A Transposon System for Random Insertion of a Gene Expression Cassette into the Chromosome of Bacillus Subtilis.

Bacillus subtilis is a robust industrial workhorse for the production of heterologous proteins. Chromosomal integration-based protein production has advantages over plasmid-based methods. Considering that the expression level of a gene is affected by its location in the chromosome, it is important to find an optimal integration site for the gene to be expressed. This work establishes a method for random insertion of a gene expression cassette into chromosomes, enabling the screening of optimal integration sites for high-level protein production. Specifically, a gene expression cassette and a chloromycetin-resistance marker are assembled into a transposon. This transposon is inserted between the promoter and the ribosomal binding site of the zeocin-resistance marker in the chromosome, which blocks the transcription of the zeocin-resistance gene. Trans-posase Himar1-mediated transposition of this transposon activates the zeocin-resistance marker, which can be selected on plates containing both chloromycetin and zeocin. The transposition frequency was over 10-5. This method was used to select proper insertion sites for the expression cassette of methyl parathion hydrolase (MPH). Compared with the common integration site amyE, the expression level of MPH was increased up to 50 % at the yjbH site.