Terpenoid Precursors Research Articles

Phaedon cochleariae and Gastrophysa viridula (Coleoptera: Chrysomelidae) produce defensive iridoid monoterpenes de novo and are able to sequester glycosidically bound terpenoid precursors

Larvae of the chrysomelid beetles Phaedon cochleariae and Gastrophysa viridula use iridoid monoterpenes for defense. The compounds are synthesized de novo in the glandular tissue and the reservoir. However, larvae feeding on leaves impregnated with thioglycosides of early precursors of iridoid biosynthesis such as the thioglycosides of 8-hydroxygeraniol, 8 and 9, rapidly accumulate in their defensive secretion. Thioglycosides combine a unique structural similarity to natural substrates with an exceptional chemical and biological stability against hydrolytic enzymes and can be, therefore, used to study transport phenomena of glycosides. The successful import suggests that the larvae possess, in addition to the de novo biosynthesis, the capability to sequester appropriate glycoside precursors that are transformed to iridoid monoterpenes in the reservoir. The uptake process displays a remarkable substrate selectivity, since the thioglycoside of geraniol 10 is not imported. From the two isomeric thioglycosides of 8-hydroxygeraniol, 8 and 9, the isomer 8 is preferred by a factor of ten. The data clearly support the existence of a highly selective transport system which enables the larvae to utilize plant derived terpenoid precursors in addition to their own de novo biosynthesis of iridoids.

Metabolic engineering of essential oil yield and composition in mint by altering expression of deoxyxylulose phosphate reductoisomerase and menthofuran synthase.

Peppermint (Mentha x piperita L.) was independently transformed with a homologous sense version of the 1-deoxy-d-xylulose-5-phosphate reductoisomerase cDNA and with a homologous antisense version of the menthofuran synthase cDNA, both driven by the CaMV 35S promoter. Two groups of transgenic plants were regenerated in the reductoisomerase experiments, one of which remained normal in appearance and development; another was deficient in chlorophyll production and grew slowly. Transgenic plants of normal appearance and growth habit expressed the reductoisomerase transgene strongly and constitutively, as determined by RNA blot analysis and direct enzyme assay, and these plants accumulated substantially more essential oil (about 50% yield increase) without change in monoterpene composition compared with wild-type. Chlorophyll-deficient plants did not afford detectable reductoisomerase mRNA or enzyme activity and yielded less essential oil than did wild-type plants, indicating cosuppression of the reductoisomerase gene. Plants transformed with the antisense version of the menthofuran synthase cDNA were normal in appearance but produced less than half of this undesirable monoterpene oil component than did wild-type mint grown under unstressed or stressed conditions. These experiments demonstrate that essential oil quantity and quality can be regulated by metabolic engineering. Thus, alteration of the committed step of the mevalonate-independent pathway for supply of terpenoid precursors improves flux through the pathway that leads to increased monoterpene production, and antisense manipulation of a selected downstream monoterpene biosynthetic step leads to improved oil composition.

Determination of metabolic rate-limitations by precursor feeding in Catharanthusroseus hairy root cultures

Precursors from the terpenoid and tryptophan branches were fed to Catharanthus roseus to determine which of the two branches limits metabolic flux to indole alkaloids. The feeding of tryptophan at 17 days of the culture cycle produced auxin-like effects. Addition of low levels of auxin or tryptophan resulted in significant increases in flux to the indole alkaloids. Conversely, feeding higher levels of auxin or tryptophan resulted in increased branching and thickening of the hairy root cultures. A dramatic reduction in flux to the alkaloids was also observed. However, feeding tryptamine or terpenoid precursors had no effect. Therefore, neither pathway tested revealed to be rate-limiting during the late growth phase. Feeding of either geraniol, 10-hydroxygeraniol, or loganin at 21 days each resulted in significant increases in the accumulation of tabersonine. The addition of tryptophan or tryptamine had no effect during the stationary phase of the growth cycle. Thus, during the early stationary phase of growth the terpenoid pathway appears to be rate-limiting. Combined elicitation with jasmonic acid and feeding either loganin or tryptamine did not further enhance the accumulation of indole alkaloids.

