Simple, fast and high-efficiency transformation system for directed evolution of cellulase in Bacillus subtilis

被引:135
作者
Zhang, Xiao-Zhou [1 ]
Zhang, Y. -H. Percival [1 ,2 ,3 ]
机构
[1] Virginia Polytech Inst & State Univ, Dept Biol Syst Engn, Blacksburg, VA 24061 USA
[2] Virginia Polytech Inst & State Univ, Inst Crit Technol & Appl Sci, Blacksburg, VA 24061 USA
[3] BioEnergy Sci Ctr, Dept Energy, Oak Ridge, TN 37831 USA
关键词
RECOMBINANT PROTEINS; ENZYMATIC-HYDROLYSIS; EXPRESSION; SECRETION; CONSTRUCTION; LIBRARIES; SELECTION; GENES; SITE; COMK;
D O I
10.1111/j.1751-7915.2010.00230.x
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
Bacillus subtilis can serve as a powerful platform for directed evolution, especially for secretory enzymes. However, cloning and transformation of a DNA mutant library in B. subtilis are not as easy as they are in Escherichia coli. For direct transformation of B. subtilis, here we developed a new protocol based on supercompetent cells prepared from the recombinant B. subtilis strain SCK6 and multimeric plasmids. This new protocol is simple (restriction enzyme-, phosphatase-and ligase-free), fast (i.e. 1 day) and of high efficiency (i.e. similar to 10(7) or similar to 10(4) transformants per mu g of multimeric plasmid or ligated plasmid DNA respectively). Supercompetent B. subtilis SCK6 cells were prepared by overexpression of the competence master regulator ComK that was induced by adding xylose. The DNA mutant library was generated through a two-round PCR: (i) the mutagenized DNA fragments were generated by error-prone PCR and linearized plasmids were made using high-fidelity PCR, and (ii) the multimeric plasmids were generated based on these two DNA templates by using overlap PCR. Both protein expression level and specific activity of glycoside hydrolase family 5 endoglucanse on regenerated amorphous cellulose were improved through this new system. To our limited knowledge, this study is the first report for enhancing secretory cellulase performance on insoluble cellulose.
引用
收藏
页码:98 / 105
页数:8
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