一种高效的多点组合突变克隆构建方法

高质量的突变方法和高效的筛选方法相结合可以提高酶定向进化的效率。文中开发了一种高效的多点组合突变(Multi-points combinatorial mutagenesis,MCM)的克隆方法。MCM方法通过引入DNA组装、融合PCR和杂交技术,实现高效多点组合突变。应用优化后的方法定向进化改造苯甲酰甲酸脱羧酶(Benzoylformate decarboxylase,BFD)来测试MCM方法的效率。通过电转至大肠杆菌感受态Escherichia coli TreliefTM 5α所获得的单菌落数量(Colony-formingunits,CFUs)超过106 CFUs/μgDNA。经验证90/100单菌落精确组装;5个位点L109、L110、H281、Q282和A460同时组合突变的效率达到88%。最后,筛选到一种kcat/Km提高10倍的突变酶(L109Y、L110D、H281G、Q282V和A460M)。因此,应用该方法可以有效地创建突变体库,促进酶的定向进化技术的快速发展。

A high efficiency cloning approach of multi-points combinational mutagenesis

The combination of high-quality mutagenesis and effective screening can improve the efficiency of enzyme directed evolution. In this study, a high efficiency cloning construction method of Multi-points Combinational Mutagenesis(MCM) was developed. Efficient multi-point combination mutations were performed in this MCM method by introducing DNA assembly, fusion PCR and hybridization techniques. After optimization, the efficiency of MCM was tested by directed evolution of benzoylformate decarboxylase. The obtained number of Colony Forming Units(CFUs) by electroporation to competent cells E. coli TreliefTM 5α exceeded 106 CFUs/μg DNA. Test results show that 90/100 clones were precisely assembled. The efficiency of simultaneous mutation at 5 sites(L109, L110, H281, Q282 and A460) was up to 88%. Finally, a mutant enzyme(L109 Y, L110 D, H281 G, Q282 V and A460 M) with a 10-fold increase in kcat/Km was obtained. Therefore, this method can effectively create diverse mutant libraries and promote the rapid development of enzyme directed evolution.