Abstract
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 language | English |
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Pages (from-to) | 2012–2020 |
Number of pages | 9 |
Journal | ACS Catalysis |
Volume | 8 |
Issue number | 3 |
DOIs | |
Publication status | Published - 24 Jan 2018 |
Keywords
- biosynthetic (−)-menthol production
- ketosteroid isomerase
- isopulegone isomerase
- enzyme engineering
- robotics
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Nicholas Rattray
- Strathclyde Institute Of Pharmacy And Biomedical Sciences - Senior Lecturer
Person: Academic