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

DOI: 10.1021/acscatal.7b04115 DOI Help

Authors: Andrew Currin (Manchester Institute of Biotechnology, University of Manchester) , Mark S. Dunstan (Manchester Institute of Biotechnology, University of Manchester) , Linus O. Johannissen (Manchester Institute of Biotechnology, University of Manchester) , Katherine A. Hollywood (Manchester Institute of Biotechnology, University of Manchester) , Maria Vinaixa (Manchester Institute of Biotechnology, University of Manchester) , Adrian J. Jervis (Manchester Institute of Biotechnology, University of Manchester) , Neil Swainston (Manchester Institute of Biotechnology, University of Manchester) , Nicholas J. W. Rattray (Manchester Institute of Biotechnology, University of Manchester) , John M. Gardiner (Manchester Institute of Biotechnology, University of Manchester) , Douglas B. Kell (Manchester Institute of Biotechnology, University of Manchester) , Eriko Takano (Manchester Institute of Biotechnology, University of Manchester) , Helen S. Toogood (Manchester Institute of Biotechnology, University of Manchester) , Nigel S. Scrutton (Manchester Institute of Biotechnology, University of Manchester)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Acs Catalysis

State: Published (Approved)
Published: January 2018
Diamond Proposal Number(s): 12788

Abstract: 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.

Journal Keywords: Biosynthetic (–)-menthol production; ketosteroid isomerase; isopulegone isomerase; enzyme engineering; robotics

Subject Areas: Biology and Bio-materials, Chemistry


Instruments: I03-Macromolecular Crystallography , I04-Macromolecular Crystallography