Liu_2021_Mol.Catal_514_111851

E. coli/gmeh3, an E. coli transformant expressing GmEH3, had the best regiocomplementarity (alphaS = 72.4% and betaR = 97.6%) for (S)- and (R)-1,2-epoxyhexanes among five tested aliphatic chain rac-1,2-epoxides (1a-5a). To prepare (R)-hexane-1,2-diol (1b) with high eep via enantioconvergent hydrolysis of rac-1a, the regioselectivity coefficient alphaS of GmEH3 for (S)-1a was improved by reshaping its substrate-binding pocket (SBP). Based on the semi-rational design, Trp102, Ile105, Ile178, Pro187 and Leu189 lining GmEH3's SBP were identified, each of which was substituted with four residues, respectively. From 17 transformants harboring single-site variants of gmeh3, E. coli/gmeh3W102L, /gmeh3W102I, /gmeh3W102V and /gmeh3P187F were selected, catalyzing the conversion of rac-1a into (R)-1b with obviously enhanced eep values of 59.3-78.3%. Then, three transformants containing double-site variants were constructed by combinatorial mutagenesis of gmeh3P187F separately with gmeh3W102L, gmeh3W102I and gmeh3W102V. Among the three transformants, E. coli/gmeh3W102V/P187F displayed the largest alphaS of 89.7% with betaR of 96.2%. The enantioconvergent hydrolysis of 500 mM rac-1a was conducted using 200 mg/mL wet cells of E. coli/gmeh3W102V/P187F at 25 degreesC for 12 h, affording (R)-1b with 83.1% eep and 91.5% yield. The molecular docking simulation analysis demonstrated that GmEH3W102V/P187F more regiopreferentially attacks Calpha in the oxirane ring of (S)-1a than GmEH3.