大肠杆菌异源合成光学纯(R,R)-2,3-丁二醇的代谢途径组装与人工调控研究

In the previous NSFC Youth Foundation, the applicant has successfully optimized the physiological function of 2,3-butanediol producing microbe by cofactor metabolic engineering. In the present proposal, the applicant intend to use the emerging synthetic biology method to optimize the microbial metabolism in related to 2,3-butanediol fermentation based on the previous achievement. A synthetic metabolic pathway of enantiomerically pure (R, R)-2,3-butanediol is intended to be constructed in Escherichia coli. The synthetic metabolic pathways harboring promoters with different strength were first constructed quickly using the DNA assembler technology. This would be beneficial for regulating the enzyme expression level accurately. Then the selected most efficient synthetic metabolic pathway will be integrated into the E. coli chromosome, fulfilling the expression of exogenous genes coordinately. The accurate regulation upon promoter combinations is supposed to optimize the adaptation between artificial regulatory elements and functional modules as well as that between functional modules and host cell. The object is to enhance the (R, R)-2,3-butanediol synthetic ability, and analyze the relationship and adaption mechanism between the synthetic metabolic pathway and the host. At the same time, in the present proposal, the artificial synthetic pathway would be integrated into the chromosome via the two-step homologous recombination method, thus, E. coli can be assigned the ability of stable enantiomerically pure (R, R)-2,3-butanediol production, and the physiological function of the synthetic metabolic pathway based on the module assembly may also be dissected.

围绕青年基金中采用辅酶工程优化产2,3-丁二醇微生物生理功能取得的进展，以生物合成光学纯(R,R)-2,3-丁二醇为研究对象，针对天然微生物不能专一合成光学纯(R,R)-2,3-丁二醇的难题，拟进一步采用合成生物学方法，在大肠杆菌中人工构建专一合成光学纯(R,R)-2,3-丁二醇的代谢途径。首先，基于DNA assembler技术一步组装并筛选不同强度启动子组合的(R,R)-2,3-丁二醇合成途径，通过将人工构建的代谢途径中每个酶的表达量加以限制并精确调控，实现最优的多基因联合协同表达；期望通过启动子精细调控，提高(R,R)-2,3-丁二醇合成效率的同时，剖析该人工合成代谢途径与宿主细胞的互作关系及适配机制。其次，通过两步同源重组方法实现人工合成代谢途径在染色体上精确敲入，赋予大肠杆菌稳定专一合成(R,R)-2,3-丁二醇代谢功能的同时，剖析这种基于模块组装获得的代谢途径的生理学功能。

以生物合成光学纯(R, R)-2,3-丁二醇为研究对象，针对天然微生物不能专一合成光学纯(R, R)-2,3-丁二醇的难题，采用合成生物学方法，在大肠杆菌中人工构建了专一合成光学纯(R, R)-2,3-丁二醇的代谢途径，通过启动子工程的手段实现了最优的多基因联合协同表达，提高了(R, R)-2,3-丁二醇合成效率的同时，剖析该人工合成代谢途径与宿主细胞的适配机制。并通过两步同源重组方法实现了人工合成代谢途径在染色体上精确敲入，赋予了大肠杆菌稳定专一合成(R, R)-2,3-丁二醇代谢功能的同时，剖析了这种基于模块组装获得的代谢途径的生理学功能。项目总体研究进展基本按照计划进行，利用合成生物学的技术手段在大肠杆菌中建立了(R, R)-2,3-丁二醇专一合成途径，使该化合物的产量达到了115 g/L，ee值>99%，并解析了人工合成代谢途径适配宿主细胞的机制。相关成果发表在包括生物化工顶级刊物Biotechnology and Bioengineering在内的期刊上。项目执行期间，共发表SCI论文16篇（第一/通讯），申请专利5项，授权2项，主编出版译著1部；受邀撰写Wiley出版社专著1个章节，国际会议邀请报告2次。