Engineering the "Missing Link" in Biosynthetic (-)-Menthol Production: Bacterial Isopulegone Isomerase

ACS Catalysis
Andrew CurrinNigel S Scrutton

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.

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Citations

May 1, 2020·PloS One·Gajendar Komati ReddyEriko Takano
Sep 28, 2018·Scientific Reports·Aitor Hernandez-OrtegaNigel S Scrutton
May 28, 2019·Applied Microbiology and Biotechnology·Sandra Moser, Harald Pichler
Apr 23, 2020·Biotechnology for Biofuels·Chien-Yuan Lin, Aymerick Eudes
Feb 19, 2019·Technological Forecasting and Social Change·Barbara Ribeiro, Philip Shapira
Nov 20, 2019·Biochimica Et Biophysica Acta. Proteins and Proteomics·Rhys CutlanNicholas J Harmer
Dec 24, 2021·Biotechnology for Biofuels·Sasa LiuXiaobing Yang

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Methods Mentioned

BETA
PCR
X-ray
targeted mutations

Software Mentioned

Gaussian09
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GeneGenie
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Gromacs
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