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

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。cimA编码的甲基苹果酸合成酶可以催化丙酮酸和乙酰-CoA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 YILWPtacpXMJ19cimA，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。

Abstract:

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 ilvBN) 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 ilvBN expression. Citramalate synthase, encoded by cimA, catalyzes the generation of α-ketobutyrate from pyruvate and acetyl-CoA. α-Ketobutyrate is a direct precursor of L-isoleucine; thus, increasing cimA expression increases the metabolic flux of L-isoleucine synthesis. In the present study, the promoter of the ilvBNC 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 ilvBNC. 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 cimA gene under the control of a strong promoter was introduced into C. glutamicum YILWPtac to construct C. glutamicum YILWPtacpXMJ19cimA. Compared to the original strain, in C. glutamicum YILWPtacpXMJ19cimA, 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.