Engineering an alcohol dehydrogenase from Gluconobacter oxydans for improved production of a bulky Ezetimibe intermediate

(4S)-3-[(5S)-5-(4-fluorophenyl)-5‑hydroxy-valyl]-4-phenyl-1,3-oxazacyclopentane-2-one ((S)-Fop alcohol) is a key chiral intermediate for the synthesis of ezetimibe, and could be synthesized via asymmetric reduction of (S)-4-phenyl-3-[5-(4-fluorophenyl)-5-oxopentanoyl]-2-oxazolidione (Fop dione). However, discovering and engineering of ketoreductases toward bulky-bulky (diaryl) ketones is still challenging. Previously, we identified an alcohol dehydrogenase Gox0525 from Gluconobacter oxydans DSM2343 which possessed strict diastereoselectivity (d.e. value > 99%) but low activity toward Fop dione. In this study, a semi-rational design based on the focused rational iterative site-specific mutagenesis (FRISM) based on site-directed saturation mutagenesis was performed to improve the catalytic efficiency of Gox0525. The variant M4 (Y92G/P93M/Y94P/L151V) shows a 64-fold enhanced catalytic efficiency (K_cat/K_m) and 47-fold in specific activity compared with the wild type Gox0525. Engineered Escherichia coli cells co-expressing the variant M4 and glucose dehydrogenase from Bacillus subtilis (BsGDH) for NADPH regeneration were employed as biocatalysts for gram-scale reaction of Fop dione. As a result,95 mM (33.76 g/L) Fop dione was completely transformed within 4 h, affording (S)-Fop alcohol with > 99% d.e. value, the yield of 96%, and the space-time yield of 195.6 g/L/d.