Premium
CompassR Yields Highly Organic‐Solvent‐Tolerant Enzymes through Recombination of Compatible Substitutions
Author(s) -
Cui Haiyang,
Jaeger KarlErich,
Davari Mehdi D.,
Schwaneberg Ulrich
Publication year - 2021
Publication title -
chemistry – a european journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.687
H-Index - 242
eISSN - 1521-3765
pISSN - 0947-6539
DOI - 10.1002/chem.202004471
Subject(s) - recombination , directed evolution , in silico , chemistry , acetone , bacillus subtilis , molecular dynamics , homologous recombination , lipase , enzyme , solvent , gene , stereochemistry , mutant , biology , biochemistry , genetics , computational chemistry , bacteria
The CompassR (computer‐assisted recombination) rule enables, among beneficial substitutions, the identification of those that can be recombined in directed evolution. Herein, a recombination strategy is systematically investigated to minimize experimental efforts and maximize possible improvements. In total, 15 beneficial substitutions from Bacillus subtilis lipase A (BSLA), which improves resistance to the organic cosolvent 1,4‐dioxane (DOX), were studied to compare two recombination strategies, the two‐gene recombination process (2GenReP) and the in silico guided recombination process (InSiReP), employing CompassR. Remarkably, both strategies yielded a highly DOX‐resistant variant, M4 (I12R/Y49R/E65H/N98R/K122E/L124K), with up to 14.6‐fold improvement after screening of about 270 clones. M4 has a remarkably enhanced resistance in 60 % (v/v) acetone (6.0‐fold), 30 % (v/v) ethanol (2.1‐fold), and 60 % (v/v) methanol (2.4‐fold) compared with wild‐type BSLA. Molecular dynamics simulations revealed that attracting water molecules by charged surface substitutions is the main driver for increasing the DOX resistance of BSLA M4. Both strategies and obtained molecular knowledge can likely be used to improve the properties of other enzymes with a similar α/β‐hydrolase fold.