组合代谢调控提高大肠杆菌对氨基苯甲酸产量

Improving the production of 4-aminobenzoic in engineered Escherichia coli by combinatorial regulation

摘要:

对氨基苯甲酸是一种重要的有机合成中间体，广泛应用于医药、染料等行业。近年来对氨基苯甲酸作为一种潜在的高强度共聚物单体越来越受到重视。对氨基苯甲酸作为叶酸合成的前体之一，其合成在大肠杆菌体内由叶酸合成途径的pabA、pabB和pabC三个基因负责，催化分支酸合成对氨基苯甲酸。本研究以实验室构建的酪氨酸高产工程菌 TYR002作为出发菌株，首先弱化双功能分支酸突变酶/预苯酸脱氢酶 TyrA的表达，以减少酪氨酸积累，然后利用3种不同强度的组成型启动子分别调控pabA、pabB和pabC的表达。摇瓶发酵表明不同的组合调控模式下大肠杆菌发酵培养基中的对氨基苯甲酸积累量存在显著差异，最高可获得0.67 g/L的摇瓶发酵产量。进一步通过发酵条件优化和分批补料发酵，在5 L发酵罐中获得了6.4 g/L的对氨基苯甲酸产量。本研究为改善对氨基苯甲酸生物合成效率提供了重要理论参考。

关键词:对氨基苯甲酸，大肠杆菌，组合调控，代谢工程，叶酸合成途径

Abstract:

Para-aminobenzoate (PABA) is an important chemical for organic synthesis and extensively used in pharmaceutical and dye industry. In recent years, PABA has received increasing attention as a potential component of high-strength polymer. In Escherichia coli, three genes of pabA, pabB and pabC are responsible for PABA production from chorismate in folate synthetic pathway. However, E. coli does not accumulate or accumulates very few amounts of PABA under normal growth condition. In this study, the tyrosine-producing E. coli TYR002 constructed previously was used as the starting strain for developing PABA-producing strain. First, the activity of bifunctional chorismate mutase/prephenate dehydrogenase TyrA in E. coli TYR002 was weakened to reduce the production of tyrosine. Then, three different constitutive promoters were used to regulate the expression of pabA, pabB and pabC in recombinant plasmid which was transformed into E. coli for improving PABA production. The shake-flask fermentation showed that the different combination of constitutive promoters significantly affected the production of PABA, and the highest shake-flask fermentation titer was 0.67 g/L. After further condition optimization, the engineered E. coli produced 6.4 g/L PABA under 5 L fed-batch fermentation. This study could be a good reference for improving microbial production of PABA.

Key words:para-aminobenzoate, Escherichia coli, combinatorial regulation, metabolic engineering, folate synthetic pathway