No abstract

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.

Microbial synthesis of (−)‐menthol, a compound of plant origin, is of great importance because of the high demand for this product and related sustainability issues. However, the total biosynthesis of (−)‐menthol from easily available feedstocks like (−)‐limonene by engineered microbial hosts is stalled by the poor protein expression or activity of several enzymes from the native (−)‐menthol biosynthesis pathway of mint (Mentha piperita). Among these unsatisfied steps, (−)‐isopiperitenol dehydrogenase (IPDH) catalyzed oxidation reaction of (−)‐trans‐isopiperitenol was one of the bottlenecks that need to be optimized. In this work, two novel bacterial enzymes with IPDH activity were discovered to replace their inefficient counterpart from plant cells in microbial (−)‐menthol synthesis. Two key residues in PaIPDH from Pseudomonas aeruginosa were mutated to PaIPDHE95F/Y199V with 4.4‐fold improved specific activity than PaIPDH. The mechanism for the beneficial mutations was elucidated by molecular dynamics simulations. PaIPDHE95F/Y199V was used to synthesize (−)‐isopiperitenone from (−)‐limonene in vivo via a self‐sufficient cofactor cascade enzyme reaction, affording a 3.7‐fold enhanced titer of (−)‐isopiperitenone compared with that obtained using the original mint IPDH (MpIPDH). The bacterial enzyme PaIPDHE95F/Y199V can be applied in the future for constructing a more efficient artificial pathway to biosynthesize (−)‐menthol in a microbial whole‐cell system.