Terpenoid Biosynthetic Pathway Research Articles

Terpenoids in Grapes and Wines: Origin and Micrometabolism during the Vinification Process

Terpenoids, which are typical components of the essential oils of flowers and fruits, are also present as free and glycosylated conjugates amongst the secondary metabolites of wine grape varieties of Vitis vinifera. Hence, when these compounds are present in wine, they are considered to originate from the grapes and not from fermentation. However, the biosynthesis of monoterpenes by Saccharomyces cerevisiae in the absence of grape derived precursors was shown recently to be of de novo origin in wine yeast strains. The contribution of yeast and bacterial fermentation metabolites to the aromatic profile of wine is well documented. However, the biotechnological application of this knowledge is still rather limited and often contradictory. Redox conditions, size of inoculums, temperatures of fermentation, osmotic pressure and the medium nutritional content can profoundly affect the profile of yeast and bacterial metabolites produced or their biotransformation capacity in wine. Results obtained in the last decades in relation to microbial micrometabolism of aroma compounds measured with more sophisticated GC-MS methods are discussed in relation to the known terpenoid biosynthetic pathways and wine composition. Further development of metabolic footprinting techniques for the discrimination of wine quality must be one of the main challenges for wine biotechnologists in the near future.

Significance of terpenoids in induced indirect plant defence against herbivorous arthropods

Many plants respond to herbivory by arthropods with an induced emission of volatiles such as green leaf volatiles and terpenoids. These herbivore-induced plant volatiles (HIPVs) can attract carnivores, for example, predators and parasitoids. We investigated the significance of terpenoids in attracting herbivores and carnivores in two tritrophic systems where we manipulated the terpenoid emission by treating the plants with fosmidomycin, which inhibits one of the terpenoid biosynthetic pathways and consequently terpenoid emission. In the 'lima bean' system, volatiles from spider-mite-infested fosmidomycin-treated plants were less attractive to the predatory mite Phytoseiulus persimilis than from infested control plants. In the 'cabbage' system, fosmidomycin treatment did not alter the attractiveness of Brussels sprouts to two Pieris butterflies for oviposition. The parasitoid Cotesia glomerata did not discriminate between the volatiles of fosmidomycin-treated and water-treated caterpillar-infested cabbage. Both P. persimilis and C. glomerata preferred volatiles from infested plants to uninfested ones when both were treated with fosmidomycin. Chemical analysis showed that terpenoid emission was inhibited more strongly in infested lima bean plants than in Brussels sprouts plants after fosmidomycin treatment. This study shows an important role of terpenoids in the indirect defence of lima bean, which is discussed relative to the role of other HIPVs.

Downregulation of terpenoid indole alkaloid biosynthetic pathway by low temperature and cloning of a AP2 type C-repeat binding factor (CBF) from Catharanthus roseus (L). G. Don

Plants produce secondary metabolites in response to various external signals. Coordinated transcriptional control of biosynthetic genes emerges as a major mechanism dictating the accumulation of secondary metabolites in plant cells. However, information about stress regulation of secondary metabolites and the molecular mechanisms regulating these specialized pathways are poorly understood. Here, we show that terpenoid indole alkaloid (TIA) biosynthetic pathway is differentially regulated in response to different abiotic stresses in Catharanthus roseus, a model medicinal plant producing important anticancer and antihypertensive drugs. Semiquantitative RT-PCR analysis of TIA and related primary pathway genes in response to dehydration, low temperature, salinity, UV-light and wounding revealed their negative regulation in response to low temperature. HPLC analysis further supports the notion that TIA biosynthetic pathway is negatively controlled by low temperature stress. Furthermore, we report the cloning of a C-repeat binding transcription factor from C. roseus (CrCbf), belonging to AP2 class of transcription factor and possessed the NLS and CBF signature sequence characteristic of CBFs. CrCbf was found to be similar to Brassica Cbfs, whereas it was distant to monocot Cbfs. Southern analysis of CrCbf revealed the presence of more than one copy of CrCbf gene or other Cbf homologues in C. roseus genome. The transcription of CrCbf was found to be constitutive in response to low temperature but it showed differential distribution. The need for identifying novel transcription factors in understanding secondary metabolite biosynthesis is discussed.