(−)-2C-Methyl- d-erythrono-1,4-lactone is formed after application of the terpenoid precursor 1-deoxy- d-xylulose

Application of [1,2- 14C]1-deoxy- d-xylulose, the committed precursor of terpenoids, thiamine and pyridoxol, to a variety of plant species resulted in the labelling of an unknown metabolite. The isolation and purification of this metabolite from Ipomoea purpurea plants fed with 1-deoxy- d-xylulose (DX), followed by NMR analysis, resulted in the identification of its structure as (−)-2C-methyl- d-erythrono-1,4-lactone (MDEL). MDEL has been previously isolated as a stress metabolite of certain plants. A hypothetical biosynthetic scheme is given.

Biosynthesis of 2-C-methyl-D-erythritol in plants by rearrangement of the terpenoid precursor, 1-deoxy-D-xylulose 5-phosphate

Leaves of Liriodendron tulipifera convert 1-deoxy-D-xylulose to 2-C-methyl-D-erythritol via a skeletal rearrangement reminiscent of the formation of terpene precursors from 1-deoxy-D-xylulose. The data suggest that 2-C-methyl-D-erythritol 4-phosphate is either an intermediate or a side product of the deoxyxylulose 4-phosphate pathway of terpenoid biosynthesis. The incorporation of 13 C-labelled 1-deoxy-D-xylulose into 2-C-methyl-D-erythritol of Liriodendron tulipifera demonstrates that the branched polyol is formed from the terpenoid precursor 1-deoxy-D-xylulose by an intramolecular rearrangement.

Inhibition of alkaloid accumulation by 2,4-D in Catharanthus roseus cell suspension is overcome by methyl jasmonate

Summary In Catharanthus roseus cells (strain C20) 2,4-D exerted a dramatic inhibition on alkaloid terpenoid precursor availability. In this condition, ajmalicine content is less than 2μg/gDW. During one subculture the auxin depletion enhanced greatly the ajmalicine content: about 300μg/gDW. In presence of 2,4-D, exogenous methyl jasmonate (MJ) triggered an ajmalicine accumulation corresponding to 80 % of the one observed in a 2,4-D free medium. Thus MJ takes the auxin depletion signal place. In a 2,4-D free medium, inhibition of MJ synthesis by salicylhydroxamic acid or salicylic acid limits greatly alkaloid content which could be restored by exogenous MJ. Thus, MJ can be regarded as a secondary messenger in transduction of the auxin depletion signal.

Pravastatin: A tool for investigating the availability of mevalonate metabolites for primary and secondary metabolism in Catharanthus roseus cell suspensions

In the C20 strain of Catharanthus roseus, 2,4‐dichlorophenoxyacetic acid (2,4‐D) reduces alkaloid accumulation by inhibiting the synthesis of precursors of alkaloid terpenoids. However, the presence of this growth regulator is necessary to promote growth, as measured in terms of dry weight and sterol content. The terpenoid metabolism implicated in the accumulation of alkaloids would therefore be the target of 2,4‐D inhibition and not the metabolism leading to sterol biosynthesis. The specific inhibition by pravastatin of 3‐hydroxy‐3‐methylglutaryl CoA reductase (EC 1.1.1.34), the enzyme that catalyses mevalonate synthesis, enables the limitation of mevalonate on sterol content and on alkaloid accumulation to be studied. In presence of pravastatin cells are supplied with labelled mevalonate. Under these conditions the mevalonate is incorporated into sterols but not into alkaloids accumulated in the absence of 2,4‐D. The inhibition of sterol biosynthesis induced by pravastatin is not overcome by zeatin or a cytokinin‐like compound, whereas the inhibition of alkaloid accumulation can be partially overcome. The use of pravastatin shows that the availability of mevalonate for primary and secondary metabolism is differently regulated in Catharanthus roseus cells.

Molecular taphonomy of membrane constituents the search for the most primitive membranes

