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 (kcat/Km) of plant origin PGRs. In this study, a novel bacterial PGR from Pseudomonas resinovorans (PrPGR) was identified, and the most successful variant, PrPGRM2‐1 (A50V/G53W), was obtained, showing respective 20‐fold and 204‐fold improvements in specific activity and catalytic efficiency. PrPGRM2‐1 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 PrPGRM2‐1, 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.