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

  • Identification data

    Identifier: imarina:3715456
    Authors:
    Currin ADunstan MJohannissen LHollywood KVinaixa MJervis ASwainston NRattray NGardiner JKell DTakano EToogood HScrutton N
    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.
  • Others:

    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: 2024-09-07
    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: https://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
  • Keywords:

    Catalysis,Chemistry (Miscellaneous),Chemistry, Physical
    Robotics
    Ketosteroid isomerase
    Isopulegone isomerase
    Enzyme engineering
    Biosynthetic (-)-menthol production
    Química
    Materiais
    Interdisciplinar
    General chemistry
    Engenharias ii
    Ciências agrárias i
    Chemistry, physical
    Chemistry (miscellaneous)
    Chemistry (all)
    Catalysis
    Astronomia / física
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