Homologous overexpression of a lipase from <Emphasis Type="Italic">Burkholderia cepacia</Emphasis> using the lambda Red recombinase system |
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Authors: | Bin Jia Jiang-Ke Yang Wen-Shan Liu Xu Li Yun-Jun Yan |
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Institution: | (1) Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, 430074 Wuhan, People’s Republic of China; |
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Abstract: | Red recombinase system of the λ 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. |
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Keywords: | |
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