Purification and structural analysis of volatile sesquiterpenes produced by Escherichia coli carrying unidentified terpene synthase genes from edible plants of the family Araliaceae

Abstract

A simple method to purify volatile sesquiterpenes from recombinant Escherichia coli was developed using the cells that carried known sesquiterpene synthase (Tps) genes ZzZss2 (ZSS2) and ZoTps1. This method was applied for the purification and structural analyses of volatile sesquiterpenes produced by E. coli cells that carried unidentified Tps genes, which were isolated from the Aralia-genus edible plants belonging to the family Araliaceae. Recombinant cells carrying each Tps gene were cultured in the two-layer medium (n-octane/TB medium), and volatile sesquiterpenes trapped in n-octane were purified through two-phase partition, silica gel column chromatography, and reversed-phase preparative high-performance liquid chromatography, if necessary. Further, their structures were confirmed by nuclear magnetic resonance, [α], and gas chromatography-mass spectrometry analyses. Herein, the products of E. coli cells that carried two Tps gene (named AcTps1 and AcTps2) in Araria cordata "Udo" and a Tps gene (named AeTps1) in Aralia elata "Taranoki" were studied resulting in identifying functionalities of these cryptic Tps genes.

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.…”

“…It was then concentrated to 3-5 mL in vacuo. The produced volatile sesquiterpenes in the alkane layer were monitored by silica gel thinlayer chromatography (TLC) (Merck) developed with n-pentane or n-pentane-EtOAc (5:1) and visualized as blue spots with molybdophosphoric acid color development [15]. In the silica gel TLC analyses developed with n-pentane, hydrocarbon volatile sesquiterpenes showed Rf values of 0.5-0.8, and volatile sesquiterpenes with OH functionalities showed Rf ~0.…”

“…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.…”

Isolation and Functional Characterization of New Terpene Synthase Genes from Traditional Edible Plants

“…The analysis of the amino acid sequence similarity of TPSs and their accession numbers were described previously 21,36 , except for AtTPS10 AF178535 .…”