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. 2017 Mar 17;6(3):416-420.
doi: 10.1021/acssynbio.6b00297. Epub 2017 Jan 4.

A Simple Combinatorial Codon Mutagenesis Method for Targeted Protein Engineering

Ketaki D Belsare  1 , Mary C Andorfer  1 , Frida S Cardenas  1 , Julia R Chael  1 , Hyun June Park  1 , Jared C Lewis  1
Affiliations

A Simple Combinatorial Codon Mutagenesis Method for Targeted Protein Engineering

Ketaki D Belsare et al. ACS Synth Biol. .

Abstract

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 P450BM3, 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.

Keywords: codon mutagenesis; cytochrome P450; directed evolution; halogenase; prolyl oligopeptidase.

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Figures

Figure 1
Figure 1
Overview of A) directed evolution and three common mutagenesis strategies; B) combinatorial codon mutagenesis involving iterative rounds of fragment PCR using mutagenic primer pools followed by joining PCR.
Figure 2
Figure 2
A) Codon changes per gene for 50 randomly selected clones from a BM3 CCM library. B) Percentage of the 12 possible amino acid residues at mutated sites. C) The 22 targeted sites are all mutated with similar efficiency.
Figure 3
Figure 3
A) Crystal structures of RebH and BM3, and a homology model of POP containing a covalently-linked dirhodium cofactor. Cofactors shown as red sticks. Targeted residues shown as blue and green spheres; green indicates mutations in optimized variants. B) Codon changes per gene for each library. C) Reactions catalyzed by CCM library members (Ar=4-methoxyphenyl).
Figure 4
Figure 4
Targeted residues for five variants from a POP-E7 deconvolution library. Residues at targeted sites in POP-ZA2 and E7 are shown in grey. Residues shown in blue were targeted but not reverted; residues shown in green were reverted.
See this image and copyright information in PMC

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