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

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A new system is presented for the generation ofrecombinant Bacillus subtilis strains without antibioticmarkers. This system is based on two plasmids constructed in Escherichia coli. The first plasmid pHM30contains an incomplete hisI gene, the last gene in thehistidine biosynthesis operon of B. subtilis and part ofthe genes yvcA and yvcB of unkown function flankinghisI at the 3´-end. The spectinomycin resistance geneis inserted between hisI and the downstream yvcABregion. Transformation of B. subtilis with this plasmidpHM30 led to spectinomycin resistant, histidine auxotrophic strains. The integrated parts of pHM30 act likea docking station for the second plasmid pHM31. Theplasmid pHM31 contains the same yvcAB region but acomplete copy of the hisI gene and no antibiotic resistance marker. Heterologous genes to be expressed inB. subtilis were inserted into a multiple cloning sitebetween 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 spectinomycinresistance gene. The plasmids were also successfullyapplied in the chromosomal integration of the lipasegene of Bacillus thermocatenulatus under a B. subtilisglucose regulated promotor/antiterminator system.