Fragment exchange (FX) plasmid tools for CRISPR/Cas9-mediated gene integration and protease production in Bacillus subtilis.

Since its discovery as part of the bacterial adaptative immune system, CRISPR/Cas has emerged as the most promising tool on targeted genome-editing over the past few years. Various tools for genome editing in Bacillus subtilis have recently been developed, expanding and simplifying its potential development as an industrial strain. A collection of vectors compatible with high-throughput fragment exchange (FX) cloning for heterologous expression in E. coli and Bacillus were previously developed. This vector catalogue was through this work supplemented with editing plasmid for genome engineering in Bacillus by adapting two CRISPR/Cas plasmids to the cloning technology. The customized tools allow versatile editing at any chosen genomic position (single-plasmid strategy) or at a fixed genomic locus (double-plasmid strategy). The single-plasmid strategy was validated by deleting the spoIIAC gene, which has an essential role in sporulation. Using the double-plasmid strategy, we demonstrate the quick transition from plasmid-based subtilisin expression to a stable integration of the gene into the amyE locus of a seven-protease deficient KO7 strain. The newly engineered B. subtilis strain allowed for a successful production of a functional enzyme. The customized tools provide improvements to the cloning procedure, should be useful for versatile genomic engineering and contributes to a cloning platform for quick transition from HTP enzyme expression to production through fermentation of the industrially relevant B. subtilis and related strains.Importance We complemented a cloning platform with new editing plasmids that allows a quick transition from high-throughput cloning and expression of new enzymes to stable integration of genes for production of enzymes through B. subtilis fermentation. We present two systems for the effective assembly cloning of any genome editing cassette that shortens the engineering procedure to obtain the final editing constructs. The utility of the customized tools is demonstrated by disrupting Bacillus' capacity to sporulate, and by introducing stable expression of subtilisin. The tools should be useful to engineered B. subtilis strains by a variety of recombination events to ultimately improve the application range of this industry relevant host.