Discovery and Engineering of a Bacterial (+)‐Pulegone Reductase for Efficient (−)‐Menthol Biosynthesis

Abstract The biosynthesis of valuable plant‐derived monoterpene (−)‐menthol from readily available feedstocks ( e. g ., (−)‐limonene) is of great significance because of the high market demand for this product. However, biotransforming (+)‐pulegone into (−)‐menthone, the (−)‐menthol precursor, through (+)‐pulegone reductase (PGR) catalysis is inefficient because of the poor protein expression or catalytic efficiency ( k cat / K m ) of plant origin PGRs. In this study, a novel bacterial PGR from Pseudomonas resinovorans ( Pr PGR) was identified, and the most successful variant, Pr PGR M2‐1 (A50 V/G53 W), was obtained, showing respective 20‐fold and 204‐fold improvements in specific activity and catalytic efficiency. Pr PGR M2‐1 was employed to bioreduce (+)‐pulegone, resulting in 4.4‐fold and 35‐fold enhancements in (−)‐menthone titers compared with the bioreductions catalyzed by wild‐type (WT) Pr PGR and Mp PGR, respectively. Furthermore, a whole‐cell biocatalyst containing Pr PGR M2‐1 , Mp MMR, and Bst FDH was constructed and achieved the highest (−)‐menthol titer reported to date without externally supplemented NADPH/NADP + . Overall, this study details an efficient PGR with high catalytic efficiency that possesses great potential for (−)‐menthol biosynthesis.