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Genome‐Wide CRISPRi Screening of Key Genes for Recombinant Protein Expression in Bacillus Subtilis
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Genome‐Wide CRISPRi Screening of Key Genes for Recombinant Protein Expression in Bacillus Subtilis
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Journal Article
Genome‐Wide CRISPRi Screening of Key Genes for Recombinant Protein Expression in Bacillus Subtilis
2024
Overview
Bacillus subtilis is an industrially important microorganism that is often used as a microbial cell factory for the production of recombinant proteins due to its food safety, rapid growth, and powerful secretory capacity. However, the lack of data on functional genes related to recombinant protein production has hindered the further development of B. subtilis cell factories. Here, a strategy combining genome‐wide CRISPRi screening and targeted CRISPRa activation to enhance recombinant protein expression is proposed. First, a CRISPRi library covering a total of 4225 coding genes (99.7%) in the B. subtilis genome and built the corresponding high‐throughput screening methods is constructed. Twelve key genes for recombinant protein expression are identified, including targets without relevant functional annotations. Meanwhile, the transcription of recombinant protein genes by CRISPRa is up‐regulated. These screened or selected genes can be easily applied to metabolic engineering by constructing sgRNA arrays. The relationship between differential pathways and recombinant protein expression in engineered strains by transcriptome analysis is also revealed. High‐density fermentation and generalisability validation results prove the reliability of the strategy. This method can be extended to other industrial hosts to support functional gene annotation and the design of novel cell factories. Based on genome‐wide CRISPRi screening, 12 repressor genes favorable for recombinant protein production are identified. sgRNA arrays are constructed to combine these novel targets with a CRISPRa‐driven activation strategy to achieve comprehensive multi‐gene regulation. The high‐yielding strains that are obtained have improved the yield of a variety of recombinant proteins.
Publisher
John Wiley & Sons, Inc,John Wiley and Sons Inc,Wiley
Subject
/ Bacillus subtilis - genetics
/ Bacillus subtilis - metabolism
/ Clustered Regularly Interspaced Short Palindromic Repeats - genetics
/ CRISPR
/ CRISPR-Cas Systems - genetics
/ Genome, Bacterial - genetics
/ Genomes
/ genome‐wide CRISPRi screening
/ Metabolic Engineering - methods
/ Plasmids
/ Proteins
/ recombinant protein expression
/ Recombinant Proteins - genetics
