食品与发酵工业 >

2023 , Vol. 49 >Issue 21: 9 - 15

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.034924

研究报告

系统代谢工程改造谷氨酸棒杆菌高产L-丙氨酸

  • 吴晨 ,
  • 王泽婷 ,
  • 赵桂红 ,
  • 吕庚承 ,
  • 王非傲 ,
  • 刘月香 ,
  • 陈宁 ,
  • 李燕军
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  • 1(天津科技大学 生物工程学院,天津,300457)
    2(天津科技大学 工业发酵微生物教育部重点实验室,天津,300457)
    3(天津科技大学 代谢控制发酵技术国家地方联合工程实验室,天津,300457)
第一作者:硕士研究生(李燕军副教授为通信作者,E-mail:yjli@tust.edu.cn)

收稿日期: 2023-01-17

  修回日期: 2023-02-16

  网络出版日期: 2023-12-08

基金资助

国家重点研发计划“绿色生物制造”专项(2021YFC2100900)

收起

Systems metabolic engineering of Corynebacterium glutamicum for the efficient production of L-alanine

  • WU Chen ,
  • WANG Zeting ,
  • ZHAO Guihong ,
  • LYU Gengcheng ,
  • WANG Feiao ,
  • LIU Yuexiang ,
  • CHEN Ning ,
  • LI Yanjun
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  • 1(College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China)
    2(Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin University of Science and Technology, Tianjin 300457, China)
    3(National and Local United Engineering Lab of Metabolic Control Fermentation Technology, Tianjin University of Science and Technology, Tianjin 300457, China)

Received date: 2023-01-17

  Revised date: 2023-02-16

  Online published: 2023-12-08

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摘要

L-丙氨酸是一种重要的天然氨基酸,广泛应用于医药、食品、饲料等领域。目前,L-丙氨酸主要通过大肠杆菌来生产,开发安全、工业稳定的谷氨酸棒杆菌生产菌株具有重要的意义。该研究筛选了外源丙氨酸脱氢酶,在不以其他氨基酸为氨基供体的条件下高效率合成L-丙氨酸。强化非磷酸转移酶系统(phosphotransferase system,PTS)葡萄糖摄取途径,极大地促进了细胞的生长和L-丙氨酸的合成。引入异源Entner-Dondoroff途径,通过4步反应即可实现糖酵解途径10步反应供应前体物丙酮酸,进一步提高了L-丙氨酸的产量。此外,为了避免抗生素的使用和保证菌株的遗传稳定性,将关键基因整合到基因组上。最终工程菌株在5 L发酵罐发酵48 h产生104 g/L L-丙氨酸。该研究为利用谷氨酸棒杆菌工业化生产L-丙氨酸奠定了基础,同时为其他丙酮酸族产品的代谢工程研究提供了参考。

关键词： 谷氨酸棒杆菌; L-丙氨酸; 丙氨酸脱氢酶; 非PTS系统; EMP途径; ED途径

本文引用格式

吴晨 , 王泽婷 , 赵桂红 , 吕庚承 , 王非傲 , 刘月香 , 陈宁 , 李燕军 . 系统代谢工程改造谷氨酸棒杆菌高产L-丙氨酸[J]. 食品与发酵工业, 2023 , 49(21) : 9 -15 . DOI: 10.13995/j.cnki.11-1802/ts.034924

Abstract

L-alanine is an important natural amino acid, which is widely used in medicine, food and feed industries. At present, L-alanine is mainly produced by Escherichia coli. It is of great significance to develop safe and industrially robust Corynebacterium glutamicum production strains. In this study, exogenous alanine dehydrogenase was optimized to synthesize L-alanine efficiently without using other amino acids as an amino donor. The non-phosphotransferase system glucose uptake pathway was strengthened by the simultaneous inactivation of the transcriptional repressor IolR and overexpression of the glucose kinase, which drastically promote the cell growth and L-alanine synthesis. The introduction of exogenous Entner-Doudoroff pathway that supply pyruvate only through 4-step reactions, an alternative to the 10-step Embden-Meyerhof-Parnas (EMP) pathway, further increased the production of L-alanine. In addition, in order to avoid the use of antibiotics and guarantee the genetic stability of the producer, essential genes were integrated into the genome of the strain. The final engineered strain produced 104 g/L L-alanine after 48 h fermentation in a 5 L fermenter. The study lays a solid foundation for the industrial production of L-alanine by C. glutamicum, and provides a reference for the metabolic engineering of other microbes to produce products derived from pyruvate.

Key words： Corynebacterium glutamicum; L-alanine; alanine dehydrogenase; non-phosphotransferase system; embden-meyerhof-parnas (EMP) pathway; entner-doudoroff pathway

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