新金色分枝杆菌转化植物甾醇合成二去甲胆烯醛的研究

二去甲胆烯醛（Bisnorcholenaldehyde，BA）是黄体酮等甾体药物合成的优良中间体。本项目前期筛选得到能以廉价易得的植物甾醇为原料合成BA新金色分枝杆菌（Mycobacterium neoaurum）DW01。生物转化植物甾醇合成BA的过程可分为母核修饰和侧链降解两部分，目前母核修饰机制较明确，而侧链降解机制尚不清晰。本项目拟将M. neoaurum DW01基因组和转录组数据与该菌对不同侧链类型甾体的降解特征进行综合分析，理论预测BA合成途经和关键基因；对预测的关键基因，通过考察单个或多个基因敲除和表达对M. neoaurum DW01甾体降解特征的影响，及测定基因编码蛋白的酶学性质，实验分析BA合成关键基因和途经；在BA合成途径研究基础上，运用多种策略对菌株进行代谢工程改造和对发酵工艺进行优化，以提高M. neoaurum DW01的BA产量。

Bisnorcholenaldehyde (BA) is an important intermediate for the synthesis of some steroid hormone drugs, like progesterone. In our previous researches, Mycobacterium neoaurum strain DW01 that could biotransform the low-cost phytosterol to BA was isolated. The biosynthesis pathway of BA from phytosterol can be separated into the modification of steroid ring and the degradation of side chain. According to the previous studies, the mechanism of steroid ring modification was clear, while the process for how the side-chain of phytosterol was biodegraded to the side-chain of BA was still unknown. In this study, the pathway and key genes for BA biosynthesis in M. neoaurum DW01 will be speculated in theory, by genome and transcriptome sequencing, as well as the characterization of the strains on biodegradation of steroids with different types of side-chain. Subsequently, in order to experimentally understand the functions of theoretically speculated key genes in the BA biosynthesis pathway, the effects of single or multiple deletions or expressions of speculated gene on the steroid biodegradation of M. neoaurum DW01 will be investigated and the properties of enzymes coded by the genes will be characterized. After the understanding of the BA biosynthesis pathway, multiple strategies on metabolic engineering and fermentation optimization will be employed to enhance the BA production in M. neoaurum DW01.