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Bacillus subtilis  ATCC 6051a is an undomesticated strain used in the industrial production of enzymes. Because it is poorly transformable, genetic manipulation in this strain requires a highly efficient genome editing method. In this study, a  Streptococcus pyogenes  CRISPR/Cas9 system consisting of an all-in-one knockout plasmid containing a target-specific guide RNA,  cas9 , and a homologous repair template was established for highly efficient gene disruption in  B. subtilis  ATCC 6051a. With an efficiency of 33% to 53%, this system was used to disrupt the  srfC ,  spoIIAC ,  nprE ,  aprE  and  amyE  genes of  B. subtilis  ATCC 6051a, which hamper its use in industrial fermentation. Compared with  B. subtilis  ATCC 6051a, the final mutant, BS5 (Δ srfC , Δ spoIIAC , Δ nprE , Δ aprE , Δ amyE ), produces much less foam during fermentation, displays greater resistant to spore formation, and secretes 2.5-fold more β-cyclodextrin glycosyltransferase into the fermentation medium. Thus, the CRISPR/Cas9 system proved to be a powerful tool for targeted genome editing in an industrially relevant, poorly transformable strain.

Multigene disruption in undomesticated Bacillus subtilis ATCC 6051a using the CRISPR/Cas9 system