Integration of A Lipase Gene into the Bacillus subtilis Chromosome: Recombinant Strains Without Antibiotic Resistance Marker

A new system is presented for the generation of recombinant Bacillus subtilis strains without antibiotic markers. This system is based on two plasmids constructed in Escherichia coli. The first plasmid pHM30 contains an incomplete hisI gene, the last gene in the histidine biosynthesis operon of B. subtilis and part of the genes yvcA and yvcB of unkown function flanking hisI at the 3´-end. The spectinomycin resistance gene is inserted between hisI and the downstream yvcAB region. Transformation of B. subtilis with this plasmid pHM30 led to spectinomycin resistant, histidine auxotrophic strains. The integrated parts of pHM30 act like a docking station for the second plasmid pHM31. The plasmid pHM31 contains the same yvcAB region but a complete copy of the hisI gene and no antibiotic resistance marker. Heterologous genes to be expressed in B. subtilis were inserted into a multiple cloning site between hisI and the downstream region. Transformants of B.subtilis/pHM30 with pHM31 derivatives were selected on minimal medium without histidine. By double crossovers during homologous recombination the heterologous genes were integrated, replacing the defect copy of hisI and the spectinomycin resistance gene. The plasmids were also successfully applied in the chromosomal integration of the lipase gene of Bacillus thermocatenulatus under a B. subtilis glucose regulated promotor/antiterminator system.