Engineering the “missing link” in biosynthetic (−)-menthol production: bacterial isopulegone isomerase

Andrew Currin, Mark S. Dunstan, Linus O. Johannissen, Katherine A. Hollywood, Maria Vinaxia, Adrian J. Jervis, Neil Swainston, Nicholas J. W. Rattray, John M. Gardiner, Douglas B. Kell, Eriko Takano, Helen S. Toogood, Nigel S. Scrutton

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21 Citations (Scopus)
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The realization 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 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 3 binding to identify key active site residue targets. The KSI variant was demonstrated to function efficiently within cascade biocatalytic reactions 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
Original languageEnglish
Pages (from-to)2012–2020
Number of pages9
JournalACS Catalysis
Issue number3
Publication statusPublished - 24 Jan 2018


  • biosynthetic (−)-menthol production
  • ketosteroid isomerase
  • isopulegone isomerase
  • enzyme engineering
  • robotics


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