Dataset Information

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

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 ?5-3-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.

SUBMITTER: Currin A

PROVIDER: S-EPMC5937688 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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

Currin Andrew A Dunstan Mark S MS Johannissen Linus O LO Hollywood Katherine A KA Vinaixa Maria M Jervis Adrian J AJ Swainston Neil N Rattray Nicholas J W NJW Gardiner John M JM Kell Douglas B DB Takano Eriko E Toogood Helen S HS Scrutton Nigel S NS

ACS catalysis 20180124 3

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 ...[more]

PMID: 29750129

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Dataset Information

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

Publications

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

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