耐正丁醇乳酸菌NADH与乙酰辅酶A主代谢途径解析

n-butanol, an important bulk chemical, is produced by Clostridium species. Various efforts have been made to transfer the clostridial n-butanol pathway into other microorganisms. However, the titers of heterologous n-butanol production was much lower than native host. Mixed-acid fermentation pathway deficientEscherichia coli strain lost its ability to grow anaerobically due to the lack of NADH-consuming pathways as an electron sink. Further deleted the phosphate acetyltransferase(pta), the flux was directed to the NADH-consuming and a modified clostridial n-butanol pathway provided an irreversible reaction catalyzed by trans-enoyl-coenzyme A (CoA) reductase (Ter). The anaerobic growth was rescued and the n-butanol production achieved a level comparable to that of Clostridium species with titers of 15g/liter in flasks, reaching the limits. Lactica acid bacteria can be a more potential host, because of higher limits. But its NADH and acetyl-CoA primary metabolisms are not identified and characterized absolutely, difficult to construct anaerobic growth rescue platform as before. This work aims to reveal homo-/hetero-lactic acid fermentation pathways mainly consumed NADH by gene-konckouts, genetic complements, enzymology analysis, and to select suitable (mutant)pyruvate dehydrogenase complex or pyruvate formate lyase combined formate dehydrogenase for building NADH and acetyl-CoA driving forces to direct the flux to n-butanol synthesis,thus laying the theoretical foundation of high-titer 1-butanl productionl.

正丁醇为重要大宗化学品，可由梭菌生产。将其合成途径移至其它微生物，产物丁醇浓度绝大部分远不及梭菌。大肠杆菌主消耗NADH的混合酸发酵途径被阻断后无法厌氧生长；再阻断消耗乙酰辅酶A的磷酸乙酰基转移酶基因，代谢流将被导向消耗NADH的、基于反式烯酰辅酶A还原酶的重组丁醇合成途径，解救宿主菌厌氧生长，产丁醇浓度与梭菌相当，达到其耐受极限。乳酸菌以更高丁醇耐受性，有望成为更好丁醇发酵宿主，但其NADH与乙酰辅酶A的主代谢途径尚未在基因水平上得到完全解析，尚难建立前述厌氧生长解救平台。拟通过基因敲除、代谢产物谱与酶学分析等手段阐明耐丁醇乳酸菌主消耗NADH的同型/异型乳酸发酵途径；甄选能在厌氧条件下催化丙酮酸生成NADH与乙酰辅酶A的丙酮酸脱氢酶复合体或其变体或丙酮酸甲酸裂解酶/甲酸脱氢酶组合；从而构造NADH与乙酰辅酶A驱动力，为引导代谢流向丁醇合成途径，在乳酸菌中实现高浓度丁醇发酵打好理论基础。

正丁醇为重要大宗化学品，可由梭菌生产。将其合成途径移至其它微生物，产物丁醇浓度绝大部分远不及梭菌。大肠杆菌主消耗NADH的混合酸发酵途径被阻断后无法厌氧生长；再阻断消耗乙酰辅酶A的磷酸乙酰基转移酶基因，代谢流将被导向消耗NADH的、基于反式烯酰辅酶A还原酶的重组丁醇合成途径，解救宿主菌厌氧生长，产丁醇浓度与梭菌相当，达到其耐受极限。乳酸菌以更高丁醇耐受性，有望成为更好丁醇发酵宿主，但其NADH与乙酰辅酶A的主代谢途径尚未在基因水平上得到完全解析，尚难建立前述厌氧生长解救平台。.我们开发了短小乳杆菌和植物乳杆菌CRISPR-Cas基因组编辑工具，鉴定并敲除了短小乳杆菌CGMCC1.2028和植物乳杆菌WCFS1的多个乳酸脱氢酶和乙酰乳酸合酶基因，阻断了乳酸与乙偶姻的合成，得到丙酮酸大量积累的工程菌株。在其中的短小乳杆菌中过表达运动发酵单胞菌来源的丙酮酸脱羧酶基因可将积累的丙酮酸转变为同型乙醇发酵，说明绝大部分葡萄糖经丙酮酸到乙醛最终转化为乙醇。另一方面，在积累丙酮酸的植物乳杆菌中过表达丙酮酸脱羧酶基因和大肠杆菌乙酰化乙醛脱氢酶基因使好氧条件下乙酸合成从0.12提高到0.4克/克葡萄糖，暗示流向乙酰辅酶A合成的碳流增加。最后表达乙酰辅酶A到正丁醇合成途径，厌氧发酵产物中检测到150 mg/L正丁醇。然而，碳代谢流分析发现葡萄糖并非先后经丙酮酸、乙醛和乙酰辅酶A转化为正丁醇（由丙酮酸脱羧酶和乙酰化乙醛脱氢酶催化），推测是经丙酮酸由丙酮酸甲酸裂解酶催化产生乙酰辅酶A后转化为正丁醇。 .我们的工作为引导代谢流向丁醇合成途径，在乳酸菌中实现高浓度丁醇发酵打好理论基础。有望通过敲除短小乳杆菌和植物乳杆菌中的多个乙醇脱氢酶基因，将它们转变为同型正丁醇发酵的菌株，再通过基因整合、启动子优化和驯化等方式，最终获得稳定高效的正丁醇发酵菌株。