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

Autor:	Shen P; College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China., Niu D; Department of Biological Chemical Engineering, College of Chemical Engineering and Materials Science, Tianjin University of Science and Technology, Tianjin 300457, China., Liu X; Department of Biological Chemical Engineering, College of Chemical Engineering and Materials Science, Tianjin University of Science and Technology, Tianjin 300457, China., Tian K; Department of Biological Chemical Engineering, College of Chemical Engineering and Materials Science, Tianjin University of Science and Technology, Tianjin 300457, China., Permaul K; Department of Biotechnology and Food Science, Faculty of Applied Sciences, Durban University of Technology, PO Box 1334, Durban 4001, South Africa., Singh S; Department of Biotechnology and Food Science, Faculty of Applied Sciences, Durban University of Technology, PO Box 1334, Durban 4001, South Africa., Mchunu NP; Biotechnology Platform, Agricultural Research Council, Private Bag X5, Onderstepoort 0110, South Africa., Wang Z; College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China.; Department of Biological Chemical Engineering, College of Chemical Engineering and Materials Science, Tianjin University of Science and Technology, Tianjin 300457, China.
Jazyk:	angličtina
Zdroj:	Journal of industrial microbiology & biotechnology [J Ind Microbiol Biotechnol] 2022 May 25; Vol. 49 (3).
DOI:	10.1093/jimb/kuac009
Abstrakt:	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. Here, a pair of plasmids, pUB-MazF and pUB'-EX1, were firstly constructed. pUB-MazF is a thermosensitive, 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 nonreplicative 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 in the 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, genetically stable recombinants integrated multiple copies of amyS, from Geobacillus stearothermophilus ATCC 31195 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, in a 50-l bioreactor reached 50 753 U/ml after 72 hr fermentation. This strategy therefore has potential for production of other enzymes in B. licheniformis and for genetic modification of other Bacillus species. (© The Author(s) 2022. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.)
Databáze:	MEDLINE
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