Homologous overexpression of a lipase from Burkholderia cepacia using the lambda Red recombinase system
Homologous overexpression of a lipase from Burkholderia cepacia using the lambda Red recombinase system
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DOI：
10.1007/s10529-009-0189-9
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发表时间：
2010-04-01
影响因子：
2.7
通讯作者：
Yan, Yun-Jun
中科院分区：
文献类型：
--
作者：
Jia, Bin;Yang, Jiang-Ke;Yan, Yun-Jun
噬菌体的红色重组酶系统被广泛用于短线性DNA片段和基因组的重组。利用该系统，我们获得了洋葱伯克霍尔德菌T7 RNA聚合酶（RNAP）替代突变体。为了测试T7突变体的表达能力，我们转化了4种不同的脂肪酶表达载体，并对这些重组体的脂肪酶活性进行了评估。我们的研究结果表明，该单位与基因组之间的500 nt同源性足以产生突变，这种策略能够以85%的效率快速建立突变株。经表达纯化后，获得的最高纯化脂肪酶活性为3990 U/l，接近野生型的3倍。
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.