A rapid and efficient strategy for combinatorial repression of multiple genes in Escherichia coli

• Published in: Microbial cell factories Vol. 24; no. 1; pp. 74 - 12
• Main Authors: Zheng, Yi, Mo, Yuxia, Yuan, Yingbo, Su, Tianyuan, Qi, Qingsheng
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 28.03.2025; BMC
• Subjects: Analysis; Biosynthesis; Combinatorial repression; CRISPR-Cas Systems; CRISPRi; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Gene expression; Gene Expression Regulation, Bacterial; Genetic aspects; Identification and classification; Inducible promoters; Metabolic Engineering - methods; Metabolic flow; Multiple genes; Plasmids - genetics; Plasmids - metabolism; Promoter Regions, Genetic; RNA, Guide, CRISPR-Cas Systems - genetics; Japan; Combinatorial repression; Multiple genes; Inducible promoters; CRISPRi; Metabolic flow
• ISSN: 1475-2859; 1475-2859
• DOI: 10.1186/s12934-025-02697-x

The regulation of multiple gene expression is pivotal for metabolic engineering. Although CRISPR interference (CRISPRi) has been extensively utilized for multi-gene regulation, the construction of numerous single-guide RNA (sgRNA) expression plasmids for combinatorial regulation remains a significant challenge. In this study, we developed a combinatorial repression system for multiple genes by optimizing the expression of multi-sgRNA with various inducible promoters in Escherichia coli. We designed a modified Golden Gate Assembly method to rapidly construct the sgRNA expression plasmid p3gRNA-LTA. By optimizing both the promoter and the sgRNA handle sequence, we substantially mitigated undesired repression caused by the leaky expression of sgRNA. This method facilitates the rapid assessment of the effects of various inhibitory combinations on three genes by simply adding different inducers. Using the biosynthesis of N-acetylneuraminic acid (NeuAc) as an example, we found that the optimal combinatorial inhibition of the pta, ptsI, and pykA genes resulted in a 2.4-fold increase in NeuAc yield compared to the control. We anticipate that our combinatorial repression system will greatly simplify the regulation of multiple genes and facilitate the fine-tuning of metabolic flow in the engineered strains.