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 Trelief™ 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 (L109Y, L110D, H281G, Q282V and A460M) 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.
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 Trelief™ 5α 所获得的单菌落数量(Colony-forming units，CFUs) 超过10⁶ CFUs/μg DNA。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。.
Keywords: benzoylformate decarboxylase; directed evolution; high-throughput screening; multi-points combinatorial mutagenesis.