Identification of novel sesquiterpene synthase genes that mediate the biosynthesis of valerianol, which was an unknown ingredient of tea

Abstract

Seven cDNA clones encoding terpene synthases (TPSs), their structures closely related to each other, were isolated from the flower of Camellia hiemalis (‘Kantsubaki’). Their putative TPS proteins were phylogenetically positioned in a sole clade with the TPSs of other Camellia species. The obtained Tps genes, one of which was designated ChTps1 (ChTps1a), were introduced into mevalonate-pathway-engineered Escherichia coli, which carried the genes for utilizing acetoacetate as a substrate, and cultured in a medium including lithium acetoacetate. Volatile products generated in the E. coli cells transformed with ChTps1 were purified from the cell suspension culture, and analyzed by NMR. Consequently, the predominant product with ChTPS1 was identified as valerianol, indicating that the ChTps1 gene codes for valerianol synthase. This is the first report on a gene that can mediate the synthesis of valerianol. We next synthesized a Tps ortholog encoding ChTPS1variant R477H (named CsiTPS8), whose sequence had been isolated from a tea tree (Camellia sinensis), carried out similar culture experiment with the E. coli transformant including CsiTps8, and consequently found valerianol production equally. Furthermore, GC-MS analysis of several teas revealed that valerianol had been an unknown ingredient in green tea and black tea.

Biochemical Fingerprint of Greek Sideritis spp.: Implications for Potential Drug Discovery and Advanced Breeding Strategies

“…perfoliata had the most diverse and rich terpene profile among the studied species, with a-elemol being the most abundant compound ( Table 3). The high concentration of elemol could be attributed to a heat induced rearrangement of hedycaryol [43], however this hypothesis is not supported since its presence was also detected as a TMS derivative formed under mild conditions (Figure 2).…”

A modern purification method for volatile sesquiterpenes produced by recombinant Escherichia coli carrying terpene synthase genes

“…We further demonstrated the usefulness of the developed purification method of volatile sesquiterpenes for determining their absolute structures, i.e. their structures were confirmed using NMR, [α] D , and GC-MS analyses, exhibiting their physicochemical data, which were previously unreported in many cases [14][15][16][17]. In addition, we evaluated the antioxidant activities of several of the purified volatile sesquiterpenes.…”

“…Tps genes that are introduced in this review were isolated from the flowers of the genera Humulus (HILIS/NES) and Camellia (CbTps1 and ChTps1), the rhizomes of the genus Zingiber (ZzZss2 (ZSS2) and ZoTps1), and the young leaves of the genus Aralia (AcTps1, AcTps2, and AeTps1), which have been described [14,15] or will soon be submitted [16,17]. These isolated Tps genes (usually the full length cDNAs) were individually transferred into a mevalonate-pathway-engineered E. coli using the E. coli expression vector pETDuet-1 (Merck) without their codon-usage optimization.…”

“…For this study, we needed to establish a method to purify the volatile sesquiterpenes produced with high recovery yields in order to unambiguously determine their structures and explore their biological activities. Consequently, we found that a two-layer system with n-octane/TB medium can trap the resultant volatile sesquiterpenes in the n-octane layer without harming E. coli cells, and purified them through two-phase partitioning (n-octane + n-hexane/alkaline 50% MeOH), silica gel column chromatography, and reversed-phase preparative high-performance liquid chromatography (HPLC) (if necessary) [14][15][16][17]. We further demonstrated the usefulness of the developed purification method of volatile sesquiterpenes for determining their absolute structures, i.e.…”

Changes of Crocin and Other Crocetin Glycosides in Saffron Through Cooking Models, and Discovery of Rare Crocetin Glycosides in the Yellow Flowers of Freesia Hybrida

“…Each 0.1 g of their dried materials was ground with an addition of 0.3 ml of water (H 2 O) and 0.3 ml of MeOH, subsequently extracted with 0.6 ml of chloroform (by mixing and centrifugation), and the chloroform extraction was repeated by adding 0.5 ml of H 2 O, and finally, each chloroform phase was concentrated to threefold by decompression. The prepared samples were subjected to GC-MS using Shimadzu GCMS-QP5050 (Shimadzu, Kyoto, Japan), as described ( 15 ).…”