食品科学 ›› 2020, Vol. 41 ›› Issue (18): 98-104.doi: 10.7506/spkx1002-6630-20190712-173

• 生物工程 • 上一篇    下一篇

代谢工程改造谷氨酸棒杆菌生产甲硫氨酸

赵兰,刘诗梦,秦汉雄,王亚南,樊占青,闵伟红   

  1. (吉林农业大学食品科学与工程学院,小麦和玉米深加工国家工程实验室,吉林?长春 130118)
  • 出版日期:2020-09-25 发布日期:2020-09-18
  • 基金资助:
    国家自然科学基金面上项目(31771957)

Metabolic Engineering of Corynebacterium glutamicum for the Production of Methionine

ZHAO Lan, LIU Shimeng, QIN Hanxiong, WANG Yanan, FAN Zhanqing, MIN Weihong   

  1. (National Engineering Laboratory for Wheat and Corn Further Processing, College of Food Science and Engineering, Jilin Agricultural University, Changchun 130118, China)
  • Online:2020-09-25 Published:2020-09-18

摘要: 目的:通过构建新型微生物菌株提高微生物发酵法生产甲硫氨酸产量。方法:对甲硫氨酸合成途径关键酶天冬氨酸激酶(aspartate kinase,AK)、高丝氨酸脱氢酶(homoserine dehydrogenase,HSD)和高丝氨酸乙酰基转移酶(homoserine acetyltransferase,HAT)进行定向改造及过表达研究,通过实时聚合酶链式反应和高效液相色谱分析代谢流情况,并对竞争途径关键酶高丝氨酸激酶(homoserine kinase,HSK)A20底物结合位点进行定点突变及酶学性质表征,降低苏氨酸途径碳流的消耗。结果:菌株WTg1/PEC-lysCm-SD-homm-SD-metX中lysC、hom和metX相比于原菌AK基因表达量提高6.896、2.378?倍及1.659?倍。液相色谱分析发现甲硫氨酸产量相比于原菌提高到2.26?倍,达到4.14?g/L。同时获得HSK突变体thrB-A20Y、thrB-A20H、thrB-A20L是原菌HSK活性的39%、43%及49%。结论:本实验通过过表达干路关键酶并筛选弱化支路关键酶位点两方面,对谷氨酸棒杆菌天冬氨酸下游代谢进行调控,强化甲硫氨酸碳流运输系统,增强了碳流量的积累,有效提高了甲硫氨酸产量。研究可为构建甲硫氨酸工程菌提供一定理论参考。

关键词: 谷氨酸棒杆菌;菌株构建;过表达;筛选钝化酶;代谢流分析

Abstract: Objective: To a new strain for improved microbial production of methionine. Methods: Aspartate kinase (AK), homoserine dehydrogenase (HSD) and homoserine acetyltransferase (HAT), the key enzymes of methionine synthesis pathway, were modified and overexpressed in vitro. In addition, site-directed mutagenesis and characterization of the substrate binding site of the key enzyme, homoserine kinase (HSK) A20, were carried out to reduce the enzymatic activity and to prevent carbon flow from entering the threonine pathway. Results: Compared with the original strain, the expression levels of lysC, hom and metX in strain WTg1/PEC-lysCm-SD-homm-SD-metX increased by 6.896, 2.378 and 1.659 times, respectively. The liquid chromatographic analysis showed that the production of methionine was 4.14 g/L, 2.26 times higher than that of the original strain. The activities of HSK mutants thrB-A20Y, thrB-A20H and thrB-A20L were 39%, 43% and 49% as compared to HSK. Conclusion: In this experiment, the downstream metabolism of aspartic acid in Corynebacterium glutamicum was regulated by overexpression of the key enzymes in the trunk and weakening of the key enzymes in the branch. As a result, the methionine carbon transport system was strengthened, the accumulation of carbon flow was enhanced, and the production of methionine was effectively increased. This study provides theoretical reference for the construction of engineered bacteria for methionine production.

Key words: Crynebacterium glutamicum; strain construction; overexpression; screening of passivating enzymes; metabolic flux analysis

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