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

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 (20⁴ =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.