Efficient Production of δ-Guaiene, an Aroma Sesquiterpene Compound Accumulated in Agarwood, by Mevalonate Pathway-Engineered <i>Escherichia coli</i> Cells

Abstract

Mevalonate pathway for isoprenoid biosynthesis was constructed in Escherichia coli cells by the transformation with a gene cluster isolated from Streptomyces sp., and farnesyl diphosphate synthase and δ-guaiene synthase genes were coexpressed in this strain. This transformant was capable of liberating an appreciable amount of δ-guaiene, an aroma sesquiterpene compound accumulated in agarwood, and its concentration was elevated to more than 30 μg/ml culture by the incubation with mevalonolactone as an isoprene precursor in a nutrient-enriched Terrific broth. Coexpression of type 1 isopentenyl diphosphate isomerase plus acetoacetyl-CoA ligase genes also enhanced δ-guaiene production, and the concentration of the compound was approximately 38 -42 μg/ml culture in the presence of mevalonolactone or lithium acetoacetate. These results clearly indicate that mevalonate pathway-engineered E. coli cells showed an appreciable δ-guaiene producing activity in the enriched medium in the presence of appropriate isoprene precursors.

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

“…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…”

“…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]. In many cases, 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]. However, such methods are sometimes insufficient for the unambiguous determination of their chemical structures and consequent identification of the Tps genes.…”

High Level Production of δ-Guaiene, a Bicyclic Sesquiterpene Accumulated in Agarwood, by Co-expression of δ-Guaiene Synthase and Farnesyl Diphosphate Synthase Genes in Tobacco BY-2 Cells

“…In the total ion chromatogram (Figure 3 A), two major peaks were found in the sample released from the transformed tobacco cultures, and they were identified as α-guaiene and δguaiene by the retention indices and mass spectra (Figure 3 B) in the data base, respectively [12,13]. We have previously reported [8,9] that sesquiterpene compounds produced by E. coli cells transformed with GS plus FPS were detected only in the head space of the cultures. The similar situation was also observed in the transformed tobacco BY-2 cells, and α-guaiene and δ-guaiene were found to be accumulated only in the head space of the vials containing the transformed tobacco culture but not in the medium or the cells.…”

“…More recently, we transformed E. coli with a gene cluster isolated from Streptomyces sp. which contains mevalonate pathway-related genes, and demonstrated [9] that, in this isoprenoid pathway-engineered E. coli, 35 -42 mg δ-guaiene/L culture was accumulated in the presence of appropriate precursors. However, productivity of δ-guaiene in E. coli cell system was not satisfactory, and, therefore, we attempted Figure 2: Schematic presentation of the expression vectors of GS and FPS genes in tobacco BY-2 cell cultures.…”

“…To our knowledge, δ-guaiene cannot be purchased from commercial venders, and, as described above, this compound is accumulated in Aquilaria plants only under very limited condition. Therefore, we attempted to construct the bio-production system of this compound employing Escherichia coli cells [8,9]. A GS gene and a farnesyl diphosphate synthase (FPS) gene isolated from A. microcarpa [10] were co-expressed in E. coli [8], and production of a low concentration of δ-guaiene (less than 1 mg/L culture) was observed in the transformed cells.…”