SeSaM‐Tv‐II Generates a Protein Sequence Space that is Unobtainable by epPCR

Abstract

Generating high-quality mutant libraries in which each amino acid is equally targeted and substituted in a chemically diverse manner is crucial to obtain improved variants in small mutant libraries. The sequence saturation mutagenesis method (SeSaM-Tv(+) ) offers the opportunity to generate such high-quality mutant libraries by introducing consecutive mutations and by enriching transversions. In this study, automated gel electrophoresis, real-time quantitative PCR, and a phosphorimager quantification system were developed and employed to optimize each step of previously reported SeSaM-Tv(+) method. Advancements of the SeSaM-Tv(+) protocol and the use of a novel DNA polymerase quadrupled the number of transversions, by doubling the fraction of consecutive mutations (from 16.7 to 37.1 %). About 33 % of all amino acid substitutions observed in a model library are rarely introduced by epPCR methods, and around 10 % of all clones carried amino acid substitutions that are unobtainable by epPCR.

To get what we aim for – progress in diversity generation methods

“…SeSaM-methods MCST [57] dITP-PCR [62] POE-PCR [63] MegAnneal [60] TRINS [56] epRCA [58] SeSaM-Tv-II [59] SeSaM-III [61] Chemical extraction steps in which significant amounts of DNA are lost [33,35] and/or the need for chemo-or enzymatic DNA modifications, such as phosphorylation [31,35] and biotinylation [34]. Broadly employed focused mutagenesis methods (e.g.…”

“…• TrimerDimer [38] • SILM [40] • OmniChange [9] • SySM [44] • Amber Codon [49] • MCST [55] • dITP-epPCR [60] • POE-PCR [61] • MegAnneal [58] • TRINS [54] • epRCA [56] • SeSaM-III [59] • TMGS-PCR [92] • USERec [96] • GoldenGate Shuffling [95] • PTRec [91] • Integron [94] Covered only 48 clones revealed 66-84% coverage of all possible codons at all five targeted positions (up to 27 out of 32 possible codons). OmniChange fulfills all the requirements in terms of robustness and simplicity with respect to becoming a successful method for focused multisite saturation mutagenesis.…”

“…Random mutagenesis methods have been grouped into three mutagenesis categories: (a) PCR-based; (b) chemical mutagens; and (c) whole cell methods [1,18]. Progress in the last 3 years is summarized by covering multicodon scanning mutagenesis (MCST), 2′-deoxyinosine 5′-triphosphate (dIT)P-PCR, prolonged overlap extension-PCR (POE-PCR), MegAnneal, tandem repeat insertion (TRINS), error-prone multiply-primed rolling circle amplification (epRCA), sequence saturation mutagenesis (SeSaM)-Tv-II and SeSaM-III [56][57][58][59][60][61][62][63] (Fig. 1).…”

“…Progress in the diversity of mutant libraries has been achieved for the MCST [57], MegAnneal [60] and SeSaM [59,61,67] methods by increasing the number of transversions and subsequent mutations. SeSaM outperforms epPCR and transposon-based methods in terms of diversity and at the expense of simplicity, as well as time requirements (Megawhop < epPCR < transposon < SeSaM) for generating a random mutant library.…”

A roadmap to directed enzyme evolution and screening systems for biotechnological applications

“…The addition of nucleotide analogues to bypass mutational bias, Random Insertion and Deletion (RID) mutagenesis, Random Insertional-deletional Strand Exchange (RAISE) mutagenesis and Sequence Saturation Mutagenesis (SeSaM) off er libraries with enhanced diversity . In particular, SeSaM (Wong et al, 2004) and its transversion enriched version SeSaM-Tv (Mundhada et al, 2011, Wong et al, 2008 offer the additional benefits of generating transversion enriched libraries and the ability of incorporate subsequent mutations in a codon, which increases the obtained codon diversity from a 39.5% (single mutation in a codon) to 83% coverage of the natural diversity (Shivange et al, 2009).…”