食品与发酵工业2025，Vol.51Issue(24)：35-42,8.DOI:10.13995/j.cnki.11-1802/ts.042725
枯草芽孢杆菌中基于CRISPR的双向多基因调节系统构建和应用
Construction of a CRISPR-based bidirectional transcriptional regulation system in Bacillus subtilis for metabolic network controls
摘要
Abstract
CRISPR-based transcriptional regulation systems have been widely employed for precise metabolic network control in mi-crobial cell factories.However,the development of CRISPR activation(CRISPRa)systems in Gram-positive bacteria,particularly Bacillus subtilis,remains limited due to unclear activation domains and narrow effective windows.In this study,we constructed a bidirectional CRISPR-based regulation system in B.subtilis,capable of simultaneously activating and repressing multiple target genes.First,we estab-lished CRISPRa and CRISPR interference(CRISPRi)systems based on dCpf1 fused with activation domains(dCpf1-AD)and identified RemA as a highly efficient activation domain,enabling bidirectional regulation of multiple target genes.Second,we improved the tran-scriptional activation efficiency by optimizing protein scaffold structures,enhancing expression levels,and introducing random mutagenesis within the activation domain,resulting in a 36.7％and 40.5％increase in sfGFP and mCherry expression,respectively.Finally,we ap-plied this system to a riboflavin-producing strain and simultaneously activated and repressed three key genes involved in riboflavin biosyn-thesis,leading to an 8.7-fold increase in riboflavin production,reaching 227.5 mg/L.This study provides a novel approach for construc-ting B.subtilis-based microbial cell factories with enhanced metabolic productivity.关键词
枯草芽孢杆菌/基因调控/CRISPR激活/CRISPR抑制/核黄素Key words
Bacillus subtilis/gene regulation/CRISPR activation/CRISPR inhibition/riboflavin引用本文复制引用
刘济辅,于文文,徐显皓,武耀康,刘延峰,李江华,堵国成,吕雪芹,刘龙..枯草芽孢杆菌中基于CRISPR的双向多基因调节系统构建和应用[J].食品与发酵工业,2025,51(24):35-42,8.基金项目
国家自然科学基金项目(32372301) （32372301）
