title: Bacterial Production of Pinene by a Laboratory-Evolved Pinene-Synthase.
abstract: Successful feeding of the substrate geranylpyrophosphate (GPP) to monoterpene synthase is critical to the efficient microbial production of monoterpenes. Overexpression of GPP synthases, metabolic channeling from GPP synthase to terpene synthases, and down-tuning of endogenous competitors have been successfully used to increase the production of monoterpene. Nevertheless, the production of monoterpenes has remained considerably lower than that of hemi-/sesqui-terpenoids. We tested whether it is effective to improve the cellular activity of monoterpene synthases. To this end, we developed a high-throughput screening system to monitor for elevated GPP consumption. Through a single round of mutagenesis and screening, we isolated a pinene synthase variant that outperformed the wild-type (parent) enzyme in multiple contexts in Escherichia coli and cyanobacteria. The purified variant exhibited drastically altered metal dependency, enabling to keep the activity in the cytosol that is manganese-deficient. Coexpression of this variant with mevalonate pathway enzymes, isopentenylpyrophosphate isomerase, and GPP synthase yielded 140 mg/L pinene in a flask culture.
authors.affiliation: Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Chiba University , 1-33 Yayoi-Cyo, Inage-ku, Chiba 263-8522, Japan.
authors.forename: Miki
authors.initials: M
authors.lastname: Tashiro
authors.affiliation: Department of Biological Sciences, Graduate School of Science, University of Tokyo , 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kiyota
authors.affiliation: Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Chiba University , 1-33 Yayoi-Cyo, Inage-ku, Chiba 263-8522, Japan.
authors.forename: Shigeko
authors.initials: S
authors.lastname: Kawai-Noma
authors.affiliation: Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Chiba University , 1-33 Yayoi-Cyo, Inage-ku, Chiba 263-8522, Japan.
authors.forename: Kyoichi
authors.initials: K
authors.lastname: Saito
authors.affiliation: Department of Biological Sciences, Graduate School of Science, University of Tokyo , 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.|Department of Life Sciences (Biology), Graduate School of Arts and Science, University of Tokyo , 3-8-1 Meguro-ku, Tokyo 153-8902, Japan.
authors.forename: Masahiko
authors.initials: M
authors.lastname: Ikeuchi
authors.affiliation: Department of Nutrition and Life Science, Kanagawa Institute of Technology , 1030 Shimo-ogino, Atsugi, Kanagawa 243-0292, Japan.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Iijima
authors.affiliation: Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Chiba University , 1-33 Yayoi-Cyo, Inage-ku, Chiba 263-8522, Japan.|Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST) , 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Umeno
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000378:metabolism*; D019748:Carbon-Carbon Double Bond Isomerases / Q000378:metabolism; D000458:Cyanobacteria / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D045782:Hemiterpenes; D039821:Monoterpenes / Q000378:metabolism; D016296:Mutagenesis / Q000502:physiology; D011106:Polyisoprenyl Phosphates / Q000378:metabolism
chemical_list: D045782:Hemiterpenes; D039821:Monoterpenes; D011106:Polyisoprenyl Phosphates; C015234:geranyl pyrophosphate; D019883:Alkyl and Aryl Transferases; C108877:terpene synthase; D019748:Carbon-Carbon Double Bond Isomerases; C021453:isopentenyldiphosphate delta-isomerase
keywords: carotenoid; cyanobacteria; directed evolution; monoterpene bioproduction; terpene synthase