Enhancing terpenoid indole alkaloid production by inducible expression of mammalian Bax in Catharanthus roseus cells

Bax, a mammalian pro-apoptotic member of the Bcl-2 family, triggers hypersensitive reactions when expressed in plants. To investigate the effects of Bax on the biosynthesis of clinically important natural products in plant cells, we generate transgenic Catharanthus roseus cells overexpressing a mouse Bax protein under the beta-estradiol-inducible promoter. The expression of Bax in transgenic Catharanthus roseus cells is highly dependent on beta-estradiol concentrations applied. Contents of catharanthine and total terpenoid indole alkaloid of the transgenic cells treated with 30 micromol/L beta-estradiol are 5.0-and 5.5-fold of the control cells. Northern and Western blotting results show that expression of mammalian Bax induces transcriptional activation of Tdc and Str, two key genes in terpenoid indole alkaloid biosynthetic pathway of Catharanthus roseus cells, and stimulates the accumulation of defense-related protein PR1 in the cells, showing that the mouse Bax triggers the defense responses of Catharanthus roseus cells and activates the terpenoid indole alkaloid biosynthetic pathway. Thus, our data suggest that the mammalian Bax might be a potential regulatory factor for secondary metabolite biosynthesis in plant cells and imply a new secondary metabolic engineering strategy for enhancing the metabolic flux to natural products by activating the whole biosynthetic pathway rather than by engineering the single structural genes within the pathways.

Effect of supplementing terpenoid biosynthetic precursors on the accumulation of bilobalide and ginkgolides in Ginkgo biloba cell cultures

The effect of precursor feeding on the production of bilobalide and ginkgolides was studied with suspension cell cultures of Ginkgo biloba. The precursors greatly influenced the productivity of bilobalide and ginkgolides. Precursor supplementation increased the accumulation of both bilobalide and ginkgolides, and with positive effect on cell growth. The GA accumulation by cell cultures was influenced by precursors upstream in the metabolism, whereas the BB accumulation was under the influence of downstream precursors of the terpenoid biosynthetic pathway. Furthermore, precursor feeding modified the ratios of the BB, GA and GB in cells and cell cultures of G. biloba. The studies also aid in understanding effect of precursor feeding on the bilobalide and ginkgolides biosynthetic pathway.

(2,3,4,4-Tetramethylcyclopentyl)Methyl Acetate, a Sex Pheromone from the Obscure Mealybug: First Example of a New Structural Class of Monoterpenes

The sex pheromone of the obscure mealybug, Pseudococcus viburni, consists of (1R*,2R*,3S*)-(2,3,4,4-tetramethylcyclopentyl)methyl acetate, the first example of a new monoterpenoid structural motif in which the two isoprene units forming the carbon skeleton are joined by 2'-2 and 3'-4 connections rather than the usual 1'-4, head-to-tail connections. This highly irregular terpenoid structure, and the irregular terpenoid structures of related mealybug species, suggest that these insects may have unique terpenoid biosynthetic pathways.

Biosynthesis of chloroplastidic and extrachloroplastidic terpenoids in liverwort cultured cells: 13C serine as a probe of terpene biosynthesis via mevalonate and non-mevalonate pathways.

Two terpenoid biosynthetic pathways, the mevalonate and non-mevalonate (glyceraldehyde phosphate-pyruvate) routes, were examined by feeding (13)C-labeled serines ([1-(13)C]- and [3-(13)C]-) to the cultured cells of the liverwort, Heteroscyphus planus. The labeling patterns observed in the isoprenoid unit of the biosynthetically (13)C-labeled stigmasterol corresponded to those expected from the mevalonate pathway, while those of the phytyl side chain corresponded to those from the non-mevalonate pathway. Thus, serine is a potential probe to determine the origin of terpenoid biosynthesis, in either the mevalonate or non-mevalonate pathway.

