高产L-丙氨酸大肠杆菌细胞工厂的构建与发酵优化  

Construction and Fermentation Process Optimization of the Escherichia coli Cell Factory with High-titer Production of L-alanine

作　　者：聂玉朋 郭亮 刘立明[2] 刘佳[2] 徐慧 姜国政 田延军 Nie Yupeng;Guo Liang;Liu Liming;Liu Jia;Xu Hui;Jiang Guozheng;Tian Yanjun(Qilu University of Technology(Shandong Academy of Sciences),Shandong Food Ferment Industry Research&Design Institute,Jinan 250014;State Key Laboratory of Food Science and Technology,Jiangnan University,Wuxi 214122,Jiangsu;Yantai Hengyuan Biological Co.,Ltd.,Yantai 265709,Shandong)

机构地区：[1]齐鲁工业大学(山东省科学院)山东省食品发酵工业研究设计院,济南250014 [2]江南大学食品科学与技术国家重点实验室,江苏无锡214122 [3]烟台恒源生物股份有限公司,山东烟台265709

出　　处：《中国食品学报》2024年第6期57-73,共17页Journal of Chinese Institute Of Food Science and Technology

基　　金：泰山产业领军人才专项(鲁政办字<2019>190号)。

摘　　要：L-丙氨酸作为最小的手性化合物之一,被广泛应用于食品、医药和日化领域。目前,微生物法生产L-丙氨酸存在发酵周期长,生产强度低等问题。为此,通过强化前体供给、启动子工程和转运工程等代谢工程策略,构建高产L-丙氨酸的大肠杆菌细胞工厂。进一步通过生化工程策略优化L-丙氨酸生产工艺,提高L-丙氨酸大肠杆菌细胞工厂的生产性能。结果:过表达gapA(3-磷酸甘油醛脱氢酶基因)强化前体丙酮酸供给,使L-丙氨酸产量和转化率分别提高了5.1%和15.6%。启动子工程优化gapA表达,使L-丙氨酸产量和转化率进一步提高到18.3 g/L和0.55 g/g。过表达L-丙氨酸外运蛋白(AlaE),增加L-丙氨酸转运,使L-丙氨酸产量提高到20.4 g/L。生化工程策略优化培养基组分,得到最佳碳源为葡萄糖40 g/L,最佳氮源为(NH4)2SO425 g/L。最佳发酵条件为:接种量15%,10 h转厌氧发酵和变速补料发酵。5 L发酵罐发酵36 h,大肠杆菌菌株W-135 L-丙氨酸产量为127.2 g/L,转化率为0.83 g/g,生产强度为3.53 g/L/h,比优化前分别提高了64.3%,50.9%和64.2%。本研究利用系统代谢工程和生化工程策略,构建了发酵周期短和发酵工艺简单的L-丙氨酸高产菌株,为L-丙氨酸的工业化生产提供了理论基础。L-alanine is one of the smallest chiral compounds and has been widely used in food,medicine,and daily chemical fields.Microbial production of L-alanine has problems such as long fermentation period and low productivity.In this study,metabolic engineering strategies such as strengthen precursors supply,promoter engineering and transporter engineering was used to construct an Escherichia coli cell factory with high-titer production of L-alanine.The production process of L-alanine was optimized by biochemical engineering strategies to improve the production performance of the Escherichia coli cell factory with production of L-alanine.Results:Overexpression of gapA(Glyceraldehyde 3-phosphate dehydrogenase gene)enhanced precursor supply,increased the titer of L-alanine and the conversion of glucose by 5.1%and 15.6%,respectively.Through the optimization of gapA expression by promoter engineering,the titer of L-alanine and the conversion of glucose reached 18.3 g/L and 0.55 g/g,respectively.Overexpression of L-alanine transporter(AlaE)enhanced L-alanine transport,the titer of L-alanine reached 20.4 g/L.The composition of culture medium was optimized by biochemical engineering strategy,the best carbon source was glucose 40 g/L and the best nitrogen source was(NH4)2SO425 g/L.The optimum fermentation conditions were as follows:inoculum size:15%,fermentation mode:10 h conversion to anaerobic fermentation and feeding mode:variable speed feed.After 36 h fermentation in a 5 L bioreactor,the constructed strain E.coli W-135 produced 127.2 g/L of L-alanine,with a yield of 0.83 g/g glucose and a productivity of 3.53 g/L/h,which were 64.3%,50.9% and 64.2% higher than those before optimization,respectively.In this study,a L-alanine high-titer strain with short fermentation period and simple fermentation process was constructed by using the strategy of system metabolic engineering and biochemical engineering,which provided a theoretical basis for the industrial production of L-alanine.

关 键 词：L-丙氨酸 大肠杆菌 代谢工程 外排 生化工程 

分 类 号：TQ922.2[轻工技术与工程—发酵工程]