Engineering the "Missing Link" in Biosynthetic (-)-Menthol Production: Bacterial Isopulegone Isomerase.

The realization of a synthetic biology approach to microbial (1,2,5)-()-menthol () production relies on the identification of a gene encoding an isopulegone isomerase (IPGI), the only enzyme in the biosynthetic pathway as yet unidentified. We demonstrate that Δ5-3-ketosteroid isomerase (KSI) from can act as an IPGI, producing ()-(+)-pulegone (()-) from (+)--isopulegone (). Using a robotics-driven semirational design strategy, we identified a key KSI variant encoding four active site mutations, which confer a 4.3-fold increase in activity over the wild-type enzyme. This was assisted by the generation of crystal structures of four KSI variants, combined with molecular modeling of binding to identify key active site residue targets. The KSI variant was demonstrated to function efficiently within cascade biocatalytic reactions with downstream enzymes pulegone reductase and (-)-menthone:(-)-menthol reductase to generate from . This study introduces the use of a recombinant IPGI, engineered to function efficiently within a biosynthetic pathway for the production of in microorganisms.