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

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 str...

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Bibliographic Details
Published in:PloS one 2014-03, Vol.9 (3), p.e93317
Main Authors: Furubayashi, Maiko, Ikezumi, Mayu, Kajiwara, Jun, Iwasaki, Miki, Fujii, Akira, Li, Ling, Saito, Kyoichi, Umeno, Daisuke
Format: Article
Language:English
Subjects:
Alkyl and Aryl Transferases - chemistry
Amino Acid Sequence
Analysis
Binding sites
Biology and Life Sciences
Biosynthesis
Biotechnology
Breeding
Carbon-carbon composites
Carotenoids
Carotenoids - chemistry
Colorimetry
Colorimetry - methods
Consumption data
Directed evolution
E coli
Engineering
Enzymes
Escherichia coli
Escherichia coli - chemistry
Evolution
Genomes
High-Throughput Screening Assays - methods
Medical screening
Metabolic engineering
Metabolism
Molecular Sequence Data
Mutagenesis
Mutation
Pathways
Proteins
Screening
Substrate Specificity
Substrates
Terpene synthase
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Summary: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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0093317