Taphonomy is the study of fossilization processes, and includes "reconstruction" of past animals from their fossils. At the molecular level, the study of the organic constituents of sediments (crude oils and coals, but also clays, shales, etc) has yielded hundreds of structures, essentially in the hands of P.Albrecht and his co-workers in Strasbourg. These are sometimes hardly modified molecular fossils of known natural products. For instance, cholestane "must" come from cholesterol : reductions are frequent molecular taphonomic processes. Diacholestane, also frequently present in sediments, might be derived from "diasteror', an unknown but biosynthetically reasonable rearranged cholesterol ; however, the ease of rearrangement of cholesterol to diasterane derivatives by moderate heating with clays suggests rather that diacholestane derives also from cholesterol ; Wagner-Meerwein rearrangements are reasonable taphonomic processes. To interpret data from organic geochemistry, one must therefore collect trustworthy precise structural data, rely on the extensive catalogs of natural products structures and of biosynthetic process, and know which molecular taphonomic processes are plausible. Known molecular fossils belong to several natural products families, including porphyrins obviously derived from bacterial pigments ; by far most of them are derived from terpenoid precursors, as expected as biodegradation of highly branched structures is notoriously difficult. It was also not unexpected to find, frequently, molecular fossils of some arch~eal lipids : phytane and pristane, which could also have come from chlorophyll or from tocopherols, but also bisphytane, the structure of which became meaningful only once the C40 chains of phospholipid ethers of thermophilic and of methanogenic arch~ea became known. In other cases, while the structures of the precursors could be derived without doubt, they did not tally with any known one. The most spectacular case of these "orphan fossils" has been that of the ubiquitous geohopanoids, triterpene derivatives carrying an additional n-C 5 chain, for which more than 250 structures have been fully demonstrated ; their ubiquity and abundance in sediments make them probably the most abundant natural products on Earth ! Their precursors, the bacterial hopanoids, were discovered initially in two particular bacteria, but later, by M.Rohmer, in many very varied bacteria, with very varied structures (more than 40 established so far, including the fantastic adenosylhopane). Based on the molecular dimensions and amphiphilic character of bacterial hopanoids, we postulated that they reinforce bacterial membranes like

Formation of terpenoid products in Ginkgo biloba L. cultivated cells

Ginkgolides are diterpenes arising from the terpenoid precursor: geranylgeranyl pyrophosphate (GGPP). Incorporation of [1-(14)C] isopentenylpyrophosphate ([1-(14)C]IPP) into GGPP was monitored throughout the cultivation cycle of G. biloba L. cultivated cells. Because incorporation of [1-(14)C]IPP into GGPP had never been monitored in G. biloba, in either the whole plant or cultivated cell system, modifications to existing protocols were necessary. Modifications consisted of extracting the cells with an extraction buffer supplemented with Triton-X-100. Farnesylpyrophosphate (FPP) was the major product formed. The amount of GGPP detected was about one tenth that of FPP.

Terpenoid indole alkaloid biosynthesis and enzyme activities in two cell lines of Tabernaemontana divaricata

The possible limitation of the rate of biosynthesis of terpenoid indole alkaloids by low enzyme levels was investigated in two cell lines of Tabernaemontana divaricata with different terpenoid indole alkaloid biosynthetic capacities. The activities of tryptophan decarboxylase (TDC), strictosidine synthase (SSS), strictosidine glucosidase (SG), isopentenyl pyrophosphate isomerase (IPP isomerase) and geraniol 10-hydroxylase (G10H) of both cell lines were compared. The activities of TDC, SSS and IPP isomerase did not show a direct relationship with the biosynthetic capacity but SG and G10H might be limiting. In order to test whether the availability of the terpenoid precursor limits the biosynthesis of the terpenoid indole alkaloids, loganin was fed to the cultures. Loganin-feeding did not influence any of the measured enzyme activities but increased the terpenoid indole alkaloid accumulation of both cell lines to similar levels. A five-fold increase was observed for the accumulating line and a more than 100-fold increase for the low-accumulating one. Strictosidine accumulated mainly in the low-accumulating cell line which has high TDC and low SG activity; the amounts and types of the other terpenoid indole alkaloids which accumulated were similar in both lines. From this it can be concluded that the biosynthesis of terpenoid indole alkaloids in both cultures is limited by the availability of terpenoid precursors; this pathway is not saturated with substrates under normal culture conditions.

Conversion of myrcene by submerged cultured basidiomycetes

Among 15 species screened, strains of Ganoderma applanatum, Pleurotus flabellatus and Pleurotus sajor-caju were found to be most suitable for the production of volatile, oxygenated metabolites from myrcene. Myrcenol and further acyclic monoterpenes as well as monocyclic compounds were identified as a result of hydroxylation at allylic positions, oxygenation and hydration of double bonds, oxidation of alcohols to carbonyl compounds, and formation of carbon-carbon bonds. Types and concentrations of the products depended on strain and conditions of incubation. The low conversion yields were supposedly due to the limited water solubility of the terpenoid precursor substrate.

