Engineering the ‘Missing Link’ in Biosynthetic (–)-Menthol Production: Bacterial Isopulegone Isomerase

Research output: Contribution to journalArticle

  • External authors:
  • Andrew Currin
  • Mark Dunstan
  • Linus Johannissen
  • Katherine Hollywood
  • Maria Vinaixa
  • Neil Swainston
  • Nicholas Rattray
  • Helen Toogood


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.

Bibliographical metadata

Original languageEnglish
JournalACS Catalysis
Early online date24 Jan 2018
Publication statusPublished - 2018

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