基于启动子和信号肽优化提高谷氨酰胺酶在枯草芽孢杆菌中的分泌表达  

Promoter and signal peptide optimization increases the secretory expression of glutaminase in Bacillus subtilis

作　　者：毛泽敬 张祖政 陈铭 望松柏 牛硕 余华顺[1,2,3] 张彦 郑贤良[1,2,3] MAO Zejing;ZHANG Zuzheng;CHEN Ming;WANG Songbai;NIU Shuo;YU Huashun;ZHANG Yan;ZHENG Xianliang(Angel Yeast Limited Company,Yichang 443003,Hubei,China;Hubei Provincial Key Laboratory of Yeast Function,Yichang 443003,Hubei,China;National Key Laboratory of Agricultural Microbiology,Yichang 443003,Hubei,China;College of Biological and Pharmaceutical Sciences,China Three Gorges University,Yichang 443002,Hubei,China)

机构地区：[1]安琪酵母股份有限公司,湖北宜昌443003 [2]酵母功能湖北省重点实验室,湖北宜昌443003 [3]农业微生物挖掘与利用全国重点实验室,湖北宜昌443003 [4]三峡大学生物与制药学院,湖北宜昌443002

出　　处：《微生物学通报》2024年第5期1536-1549,共14页Microbiology China

基　　金：山东省重点研发计划(2022CXGC020712)。

摘　　要：【背景】谷氨酰胺酶(glutaminase)是一种能够催化L-谷氨酰胺水解生成L-谷氨酸和氨的酶,在食品、医药等领域有重要的应用价值。【目的】研究不同启动子和信号肽对谷氨酰胺酶分泌表达的影响、构建能高效分泌表达谷氨酰胺酶的枯草芽孢杆菌(Bacillus subtilis)工程菌株,提高谷氨酰胺酶的产量。【方法】选用枯草芽孢杆菌SCK6作为表达宿主,构建带有不同启动子和信号肽的谷氨酰胺酶表达载体及重组菌株,分析不同基因元件对谷氨酰胺酶表达及分泌的影响,筛选最优启动子和信号肽,组合优化,构建高效分泌表达谷氨酰胺酶的工程菌株。【结果】实现了谷氨酰胺酶在枯草芽孢杆菌中的分泌表达,在以P_(HpaII)-P_(aprE)为启动子、YndA为信号肽的条件下,摇瓶发酵胞外最高酶活达到6.7U/mL。【结论】通过对启动子、信号肽关键基因元件进行筛选组合,构建了一株高效分泌表达谷氨酰胺酶的菌株,为酶的高效分泌表达提供了一种有效的方法。[Background]Glutaminase is an enzyme which can catalyzes the hydrolysis of L-glutamine to L-glutamic acid and ammonia,and it has been well used in the food and medicine fields.[Objective]To study the influences of different promoters and signal peptides on the secretory expression of glutaminase and construct recombinant Bacillus subtilis strains with efficient secretory expression of glutaminase to improve the production of glutaminase.[Methods]B.subtilis SCK6 was selected as the host,and the glutaminase expression vectors and recombinant strains with different promoters and signal peptides were constructed.The effects of different gene elements on the expression and secretion of glutaminase were studied,and the optimal promoter and signal peptide were selected and combined to construct an efficient and robust glutaminase-producing strain.[Results]A recombinant strain with efficient secretory expression of glutaminase was constructed.With P_(HpaII)-P_(aprE) as the promoter and YndA as the signal peptide,the strain showed the extracellular glutaminase activity reaching 6.7 U/mL in shake flask fermentation.[Conclusion]By screening and combining the promoter and signal peptide,we constructed a recombinant strain with robust production of glutaminase,providing an effective method for the efficient secretory expression of enzymes.

关 键 词：枯草芽孢杆菌 谷氨酰胺酶 启动子 信号肽 分泌表达 

分 类 号：Q78[生物学—分子生物学] Q936