代谢工程改造大肠杆菌生产戊二酸  

Metabolic engineering of Escherichia coli to produce glutaric acid

作　　者：张芝兰 高聪 郭亮 陈修来[1,2] 魏婉清 吴静 宋伟 刘立明 Zhilan ZHANG;Cong GAO;Liang GUO;Xiulai CHEN;Wanqing WEI;Jing WU;Wei SONG;Liming LIU(State Key Laboratory of Food Science and Technology,Jiangnan University,Wuxi,Jiangsu 214122,China;International Joint Laboratory on Food Safety,Wuxi,Jiangsu 214122,China;School of Life Science and Health Engineering,Jiangnan University,Wuxi,Jiangsu 214122,China)

机构地区：[1]江南大学食品科学与技术国家重点实验室,江苏无锡214122 [2]江南大学国际食品安全联合实验室,江苏无锡214122 [3]江南大学生命科学与健康工程学院,江苏无锡214122

出　　处：《过程工程学报》2023年第9期1340-1351,共11页The Chinese Journal of Process Engineering

基　　金：国家重点研发计划(编号:2020YFA0908500);天津市合成生物学科技攻关项目(编号:TSBICIP-KJGG-015)。

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,将酶工程与代谢工程相结合,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Escherichia coli(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Kp ALDH进行理性分析和蛋白质改造,使酶的酶活提高了61.0倍;在此基础上,通过代谢工程强化限速酶Kp ALDH的表达并阻断副产物乙酸代谢支路,使戊二酸得率提高了1.0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1.7(g/L)/h和0.3 g/g葡萄糖。Glutaric acid is an important industrial material intermediate,which is widely used in the chemical industry,agriculture,medicine,and other fields.At present,glutaric acid has been synthesized by the biological method.However,the glutaric acid biosynthesis pathway has many problems,such as the lengthy synthesis pathway,high cofactor consumption,and low product yield.To develop an efficient biosynthesis method to synthesize glutaric acid,a new method to produce glutaric acid using glucose as substrate was constructed in microbial cell factories by combining enzyme engineering and metabolic engineering.First,through database mining,we designed a new catalytic pathway consisting of lysineα-oxidase(LO),monoamine oxidase(MAO),α-keto acid decarboxylase(KDC),and aldehyde dehydrogenase(ALDH).Through the detection of the product,it is proven that this path can convert lysine into glutaric acid through four-step reaction catalysis in vitro.Next,the metabolic pathway was introduced into the lysine-producing strain Escherichia coli(E.coli)CCTCC M2019435 to realize the de novo synthesis of glutaric acid.To further improve the synthesis efficiency of this pathway,we analyzed the metabolic pathway and determined the rate-limiting catalytic reaction steps.Then,protein engineering was carried out for the rate-limiting enzyme KpALDH based on structural analysis,and the activity of the enzyme was increased by 61.0 fold.On this basis,the expression of the rate-limiting enzyme KpALDH was enhanced by ribosomebinding site(RBS)engineering in a lysine-producing strain,and the byproduct metabolic pathway was blocked,which increased the yield of glutaric acid by 1.0 fold than before.Finally,the fermentation conditions of glutaric acid were optimized,including temperature,inducer concentration,and ammonia nitrogen content,and the yield of glutaric acid was further increased to 62.0 g/L,with productivity and yield reaching 1.7(g/L)/h and 0.3 g/g glucose,respectively.

关 键 词：戊二酸 新路径 蛋白质改造 核糖体结合位点(RBS)调控 

分 类 号：TQ921[轻工技术与工程—发酵工程]