基于组合生物学强化Mycobacterium neoaurum降解植物甾醇侧链的代谢改造

As the most common steroid, 4-androstene-3, 17-dione (AD) and androst-1, 4-diene-3, 17-dione (ADD), are the most important steroidal intermediates for steroid drugs. Studies of steroid modifications catalyzed by microbial whole cells to AD/ADD have become a hot area for pharmaceutical and biotechnology industry. Previously, one Mycobacterium neoaurum JC-12 strain which is capable of transforming phytosterol into AD and ADD with conversion rate of 90% by isolation and mutatant breeding. The ADD production can reach 15.2 g/L with 20g/L phytosterol as substract. However, the low productivity of steroids form sterols seriously limits the commercial use of most sterol-transforming mycobacteria. This can be partially attributed to the low rate of the side chain degradation. The steroid oxidation and degradation process need lots of FAD/NAD as coenzyme. Alcanivorax borkumensis SK2’s most distinctive feature is its ability to grow efficiently and almost exclusively on alkanes. This project intends to introduce the efficient alkane degradation key enzymes cluster from A. borkumensis and intensify the degradation ability of phytosterol side chain in M.neoaurum JC-12, by using the combinatorial biology method. Furthermore, the CoQ synthesis key enzymes from Agrobacterium tumefaciens were cloned and heterologous expressed in M. neoaurum JC-12 in order to intensify the regeneration capacity of the FAD/NAD+ coenzymes. Both of these two strategies can enhance the productivity of AD/ADD from phytosterol significantly. The related research has not been reported.

雄甾-4-烯-3,17-二酮（AD）和雄甾-1,4-二烯-3,17-二酮（ADD）是生产甾体药物重要中间体。微生物转化法生产AD/ADD是研究热点。项目组前期选育获得一株高效转化植物甾醇为AD/ADD的新金色分枝杆菌JC-12，能转化20g/L底物合成15.2 g/L AD/ADD，为目前国内报道最高水平之一（ZL200810155130.7）。但存在甾醇侧链降解速率不高、降解周期长（6-7d）,侧链降解过程中消耗5分子FAD和2分子NAD+、导致氧化型辅酶供应不足和酶催化效率降低等瓶颈问题。项目拟采用组合生物学方法，先在底盘微生物新金色分枝杆菌中引入超级石油降解菌烷烃高效降解关键酶操纵子，在分枝杆菌特有的甾环氧化基础上强化其侧链降解；同时组合引入根癌农杆菌CoQ合成关键酶操纵子，强化其氧化型辅酶循环再生，整体提高新金色分支杆菌降解植物甾醇侧链合成AD/ADD的生产强度。相关研究未见报道。

项目对新金色分枝杆菌转化植物甾醇过程中的关键酶进行功能鉴定，利用组合生物学策略结合辅酶循环再生体系，实现了甾体药物中间体雄甾-4-烯-3,17-二酮（AD）、雄甾-1,4-二烯-3,17-二酮（ADD）、睾酮（TS）等的高效积累。得到如下结论：.1. 通过对诱变前后菌株3-甾酮-Δ1-脱氢酶（KSDD）氨基酸序列比对，发现氨基酸序列V366S的突变，该突变直接影响了KSDD活性。进一步对KSDD催化活性中心结构分析，发现V366S的突变能够减少底物与酶催化中心的空间位阻提高了KSDD的催化活性和稳定性，进而提高其酶活性。.2. 克隆并鉴定了新金色分枝杆菌的两个胆固醇氧化酶ChoM1和ChoM2，经纯化后对其酶学性质进行研究，结果表明和ChoM1相比，ChoM2对底物的亲和力更强，从而表现出更高的酶活性。转化胆固醇合成4-胆甾烯-3-酮对重组菌进行验证从而鉴定了ChoM在甾醇母核修饰中的功能。.3. 鉴定了来源于新金色分枝杆菌的三个SMO同工酶SMO1，SMO2和SMO3具有甾醇侧链末端氧化功能。对其进行异源表达与纯化，酶动力学分析表明SMO2对底物亲和性更强，催化效率更高。.4. 以新金色分枝杆菌JC-12为目的菌株，通过组合生物学，缺失KSH活性阻断降解途径，通过组合石油降解菌烷烃高效降解关键酶和根瘤农杆菌辅酶循环关键酶，增加甾醇代谢通量和氧化还原力进而提高产量。.5. 为了缩短发酵周期，提高ADD的生产效率，在5-L发酵罐上对重组菌株的发酵工艺进行优化。以果糖作为初始碳源，明显消除重组菌生长延滞期，然后结合多阶段发酵策略，最终ADD的生产效率从0.074 g/L/h提高到0.112 g/L/h。.本研究鉴定关键酶的功能并通过组合生物学和辅酶循环再生，获得了高产ADD等菌株。项目资助期间，相关研究成果发表在ACS Catalysis、Green Chemistry、Scientific Reports、Molecules、J Ind Microbiol Biotechnol等期刊（SCI论文14篇，其中IF>6论文3篇，单篇最高IF为12.221，累计IF>60），获授权中国发明专利3项，为工业化利用新金色分枝杆菌转化植物甾醇合成甾体药物中间体提供借鉴。