Engineering the “missing link” in biosynthetic (−)-menthol production : bacterial isopulegone isomerase
Currin, Andrew and Dunstan, Mark S. and Johannissen, Linus O. and Hollywood, Katherine A. and Vinaxia, Maria and Jervis, Adrian J. and Swainston, Neil and Rattray, Nicholas J. W. and Gardiner, John M. and Kell, Douglas B. and Takano, Eriko and Toogood, Helen S. and Scrutton, Nigel S. (2018) Engineering the “missing link” in biosynthetic (−)-menthol production : bacterial isopulegone isomerase. ACS Catalysis, 8 (3). 2012–2020. ISSN 2155-5435 (https://doi.org/10.1021/acscatal.7b04115)
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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
ORCID iDs
Currin, Andrew, Dunstan, Mark S., Johannissen, Linus O., Hollywood, Katherine A., Vinaxia, Maria, Jervis, Adrian J., Swainston, Neil, Rattray, Nicholas J. W. ORCID: https://orcid.org/0000-0002-3528-6905, Gardiner, John M., Kell, Douglas B., Takano, Eriko, Toogood, Helen S. and Scrutton, Nigel S.;-
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Item type: Article ID code: 65892 Dates: DateEvent24 January 2018Published15 January 2018AcceptedSubjects: Science > Chemistry Department: Faculty of Science > Strathclyde Institute of Pharmacy and Biomedical Sciences Depositing user: Pure Administrator Date deposited: 30 Oct 2018 14:53 Last modified: 03 Oct 2024 00:29 Related URLs: URI: https://strathprints.strath.ac.uk/id/eprint/65892