Overexpression of Thermophilic α-amylase in Bacillus Licheniformis using a High Efficiency Chromosomal Integration and Amplification Strategy
Highly efficient preparation of industrially important enzymes depends on development of the genetically stable and high-yield microbial cell factories, which is often a challengeable laboratory hard work. In aims to simplify strain development with high efficiency for enzyme overproduction, a new strategy based on chromosomal integration and amplification in Bacillus sp . was developed. A pair of plasmids, an integrated expression plasmid pUB'-Ex1 and a thermosensitive replicable plasmid
... zF, were constructed. pUB'-Ex1 conditionally self-replicated in Bacillus sp . when the RepF in pUB-MazF expressed. pUB-MazF thermosensitively self-replicated in Bacillus sp . , which was easily cured from the host by inducing MazF expression with IPTG. Bacillus licheniformis BL-UBM that integrated with pUB-MazF was then transformed with pUB'-amyS derived from pUB'-Ex1 by in-frame cloning of amyS encoding a thermophilic α-amylase from Geobacillus stearothermophilus ATCC 31195. The transformant of B. licheniformis BL-UBM with pUB'-amyS was cultivated at 42 o C with the existence of 1 mmol/l IPTG and 500 μg/ml kanamycin and the recombinants with high α-amylase activities were selected. All tested recombinants were extremely high genetic stability. One of which, recombinant BLiS-002, carried five copies of amyS and produced the highest yield of α-amylase. It could yield 50,753 U/ml of α-amylase in a 50-l bioreactor. The strategy developed in this study is of application potential for convenient and quick strain development for industrially important enzyme overexpression.