Regioselective inversion of an epoxide hydrolase by fine-tune its substrate-binding pocket for the production of (R)- and (S)-phenyl-1,2-ethanediol

•E. coli/rpehL360V exhibited an increase in αS from 77.0 % to 96.9 %.•E. coli/rpehI194F demonstrated a βS increase from 23.0 % to 98.8 %.•E. coli/rpehL360V or /rpehI194F catalyzed 200 mM (S)-SO to produce (R)- or (S)-PED in high eep values.•MD simulations elucidated the origin of the regioselectivity inversion in RpEHI194F. Molecular modification of key residues within substrate-binding pocket (SBP) of epoxide hydrolases (EHs) effectively regulates their regioselectivity. In this study, the regioselectivity of an EH from Rhodotorula paludigensis (RpEH) was almost inverted toward (S)-styrene oxide (SO) and its derivatives by finely tuned its SBP. Utilizing computer-aided design, two residues within the RpEH’ SBP were identified and replaced with residues of varying polarity and steric hindrance. Screening of 16 single-site mutant strains revealed that E. coli/RpehL360V had the remarkably improved regioselectivity toward (S)-SO with αS increased from 77.0 % to 96.9 %, interestingly, E. coli/RpehI194F displayed a complete inverted regioselectivity with a corresponding βS increased from 23.0 % to 98.8 %. In addition, the inverted regioselectivity betwen E. coli/RpehL360V and E. coli/RpehI194F were also observed toward (S)-p-nitrostyrene oxide (αS > 98.6 %, βS = 96.7 %) and (S)-p-chlorostyrene oxide (αS > 99 %, βS =91.4 %). Then, both E. coli/RpehL360V and E. coli/RpehI194F whole cells were used for a gram-scale hydrolysis of 200 mM (S)-SO in phosphate buffer (100 mM, pH 7.0) at 0 °C, producing both (R)- and (S)-phenyl-1,2-ethanediol with eep values of 97.2 % and 97.6 %, and space-time yields (STY) of 5.4 and 3.3 g/L/h, respectively. Further analysis by molecular dynamics (MD) simulations revealed that changes in intermolecular interactions between SBP residues and the substrate allowed RpEHI194F to more regiopreferentially attack the Cβ of the oxirane ring in (S)-SO, thereby inverting its regioselectivity. [Display omitted]