Configurational analysis of new furanosesquiterpenes from Dysidea herbacea. Assignment of absolute stereochemistry.

Two new marine furanoterpenes (+)-(4a S, 6 R,8a S)-4,4,7-trimethyl-4,4a,5,6,8,8a,9-hexahydronaphtho[2,3- b]furan-6-yl acetate ( 1) and (+)-(4a S,7 R,8a S)-6,9,9-trimethyl-4,4a,7,8,8a,9-hexahydronaphthol[2,3 b]furan-7-yl acetate ( 2), were isolated sample of Dysidea herbacea collected from Harrier Reef on the Great Barrier Reef, Australlia. The enantiomer of 1, (−)-(4a R,6 S,8a R)-4,4,7–trimethyl– 4,4a,5,6,8,8a,9–hexahydronaptho[2.3–b]furan–6–yl acetate (17) was found in another sample of D. herbacea collected nearby from Norman Reef. The structures were determined by spectroscopic methods, in particular 2D NMR, and the absolute configurations of the parent rings of 1 and 2 defined by chemical correlation and application of Kakisawa's of the Mosher ester method. Compounds 1 and 2 are diastereomers of acetates 5 and 6, reported earlier from D. herbacea, but have opposite configuration with respect to the ring junctions. These results and others suggest D. herbacea, may possess a dual capacity for antipodal cyclization pathways from a common achiral terpenoid precursor.

Effect of terpenoid precursor feeding and elicitation on formation of indole alkaloids in cell suspension cultures of Catharanthus roseus

The effects of terpenoid precursor feeding and elicitation by a biotic elicitor on alkaloid production of Catharanthus roseus suspension cultures were studied. After addition of secologanin, loganin or loganic acid an increase in the accumulation of ajmalicine and strictosidine and a decrease of tryptamine level was observed in non-elicited cells. Elicitation increased tryptamine accumulation in non-fed cells but it did not further increase alkaloid accumulation in precursor-fed cells. A decrease of tryptamine level was also observed, despite the induction of the tryptamine pathway after elicitation. Feeding mevalonic acid did not increase alkaloid accumulation in any studied case.

Terpenoid precursors via steroid degradation: synthesis of (–)-warburganal

The sesquiterpene (–)-warburganal was prepared from a (1S)-1-methoxycarbonyl-2,5,5,8a-tetramethyl-decahydronaphthalene (4) obtained by degradation of commercial glycyrrhetinic acid.

Incorporation of2H from2H2O into ABA in Tomato Shoots: Evidence for a Large Pool of Precursors

Incorporation of²H from²H₂O into ABA in Tomato Shoots: Evidence for a Large Pool of Precursors

Demonstration that limonene is the first cyclic intermediate in the biosynthesis of oxygenated p-menthane monoterpenes in Mentha piperita and other Mentha species

The volatile oil of mature Mentha piperita (peppermint) leaves contains as major components the oxygenated p-menthane monoterpenes l-menthol (47%) and l-menthone (24%) as well as very low levels of the monoterpene olefins limonene (1%) and terpinolene (0.1%), which are considered to be probable precursors of the oxygenated derivatives. Immature leaves, which are actively synthesizing monoterpenes, produce an oil with comparatively higher levels of limonene (~3%), and isolation of the pure olefin showed this compound to consist of ~80% of the l-(4 S)-enantiomer and ~20% of the d-(4 R)-enantiomer. The time course of incorporation of [U- 14C]sucrose into the monoterpenes of M. piperita shoot tips was consistent with the initial formation of limonene and its subsequent conversion to menthone via pulegone. d,l-[9- 3H]Limonene and [9,10- 3H]terpinolene were prepared and tested directly as precursors of oxygenated p-menthane monoterpenes in M. piperita shoot tips. Limonene was readily incorporated into pulegone, menthone, and other oxygenated derivatives, whereas terpinolene was not appreciably incorporated into these compounds. Similarly, d,l-[9- 3H]limonene was specifically incorporated into pulegone in Mentha pulegium and into the C-2-oxygenated derivative carvone in Mentha spicata, confirming the role of this olefin as the essential precursor of oxygenated p-menthane monoterpenes. Soluble enzyme preparations from the epidermis of immature M. piperita leaves converted the acyclic terpenoid precursor [1- 3H]geranyl pyrophosphate to limonene as the major cyclic product, providing a further indication that this olefin plays a central role in the formation of oxygenated monoterpenes in Mentha. No free intermediates were detected in the cyclization of geranyl pyrophosphate to limonene, suggesting that the olefin is the first cyclic intermediate to arise in the pathway, and resolution of the biosynthetic limonene, by crystallization of the derived d- and l-carvoximes, indicated an enantiomer mixture nearly identical to that isolated from the leaf oil.

