Clarifying the Difference between Iterative Saturation Mutagenesis as a Rational Guide in Directed Evolution and OmniChange as a Gene Mutagenesis Technique

Abstract

A recent directed‐evolution study by Schwaneberg and co‐workers comparing the widely used iterative saturation mutagenesis (ISM) with the OmniChange version of saturation mutagenesis (SM) prompts us to point out some flaws in the conclusions presented therein. Most importantly, ISM is a semirational strategy in directed evolution that is independent of the particular type of SM that the experimenter may choose; this means that OmniChange should not be compared with ISM. When aiming to improve enzyme selectivity or activity by the ISM strategy, the state‐of‐the‐art calls for SM at randomization sites lining the enzyme binding pocket as part of the combinatorial active‐site saturation test (CAST). Our recent studies focusing on the refinement of CAST/ISM have shown that this approach works best when using multiresidue randomization sites as opposed to single‐residue sites owing to the possibility of cooperative mutational effects. This advance was not considered by Schwaneberg and co‐workers, thus leading to questionable conclusions when pitching CAST/ISM against OmniChange.

The Crucial Role of Methodology Development in Directed Evolution of Selective Enzymes

“… Systematization of CAST/ISM . a) Randomization sites A, B, C, etc, each comprising one or more residues.…”

“…Thec rucial systematization was achieved in 2005 with the emergence of the Combinatorial Active-site Saturation Te st (CAST) (Figure 2a), [43] ac onvenient acronym to distinguish it from SM at remote sites for other purposes such as thermostabilization [44] or induction of allostery. [45] Figure 2b shows arecently modified schematic representation of CAST, [46] in which only as elect few CAST residues are considered (B,C ,E ,a nd H). Individual residues can be grouped into larger randomization sites.F irst-and secondsphere CAST residues are included.…”

“…Systematization of CAST/ISM. [46] a) Randomization sites A, B, C, etc, each comprising one or more residues. b) Reduced number of CAST sites chosen by structural, consensus and computational guides.…”

Die zentrale Rolle der Methodenentwicklung in der gerichteten Evolution selektiver Enzyme

“…B. Thermostabilisierung oder Allosterie‐Induktion . Abbildung b zeigt eine kürzlich modifizierte schematische Darstellung von CAST, in der nur einige wenige ausgewählte CAST‐Reste berücksichtigt sind (B, C, E und H). Einzelne Aminosäure‐Positionen können zu größeren Randomisierungsstellen gruppiert werden.…”

“…Mehrere sehr unterschiedliche SM‐Varianten wurden ebenfalls beschrieben, darunter OmniChange, welches eine gleichzeitige Sättigung von bis zu fünf Codonen in einem allerdings vierstufigen Prozess ermöglicht: a) Vektor‐ und Insertbildung durch klassische PCR mittels phosphorothiolierter Oligonukleotide, die degenerierte Codons enthalten; b) Verwendung von I 2 zur Spaltung der Phosphorothiodiesterbindungen zur Bildung komplementärer 5′‐Überhänge in Doppelstrang‐DNA; c) komplementäre DNA‐Hybridisierung zur Assembly der mutierten Plasmide; und d) Transformation in E. coli und Reparatur der Einzelstrangbrüche (“nick repair”). In einem alternativen, fünfstufigen Ansatz, genannt OSCARR, werden gezielt entworfene mutagene Kassetten und eine Megaprimer‐PCR verwendet, ebenfalls unter Bildung fokussierter Bibliotheken.…”