CRISPR-Cas系统已被广泛用于细胞代谢网络的精细调控。然而革兰氏阳性菌中的CRISPR激活(CRISPRa)系统仍存在激活效果差、作用窗口较小的问题。该研究在模式工业微生物枯草芽孢杆菌中构建了一种基于CRISPR-dCpf1的基因表达双向调控系统,可实现同时对多个基因的原位激活和抑制。首先,通过将不同类型的转录激活结构域与dCpf1融合,发现dCpf1-RemA可同时激活多个不同的启动子;此外,通过优化crRNA的靶向位点,dCpf1-RemA还具有抑制基因表达的功能,可用于构建CRISPR抑制(CRISPRi)系统。其次,通过构建蛋白支架结构和优化dCpf1-RemA表达量等方法,提高了CRISPRa系统的转录激活效果,使sfGFP和mCherry的表达量分别提高了36.7%和40.5%。最后,使用该系统全局调控枯草芽孢杆菌中核黄素的代谢网络,使核黄素产量提高了8.7倍,达到227.5 mg/L。该研究为枯草芽孢杆菌中代谢网络的全局调控提供了新工具。
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
.
DOI: 10.13995/j.cnki.11-1802/ts.042725
CRISPR-based transcriptional regulation systems have been widely employed for precise metabolic network control in microbial 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 established 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 transcriptional 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 applied this system to a riboflavin-producing strain and simultaneously activated and repressed three key genes involved in riboflavin biosynthesis, leading to an 8.7-fold increase in riboflavin production, reaching 227.5 mg/L.This study provides a novel approach for constructing B.subtilis-based microbial cell factories with enhanced metabolic productivity.
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