The realisation of a synthetic biology approach to microbial (1R, 2S, 5R)-(–)-menthol (1) production relies on the identification of a gene encoding an isopulegone isomerase (IPGI), the only enzyme in the Mentha piperita biosynthetic pathway as yet unidentified. We demonstrate that Δ5-3-ketosteroid isomerase (KSI) from Pseudomonas putida can act as an IPGI, producing (R)-(+)-pulegone (R)-2 from (+)-cis-isopulegone (3). Using a robotics-driven semi-rational 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 modelling of 3 binding to identify key active site residue targets. The KSI variant was demonstrated to function efficiently within cascading biotransformations with downstream Mentha enzymes pulegone reductase and (–)-menthone:(–)-menthol reductase to generate 1 from 3. This study introduces the use of a recombinant IPGI, engineered to function efficiently within a biosynthetic pathway for the production of 1 in microorganisms.