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

Kazutoshi Shindo,Yurika Shibuya,Arisa Watanabe,Norihiko Misawa,Yuri Nakamura,Jun-Ichiro Hattan,Tomoko Ito,Mariko Kato,Maki Sugiyama,Miho Sato

doi:10.1080/09168451.2017.1386085

Kazutoshi Shindo, Yurika Shibuya + Show 8 more

Open Access

https://doi.org/10.1080/09168451.2017.1386085

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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]D, 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.

Highlights

Functional identification of cryptic terpene synthase (Tps) genes have often been performed using recombinant micro-organisms containing Escherichia coli that can functionally express the corresponding Tps genes and generate the sesquiterpene synthase (TPS) products [1,2,3,4]
To recover volatile sesquiterpenes – TPS protein products that are produced by E. coli carrying the corresponding Tps genes—the recombinant E. coli had often been cultured with two phases (n-dodecane (C12 alkane)/ H2O; 20% (v/v)) to recover the products in n-dodecane [5,6,8]
Since 2008, Misawa et al have identified several novel Tps genes for the production of volatile sesquiterpenes using recombinant E. coli cells carrying the corresponding unidentified Tps genes derived from edible or flavor plants [2,3,5,6,8,18]. They identified volatile sesquiterpenes produced by E. coli cells that carried the ZzZss2 gene and the ZoTps1 gene by gas chromatography–mass spectrometry (GC–MS) analyses to be β-eudesmol [5] (1) and (S)- β-bisabolene [8] (2), respectively

Summary

Introduction

Functional identification of cryptic terpene synthase (Tps) genes have often been performed using recombinant micro-organisms containing Escherichia coli that can functionally express the corresponding Tps genes and generate the sesquiterpene synthase (TPS) products [1,2,3,4]. The generated sesquiterpenes (the TPS products) have been analyzed by gas chromatography–mass spectrometry (GC–MS) using the MS library or co-chromatography with the authentic samples [2,3,5,6,7,8]. Such methods are sometimes insufficient for the unambiguous determination of their chemical structures and consequent identification of the Tps genes. The present report shows a simple method to purify volatile sesquiterpenes from recombinant E. coli followed by functional identification of Aralia-derived cryptic Tps genes and structural identification of the generated products

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