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。
The starting strain,Escherichia coli MG1655(M0),was selected for this study,and the CRISPR/Cas9 genome editing technology was utilized to optimize the L-arginine synthesis gene cluster.Initially,the ornithine decarboxylase genes speC and speF were knocked out to enhance the ornithine supply,a precursor of arginine.Subsequently,integration of the argCJBDF gene cluster from Corynebacterium glutamate was used to deactivate the arginine decarboxylase gene adiA,blocking L-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-yield L-arginine engineering strains.
作者
林子静
陈允亮
张蔓
李永臻
LIN Zijing;CHEN Yunliang;ZHANG Man;LI Yongzhen(Research Center for Basic Medical Science,Medical College,Qinghai University,Xining 810016,China;Institute of Pharmaceutical Biotechnology,Chinese Academy of Medical Sciences,Beijing 100000,China)
出处
《生物学杂志》
北大核心
2025年第5期40-45,共6页
Journal of Biology
基金
国家自然科学基金项目(21967018)。
