Homologous overexpression of a lipase from Burkholderia cepacia using the lambda Red recombinase system.
Red recombinase system of the lambda phage is widely used for recombination of short linear DNA fragments and genome. Using this system, we obtained T7 RNA polymerase (RNAP) substitution mutants in Burkholderia cepacia. To test the expression abilities of the T7 mutants, four different lipase expression vectors were transformed and the lipase activity of these recombinants was evaluated. Our results suggest that 500 nt homology between the unit and the genome is sufficient to generate mutations and this strategy enables the rapid establishment of mutant strains with efficiencies of 85%. After expression and purification, the highest purified lipase activity obtained was 3,990 U/l, nearly triple that of the wild-type organism.
Jia
Bin
B
Key Laboratory of Molecular Biophysics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, 430074 Wuhan, People's Republic of China.
Yang
Jiang-Ke
JK
Liu
Wen-Shan
WS
Li
Xu
X
Yan
Yun-Jun
YJ
D010582:Bacteriophage lambda / Q000235:genetics*; D016956:Burkholderia cepacia / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D008049:Lipase / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D045522:Recombinases / Q000235:genetics; D012988:Soil Microbiology; D014764:Viral Proteins / Q000235:genetics
D011994:Recombinant Proteins; D045522:Recombinases; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D008049:Lipase