title: Amino acid substitutions in random mutagenesis libraries: lessons from analyzing 3000 mutations.
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.
authors.affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany.|Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhao
authors.affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany.
authors.forename: Victorine Josiane
authors.initials: VJ
authors.lastname: Frauenkron-Machedjou
authors.affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany.
authors.forename: Tsvetan
authors.initials: T
authors.lastname: Kardashliev
authors.affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany.
authors.forename: Anna Joëlle
authors.initials: AJ
authors.lastname: Ruff
authors.affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany.
authors.forename: Leilei
authors.initials: L
authors.lastname: Zhu
authors.affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany.
authors.forename: Marco
authors.initials: M
authors.lastname: Bocola
authors.affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany. u.schwaneberg@biotec.rwth-aachen.de.
authors.forename: Ulrich
authors.identifier: http://orcid.org/0000-0003-2285-4202
authors.initials: U
authors.lastname: Schwaneberg
mesh_terms: D019943:Amino Acid Substitution; D001412:Bacillus subtilis / Q000201:enzymology; D004252:DNA Mutational Analysis; D019020:Directed Molecular Evolution / Q000379:methods; D015723:Gene Library; D008049:Lipase / Q000235:genetics; D016296:Mutagenesis; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D015202:Protein Engineering / Q000379:methods
chemical_list: D008049:Lipase
keywords: Directed evolution; Lipase; Protein engineering; Random mutagenesis; SeSaM; epPCR