Sesquiterpenoids are very common among natural products. A large number of sesquiterpene synthase genes have been cloned and functionally characterized. However, until now there is no report about the δ-cadinol synthase predominantly forming δ-cadinol (syn. torreyol) from farnesyl diphosphate. Sesquiterpenoids boreovibrins structurally similar to δ-cadinol were previously isolated from culture broths of the basidiomycete fungus Boreostereum vibrans. This led us to expect a corresponding gene coding for a δ-cadinol synthase that may be involved in the biosynthesis of boreovibrins in B. vibrans. Here we report the cloning and heterologous expression of a new sesquiterpene synthase gene from B. vibrans. The crude and purified recombinant enzymes, when incubating with farnesyl diphosphate as substrate, gave δ-cadinol as its principal product and thereby identified as a δ-cadinol synthase.

Unprecedented dimeric Erythrina alkaloids, spirocyclic (6/5/6/6) erythrivarine A (1) and spiro-fused (6/5/7/6) rings erythrivarine B (2), were isolated from the cultivated plant, E. variegata. The structures were determined on the basis of 1D and 2D NMR, FTIR, UV, and mass spectroscopic data and X-ray crystal diffraction. The biogenetic relationship of 1 and 2 is proposed.

This review provides an overview of diterpene synthases which were initially identified via genetic and/or biochemical means, traversing all organisms researched to date.

The traditional Chinese medicinal plant, Isodon L., is remarkably rich in pharmacologically active ent-kaurane diterpenoids of diverse carbon skeletons. In an effort to create a resource for gene discovery and elucidate the biosynthesis of Isodonent-kaurane diterpenoids, three cDNAs (named IeCPS1, IeCPS2 and IeCPS2a) were isolated putatively encoding copalyl diphosphate synthases from Isodoneriocalyx leaves. Recombinant proteins of IeCPS1 and IeCPS2 were expressed, respectively, in Escherichia coli, and were shown to specifically convert geranylgeranyl diphosphate to copalyl diphosphate as demonstrated by GC–MS analyses. Based on tissue-specific expression and metabolic localization studies, the IeCPS2 transcripts were detected in young and mature leaves where the dominant ent-kaurane diterpenoid maoecrystal B accumulates, whereas no detectable expression of IeCPS2 was observed in germinating seeds where the gibberellin biosynthetic pathway is usually active. In addition, no evidence for maoecrystal B was found in germinating seeds. On the other hand, IeCPS1 transcripts significantly accumulated in germinating seeds as well as in leaves. The biochemical and molecular genetic evidence thus indicated that IeCPS2 is a copalyl diphosphate synthase potentially involved in the biosynthesis of Isodon diterpenoids in leaves, while IeCPS1 is more probably relevant to gibberellin formation and may, in addition, participate in Isodonent-kaurane diterpenoid production.Graphical abstractRecombinant IeCPS2 converts geranylgeranyl diphosphate to copalyl diphosphate. IeCPS2 expression occurs in Isodon eriocalyx leaves where ent-kaurane diterpenoids accumulate, this being considered highly relevant to the biosynthesis of Isodon ent-kauranoids.Highlights► Three cDNAs from I.eriocalyx leaves putatively encoded copalyl diphosphate synthases. ► Recombinant IeCPSs convert geranylgeranyl diphosphate to copalyl diphosphate. ► IeCPS2 expression occurs in leaves, where Isodonent-kaurane diterpenoids accumulate. ► Germinating seeds have neither detectable IeCPS2 transcripts nor Isodon diterpenoids. ► IeCPS1 is expressed in germinating seeds as well as in leaves.

The reduction of geraniol to citronellol is the first step for the synthesis of natural phytol in the production of tocopherols and natural vitamin K. Baker’s yeast was used in the bioreduction described above as a whole-cell biocatalyst. However, the enzyme responsible for the reduction of geraniol to citronellol is not yet known. Four old yellow enzyme (OYE) genes were cloned from yeast and plants, and expressed in Escherichia coli for a high level of recombinant proteins. The recombinant protein displayed a catalytic activity of converting geraniol to citronellol as a sole product verified by GC-MS analyses. The recombinant OYE2 intact cells were found to show 3.7 and 1.9-fold higher activity than that of yeast cells and the recombinant crude extracts, respectively. Compared to the recombinant fusion enzyme, the entrokinase-cleaved enzyme displayed nearly identical activity for geraniol reduction. To our knowledge, this is the first enzyme identified to catalyze the formation of citronellol from geraniol by reducing the allylic alcohol double bond, which is normally known as inactivating group for the old yellow enzymes. Open image in new window

Gastrodia elata Bl. is an achlorophyllous orchid plant feeding on the fungus Armillaria mellea. The plant lives underground during its life cycle except for florescence. Gastrodianins, members of the superfamily of monocot mannose-binding protein (MBP), have been identified from Gastrodia elata, yet their physiological functions in the plant are rarely understood. Aspects of expression of gastrodianins in growth and development of the plant will be helpful to dissect their functional roles. Two types of cDNA clones with complete cDNA sequences matching the known gastrodianins were obtained from G. elata Bl.f.glauca S.chow (Orchidaceae) and designated gastrodianin-4A (ga4A) and gastrodianin-4B (ga4B), respectively. But only one isoform was found to be expressed in all different parts of a single plant. Based on the RNA gel blot analysis, gastrodianins were much more abundantly expressed in the fully opened flowers than the underground corms where an enhanced expression was found in the out layers of secondary corms. By RNA in situ hybridization gastrodianin transcripts were distinctly detected in the cortical cells and vascular cells of corms. Strong transcript accumulations were observed in two to eight layers of cortical cells in secondary corms. From its peripheral tissue expression pattern and level in corms and flowers, the gastrodianin may account for a possible defense against phytopathogens or insects.

Geraniol may accumulate up to 86–98% of the leaf essential oils in geraniol chemotypes of the evergreen camphor tree Cinnamomum tenuipilum. A similarity-based cloning strategy yielded a cDNA clone that appeared to encode a terpene synthase and which could be phylogenetically grouped within the angiosperm monoterpene synthase/subfamily. After its expression in Escherichia coli and enzyme assay with prenyl diphosphates as substrates, the enzyme encoded by the putative C. tenuipilum monoterpene synthase gene was shown to specifically convert geranyl diphosphate to geraniol as a single product by GC–MS analysis. Biochemical characterization of the partially purified recombinant protein revealed a strong dependency for Mg2+ and Mn2+, and an apparent Michaelis constant of 55.8 μM for geranyl diphosphate. Thus, a new member of the monoterpene synthase family was identified and designated as CtGES. The genome contains a single copy of CtGES gene. Expression of CtGES was exclusively observed in the geraniol chemotype of C. tenuipilum. Furthermore, in situ hybridization analysis demonstrated that CtGES mRNA was localized in the oil cells of the leaves.A cDNA clone from Cinnamomum tenuipilum was isolated, functionally expressed in Escherichia coli and thereby identified as a single copy gene coding for a geraniol synthase. The CtGES is more abundantly expressed in leaves of a geraniol chemotype than in those of either linalool or farnesol chemotypes.