A Comparative Reengineering Study of cpADH5 through Iterative and Simultaneous Multisite Saturation Mutagenesis

Abstract

Positions identified in directed evolution campaigns or by (semi)rational design can be recombined iteratively or simultaneously. Iterative recombination has yielded many success stories and is beneficially used if screening capabilities are limited (four iterative SSMs generate 20×4=80 different enzyme variants). Simultaneous site saturation mutagenesis offers significantly higher diversity (20 =160 000 variants) and enables greater improvements to be found, especially if the selected positions are in close proximity to each other (cooperative effects). Here we report a first comprehensive comparison of iterative and simultaneous saturation of four residues in Candida parapsilosis alcohol dehydrogenase 5 (cpADH5) with methyl 3-hydroxyhexanoate as substrate. Screening of 7200 clones from 33 site saturation mutagenesis libraries (exploring 17 recombination paths) yielded the cpADH5 W286A variant, with a 82-fold improved initial activity toward methyl 3-hydroxyhexanoate. Screening 3500 clones from a single OmniChange library with four positions (C57, W116, L119, and W286; 1.8 % of the generated sequence space) yielded the cpADH5 C57V/W286S variant, with a 108-fold improvement in initial activity toward methyl 3-hydroxyhexanoate. A 1.8 % coverage of the sequence space of the simultaneous multisite saturation library was, in comparison to the investigated 17 recombination paths, sufficient to identify a cpADH5 variant with improved activity.

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

“…A recent directed‐evolution study by Schwaneberg and co‐workers entitled “A Comparative Reeingineering Study of cpADH5 through Iterative and Simultaneous Multisite Saturation Mutagenesis” caught our immediate attention, specifically because the conclusions presented therein are not scientifically sound. Two ways of applying saturation mutagenesis (SM) were compared in this paper on using the alcohol dehydrogenase cpADH5 as the enzyme with the aim of enhancing activity in the (reversible) oxidation of 3‐hydroxyhexanoate: CAST/ISM– versus OmniChange…”

“…In the comparative study, CAST/ISM was tested in what we consider to be a questionable procedure, specifically when pitching it against the subsequent OmniChange experiments: Four single one‐residue sites were first chosen, three from a previous study (C57, W116 and L119), and one from a computational analysis (W286). Then conventional NNK‐based SM by using QuikChange was applied individually to all four single‐residue sites, as schematically illustrated in Figure B with B, C, E, and H representing the single residues.…”

“…In distinct contrast to the CAST/ISM experiments described in the comparative study, OmniChange was applied after grouping the same four single amino acid positions into a four‐residue randomization site (C57/W116/L119/W286) . This is analogous to the scenario in Figure B; it resulted in 160 000 theoretically different mutants, of which only 1.8 % were successfully screened with no oversampling, analogous to the above mentioned lipase study .…”

“…This is analogous to the scenario in Figure B; it resulted in 160 000 theoretically different mutants, of which only 1.8 % were successfully screened with no oversampling, analogous to the above mentioned lipase study . As the authors themselves speculate, based solely on docking experiments, randomizing four residues simultaneously enables potential cooperative mutational effects with the generation of optimal results; however, in their study, the necessary deconvolution of the best double mutant into the two single mutants C57V and W286S was not performed . The fact that potential cooperative mutational effects were largely prevented by specifically designing a fundamentally different and suboptimal form of ISM also forbids any direct comparison.…”

“…[5] SM-based randomizationo fasite at the binding pocket for enhancing an enzyme's stereoselectivity and activity was first reported in 2001 (although the term CASTingw as not used at the time), the enzyme was al ipase, and NNK-based randomization was performed at af our-residues ite (NNK:Nstands for the base A, T, Co rG;Kf or TorG;t his encodes all 20 canonical amino acids theoretically resulting in 160 000 mutants), [2b] as in the case of the Schwaneberg study. [1] The subsequently introduced term CAST is simply ac onvenient acronym that can be used to distinguish it from SM at remote sites for other purposes such as increasing protein robustness. [2c, 6] Importantly, when applying the CAST strategy,S SM and CSM can be performed by using any gene mutagenesis technique, including those based mainly on PCR,s uch as the popularQ uik-Change, [7a] MegaPrimer, [7b] overlap-extension PCR [7c] and others, [7d-i] or,i np rinciple, methods based on either gene assembly such as Gibson, [8a] ADO [8b] and others, [8c, d] or gene synthesis such as Twist, [9a] Sloning [9b] and others.…”