Methodology Development in Directed Evolution: Exploring Options when Applying Triple‐Code Saturation Mutagenesis

Abstract

Directed evolution of stereo- or regioselective enzymes as catalysts in asymmetric transformations is of particular interest in organic synthesis. Upon evolving these biocatalysts, screening is the bottleneck. To beat the numbers problem most effectively, methods and strategies for building "small but smart" mutant libraries have been developed. Herein, we compared two different strategies regarding the application of triple-code saturation mutagenesis (TCSM) at multiresidue sites of the Thermoanaerobacter brockii alcohol dehydrogenase by using distinct reduced amino-acid alphabets. By using the synthetically difficult-to-reduce prochiral ketone tetrahydrofuran-3-one as a substrate, highly R- and S-selective variants were obtained (92-99 % ee) with minimal screening. The origin of stereoselectivity was provided by molecular dynamics analyses, which is discussed in terms of the Bürgi-Dunitz trajectory.

Synthesis of Trinucleotide Building Blocks in Solution and on Solid Phase

“…There are a number of techniques of combinatorial and evolutionary protein engineering available, which combine random mutagenesis or combinatorial gene synthesis with functional screening, at the phenotypic level, of the generated library of many structural variants. Successful screening relies on a library with a large number of potential hits and effective screening methods (Popova, Schubert, Bulla, Buchwald, & Kramer, ; Qu et al., ). Therefore, it is highly desirable not to waste a large proportion of a gene library on candidates that have little or no chance to pass the functional test.…”

Strategies in Protein Engineering to Evolve Proteins and Mimicking Evolution in the Laboratory Scale

“…This has been proved to be a very effective technique recently to tackle the very important challenge of the sequence-function relationship in case of directed evolution. The libraries thus generated on mutability landscapes can be used to engineer any fitness trait of user-defined interest [6,7,13]. We are entering into an era where the 'user-defined' interest has changed a lot.…”

“…Towards this goal, raised the need to develop the small and smart libraries. And hence forth different strategies including triple-code saturation mutagenesis (TCSM) at multiresidue sites of the Thermoanaerobacter brockii alcohol dehydrogenase by using distinct reduced amino acid alphabets and with requirement minimal screening have been recently reported [13][14][15]. So not only the physical labor has been greatly reduced, but also chances of false-postive results and codon-degeneracy can be minimized under this protocol [13].…”

Laboratory evolution of an alcohol dehydrogenase towards enantioselective reduction of difficult-to-reduce ketones

“…As an attractive ADH in biotechnology, the thermostable ADH from Thermoanaerobacter brockii (TbSADH) has already been repurposed to perform asymmetric reduction of prochiral ketones with the formation of enantio-pure secondary alcohols by our group and others (Heiss et al 2001;Li et al 2017;Musa et al 2009;Qu et al 2018;Sun et al 2016a, b). TbSADH is an NAD(P) H-and Zn-dependent enzyme, and its reduction mechanism has been elucidated.…”

“…TbSADH is an NAD(P) H-and Zn-dependent enzyme, and its reduction mechanism has been elucidated. In general, the carbonyl oxygen of the unsymmetrical ketone substrate coordinates to the Zn ion, and a hydride from the cofactor NADPH attacks the carbonyl carbon atom from either the Re or Si face, producing the corresponding optically active secondary alcohol (Li et al 2017;Moa and Himo 2017;Nealon et al 2015;Qu et al 2018).…”