Isolation and functional characterization of a β-eudesmol synthase, a new sesquiterpene synthase from Zingiber zerumbet Smith

Abstract 

In this paper, we have identified a new sesquiterpene synthase gene (ZSS2) from Zingiber zerumbet Smith. Functional expression of ZSS2 in Escherichia coli and in vitro enzyme assay showed that the encoded enzyme catalyzed the formation of β-eudesmol and five additional by-products. Quantitative RT-PCR analysis revealed that ZSS2 transcript accumulation in rhizomes has strong seasonal variations. To further confirm the enzyme activity of ZSS2 and to assess the potential for metabolic engineering of β-eudesmol production, we introduced a gene cluster encoding six enzymes of the mevalonate pathway into E. coli and coexpressed it with ZSS2. When supplemented with mevalonate, the engineered E. coli produced a similar sesquiterpene profile to that produced in the in vitro enzyme assay, and the yield of β-eudesmol reached 100mg/L.

Abbreviations: DMAPP, dimethylallyl pyrophosphate, DXP, 1-deoxy-d-xylulose-5-phosphate, FPP, farnesyl diphosphate, GC–MS, gas chromatography–mass spectrometry, HMGS, hydroxymethylglutaryl CoA synthase, HMGR, hydroxymethylglutaryl CoA reductase, IPP, isopentenyl pyrophosphate, IPPI, isopentenyl pyrophosphate isomerase, MK, mevalonate kinase, MeJA, methyl jasmonate, MPD, mevalonate diphosphate decarboxylase, PMK, phosphomevalonate kinase, RACE, rapid amplification of cDNA ends, TPS, terpene synthase

Keywords: Sesquiterpene synthase gene, β-Eudesmol, Seasonal variation, Metabolic engineering, Mevalonate pathway, Zingiber zerumbet Smith