新金色分枝杆菌转化植物甾醇合成二去甲胆烯醛的研究

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.

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。

本项目以新金色分枝杆菌（Mycolicibacterium neoaurum）DW01为研究对象，探究了M. neoaurum DW01转化植物甾醇合成甾体药物中间体22-羟基-23,24-二去胆甾-4-烯-3-酮（1,4-HBC）的机制和影响因素，建立了该菌的基因编辑方法和工具，并通过基因工程改造和培养条件优化有效提高了1,4-HBC产量。主要结论如下：（1）建立了基于O/W乳化的1,4-HBC发酵体系，该体系可有效提高植物甾醇在液相中的分散和促进1,4-HBC的生物合成；（2）通过不同条件下菌株的发酵特性和转录组分析，梳理推测了1,4-HBC的合成过程和影响因素，结果显示1,4-HBC的合成过程与已报道的胆固醇代谢途径密切相关，并受到辅酶NAD+合成和再生、细胞壁组成和细胞膜壁通透性、乙酰-CoA和丙酰-CoA代谢等因素的影响；（3）基于1,4-HBC代谢过程梳理和转录组分析构建了系列重组菌株，结果显示基因choM2、cyp125、mmpL5-mmpS5、IC40_RS0112640-IC40_RS0112645、greAB和nox的表达可有效提高菌株的1,4-HBC产量；其中IC40_RS0112640-IC40_RS0112645可能与脂质代谢有关，其表达对1,4-HBC产量提升最为显著，达到1.32 g/L；（4）基于同源重组-Cre/loxLR特异位点重组开发了分枝杆菌基因敲除工具，与传统p2NIL/pGOAL19敲除系统相比，可有效减少筛选的工作量和提高敲除效率；（5）探究了1,4-HBC合成过程，提示M. neoaurum DW01中1,4-HBC应该主要是通过羟醛裂解的方式合成的，Ltp3和Ltp4在该过程中具有关键的作用，以及Ltp2、IC40_RS0112640和IC40_RS0112645可能是该过程的重要参与者。