Discovery and Engineering of Bacterial (−)‐Isopiperitenol Dehydrogenases to Enhance (−)‐Menthol Precursor Biosynthesis

Abstract 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 Pa IPDH from Pseudomonas aeruginosa were mutated to Pa IPDH E95F/Y199V with 4.4‐fold improved specific activity than Pa IPDH. The mechanism for the beneficial mutations was elucidated by molecular dynamics simulations. Pa IPDH E95F/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 ( Mp IPDH). The bacterial enzyme Pa IPDH E95F/Y199V can be applied in the future for constructing a more efficient artificial pathway to biosynthesize (−)‐menthol in a microbial whole‐cell system. magnified image