Overexpression of Thermophilic α-amylase in Bacillus Licheniformis using a High Efficiency Chromosomal Integration and Amplification Strategy [post]

Peili Shen, Dandan Niu, Xuelian Liu, Kangming Tian, Permaul Kugenthiren, Suren Singh, Zhengxiang Wang
2021 unpublished
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
more » ... cable plasmid pUB-MazF, 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.
doi:10.21203/rs.3.rs-147003/v1 fatcat:mbw54was4jhaxprl6f6n4ieqcm