梭菌产丁醇途径重构与调试

Butanol is a basic bulk chemical, and is expected to become the next generation of fuel. Compared to petroleum based butanol, biobutanol manufacturing costs are higher. Increasing the butanol biosynthesis selectivity and the conversion rate of raw materials of Clostridia is an effective means to enhance the economic and competitive of the process. Preliminary studies showed that direct knockout / inhibit the production of low-value product pathways of Clostridium would cause the global effect on acid - solvent metabolic network, and could not achieve the desired results. However, the dredge of the rate-limiting step of butanol synthesis should be an effective way to enhance butanol production and weaken low value products synthesis. The project intends to dredge rate-limiting step of butanol synthesis - to replace the natural butyl-CoA dehydrogenase which use EtfAB as redox partners with trans-2-enoyl CoA which use NADH as cofactor. Based on the 2.0 A crystal structure of trans-2-enoyl CoA reductase that the applier and the collaborators has already got, we will analysis croton-coenzyme A / hexene-coenzyme A substrate binding pocket, and do rational design to improve the catalytic efficiency and croton-CoA specificity. Escherichia coli NADH-dependent enzyme screening platform will also be used to select those enzyme mutants have higher catalytic efficiency from protein irrational designe, thus to resolve trans -2 - enoyl CoA catalytic mechanism. Mutants will further import into Clostridia to test the suitability of the genes variants with butanol production and reveal the relationship of the metabolic node and metabolic pathways.

丁醇是大宗基础化学品，有望成为新一代燃料。生物丁醇相对石油基丁醇制造成本偏高。提高产丁醇梭菌丁醇合成选择性和对原料转化率是提升生物丁醇经济性的有效手段。前期研究表明，直接敲除/抑制产丁醇梭菌低值副产物途径引起产酸-产溶剂代谢网络全局效应而无法达到预期效果，而疏通丁醇合成限速步骤是增强丁醇合成弱化低值产物代谢流的有效手段。本项目拟将丁醇合成限速步骤- - 以EtfAB为氧化还原反应伴侣的丁酰辅酶A脱氢酶替换成以NADH为辅酶的反式-2-烯酰辅酶A。通过申请人和合作实验室已获得的反式-2-烯酰辅酶A 2.0 A晶体结构分析和巴豆酰辅酶A/己烯酰辅酶A的底物结合口袋，利用有理设计提高催化效率和对目标底物巴豆酰辅酶A的特异性；同时利用大肠杆菌NADH依赖酶筛选平台无理设计提高催化效率，解析该酶的催化机理。突变体进一步导入产丁醇梭菌体内调试基因变体和产丁醇途径的适配性揭示代谢节点和代谢途径的关联关系。

反式－2－烯酰辅酶 A 还原酶（TERs）是一类以 NAD（P）H 为辅酶，催化烯酰辅酶 A 生成酰基辅酶 A 的氧化还原酶。该酶被应用于疏通大肠杆菌异源丁醇合成途径的限速步骤，但其在分子水平的生物学功能和催化机制不清楚。本项目鉴定了齿垢密螺旋菌来源的反式-2-烯酰辅酶 A 还原酶（TdTER）在丙酮丁 醇梭菌中的同源蛋白（CaTER）。CaTER 与 TdTER 同样具有以 NADH 为辅因子催化巴豆酰辅酶 A 到丁酰辅酶 A 不可逆反应的功能。通过 CaTER 晶体结构解析 与突变体酶动力学分析，预测了 CaTER 的辅因子结合域及活性中心重要催化位 点；通过定点突变，得到一种对底物巴豆酰辅酶 A 亲和力提高 3 倍，催化效率提高 2 倍的 CaTER 突变体；鉴定了辅酶 NADH 或 NADPH 依赖偏好性的重要氨基酸位点。本项目开发了产溶剂梭菌基于I-SceI归巢内切酶的无痕的基因修饰系统和包含酿脓链球菌来源的Cas9 刻痕酶, 向导RNA及同源修复模板的pNICKclos系统，敲除效率最高100%。项目获得了若干可高效发酵秸秆糖液到丁醇的产溶剂梭菌，为降低利用废弃农林生物质产溶剂和丁醇的生产成本提供了新途径。