Efficient Large‐Scale and Scarless Genome Engineering Enables the Construction and Screening of Bacillus subtilis Biofuel Overproducers

Bacillus subtilis is a versatile microbial cell factory that can produce valuable proteins and value‐added chemicals. Long fragment editing techniques are of great importance for accelerating bacterial genome engineering to obtain desirable and genetically stable host strains. Herein, we develop an efficient CRISPR‐Cas9 method for large‐scale and scarless genome engineering in the Bacillus subtilis genome, which can delete up to 134.3 kb DNA fragments, 3.5 times as long as the previous report, with a positivity rate of 100%. The effects of using a heterologous NHEJ system, linear donor DNA, and various donor DNA length on the engineering efficiencies were also investigated. The CRISPR‐Cas9 method was then utilized for Bacillus subtilis genome simplification and construction of a series of individual and cumulative deletion mutants, which are further screened for overproducer of isobutanol, a new generation biofuel. These results suggest that the method is a powerful genome engineering tool for constructing and screening engineered host strains with enhanced capabilities, highlighting the potential for synthetic biology and metabolic engineering.