代谢工程改造大肠杆菌生产没食子酸

Metabolic engineering of Escherichia coli for the production of gallic acid

摘要:

没食子酸是一种具有抗氧化等多种药用价值的天然植物酚类化合物，目前主要的生产方法是通过提取植物中单宁进行化学或酶水解方式制备，存在成本高和污染大的问题。基于微生物细胞工厂的绿色合成为没食子酸生产提供了有效替代途径，然而目前的产量仍难以达到工业化要求。为了利用廉价底物实现没食子酸的从头高效合成，本研究以一株高效合成3-脱氢莽草酸的大肠杆菌为底盘，首先确定没食子酸最优合成路径的关键酶为3-脱氢莽草酸脱水酶(AroZ)及对羟基苯甲酸羟化酶(PobA)，且二者最优表达比例为1:20。基于质粒拷贝数和启动子工程实现最优路径在底盘菌株体内的构建，并结合蛋白质工程改造获得限速酶突变体PobA^M2/A45S/V47P，使得没食子酸摇瓶生产水平达到3.6 g/L。最后，在5 L发酵罐进行菌株稳定性和发酵条件优化，没食子酸产量达到26.7 g/L，糖酸转化率0.15 g/g。本研究实现了以葡萄糖为底物从头合成没食子酸产量的突破，为强化没食子酸及相关植物酚酸的生物合成提供了重要参考。

Abstract:

Gallic acid is a plant-derived phenolic compound with various medicinal values, including antioxidant properties. The primary method for producing gallic acid at present involves the chemical or enzymatic hydrolysis of tannins extracted from plants, which is associated with high costs and serious environmental pollution. Green synthesis based on microbial cell factories offers an effective alternative route for the production of gallic acid. However, the current yields of green synthesis are insufficient for industrial-scale requirements. Therefore, the development of a de novo synthesis strategy for gallic acid using low-cost substrates holds significant potential for industrial applications. In this study, an Escherichia coli strain capable of efficiently synthesizing 3-dehydroshikimic acid was used as the chassis organism. Initially, the key enzymes for the optimal synthesis pathway of gallic acid were identified as 3-dehydroshikimate dehydratase (AroZ) and 4-hydroxybenzoate hydroxylase (PobA), with an optimal expression ratio of 1:20. The optimal pathway was constructed within the chassis strain through plasmid copy number and promoter engineering. Protein engineering was further employed to obtain a mutant of the rate-limiting enzyme, PobA^M2/A45S/V47P, which enhanced the shake flask production level of gallic acid to 3.6 g/L. Finally, strain stability and fermentation condition optimization were conducted in a 5 L fermentor, resulting in a gallic acid yield of 26.7 g/L and a sugar-to-acid conversion rate of 0.15 g/g. This study achieves a breakthrough in the de novo synthesis of gallic acid from glucose, providing an important reference for enhancing the biological synthesis of gallic acid and related phenolic acids from plants.