title: A high-throughput colorimetric screening assay for terpene synthase activity based on substrate consumption.
abstract: Terpene synthases catalyze the formation of a variety of terpene chemical structures. Systematic mutagenesis studies have been effective in providing insights into the characteristic and complex mechanisms of C-C bond formations and in exploring the enzymatic potential for inventing new chemical structures. In addition, there is growing demand to increase terpene synthase activity in heterologous hosts, given the maturation of metabolic engineering and host breeding for terpenoid synthesis. We have developed a simple screening method for the cellular activities of terpene synthases by scoring their substrate consumption based on the color loss of the cell harboring carotenoid pathways. We demonstrate that this method can be used to detect activities of various terpene synthase or prenyltransferase genes in a high-throughput manner, irrespective of the product type, enabling the mutation analysis and directed evolution of terpene synthases. We also report the possibility for substrate-specific screening system of terpene synthases by taking advantage of the substrate-size specificity of C30 and C40 carotenoid pathways.
authors.affiliation: Department of Applied Chemistry and Biotechnology, Chiba University, Chiba, Japan.
authors.forename: Maiko
authors.initials: M
authors.lastname: Furubayashi
authors.affiliation: Department of Applied Chemistry and Biotechnology, Chiba University, Chiba, Japan.
authors.forename: Mayu
authors.initials: M
authors.lastname: Ikezumi
authors.affiliation: Department of Applied Chemistry and Biotechnology, Chiba University, Chiba, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Kajiwara
authors.affiliation: Department of Applied Chemistry and Biotechnology, Chiba University, Chiba, Japan.
authors.forename: Miki
authors.initials: M
authors.lastname: Iwasaki
authors.affiliation: Department of Applied Chemistry and Biotechnology, Chiba University, Chiba, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Fujii
authors.affiliation: Department of Applied Chemistry and Biotechnology, Chiba University, Chiba, Japan.
authors.forename: Ling
authors.initials: L
authors.lastname: Li
authors.affiliation: Department of Applied Chemistry and Biotechnology, Chiba University, Chiba, Japan.
authors.forename: Kyoichi
authors.initials: K
authors.lastname: Saito
authors.affiliation: Department of Applied Chemistry and Biotechnology, Chiba University, Chiba, Japan; Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), Kawaguchi, Saitama, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Umeno
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000737:chemistry*; D000595:Amino Acid Sequence; D002338:Carotenoids / Q000737:chemistry; D003124:Colorimetry / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry; D057166:High-Throughput Screening Assays / Q000379:methods*; D008969:Molecular Sequence Data; D013379:Substrate Specificity
chemical_list: D002338:Carotenoids; D019883:Alkyl and Aryl Transferases; C108877:terpene synthase