Overexpression of thermophilic a -amylase in Bacillus licheniformis using a high efficiency chromosomal integration and amplification strategy

Abstract Background: The production of industrially important enzymes depends on the development of genetically stable and high-yielding microorganisms. In order to simplify the development of strains able to efficiently overproduce enzymes, a new strategy based on chromosomal integration and amplification in Bacillus sp. was developed.Results: A thermosensitive replicable plasmid pUB-MazF and an integrated expression plasmid pUB'-Ex1 were constructed. pUB-MazF with a thermo-sensitive replicase RepF encoded by gene repF can self-replicate in Bacillus sp. if the cultivation temperature is no more than 35 oC and if the MazF encoded by mazF is not expressed. mazF is controlled by an isopropyl β-d-1-thiogalactopyranoside (IPTG)-inducing promoter and MazF is toxic to the cell. When IPTG is present and the MazF encoded by mazF in pUB-MazF is expressed, the host cell should lose the plasmid to survive. At a cultivation temperature over 37 oC, pUB-MazF will not well replicate and is not stable. pUB'-Ex1 has a disrupted repF and cannot self-replicate but can replicate when pUB-MazF is present. By using this approach, pUB'-Ex1 and constructs can replicate and will be forced to integrate into the host chromosome at 37oC or over when a selectable marker is present. The host was cured by MazF expression after induction with IPTG. Bacillus licheniformis BL-UBM integrated with pUB-MazF was transformed with pUB'-amyS derived from pUB'-Ex1 by in-frame cloning of amyS encoding a thermophilic α-amylase from Geobacillus stearothermophilus ATCC 31195. B. licheniformis BL-UBM transformed with pUB'-amyS was cultivated at 42oC with 1 mmol/l IPTG and 500 μg/ml kanamycin and the recombinants expressing high α-amylase activities were selected. All recombinants evaluated were genetically stable. The highest yield of α-amylase of 50 753 U/ml was produced by recombinant BLiS-002 carrying five copies of amyS in a 50-l fermenter. Conclusion: The strategy developed in this study has potential application for convenient and rapid development of strains overexpressing industrially important enzymes.