Construction of Bacillus subtilis chassis strain with enhanced α-amylase expression capability based on CRISPRi screening

Bacillus subtilis has been widely used in the expression of recombinant proteins due to its food safe and powerful secretion characteristic, but the current production level cannot meet the increasing industrial needs. To enhance the production of recombinant protein, we first screened target key genes that are directly or indirectly involved in protein synthesis, using CRISPRi technology targeting the whole genome, with industrial valuable Bacillus stearothermophilus α-amylase as the model protein. Then the screened key genes were combined, yielding a chassis strain that owning enhanced protein expression capability. Following overlaying molecular chaperone GroES/L and peptidoglycan glycosyltransferase PonA, α-amylase activity reached 102,893 U/mL in a 3-L fermenter, the highest level reported till now. Finally, transcriptome analysis showed that the enhanced recombinant expression may be due to more rational allocation of energy and resources. These strategies can be well implicated in engineering other microbial cell factories for higher industrial production.