基于ATP和NADH耦合调控的一步厌氧发酵产琥珀酸重组E.coli的代谢工程

The exclusively anaerobic succinic acid producers have advantages in atom economy and the whole process for CO2 fixation, and thus they become the most potential strains for bio-based succinic acid industry production in the future.However, the bottleneck of the succinic acid producers we have is the cell growth rate <0.05 h-1, succinic acid productivity <0.18 g/Loh, mass yield <80% which is much lower than the maximum theoretical yield. The main reason of low cell growth rate, poor succinic acid synthesis rate and yield is the shortage of ATP and NADH supply. Depending on the drawback and the experience foundation, this project will focus on enhancing the growth ability of E.coli and succinic acid synthetic rate under anaerobic conditions according to reforming the excretion system of succinic acid and optimizing the extracellular conditions to decrease the ATP consumption of non-growth, combining with regulating the pathways of intracellular ATP synthesis. Furthermore, an enhancing NADH supply and a suitable ratio of NADH/NAD+ will be maintained by regulation of ORP and reforming the NADH system to increase the succinic acid mass yield and decrease the by-products formation. Finally, an efficient strategy of strain construction and fermentation pattern to produce succinic acid under anaerobic conditions will be established based on reconstruction and regulation of both ATP and NADH system.

一步厌氧产琥珀酸菌株具有原子经济性高、全过程固定CO2等优点，是生物基琥珀酸最具潜力的工业化菌株。然而，目前生产菌株比生长速率<0.05 h-1，琥珀酸合成速率<0.18 g/loh，且收率仅为80%左右，远低于理论收率131%。ATP与还原力供给不足是厌氧条件下菌株生长速率缓慢、琥珀酸合成速率和收率较低的主要原因。本项目通过改造琥珀酸分泌系统，优化胞外培养环境，减少非细胞生长ATP消耗，结合胞内合成ATP途径的调控，改善菌株厌氧生长性能，提高琥珀酸合成速率；通过电化学、氧化还原电位调控从胞外供给电子，结合胞内NADH生物合成系统的改造，提高胞内NADH的总量，维持胞内合适的NADH/NAD+比例，提高琥珀酸合成途径的代谢通量，减少副产物生成。通过对菌株ATP和NADH系统的改造，最终实现琥珀酸高效生产，建立基于辅酶ATP和NADH调控的一步厌氧发酵产琥珀酸重组大肠杆菌的构建及发酵策略。

一步厌氧产琥珀酸菌株具有原子经济性高、全过程固定 CO2 等优点，是生物基琥珀酸最具潜力的工业化菌株。前期研究表明ATP 与还原力供给不足是厌氧条件下菌株生长速率缓慢、琥珀酸合成速率和收率较低的主要原因。本项目通过3年的研究，全面完成项目预期目标，筛选获得了多株能够在厌氧环境下一步厌氧发酵制备丁二酸，其中发酵性能最佳的菌株为E. coli Suc260，其基因型为E. coli C (∆ldhA, ∆adhE, ∆ackA, ∆focA, ∆pflB)，产物浓度最高66.7g/L，收率1.01g/g。项目实施期间共申请专利13项，其中授权1项，公开发表论文8篇，其中SCI论文7篇，相关技术成果获两项省部级二等奖。