生物学杂志

生物学杂志 ›› 2025, Vol. 42 ›› Issue (5): 40-.doi: 10.3969/j.issn.2095-1736.2025.05.040

• 合成生物学专题 • 上一篇    下一篇

CRISPR/Cas9优化E.coliMG1655的L-Arg合成途径及代谢组学分析

林子静1， 陈允亮2， 张　蔓1， 李永臻1   

  1. 1. 青海大学 医学院 基础医学研究中心， 西宁 810016；
    2. 中国医学科学院 医药生物技术研究所， 北京 100000
  • 出版日期:2025-10-18 发布日期:2025-10-14
  • 通讯作者: 李永臻，副教授，研究方向为微生物次级代谢产物及生物合成机制，E-mail：liyongzhen885@qhu.edu.cn
  • 作者简介:林子静，硕士生，研究方向为微生物代谢工程，E-mail：leahlinz@163.com
  • 基金资助:
    国家自然科学基金项目（21967018）

Optimization of the anabolic pathway and metabolomic analysis of L-arginine in E. coli MG1655 using CRISPR/Cas9

LIN Zijing1, CHEN Yunliang2, ZHANG Man1, LI Yongzhen1   

  1. 1. Research Center for Basic Medical Science, Medical College, Qinghai University, Xining 810016, China;
    2. Institute of Pharmaceutical Biotechnology, Chinese Academy of Medical Sciences, Beijing 100000, China
  • Online:2025-10-18 Published:2025-10-14

摘要： 以大肠杆菌MG1655为起始菌株（M0），采用CRISPR/Cas9基因组编辑技术对L-精氨酸合成基因簇进行优化。首先，敲除鸟氨酸脱羧酶基因speC和speF以增加精氨酸前体物鸟氨酸的供应，然后整合谷氨酸棒状杆菌来源基因簇argCJBDF失活精氨酸脱羧酶基因adiA以期阻断L-精氨酸的降解，同时加强L-精氨酸的外源合成途径，得到重组菌株M1（MG1655ΔspeCΔspeFΔadiA::argCJBDF）。结果显示，重组菌株M1中精氨酸产量达1.35 g/L，与M0菌株相比提高0.88 g/L。利用非靶向代谢组学技术对2个菌株的代谢产物进行比较分析，结果表明，重组菌株M1中精氨酸代谢途径及其相关代谢产物浓度发生相应的变化，影响了精氨酸、赖氨酸等氨基酸类、有机酸类等多种代谢物的合成。研究为后续针对性地调控这些旁路代谢途径，构建高产L-精氨酸工程菌株提供了研究思路和方向。

关键词: 大肠杆菌MG1655, L-精氨酸, 基因编辑, 优化工程, 代谢组学

Abstract: The starting strain，Escherichia coliMG1655 (M0)， was selected for this study, and the CRISPR/Cas9 genome editing technology was utilized to optimize theL-arginine synthesis gene cluster. Initially, the ornithine decarboxylase genesspeCandspeFwere knocked out to enhance the ornithine supply, a precursor of arginine. Subsequently, integration of theargCJBDFgene cluster fromCorynebacterium glutamatewas used to deactivate the arginine decarboxylase geneadiA, blockingL-arginine degradation while enhancing its synthesis pathway. The resulting recombinant strain M1 (MG1655ΔspeCΔspeFΔadiA::argCJBDF) achieved an arginine yield of 1.35 g/L, representing a 0.88 g/L increase compared to strain M0. Untargeted metabolomics analysis revealed significant alterations in the arginine metabolic pathway and related metabolite concentrations in recombinant strain M1, impacting amino acid synthesis such as arginine and lysine, organic acids, and other metabolites. This study offers insights into potential strategies for regulating these alternate metabolic pathways and developing high-yieldL-arginine engineering strains.

Key words: E. coliMG1655, L-arginine, gene editing, optimization engineering, metabolomics

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