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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Acknowledgments
We are grateful for the kind gift of plasmids by Dr. Laurence G. Rahme (Harvard Medical School and Massachusetts General Hospital, Boston, MA, USA) and Professor Karl-Erich Jaeger (Institute for Molecular Enzyme Technology, Heinrich Heine University Duesseldorf, Juelich, Germany). This work was funded by the National High Technology Research and Development Program of China (863 Program) (Nos 2006AA020203, 2007AA05Z417, 2007AA100703 and 2009AA03Z232) and the Program for New Century Excellent Talents in University (NCET-07-0336).
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Jia, B., Yang, JK., Liu, WS. et al. Homologous overexpression of a lipase from Burkholderia cepacia using the lambda Red recombinase system. Biotechnol Lett 32, 521–526 (2010). https://doi.org/10.1007/s10529-009-0189-9
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DOI: https://doi.org/10.1007/s10529-009-0189-9