by valine dehydrogenase, resulting the corresponding acyl-CoA derivatives.Branched-chain amino acid catabolism begins with valine dehydrogenase catalyzes the transamination of valine, leucine or isoleucine. There are two separate gene clusters encode BCDH in S.roseosporus through blast analysis. However, little known about the function of BCDH in S.roseosporus.We speculate that BCDH in S. roseosporus NRRL 15998 may supply fatty acids for the biosynthesis of A21978C mix including daptomycin. Thus, intensive study of BCDH has a significant impact on daptomycin biosynthesis in S. roseosporus through metabolic engineering approach, which would be a key to enhanced production of daptomcyin.Objective:This work focus on exploring the mechanism of the branched-chain-keto acid dehydrogenase (BCDH) and its impact on growth of S. roseosporus NRRL 15998 as well as daptomycin biosynthesis.Methods:1. Primers were designed for cloning genes encode BCDH from the S. roseosporus genome and these genes were analyzed by bioinformation analysis. Then, the transcriptional level of bkdl and bkd2 in wide type S. roseosporus were measured.2. In order to investigate the role of bkdl and bkd2 in S. roseosporus. The deletion mutant bkd1DM and bkd2DM were constructed. At the same time, bkdl overexpression strains(OM) and complemention strain(CM) were constructed. Further experiments were focus on the membrance fatty acid composition and branched-chain amino acid catabolism in vivo.3. In order to investigate the relationship between gene bkd and A21978C biosynthesis, the antibiotics production of WT, DM, CM and OM were measured through bioassay after fermentation. Besides, the role of BCDH in decanoic acid tolerance were test.Results:1. Two separate gene clusters encode BCDH were successfully cloned from S.roseosporus NRRL 15998. Here, bkdl corresponds to bkdA1B1C1 and bkd2 corresponds to bkdA2B2C2 in Streptomyces coelicolor. Each cluster contains E1α, E1β and E2 subunits. The bkd2 transcript was hardly detected relative to bkdl transcript in wide type S. roseosporus during fermentation.2. The deletion mutant bkd1DM and bkd2DM were comfirmed. At the same time, bkd1 overexpression strains (OM) with corresponding control strains and complemention strain (CM) were characterized. The knockout mutant of bkdl resulting in defective in branched chain fatty acids (BCFAs) and the phenotype of cannot utilize branch-chain amino acid as sole carbon source. These results showed Bkdl execute BCDH activity in S. roseosporus.3. The A21978C production of bkd1DM was dramatically decreased and could hardly detected. After complemention, strain restored production partly by 61.5%. The production of bkd2 DM was 34.5% lower than WT. A21978C production was increased 207.2% and 94.8% compared to the control after overexpression of bkd1 under two promoters respectively. Bkd1 mutant decreased decanoic acid tolerance, while bkd2DM showed no difference with WT.Conclusion:Two separate gene clusters encode BCDH exist in S. roseosporus NRRL 15998 and only Bkd1 is responsible for branched-chain amino acid catabolism. BCDH is required for A21978C production as the provider of precursor fatty acid by mediating branched-chain amino acid metabolize.