Cui Qianqian, Zhou Fengli, Liu Weifeng, Tao Yong
CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, No. 1-3, Beichenxi Road, Chaoyang District, Beijing, 100101, China.
University of Chinese Academy of Sciences, Beijing, 100049, China.
Biotechnol Lett. 2017 Oct;39(10):1567-1574. doi: 10.1007/s10529-017-2389-z. Epub 2017 Jun 29.
To improve the production of short branched-chain acyl-CoAs for avermectin biosynthesis, the functional expression of the branched chain α-keto acid dehydrogenase complex (BKDH) from Streptomyces avermitilis was systematically optimized by selectively regulating individual subunit expression in Escherichia coli.
Functional expression of the BKDH complex was achieved by independent and selective optimization of individual subunit genes of the complex. Codon optimization significantly improved the expression of complex component proteins BkdH and LpdA1 but expression of BkdF and BkdG depended on coexpression of the bkdH gene. The optimized BKDH complex supplied sufficient short branched-chain acyl-CoA to synthesize phlorisovalerophenone, a key intermediate in bitter acid (humulone) synthesis. We also developed a novel p15A origin-derived high-copy-number vector system for expression; the yield of PIVP was 350 ng/mOD.
Through optimization strategies, we obtained stable, functional expression of the BKDH complex in E. coli, which could be applied in the heterologous production of numerous high-value-added chemicals, especially polyketides.
为提高阿维菌素生物合成中短链支链酰基辅酶A的产量，通过在大肠杆菌中选择性调控单个亚基的表达，对阿维链霉菌的支链α-酮酸脱氢酶复合体（BKDH）的功能表达进行了系统优化。
通过对复合体单个亚基基因进行独立且选择性的优化，实现了BKDH复合体的功能表达。密码子优化显著提高了复合体组成蛋白BkdH和LpdA1的表达，但BkdF和BkdG的表达依赖于bkdH基因的共表达。优化后的BKDH复合体提供了足够的短链支链酰基辅酶A来合成间苯二酚异戊酮，这是苦味酸（蛇麻酮）合成中的关键中间体。我们还开发了一种新型的源自p15A的高拷贝数表达载体系统；间苯二酚异戊酮的产量为350 ng/mOD。
通过优化策略，我们在大肠杆菌中获得了BKDH复合体稳定的功能表达，可应用于多种高附加值化学品，尤其是聚酮化合物的异源生产。
