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

• Published in: ChemSusChem Vol. 17; no. 23; pp. e202400704 - n/a
• Main Authors: Wu, Qiong, Li, Hai‐Peng, Liu, Ya, Shou, Chao, Chen, Qi, Xu, Jian‐He, Li, Chun‐Xiu
• Format: Journal Article
• Language: English
• Published: Germany Wiley Subscription Services, Inc 06.12.2024
• Subjects: (−)-menthol; bacterial (+)-pulegone reductase; Biocatalysis; Biosynthesis; Catalysis; Cyclohexane Monoterpenes - metabolism; Efficiency; Menthol; Menthol - metabolism; molecular dynamics; Monoterpenes - metabolism; Oxidoreductases - metabolism; Protein Engineering; Pseudomonas - enzymology; Pseudomonas - metabolism; Reductases; Stereoisomerism; structure-guided mutagenesis; whole-cell biocatalysts; whole-cell biocatalysts; (−)-menthol; bacterial (+)-pulegone reductase; molecular dynamics; structure-guided mutagenesis
• ISSN: 1864-5631; 1864-564X; 1864-564X
• DOI: 10.1002/cssc.202400704

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 (A50 V/G53 W), 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. A bacterial (+)‐pulegone reductase with excellent catalytic performance was discovered, and structure‐guided mutagenesis was employed to enhance its activity for efficient (−)‐menthol biosynthesis.