Enzymatic synthesis of valerena‐4,7(11)‐diene by a unique sesquiterpene synthase from the valerian plant (Valeriana officinalis)

Abstract

Valerian (Valeriana officinalis) is a popular medicinal plant in North America and Europe. Its root extract is commonly used as a mild sedative and anxiolytic. Among dozens of chemical constituents (e.g. alkaloids, iridoids, flavonoids, and terpenoids) found in valerian root, valerena-4,7(11)-diene and valerenic acid (C15 sesquiterpenoid) have been suggested as the active ingredients responsible for the sedative effect. However, the biosynthesis of the valerena-4,7(11)-diene hydrocarbon skeleton in valerian remains unknown to date. To identify the responsible terpene synthase, next-generation sequencing (Roche 454 pyrosequencing) was used to generate ∼ 1 million transcript reads from valerian root. From the assembled transcripts, two sesquiterpene synthases were identified (VoTPS1 and VoTPS2), both of which showed predominant expression patterns in root. Transgenic yeast expressing VoTPS1 and VoTPS2 produced germacrene C/germacrene D and valerena-4,7(11)-diene, respectively, as major terpene products. Purified VoTPS1 and VoTPS2 recombinant enzymes confirmed these activities in vitro, with competent kinetic properties (K(m) of ∼ 10 μm and k(cat) of 0.01 s(-1) for both enzymes). The structure of the valerena-4,7(11)-diene produced from the yeast expressing VoTPS2 was further substantiated by (13) C-NMR and GC-MS in comparison with the synthetic standard. This study demonstrates an integrative approach involving next-generation sequencing and metabolically engineered microbes to expand our knowledge of terpenoid diversity in medicinal plants.

Functional Identification of Valerena-1,10-diene Synthase, a Terpene Synthase Catalyzing a Unique Chemical Cascade in the Biosynthesis of Biologically Active Sesquiterpenes in Valeriana officinalis

“…Compound 2, valerena-1,10-diene, was characterized from fractions 9 -11, whereas compound 1, germacrene C, from fractions 21-23 was used for identification. NMR spectra were measured either on a Bruker 500MHz or Varian Vnmr 500 ( 1 H, 500 MHz; 13 C, 125MHz) spectrometer using hexane-d 14 and CDCl 3 as solvent.…”

“…Labeling Studies-To prepare 13 C-labeled valerena-1,10-diene, six cultures of 0.5 liters of SCE-Leu liquid media were inoculated with seed train cultures as before and grown for 4 days. On the fourth day, 3.0 g of [1-13 C]sodium acetate (Sigma) was added, and incubation was continued for an additional 48 h. The cultures were then harvested and extracted with acetone and hexane, and the hexane phase was fractionated over silica gel to isolate 6.5 mg of labeled 2.…”

“…On the fourth day, 3.0 g of [1-13 C]sodium acetate (Sigma) was added, and incubation was continued for an additional 48 h. The cultures were then harvested and extracted with acetone and hexane, and the hexane phase was fractionated over silica gel to isolate 6.5 mg of labeled 2. The labeled sample and 6.5 mg of unlabeled 2 were analyzed by 13 C NMR under identical experimental conditions (standard s2pul pulse sequence with 340 scans and the delay and pulse width set to the manufacturer's default values). Enrichment values were determined by comparing the ratios of the peak heights between the two samples, then multiplying by 1.1% to give the percent enrichment relative to that occurring from the natural abundance of 13 C (23, 24).…”

“…One of the minor compounds was identified as germacrene A based on identical retention time and mass spectral characteristics to a germacrene A standard provided by Robert M. Coates (University of Illinois) (30). Compound 1 isolated from root material was identified as germacrene C based on a combination of 1 H, 13 1 H gCOSY analysis revealed four spin systems stretching from H-1 to H-2, H-4 to H-6, H-8 to H-9, H-11 to CH 3 -12 and CH 3 -13. 13 C and 13 C, 1 H HMBC correlations established the E-configuration of double bonds between C-2 and C-3 and between C-1 and C-10 ( Fig.…”

“…Using these resources, we identified seven possible terpene synthase gene candidates, examined their tissue-specific expression profiles, and correlated these with the accumulation of sesquiterpenes, then functionally characterized the terpene synthase enzymes encoded by these genes in vitro and in vivo. During the course of this work, Pyle et al (13) also reported a similar effort to identify sesquiterpene synthases from V. officinalis and described the characterization of a valerena-1,10-diene synthase. However, in the current work we have exploited the valerenadiene synthase to probe and validate an unusual catalytic cascade for the generation of isobutenyl constellations on a unique class of biologically active sesquiterpenes.…”