On the incorporation of geraniol and farnesol into cantharidin (author's transl)

On the incorporation of geraniol and farnesol into cantharidinEarlier investigations [1] have shown that cantharidin (1) is biosynthesized by the male Lytta vesicatoria L. (Meloidae, Coleoptera) from the common terpenoid precursors mevalonate and farnesol (3). To prove if geraniol (2) is incorporated via farnesol (3) into cantharidin (1) the following geraniols have been synthesized and injected into either larvae or male adult Lytta vesicatoria, partly in a mixture with synthetic 11′, 12‐[3H]‐farnesol as an internal standard: 2‐[14C]‐, 7‐[14C]‐, 7′, 8‐[14C]‐, 7′, 8‐[3H]‐geraniol. Unexpectedly, geraniol (2) was not specifically incorporated into cantharidin (1) perhaps due to its higher toxicity or its faster degradation relative to the other precursors before incorporation. The incorporation of U‐[14C]‐leucine, U‐[14C]‐isoleucine and 1‐[14C]‐glucose into cantharidin (1) via their metabolites is evident by degradation studies, whereas 1‐[14C]‐ and 2‐[14C]‐glycine do not serve as precursors for cantharidin (1).

Transfer of cantharidin (1) during copulation from the adult male to the femaleLytta vesicatoria (‘Spanish flies’)

Adult males of the ‘spanish fly’Lytta vesicatoria (Meloidae, Coleoptera) are able to biosynthesize cantharidin (1) ‘in copula’ after injection of E, E-11′-(3H,14C)-farnesol and 2-14C-methylfarnesoate. During mating, the cantharidin biosynthesized in the males is transferred to the females, who are unable to biosynthesize cantharidin from any terpenoid precursors. After injection of (3H,14C)-cantharidin into males during copulation, more than 10% is transferred into the female sex organs. Males injected with 2-(3H,14C)-mevalonate 24–30 h prior to copulation transferred 93–98% of the biosynthesized cantharidin to the females during mating. After mating, the males continue to produce cantharidin. It seems that in the male the biosynthesis of cantharidin is stimulated during copulation.

The isolation and identification of juvenile hormone from cockroach corpora allata [formula omitted

Methyl (2E, 6E) - 10, 11 - epoxy - 3, 7, 11 - trimethyl - 2, 6 - dodecadienoate (JH III) is synthesized by corpora cardiaca/allata of the American cockroach Periplaneta americana in vitro and released into the culture medium. It constitutes the principal part of the biologically detectable JH activity. A fully synthetic medium without terpenoid precursors or serum was used, which proves, that the entire molecule is synthesized de novo . The presence of JH III in orders as different as Blattodea, Orthoptera, Coleoptera and Lepidoptera indicates, that it is the most widely occuring JH of insects.

Inhibition by isonicotinyl hydrazide of pigment formation in higher plants

The inhibition of greening of illuminated etiolated maize seedlings by isonicotinyl hydrazide can be alleviated by serine or pyruvate. The similar inhibition in barley can be reversed only by pyruvate. In both plants earlier intermediates in the glycollate pathway and other related compounds were ineffective in overcoming the inhibition of greening produced by isonicotinyl hydrazide. In maize seedlings radioactivity from l-serine-[3- 14C] is poorly incorporated into β-carotene, a typical chloroplast terpenoid, unless glycine and formate or, more effectively, glycine together with isonicotinyl hydrazide are supplied. These supplementations may minimize interconversion of serine and glycine, and hence dilution of radioactivity at C-3 of l-serine by unlabelled C-1 units, before incorporation into terpenoids. The results support the view that in young greening tissue the C2-3 fragment of l-serine can give rise to acetyl-CoA, an obligatory precursor of chloroplast terpenoids.