The mevalonate-independent pathway is expressed in transformed roots of Artemisia annua and regulated by light and culture age

Artemisia annua produces a large number of unique terpenoids, making it of particular interest as a source of phytochemicals and a useful model plant for studying terpenoid metabolism. The ability to engineer fast-growing in vitro cultures to produce terpenoids in high yield would be a dramatic step towards commercial use. Two distinct pathways have been characterized in higher plants leading to the biosynthesis of isopentenyl diphosphate, the common precursor to all terpenes: the cytosolic mevalonate pathway and the plastid-localized mevalonate-independent pathway. While transformed roots of A. annua have been demonstrated to be superior to whole plants in terms of yield of the sesquiterpene artemisinin, they appear to lack functional chloroplasts, bringing into question the presence of a functional mevalonate-independent pathway. Using a cDNA library made from these roots, we isolated two clones encoding deoxy-d-xylulose-5-phosphate synthase (DXPS) and deoxy-d-xylulose-5-phosphate reductoisomerase (DXPR). The biochemical function of both enzymes was confirmed by complementing E. coli dxps- and dxpr-mutants. Northern blot analysis showed that the transformed root cultures expressed these genes at different levels during the culture cycle. In addition, cultures grown in continuous light showed substantial increases in DXPS transcript levels compared to dark-grown cultures. These results represent an important step towards demonstrating the presence of the plastid-localized terpenoid biosynthetic pathway in these easily engineered in vitro cultures.

Genetic modification of plant secondary metabolite pathways using transcriptional regulators.

Plant secondary metabolism is the source of many natural products with diverse applications, including pharmaceuticals, food colors, dyes and fragrances. Functions in plants include attraction of pollinating insects and protection against pests and pathogens. An important regulatory step in secondary metabolism is transcription of the biosynthetic genes. The aim of this chapter is to discuss results and opportunities concerning modification of secondary metabolism using transcriptional regulators. The transcriptional regulation of two well-studied secondary pathways, the phenylpropanoid pathway and its flavonoid branch, and the terpenoid indole alkaloid biosynthetic pathway, are reviewed. Some examples of successful engineering of these pathways via transcriptional regulators are discussed.

Metabolic engineering of the terpenoid biosynthetic pathway of Escherichia coli for production of the carotenoids β-carotene and zeaxanthin

Metabolic engineering of the early non-mevalonate terpenoid pathway of Escherichia coli was carried out to increase the supply of prenyl pyrophosphates as precursor for carotenoid production. Transformation with the genes dxs for over-expression of 1-deoxy-d-xylulose 5-phosphate synthase, dxr for 1-deoxy-d-xylulose 5-phosphate reductoisomerase and idi encoding an isopentenyl pyrophosphate stimulated carotenogenesis up to 3.5-fold. Co-transformation of idi with either dxs or dxr had an additive effect on s-carotene and zeaxanthin production which reached 1.6 mg g−1 dry wt.

Biochemistry and biological functions of citrus limonoids

Abstract Limonoids are one of bitter principles in citrus juices. Excessive bitterness lowers the quality and value of citrus juices. Nomilin is considered to be the precursor of all other limonoids accumulated in Citrus and related species. It is biosynthesized from acetate via the terpenoid biosynthetic pathway in the phloem region of stems and then translocated to the leaves, fruit tissues, peels, and seeds where it is further metabolized to other limonoids. The citrus limonoid aglycones are then glucosidated by limonoid UDP‐D‐glucose transferase in maturing fruit tissues and seeds. These limonoid glucosides are accumulated in such high concentrations that they are one of major secondary metabolites in citrus fruit tissues, and they play an important role in fruit quality and possibly in human health. Research on creation of transgenic citrus trees that produce fruits free of the limonoid bitterness problem is in progress. Limonoids have been shown to induce glutathione S‐transferase activity and inhib...

Effects of elicitation on different metabolic pathways in Catharanthus roseus (L.)G.Don cell suspension cultures

Cell suspension cultures of Catharanthus roseus were elicited with an-autoclaved cell-free filtrate of Pythium aphanidermatum. The regulation of alkaloid, terpenoid, and phenolic biosynthetic pathways was studied by product formation and assay of the enzyme activity of anthranilate synthase (AS), tryptophan decarboxylase (TDC), strictosidine synthase (SSS), strictosidine-β-glucosidase (SG), isopentenyl diphosphate isomerase (IPP-isomerase), geraniol-10-hydroxylase (G10H), chorismate mutase (CM), and phenylalanine ammonia lyase (PAL). After elicitation, AS and TDC activities were induced which resulted in an increase in the amount of tryptamine in the cells. For SSS and SG, no significant induction was observed. Ajmalicine accumulation was not increased compared with that in controls. An increased amount of phenolic compounds was found in the culture medium, although CM activity was not induced and PAL activity was inhibited after elicitor treatment. The activities of the enzymes IPP-isomerase and G10H were inhibited after elicitation. Also, the incorporation of [ 14C]IPP in terpenoid products such as squalene was inhibited. These findings indicate a limitation in the terpenoid pathway which could promote a shortage of secoiridoid precursors for terpenoid indole alkaloid biosynthesis.

