CompassR yields highly organic solvent-tolerant enzymes through recombination of compatible substitutions.

The CompassR rule enables to identify among beneficial substitutions the ones, which can beneficially 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 improve organic cosolvent 1,4-dioxane (DOX) resistance, were studied to compare two recombination strategies 2GenReP and InSiReP employing CompassR. Remarkably, both strategies yielded a highly DOX resistant variant M4 (I12R/Y49R/E65H/N98R/K122E/L124K) with an up to 14.6-fold improvement after screening of ~270 clones. M4 has a remarkable 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 to BSLA wild type. 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.