Mechanistic Characterisation of Two Sesquiterpene Cyclases from the Plant Pathogenic Fungus Fusarium fujikuroi.

Two sesquiterpene cyclases from Fusarium fujikuroi were expressed in Escherichia coli and purified. The first enzyme was inactive because of a critical mutation, but activity was restored by sequence correction through site-directed mutagenesis. The mutated enzyme and two naturally functional homologues from other fusaria converted farnesyl diphosphate into guaia-6,10(14)-diene. The second enzyme produced eremophilene. The absolute configuration of guaia-6,10(14)-diene was elucidated by enantioselective synthesis, while that of eremophilene was evident from the sign of its optical rotation and is opposite to that in plants but the same as in Sorangium cellulosum. The mechanisms of both terpene cyclases were studied with various (13) C- and (2) H-labelled FPP isotopomers.

Enhanced Production of δ-Guaiene, a Bicyclic Sesquiterpene Accumulated in Agarwood, by Coexpression of δ-Guaiene Synthase and Farnesyl Diphosphate Synthase Genes in Escherichia coli.

Two genes involved in δ-guaiene biosynthesis in Aquilaria microcarpa, δ-guaiene synthase (GS) and famesyl diphosphate synthase (FPS), were overexpressed in Escherichia coli cells. Immunoblot analysis revealed that the concentration of GS-translated protein was rather low in the cells transformed by solely GS while appreciable accumulation of the recombinant protein was observed when GS was coexpressed with FPS. GS-transformed cells liberated only a trace amount of δ-guaiene (0.004 µg/mL culture), however, the concentration of the compound elevated to 0.08 pg/mL culture in the cells transformed by GS plus FPS. δ-Guaiene biosynthesis was markedly activated when E. coli cells coexpressing GS and FPS were incubated in enriched Terrific broth, and the content of the compound increased to approximately 0.6 µg/mL culture. These results suggest that coexpression of FPS and GS in E. coli is required for efficient δ- guaiene production in the bacterial cells, and the sesquiterpene-producing activity of the transformant is appreciably enhanced in the nutrients-enriched medium.

Pogostol biosynthesis by the endophytic fungus Geniculosporium.

Six (13)C-labelled isotopomers of mevalonolactone were synthesised and used in feeding experiments with the endophytic fungus Geniculosporium. The high incorporation rates of (13)C-label into a sesquiterpene that was found in headspace extracts of the fungus enabled unambiguous identification of this volatile as pogostol without the need for compound purification, simply by collecting the volatile fraction with a closed-loop stripping apparatus followed by direct (13)C NMR analysis (CLSA-NMR). The feeding experiments also gave insights into the biosynthesis of pogostol, including stereochemical aspects of the terpene cyclisation reaction. The possible biological function of pogostol is discussed.

Induction, cloning and functional expression of a sesquiterpene biosynthetic enzyme, δ-guaiene synthase, of Aquilaria microcarpa cell cultures.

A homology-based cloning strategy yielded a cDNA clone presumably encoding δ-guaiene synthase, a sesquiterpene cyclase, from tissue cultures of Aquilaria microcarpa, which were treated with methyl jasmonate. Incubation of cell cultures of the plant with yeast extract also induced transcriptional activation of the sesquiterpene synthase gene. The translated protein of the gene obtained by heterologous expression in Escherichia coli catalyzed the cyclization of farnesyl diphosphate to liberate δ-guaiene with δ-guaiene and germacrene A as the minor products. The results obtained in the present study, together with the previously reported results, suggest that two classes of δ-guaiene synthase occur in Aquilaria; the enzyme proteins from A. microcarpa and A. sinensis liberate germacrene A as a minor product, while the protein from A. crassna generates α-humulene instead of germacrene A.

A comparison of headspace solid-phase microextraction and classic hydrodistillation for the identification of volatile constituents from Thapsia spp. provides insights into guaianolide biosynthesis in Apiaceae.

INTRODUCTION: Thapsia spp. (Apiaceae) are the major natural source of polyoxygenated guaianolide sesquiterpene lactones known as thapsigargins, which induce apoptosis in mammalian cells via a high affinity inhibition of the sarco/endoplasmic reticulum Ca(2+) ATPase. The mechanism of biosynthesis of thapsigargins has not been elucidated, and probable biochemical precursors such as hydrocarbon or oxygenated sesquiterpenes have not been identified in previous phytochemical analyses of essential oils from this genus. OBJECTIVE: To investigate the utility of solid phase micro-extraction (SPME), when compared with classical essential oil distillates, for identifying potential precursors of guaianolide sesquiterpene lactones from Thapsia garganica L. and Thapsia villosa L. type II. METHODOLOGY: A systematic description of the volatile components of roots, flowers, stems and fruits of T. villosa and of root, flower and fruits of T. garganica was constructed via GC-MS analyses of SPME-adsorbed compounds and of essential oils obtained through hydrodistillation of the same tissues. RESULTS: The sesquiterpenoids δ-cadinene, α- and δ-guaiene, elemol and guaiols were found to be major volatile constituents of the roots of T. garganica and T. villosa trapped using SPME. In contrast, these sesquiterpenoids were not detected or were at negligible levels in essential oils, where sesquiterpenoids are potentially converted to azulenes during hydrodistillation. CONCLUSION: The new data reported in this study demonstrates that SPME is a valuable tool for the identification of volatile sesquiterpenes when compared with analysis of essential oils, and we postulate that guaiene is the likely precursor of guaianolide sesquiterpenes from Thapsia.

Characterization of delta-guaiene synthases from cultured cells of Aquilaria, responsible for the formation of the sesquiterpenes in agarwood.

The resinous portions of Aquilaria plants, called agarwood, have been used as medicines and incenses. Agarwood contains a great variety of sesquiterpenes, and a study using cultured cells of Aquilaria showed the production of sesquiterpenes (α-guaiene, α-humulene, and δ-guaiene) to be induced by treatment with methyl jasmonate (MJ). In this study, the accumulation and production of sesquiterpenes were quantified. The amounts accumulated and produced reached a maximum at 12 h, and the most abundant product was α-humulene at 6 h and δ-guaiene after 12 h. However, a headspace analysis of the cells revealed that α-humulene is likely to be volatilized; so overall, the most abundant sesquiterpene in the cells was δ-guaiene. A cDNA library from RNA isolated from MJ-treated cells was screened using PCR methodologies to isolate five clones with very similar amino acid sequences. These clones were expressed in Escherichia coli, and enzymatic reactions using farnesyl pyrophosphate revealed that three of the clones yielded the same compounds as extracted from MJ-treated cells, the major product being δ-guaiene. These genes and their encoded enzymes are the first sesquiterpene synthases yielding guaiane-type sesquiterpenes as their major products to be reported. Expression of a fourth terpene synthase gene in bacteria resulted in the accumulation of the protein in insoluble forms. Site-directed mutagenesis of the inactive clone and three-dimensional homology modeling suggested that the structure of the N-terminal domain was important in facilitating proper folding of the protein to form a catalytically active structure.