NDLI: Amino acid substitutions in random mutagenesis libraries: lessons from analyzing 3000 mutations

Content Provider	Springer Nature Link
Author	Zhao, Jing Frauenkron Machedjou, Victorine Josiane Kardashliev, Tsvetan Ruff, Anna Joëlle Zhu, Leilei Bocola, Marco Schwaneberg, Ulrich
Copyright Year	2017
Abstract	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.
Starting Page	3177
Ending Page	3187
Page Count	11
File Format	PDF
ISSN	01757598
Journal	Applied Microbiology and Biotechnology
Volume Number	101
Issue Number	8
e-ISSN	14320614
Language	English
Publisher	Springer Berlin Heidelberg
Publisher Date	2017-01-03
Publisher Place	Berlin, Heidelberg
Access Restriction	Subscribed
Subject Keyword	Protein engineering Directed evolution Random mutagenesis epPCR SeSaM Lipase Microbiology Microbial Genetics and Genomics Biotechnology
Content Type	Text
Resource Type	Article
Subject	Medicine Applied Microbiology and Biotechnology Biotechnology

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