代谢工程改造大肠杆菌合成己二酸

Metabolic engineering of Escherichia coli for adipic acid production

摘要:

己二酸是一种具有重要应用价值的二元羧酸，是合成尼龙-66的关键前体。目前，生物法生产己二酸存在生产周期长、生产效率低的问题。本研究选择一株野生型高产琥珀酸菌株大肠杆菌(Escherichia coli) FMME N-2为底盘细胞，首先通过引入逆己二酸降解途径的关键酶，成功构建了可合成0.34 g/L己二酸的E. coli JL00菌株；接着，对合成路径限速酶进行表达优化，使E. coli JL01菌株在摇瓶发酵条件下产量达到0.87 g/L；随后，通过敲除sucD基因、过表达acs基因和突变lpd基因的组合策略平衡己二酸合成前体的供应，优化菌株E. coli JL12己二酸产量进一步提升至1.51 g/L；最后，在5 L发酵罐上对己二酸发酵工艺进行优化。工程菌株经72 h分批补料发酵，己二酸的产量达到22.3 g/L，转化率为0.25 g/g，生产强度为0.31 g/(L·h)，具备了一定的应用潜力。本研究可为包括己二酸在内的多种二元羧酸细胞工厂的构建提供理论依据和技术基础。

关键词:大肠杆菌;代谢工程;己二酸;发酵优化

Abstract:

Adipic acid is a high-value-added dicarboxylic acid which is primarily used in the production of nylon-66 for manufacturing polyurethane foam and polyester resins. At present, the biosynthesis of adipic acid is hampered by its low production efficiency. By introducing the key enzymes of adipic acid reverse degradation pathway into a succinic acid overproducing strain Escherichia coli FMME N-2, an engineered E. coli JL00 capable of producing 0.34 g/L adipic acid was constructed. Subsequently, the expression level of the rate-limiting enzyme was optimized and the adipic acid titer in shake-flask fermentation increased to 0.87 g/L. Moreover, the supply of precursors was balanced by a combinatorial strategy consisting of deletion of sucD, over-expression of acs, and mutation of lpd, and the adipic acid titer of the resulting E. coli JL12 increased to 1.51 g/L. Finally, the fermentation process was optimized in a 5 L fermenter. After 72 h fed-batch fermentation, adipic acid titer reached 22.3 g/L with a yield of 0.25 g/g and a productivity of 0.31 g/(L·h). This work may serve as a technical reference for the biosynthesis of various dicarboxylic acids.

Key words:Escherichia coli;metabolic engineering;adipic acid;fermentation optimization