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

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 (PrPGR) was identified, and the most successful variant, PrPGR_M2-1 (A50 V/G53 W), was obtained, showing respective 20-fold and 204-fold improvements in specific activity and catalytic efficiency. PrPGR_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) PrPGR and MpPGR, respectively. Furthermore, a whole-cell biocatalyst containing PrPGR_M2-1, MpMMR, and BstFDH 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.