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Bacillus licheniformis is a well-known platform strain for production of industrial enzymes. However, the development of genetically stable recombinant B. licheniformis for high-yield enzyme production is still laborious. The need for a method to efficient integration and expression of a target gene in B. licheniformis is urgent. A pair of plasmids, pUB-MazF and pUB'-EX1, were constructed from the parent plasmid pUB-EX. pUB-MazF is a thermo-sensitive, self-replicable plasmid. It was able to efficiently cure from the host cell through induced expression of an endoribonuclease MazF, which is lethal to the host cell. pUB'-EX1 is a non-replicative and integrative plasmid. Its replication was dependent on the thermosensitive replicase produced by pUB-MazF. Transformation of pUB'-EX1 into the B. licheniformis BL-UBM harboring pUB-MazF resulted in both plasmids coexisting in the host cell. At an elevated temperature, and presence of isopropyl-1-thio-β-D-galactopyranoside and kanamycin, curing of the pUB-MazF and multiple-copy integration of pUB'-EX1 occurred, simultaneously. Through this procedure, multiple copies of the α-amylase gene, amyS, from Geobacillus stearothermophilus ATCC 31 195 were successfully integrated into the chromosome of host cell. Consequently, recombinants overexpressing α-amylase were facilely obtained. The genetic stability of the recombinants was verified by repeated subculturing and shaking-flask fermentations. The production of α-amylase by recombinant BLiS-002, harboring five copies of amyS, was studied in a 50-l bioreactor. The highest α-amylase activity reached 50 753 U/ml after 72 h fermentation. This strategy therefore has potential for production of other enzymes in B. licheniformis and for genetic modification of other Bacillus species.

High-efficiency chromosomal integrative amplification strategy for overexpressing α-amylase in Bacillus licheniformis