食品科学 ›› 2017, Vol. 38 ›› Issue (4): 32-38.doi: 10.7506/spkx1002-6630-201704006

• 生物工程 • 上一篇    下一篇

基于基因转录和代谢物分析的异亮氨酸生产菌谷氨酸棒状杆菌YILW的理性改造

温 冰,麻 杰,李智祥,张成林,徐庆阳,陈 宁   

  1. 天津科技大学生物工程学院,代谢控制发酵技术国家地方联合工程实验室,天津 300457
  • 出版日期:2017-02-25 发布日期:2017-02-28
  • 基金资助:
    国家高技术研究发展计划(863计划)项目(2013AA102106);国家自然科学基金青年科学基金项目(31300069); 天津市科技特派员项目(15JCTPJC62800)

Rational Design of Corynebacterium glutamicum YILW for Isoleucine Production Based on Gene Transcription and Metabolite Analysis

WEN Bing, MA Jie, LI Zhixiang, ZHANG Chenglin, XU Qingyang, CHEN Ning   

  1. National and Local United Engineering Lab of Metabolic Control Fermentation Technology, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China
  • Online:2017-02-25 Published:2017-02-28

摘要: 为获得异亮氨酸生产菌谷氨酸棒状杆菌(Corynebacterium glutamicum)YILW的理性改造策略,考察该菌株与出发菌株C. glutamicum ATCC 13032异亮氨酸合成途径中关键酶及代谢产物的差异。结果表明,C. glutamicumYILW丙酮酸羧化酶编码基因pyc的下调表达使得其胞内草酰乙酸含量降低,过表达该基因显著增加胞内草酰乙酸含量及异亮氨酸产量(分别从1.32 μmol/g(细胞干质量,下同)和5.18 g/L提高至3.32 μmol/g和5.81 g/L),但副产物赖氨酸及胞内2-酮丁酸积累量提高。针对该问题采用强启动子替换手段过表达ilvBNC操纵子,使得其异亮氨酸产量提高至6.63 g/L。为进一步增加异亮氨酸合成,过表达输出载体编码基因brnE和brnF,其产量提高至7.31 g/L,较出发菌株C. glutamicum YILW提高41.1%,转化率提高40.0%。由此可见,在基因转录及代谢物分析结果指导下理性过表达pyc、ilvBNC操纵子及brnE和brnF能够显著提高异亮氨酸产量并降低副产物浓度。

关键词: 谷氨酸棒杆菌, 异亮氨酸, 代谢库, 草酰乙酸

Abstract: This study aimed to rationally identify new targets for improving isoleucine production. The transcription levels of the key genes and intermediate metabolite levels involved in the isoleucine synthesis pathway of Corynebacterium glutamicum ATCC 13032 and C. glutamicum YILW, a isoleucine-producing strain derived from the parental strain ATCC 13032, were compared. The gene pyc was down-regulated, which might consequently lead to reduced supply of oxaloacetate. Then pyc was overexpressed in C. glutamicum YILW (denoted as YILW-1), resulting in increased oxaloacetate concentration and isoleucine production (from 1.32 to 3.32 μmol/g (md) and from 5.18 to 5.81g/L) but higher accumulation of lysine and intracellular 2-ketobutyrate as byproduct. The ilvBNC operon was further overexpressed in YILW-1 (denoted as YILW-2), resulting in production of up to 6.63 g/L isoleucine. To enhance exportation and consequently further increase the production of isoleucine, the isoleucine exporter genes brnE and brnF was overexpressed in YILW-2 (denoted as YILW-3), leading to increased production of isoleucine (7.31 g/L) by 10.3% as compared to that of YILW-2. The strategy resulted in 41.1% higher isoleucine production (from 5.18 to 7.31 g/L) and 40.0% higher yield (from 0.10 to 0.14 g/g glucose) together with lower by-product lelvels by YILW-3 as compared to C. glutamicum YILW. It could be concluded that overexpression of the pyc, ilvBNC operon as well as brnE and brnF based on transcription and metabolite pool analysis could significantly elevate isoleucine production and decrease by-product concentration levels.

Key words: Corynebacterium glutamicum, isoleucine, metabolite pool, oxaloacetate

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