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.

Directed evolution and expression tuning of geraniol synthase for efficient geraniol production in <i>Escherichia coli</i>

“…To construct pAC-MN-idi, the region encoding the P lacidi operon was PCR-amplified from pUC-idi, which was constructed by inserting an E. coli idi gene into the XbaI/ XhoI site of a pUC18m vector, and then ligating it into the SalI site of pAC-MN (Furubayashi et al, 2014). pUCara-GES M53 was constructed by PCR-amplifying the reading frame of GES M53 from pUC-GES M53 (Furubayashi et al, 2014) and subcloning it into the EcoRI-ApaI site of the pUCara vector (Furubayashi et al, 2015).…”

“…pUCara-GES M53 was constructed by PCR-amplifying the reading frame of GES M53 from pUC-GES M53 (Furubayashi et al, 2014) and subcloning it into the EcoRI-ApaI site of the pUCara vector (Furubayashi et al, 2015).…”

“…Previously, we constructed a series of N-truncated mutants of GES to remove a transit-peptide sequence and to improve enzyme solubility (Furubayashi et al, 2014). The mutant GES M53 , which lacks the first 53 amino acids, turned out to be the best geraniol producer in Escherichia coli.…”

“…Recently, we reported a high-throughput screening method for the cellular activity of terpene synthases (Furubayashi et al, 2014). The method is based on the indirect visualization of precursor consumption for each synthase variant.…”

“…To this end, we randomly mutated the GES gene and screened for higher substrate consumption (Furubayashi et al, 2014). Analysis of the isolates revealed that the translational efficiency of GES was the major determinant of overall productivity.…”