Author, as appears in the article.: Currin A; Dunstan M; Johannissen L; Hollywood K; Vinaixa M; Jervis A; Swainston N; Rattray N; Gardiner J; Kell D; Takano E; Toogood H; Scrutton N

Department: Enginyeria Electrònica, Elèctrica i Automàtica

URV's Author/s: Vinaixa Crevillent, Maria

Keywords: Robotics Ketosteroid isomerase Isopulegone isomerase Enzyme engineering Biosynthetic (-)-menthol production

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.

Thematic Areas: Química Materiais Interdisciplinar General chemistry Engenharias ii Ciências agrárias i Chemistry, physical Chemistry (miscellaneous) Chemistry (all) Catalysis Astronomia / física

licence for use: https://creativecommons.org/licenses/by/3.0/es/

ISSN: 21555435

Author's mail: maria.vinaixa@urv.cat maria.vinaixa@urv.cat

Author identifier: 0000-0001-9804-0171 0000-0001-9804-0171

Record's date: 2023-02-18

Papper version: info:eu-repo/semantics/publishedVersion

Link to the original source: https://pubs.acs.org/doi/abs/10.1021/acscatal.7b04115

Licence document URL: http://repositori.urv.cat/ca/proteccio-de-dades/

Papper original source: Acs Catalysis. 8 (3): 2012-2020

APA: Currin A; Dunstan M; Johannissen L; Hollywood K; Vinaixa M; Jervis A; Swainston N; Rattray N; Gardiner J; Kell D; Takano E; Toogood H; Scrutton N (2018). Engineering the missing Link in Biosynthetic (-)-Menthol Production: Bacterial Isopulegone Isomerase. Acs Catalysis, 8(3), 2012-2020. DOI: 10.1021/acscatal.7b04115

Article's DOI: 10.1021/acscatal.7b04115

Entity: Universitat Rovira i Virgili

Journal publication year: 2018

Publication Type: Journal Publications