Amino acid substitutions in random mutagenesis libraries: lessons from analyzing 3000 mutations
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The quality of amino acid substitution patterns in random mutagenesis libraries is decisive for the success in directed evolution campaigns. In this manuscript, we provide a detailed analysis of the amino acid substitutions by analyzing 3000 mutations of three random mutagenesis libraries (1000 mutations each; epPCR with a low-mutation and a high-mutation frequency and SeSaM-Tv P/P) employing lipase A from Bacillus subtilis (bsla). A comparison of the obtained numbers of beneficial variants in the mentioned three random mutagenesis libraries with a site saturation mutagenesis (SSM) (covering the natural diversity at each amino acid position of BSLA) concludes the diversity analysis. Seventy-six percent of the SeSaM-Tv P/P-generated substitutions yield chemically different amino acid substitutions compared to 64% (epPCR-low) and 69% (epPCR-high). Unique substitutions from one amino acid to others are termed distinct amino acid substitutions. In the SeSaM-Tv P/P library, 35% of all theoretical distinct amino acid substitutions were found in the 1000 mutation library compared to 25% (epPCR-low) and 26% (epPCR-high). Thirty-six percent of distinct amino acid substitutions found in SeSaM-Tv P/P were unobtainable by epPCR-low. Comparison with the SSM library showed that epPCR-low covers 15%, epPCR-high 18%, and SeSaM-Tv P/P 21% of obtainable beneficial amino acid positions. In essence, this study provides first insights on the quality of epPCR and SeSaM-Tv P/P libraries in terms of amino acid substitutions, their chemical differences, and the number of obtainable beneficial amino acid positions.
KeywordsProtein engineering Directed evolution Random mutagenesis epPCR SeSaM Lipase
- Frauenkron-Machedjou VJ, Fulton A, Zhu L, Anker C, Bocola M, Jaeger KE, Schwaneberg U (2015) Towards understanding directed evolution: more than half of all amino acid positions contribute to ionic liquid resistance of Bacillus subtilis lipase A. Chembiochem 16(6):937–945. doi:10.1002/cbic.201402682 CrossRefPubMedGoogle Scholar
- LinGoerke JL, Robbins DJ, Burczak JD (1997) PCR-based random mutagenesis using manganese and reduced dNTP concentration. BioTechniques 23(3):409–412Google Scholar
- Petritis K, Kangas LJ, Yan B, Monroe ME, Strittmatter EF, Qian WJ, Adkins JN, Moore RJ, Xu Y, Lipton MS, Ii DGC, Smith RD (2006) Improved peptide elution time prediction for reversed-phase liquid chromatography-MS by incorporating peptide sequence information. Anal Chem 78(14):5026–5039. doi:10.1021/ac060143p CrossRefPubMedPubMedCentralGoogle Scholar