ilvBNC操纵子协同cimA基因过表达提高Corynebacterium glutamicum YILW L-异亮氨酸产量的研究  

Enhanced L-isoleucine Production by Corynebacterium glutamicum YILW through Overexpression of ilvBNC and cimA

作　　者：温冰[1,2] 徐国栋[1] 刘远[1] 徐庆阳[1] 谢希贤[1] 张成林[1] 陈宁[1] 

机构地区：[1]代谢控制发酵技术国家地方联合工程实验室天津市氨基酸高效绿色制造工程实验室天津科技大学生物工程学院,天津300457 [2]天津市工业微生物研究所有限公司,天津300462

出　　处：《现代食品科技》2016年第2期27-32,72,共7页Modern Food Science and Technology

基　　金：国家自然科学基金项目(31300069);天津市科技支撑计划重点项目(12ZCZDSY01900)

摘　　要：L-异亮氨酸是人和动物八种必需氨基酸之一,在生命活动中具有重要地位。乙酰羟基酸合成酶(acetohydroxyacid synthase,AHAS)是L-异亮氨酸合成途径的关键酶(由ilvBN编码),α-酮基丁酸是L-异亮氨酸合成的重要前体。因此强化ilvBN的表达以及增加α-酮基丁酸的供应理论上可提高L-异亮氨酸的合成。cim A编码的甲基苹果酸合成酶可以催化丙酮酸和乙酰-Co A快速生成L-异亮氨酸前体α-酮基丁酸,从而增强主代谢流通量。本文采用基因重组手段将L-异亮氨酸生产菌株Corynebacterium glutamicum YILW ilvBNC操纵子中的启动子替换为强启动子Ptac获得C.glutamicum YILWPtac。摇瓶发酵结果显示该菌株L-异亮氨酸产量和转化率分别较出发菌株提高了14.8%和18.6%。在此基础上过表达cimA基因,获得C.glutamicum YILWPtacp XMJ19cim A,其L-异亮氨酸酸产量和糖酸转化率分别较出发菌株提高了14.5%和42.4%。本研究可为氨基酸生产菌株的选育提供依据。L-isoleucine is one of the eight essential amino acids for humans and animals, and it plays a vital role in numerous life functions. α-Ketobutyrate is the key precursor of L-isoleucine, and acetohydroxy acid synthase（AHAS; encoded by ilv BN） is the key enzyme in L-isoleucine synthesis. Therefore, L-isoleucine production can be theoretically increased either by increasing the supply of α-ketobutyrate or ilv BN expression. Citramalate synthase, encoded by cim A, catalyzes the generation of α-ketobutyrate from pyruvate and acetyl-Co A. α-Ketobutyrate is a direct precursor of L-isoleucine; thus, increasing cim A expression increases the metabolic flux of L-isoleucine synthesis. In the present study, the promoter of the ilv BNC operon in the L-isoleucine-producing strain Corynebacterium glutamicum YILW was replaced by the potent tac promoter to construct a recombinant strain, C. glutamicum YILWPtac, which overexpresses ilv BNC. Shake flask fermentation experiments showed that L-isoleucine production and glucose（Yp/s） conversion in the recombinant strain were increased by 14.8% and 18.6%, respectively. To further increase L-isoleucine production and Yp/s, a construct containing the cim A gene under the control of a strong promoter was introduced into C. glutamicum YILWPtac to construct C. glutamicum YILWPtacp XMJ19 cim A. Compared to the original strain, in C. glutamicum YILWPtacp XMJ19 cim A, L-isoleucine production and Yp/s were increased by 14.5% and 42.4%, respectively. Thus, the findings of this study provide a basis for constructing new and improved amino acid-producing C. glutamicum strains.

关 键 词：L-异亮氨酸 谷氨酸棒杆菌 α-酮基丁酸 启动子替换 柠苹酸合成酶 

分 类 号：TQ922[轻工技术与工程—发酵工程]