Belsare Ketaki D, Andorfer Mary C, Cardenas Frida S, Chael Julia R, Park Hyun June, Lewis Jared C
Department of Chemistry, University of Chicago , Chicago, Illinois 60637, United States.
ACS Synth Biol. 2017 Mar 17;6(3):416-420. doi: 10.1021/acssynbio.6b00297. Epub 2017 Jan 4.
Directed evolution is a powerful tool for optimizing enzymes, and mutagenesis methods that improve enzyme library quality can significantly expedite the evolution process. Here, we report a simple method for targeted combinatorial codon mutagenesis (CCM). To demonstrate the utility of this method for protein engineering, CCM libraries were constructed for cytochrome P450, pfu prolyl oligopeptidase, and the flavin-dependent halogenase RebH; 10-26 sites were targeted for codon mutagenesis in each of these enzymes, and libraries with a tunable average of 1-7 codon mutations per gene were generated. Each of these libraries provided improved enzymes for their respective transformations, which highlights the generality, simplicity, and tunability of CCM for targeted protein engineering.
定向进化是优化酶的一种强大工具，而提高酶文库质量的诱变方法可以显著加快进化过程。在此，我们报告了一种用于靶向组合密码子诱变（CCM）的简单方法。为了证明该方法在蛋白质工程中的实用性，构建了细胞色素P450、嗜热栖热菌脯氨酰寡肽酶和黄素依赖性卤化酶RebH的CCM文库；针对这些酶中的每一种，有10 - 26个位点被靶向进行密码子诱变，并生成了每个基因平均具有1 - 7个密码子突变的可调文库。这些文库中的每一个都为各自的转化提供了改良的酶，这突出了CCM在靶向蛋白质工程中的通用性、简单性和可调性。
