A high-throughput colorimetric screening assay for terpene synthase activity based on substrate consumption

Maiko Furubayashi, Mayu Ikezumi, Jun Kajiwara, Miki Iwasaki, Akira Fujii, Ng Li, Kyoichi Saito, Daisuke Umeno

Research output: Contribution to journalArticlepeer-review

32 Citations (Scopus)

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.

Original languageEnglish
Article numbere93317
JournalPloS one
Volume9
Issue number3
DOIs
Publication statusPublished - 2014 Mar 28
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • General

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