基于谷氨酸棒状杆菌的茶氨酸生物合成工程菌构建  

Construction of theanine biosynthesis engineering bacteria based on Corynebacterium glutamicum

作　　者：江丽娜[1] 王荣秀[1] 贾慧艳 邓威威[1] 张正竹[1] JIANG Lina;WANG Rongxiu;JIA Huiyan;DENG Weiwei;ZHANG Zhengzhu(State Key Laboratory of Tea Plant Biology and Utilization,Anhui Agricultural University,Hefei 23003)

机构地区：[1]安徽农业大学茶树生物学与资源利用国家重点实验室,合肥230036

出　　处：《安徽农业大学学报》2018年第5期808-812,共5页Journal of Anhui Agricultural University

基　　金：国家自然科学基金项目(31300576)资助

摘　　要：将茶树谷氨酰胺合成酶基因(Cs GS1;3,GenBank登录号AB117934.1)克隆,并与穿梭表达载体pZ8-1连接,将重组质粒通过电转法转到谷氨酸棒状杆菌ATCC 13032感受态细胞,构建了谷氨酸棒状杆菌重组菌株Corynebacterium glutamicum ATCC 13022/p Z8-Cs GS1;3。再将成功构建的该重组菌株经异丙基-β-D-硫代半乳糖苷(IPTG)在30℃下诱导4 h后,进行体外酶活反应。反应产物用薄层层析(TLC)检测发现有产物茶氨酸生成,经液相色谱三重四级杆串联色谱仪(LC-MS)进一步检测确定其合成产物为茶氨酸。In this study, we selected glutamine synthetase gene （CsGS1;3） from tea transcriptome bank and obtaining its cDNA （Sequence ID： ABl17934.1） sequence by NCBI alignment which was cloned by molecular biology. The CsGS1;3 was cloned into the shuttle expression vector pZ8-1, and the recombinant plasmid was transformed into Corynebacterium glutamicum ATCC 13032 by electrotransformation, yielding the recombinant C.glutamicum strain ATCC 13032/pZ8-CsGS1;3. Then the recombinant C.glutamicum strain ATCC 13032/pZ8-CsGS1;3 was induced with IPTG for 4 h at 30℃.The supematant was collected by centrifugation at 4℃ as a crude enzyme solution, and substrates added were into the medium. The preliminary result suggested that the CsGS1;3 is capable of synthesizing theanine by TLC, then using LC-MS analysis and the result suggested that the synthesis product is theanine.

关 键 词：茶氨酸 基因克隆 谷氨酸棒状杆菌ATCC 13032 体外合成 酶活性 

分 类 号：S571.1[农业科学—茶叶生产加工] Q946.8[农业科学—作物学]