Institute of Pharmaceutical Biotechnology, Zhejiang University, Hangzhou, 310058, China.
Zhejiang Provincial Key Laboratory for Microbial Biochemistry and Metabolic Engineering, Hangzhou, 310058, China.
J Ind Microbiol Biotechnol. 2018 Apr;45(4):271-279. doi: 10.1007/s10295-018-2011-y. Epub 2018 Feb 7.
Daptomycin is a cyclic lipopeptide antibiotic produced by Streptomyces roseosporus in an acidic peptide complex A21978C. In this complex, A21978C is most abundant and contains branched-chain fatty acyl groups, while daptomycin has a straight decanoic acyl group. The branched-chain α-keto acid dehydrogenase complex (BCDH complex), encoded by bkd gene clusters in Streptomyces, is responsible for the early step of converting branched-chain amino acids into branched-chain fatty acids. In a daptomycin industrial producer S. roseosporus L30, two alleles of bkd gene clusters, bkdA1B1C1/bkdA2B2C2, and a regulatory gene bkdR located upstream of bkdA2B2C2 are identified. We show that BkdR positively regulated bkdA2B2C2 expression and was negatively auto-regulated, but is not directly involved in regulation of daptomycin gene cluster expression. However, BkdR is required for both daptomycin and A21978C production. Furthermore, deletion of bkdA2B2C2 only led to partial reduction of A21978C production, while the ΔbkdA1B1C1 mutant shows very weak production of A21978C, and the double bkd mutant has a similar production profile as the single ΔbkdA1B1C1 mutant, suggesting that bkdA1B1C1 gene cluster plays a dominant role in branched-chain fatty acid biosynthesis. So we reveal a unique regulatory function of BkdR and genetic engineered a bkd null strain for daptomycin production with reduced impurities.
达托霉素是一种由玫瑰孢链霉菌产生的环状脂肽抗生素，存在于酸性肽复合物 A21978C 中。在该复合物中，A21978C 最为丰富，含有支链脂肪酸酰基，而达托霉素则具有直链癸酰基。支链 α-酮酸脱氢酶复合物（BCDH 复合物）由链霉菌中的 bkd 基因簇编码，负责将支链氨基酸转化为支链脂肪酸的早期步骤。在达托霉素工业生产菌玫瑰孢链霉菌 L30 中，鉴定出两个 bkd 基因簇等位基因 bkdA1B1C1/bkdA2B2C2 和位于 bkdA2B2C2 上游的调控基因 bkdR。我们表明，BkdR 正向调控 bkdA2B2C2 的表达，并受到负向自身调控，但不直接参与达托霉素基因簇表达的调控。然而，BkdR 既参与达托霉素的产生，也参与 A21978C 的产生。此外，缺失 bkdA2B2C2 仅导致 A21978C 产量部分降低，而 ΔbkdA1B1C1 突变体几乎不产生 A21978C，双 bkd 突变体的产生谱与单 ΔbkdA1B1C1 突变体相似，表明 bkdA1B1C1 基因簇在支链脂肪酸生物合成中起主导作用。因此，我们揭示了 BkdR 的独特调控功能，并通过遗传工程构建了一个 bkd 缺失株，用于生产达托霉素，减少杂质。
