Removing the Obstacle to (−)‐Menthol Biosynthesis by Building a Microbial Cell Factory of (+)‐cis‐Isopulegone from (−)‐Limonene

Microbial synthesis of plant‐based (−)‐menthol is of great interest because of its high demand (≈30 kiloton per year) as well as unique odor and cooling characteristics. However, this remains a great challenge due to the yet unfilled gap between (−)‐limonene and (+)‐cis‐isopulegone. Herein, the first artificial and effective system was developed for (+)‐cis‐isopulegone biosynthesis from (−)‐limonene by recruiting two bacterial enzymes to replace their inefficient counterparts from Mentha piperita, limonene‐3‐hydroxylase, and isopiperitenol dehydrogenase. A cofactor self‐regenerative recombinant Escherichia coli strain was constructed by introducing a formate dehydrogenase for nicotinamide adenine dinucleotide phosphate (NADPH) regeneration and an engineered microbial isopiperitenol dehydrogenase. The production of (+)‐cis‐isopulegone (up to 281.2 mg L−1) was improved by 36 times compared with that of the initial strain. This work lays a reliable foundation for the microbial synthesis of (−)‐menthol. From −to +: An artificial microbial cell factory is constructed for (+)‐cis‐isopulegone biosynthesis by substituting two difficult‐to‐express plant‐based enzymes with a bacterial P450 monooxygenase (PpCamY96F/V247L) and an engineered bacterial isopiperitenol dehydrogenase (PaIPDHE95F/Y199V), which enables the microbial synthesis of (+)‐cis‐isopulegone from (−)‐limonene and lays the foundation for the large‐scale microbial manufacturing of (−)‐menthol.