A Simple Combinatorial Codon Mutagenesis Method for Targeted Protein Engineering

作者：Belsare Ketaki D; Andorfer Mary C; Cardenas Frida S; Chael Julia R; Park Hyun June; Lewis Jared C^*

来源：ACS Synthetic Biology, 2017, 6(3): 416-420.

DOI：10.1021/acssynbio.6b00297

• codon mutagenesis
• directed evolution
• halogenase
• prolyl oligopeptidase
• cytochrome P450

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(BM3), 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.

• 出版日期2017-3