Abstract: Glutaric acid is an important intermediate, which is widely used in chemical industry, agriculture, medicine and other fields. At present, there are some problems in the biosynthesis pathway of glutaric acid, such as long synthesis path, high consumption of cofactors and low yield. In order to develop an efficient method for the synthesis of glutaric acid, a new way to produce glutaric acid using glucose as substrate was constructed by combining enzyme engineering with metabolic engineering. Firstly, a novel catalytic pathway composed of lysine α-oxidase (LO), monoamine oxidase (MAO), α-ketoacid decarboxylase (KDC) and aldehyde dehydrogenase (ALDH) was designed by database mining. AB initio synthesis of glutaric acid was realized by introducing lysine producing strain E. coli CCTCC M2019435. In order to further improve the synthesis efficiency of this pathway, rational analysis and protein modification were carried out for the rate-limiting enzyme KpALDH of the pathway, and the catalytic efficiency of the enzyme was increased by 66.5 times. On this basis, the yield of glutaric acid was increased by 2.0 times through metabolic engineering to enhance the expression of rate-limiting enzyme KpALDH and block the by-product acetic acid metabolic branch. Finally, the glutaric acid fermentation conditions were optimized, the glutaric acid yield increased to 62.0 g/L at the end of fermentation, and the production intensity and yield reached 1.6 (g/L)/h and 0.3 g/g glucose, respectively.

Key words: glutaric acid, new pathway, protein engineering modification, RBS regulation

摘要： 戊二酸是一种重要的中间体，在化工、农业和医药等领域有着广泛的用途。目前，戊二酸的生物合成途径存在合成路径冗长、辅因子消耗多和产物得率低等问题。为开发高效的戊二酸合成方法，将酶工程和代谢工程相结合，构建了一条以葡萄糖为底物生产戊二酸的新途径。首先，通过数据库挖掘设计了一条由赖氨酸α氧化酶(LO)、单胺氧化酶(MAO)、α-酮酸脱羧酶(KDC)和醛脱氢酶(ALDH)组成的新型催化途径，引入赖氨酸生产菌株E. coli CCTCC M2019435后实现了戊二酸的从头合成；为进一步提高该路径的合成效率，针对路径的限速酶KpALDH进行理性分析和蛋白质改造，使酶的催化效率提高了66.5倍；在此基础上，通过代谢工程强化限速酶KpALDH的表达并阻断副产物乙酸代谢支路，使戊二酸得率提高了2.0倍；最后，优化戊二酸发酵条件，发酵结束时戊二酸产量提高到62.0 g/L，生产强度和得率分别达到1.6 (g/L)/h和0.3 g/g葡萄糖。

关键词: 戊二酸, 新路径, 蛋白质改造, RBS调控