国家自然科学基金(22208124， 32000037)；江苏省自然科学基金(BK20211529， BK20200614)
琥珀酸作为一种重要的C4平台化合物，广泛应用于食品、化学、医药等领域。利用大肠杆菌(Escherichia coli)发酵生产琥珀酸受胞内辅因子不平衡的影响，存在产率低、生产强度低、副产物多等问题。为此，对不同氧气条件下琥珀酸产量和化学计量学分析发现，微厌氧条件下E. coli FMME-N-26高效积累琥珀酸需要借助三羧酸循环(tricarboxylic acid cycle,TCA)为还原性三羧酸途径(reductive tricarboxylic acid pathway,r-TCA)提供足够的ATP和NADH。通过减少ATP消耗、强化ATP合成、阻断NADH竞争途径和构建NADH回补路径等代谢工程策略，组合调控胞内ATP与NADH含量，获得工程菌株E. coli FW-17。通过发酵条件优化，菌株E. coli FW-17在5 L发酵罐能积累139.52 g/L琥珀酸，比出发菌株提高了17.81%，乙酸浓度为1.40 g/L，降低了67.59%。进一步在1 000 L发酵罐中进行放大实验，琥珀酸产量和乙酸浓度分别为140.2 g/L和1.38 g/L。
Succinic acid is an important C4 platform chemical that is widely used in food, chemical, medicine sectors. The bottleneck of fermentative production of succinic acid by engineered Escherichia coli is the imbalance of intracellular cofactors, which often leads to accumulation of by-products, lower yield and low productivity. Stoichiometric analysis indicated that an efficient production of succinic acid by E. coli FMME-N-26 under micro-aeration conditions might be achieved when the TCA cycle provides enough ATP and NADH for the r-TCA pathway. In order to promote succinic acid production, a serial of metabolic engineering strategies include reducing ATP consumption, strengthening ATP synthesis, blocking NADH competitive pathway and constructing NADH complementary pathway were developed. As result, an engineered E. coli FW-17 capable of producing 139.52 g/L succinic acid and 1.40 g/L acetic acid in 5 L fermenter, which were 17.81% higher and 67.59% lower than that of the control strain, was developed. Further scale-up experiments were carried out in a 1 000 L fermenter, and the titer of succinic acid and acetic acid were 140.2 g/L and 1.38 g/L, respectively.