Interference of Dimethazone with Formation of Terpenoid Compounds

Abstract Dimethazone is a bleaching herbicide without any peroxidative activity. In addition to the inhibition of chlorophylls, carotenes, and xanthophylls, decreased formation of other prenyl lipids (phytol and α-tocopherol) can be observed in the presence of dimethazone. Application of this herbicide to pea plants results in the inhibition of longitudinal growth of the newly formed internodes which can be reversed by gibberellic acid. Apparently, dimethazone also decreases the endogenous gibberellin levels. As the formation of all the compounds assayed is inhibited to the same extent and as I50 -values for chlorophyll and carotenoid biosynthesis are identical (about 10 µᴍ dimethazone), the various effects of dimethazone are explained by a single enzyme target in the terpenoid biosynthetic pathway between acetate and geranylgeranyl pyrophosphate.

Biosynthesis of austalide D, a meroterpenoid mycotoxin from Aspergillus ustus

Incorporation of [1-13C]- and [1,2-13C2]-acetate, and (3RS)-[2-13C]mevalonate into austalide D, a metabolite of Aspergillus ustus, and the isolation of the cometabolites austalide J, K, and L, indicate its formation via a mixed polyketide–terpenoid biosynthetic pathway.

Alachlor influence on sorghum growth and gibberellin precursor synthesis

Growth (14 days) of sorghum ( Sorghum bicolor L. cv G522 DR) from seed planted in sand into which alachlor [2-chloro-2′,6′-diethyl- N-(methoxymethyl)acetanilide] was uniformly incorporated (0, 0.07, 0.14, 0.28, 0.56, 1.12, 2.24, or 4.48 kg/ha) was reduced by 0.14 kg/ha and severely inhibited (88%) by 0.56 kg/ha while cellular water cotent was not greatly influenced by 0.56 kg/ha. When added into the nutrient solution bathing the roots of 96-hr sorghum seedlings, alachlor (0, 0.0156, 0.0312, 0.0625, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, or 128 ppmw) was not lethal to 14-day-old sorghum at rates up to 32 ppmw (92% survival); however, shoot and root lengths were reduced 43 and 58%, respectively. Alachlor inhibition of sorghum growth appears to be closely associated with inhibition of cell enlargement; the coleoptile is the most susceptible stage of sorghum growth to alachlor. This situation closely resembles growth where gibberellic acid (GA) synthesis is inhibited. [2- 14C]Mevalonic acid ([2- 14C]MVA) incorporation into terpenoid GA precursors was evaluated using a cell-free enzyme system from etiolated sorghum coleoptiles. Alachlor did not inhibit total 14C incorporation but incorporation of 14C into kaurenol and sterols was decreased ca 80 and 75%, respectively, by 10 −6 M alachlor. Analyses for [ 14C]geranylgeraniol (GG), [ 14C]farnesol, and [ 14C]geraniol contents showed accumulation of [ 14C]farnesol and [ 14C]GG, and decreased [ 14C]geraniol. When seeds to which CGA-43089 [α-(cyanomethoximino)-benzacetonitrile] was applied 8 weeks prior to planting were substituted for untreated seeds, incorporation of [2- 14C]MVA into [ 14C]kaurenol was increased by alachlor while [ 14C]GG and [ 14C]farnesol accumulated and [ 14C]geraniol was absent at 10 −6 M alachlor. Additionally, sterol content increased in “safened” systems but was still decreased by alachlor. These data demonstrate multiple sites of alachlor activity in the GA and terpenoid biosynthetic pathway.

Biosynthesis of viridicatumtoxin, a mycotoxin from Penicilium expansum

Incorporation of sodium [1-13C]-, [2-13C]-, [1,2-13C2]-, and [1-13C, 18O]-acetate, sodium [1,2,3-13C3]- malonate, and (2S)-[Me-13C]methionine as well as sodium hydrogen[14C]carbonate and sodium[1-14C]pyruvate into viridicatumtoxin, a metabolite from Penicillium expansum, indicates its formation by a mixed polyketide–terpenoid biosynthetic pathway.