利用CRISPR-Cas9和Cre/loxP系统删除34个非必需基因构建L-苏氨酸生产菌  

Construction of L-Threonine-Producing Escherichia coli by Deleting 34 Non-Essential Genes Using CRISPR-Cas9 and Cre/loxP System

作　　者：丁志祥 胡晓清[1,2] 柳亚迪 王小元[1,2,3] DING Zhixiang;HU Xiaoqing;LIU Yadi;WANG Xiaoyuan(State Key Laboratory of Food Science and Technology,Jiangnan University,Wuxi 214122,China;School of Biotechnology,Jiangnan University,Wuxi 214122,China;International Joint Laboratory on Food Safety,Jiangnan University,Wuxi 214122,China)

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

出　　处：《食品与生物技术学报》2020年第11期71-80,共10页Journal of Food Science and Biotechnology

基　　金：国家轻工技术与工程一流学科自主课题项目(LITE2018-10)。

摘　　要：L-苏氨酸是一种被广泛应用于食品、饲料及医药等领域的必需氨基酸。大肠杆菌以葡萄糖为碳源合成L-苏氨酸的过程中,一些非必需基因的转录和翻译会消耗碳源。利用CRISPR-Cas9和位点特异性重组系统Cre/loxP,敲除了大肠杆菌MG1655基因组中puuE和ynaI区间的34个非必需基因(共30.372 kb),获得了突变菌MG003,然后通过高表达L-苏氨酸合成途径中关键基因thrA*、thrB和thrC以及L-苏氨酸转运酶编码基因rhtA和rhtC提高L-苏氨酸产量。与对照菌MG1655/pFW01-thrA*BC相比,MG003/pFW01-thrA*BC生长加快,L-苏氨酸产量提高25.5%;MG003/pFW01-thrA*BC-rhtA的L-苏氨酸产量提高了43.3%;MG003/pFW01-thrA*BC-rhtC的L-苏氨酸产量提高了74.5%。研究结果表明,大肠杆菌基因组中34个非必需基因的删除有利于其提高L-苏氨酸合成能力。L-threonine is an essential amino acid widely used in food,feed and medicine.In the process of L-threonine synthesis from glucose,transcription and translation of some non-essential genes consume carbon source.In this study,CRISPR-Cas9 and site-specific recombination system Cre/loxP were used to delete 34 non-essential genes(30.372 kb)between puuE and ynaI genes in the genome of E.coli MG1655,resulting the mutant strain MG003.The genes thrA*,thrB and thrC,the key genes in the L-threonine biosynthetic pathway,and the genes rhtA and rhtC encoding the L-threonine transporters,were then overexpressed in MG003 to increase the L-threonine production.Compared to the control strain MG1655/pFW01-thrA*BC,MG003/pFW01-thrA*BC grew faster and produced 25.5%more L-threonine,MG003/pFW01-thrA*BC-rhtA produced 43.3%more L-threonine,and MG003/pFW01-thrA*BC-rhtC produced 74.5%more L-threonine.The results indicate that deletion of the 34 non-essential genes in E.coli could improve L-threonine production.

关 键 词：大肠杆菌 L-苏氨酸 CRISPR-Cas9 Cre/loxP puuE ynaI 

分 类 号：Q933[生物学—微生物学]