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The  ganA  gene from  Bacillus subtilis  encoding a β-galactosidase for degradation of the galactomannan was integrated in different loci of the  B. subtilis  chromosome employing the CRISPR/Cas9 system. Hereby a total of five copies of  ganA  cassettes in which the  ganA  gene was fused with the glucitol-promoter were inserted in the recipient chromosome wherein hypothetical, sporulation and protease genes were deleted. The strain with five copies of  ganA  expression cassette showed a β-galactosidase activity similar to the one with the same gene on a pUB110 derived multi-copy plasmid and under the same regulatory control of the glucitol promoter and GutR activator. The production of β-galactosidase in the strain with the multi-copy plasmid decreased rapidly when growth was performed under induced conditions and without antibiotic selection. In contrast, the strain with the five copies of  ganA  in the chromosome produced β-galactosidase for at least 40 generations. This demonstrates that the CRISPR/Cas9 system is a valuable and easy tool for constructing stable producer strains. The bigger efforts that are needed for the multiple target gene integration into the chromosome compared to cloning in expression vectors were justified by the higher stability of the target genes and the lack of antibiotic resistance genes.

Multiple integration of the gene ganA into the Bacillus subtilis chromosome for enhanced β-galactosidase production using the CRISPR/Cas9 system