Furanocoumarin biosynthesis in Ruta graveolens cell cultures

The biosynthetic routes to four linear furanocoumarins—psoralen, xanthotoxin, bergapten. isopimpinellin-co-occurring in Ruta graveolens cell cultures have been investigated with six ¹⁴C-labelled compounds. Mevalonic acid was only poorly incorporated, in contrast to umbelliferone. In support of previous suggestions, 7-demethylsuberosin and (±)-marmesin were very good precursors of the linear furanocoumarins. 7-O-Prenylumbelliferone also was fairly well utilized, but this was probably owing to a prior ether cleavage yielding umbelliferone. Psoralen was well incorporated into bergapten and xanthotoxin, but not into the dimethoxylated isopimpinellin. Differences exist between the organized plant and its cell culture in terms of metabolic products and, by implication, precursor utilization. S(+)-Marmesin was obtained in small quantity from an acid-hydrolysable conjugate present in the culture medium. Syntheses of [2-¹⁴C]7-demethylsuberosin, [2-¹⁴C]osthenol, [2-¹⁴C]7-O-prenylumbelliferone, [3-¹⁴C] (±)-marmesin, and [3-¹⁴C]psoralen are described, as well as an improved method for separation of furanocoumarin mixtures by TLC and GLC.     

Aldoximes and nitriles as intermediates in the biosynthesis of cyanogenic glucosides

Young sorghum shoots have been shown to convert tyrosine, p-hydroxyphenylacetaldoxime and p-hydroxyphenylacetonitrile to dhurrin, the cyanogenic glucoside characteristic of this plant. Evidence for the in vivo formation of p-hydroxyphenylacetaldoxime but not p-hydroxyphenylacetonitrile from tyrosine has been obtained from a 'trapping experiment.'     

Cinnamic acid intermediates as precursors to mesembrine and some observations on the late stages in the biosynthesis of the mesembrine alkaloids

Experiments with various labelled cinnamic acid derivatives establish, in conjunction with previous work, that the incorporation of phenylalanine into the 3a-aryl octahydroindole ring system of the mesembrine alkaloids occurs via the intermediacy of cinnamic acid and 4′-hydroxycinnamic acid. The major pathway to the 3′,4′-di-oxyaryl substituted alkaloids proceeds via 4′-hydroxydihydrocinnamic acid (4′-phloretic acid), and 3′4′-dioxy-genated cinnamic acids are not involved as intermediates on this major pathway. In accord with this latter finding, the 3′-aryl oxygen substituent is introduced at a late state in the biosynthesis as evidenced by the bioconversion in S. strictum of sceletenone to mesembrenol and other related alkaloids. The late stages in the biosynthesis of the alkaloids are shown to involve the sequence: sceletenone, 4′-O-demethylmesembrenone, mesembrenone. Mesembrenone is converted to mesembrine, mesembrenol and mesembranol.     

Characterization of medium-chain-length polyhydroxyalkanoate biosynthesis by Pseudomonas mosselii TO7 using crude glycerol

Polyhydroxyalkanoates (PHAs) are biopolyesters produced by microorganisms that are environmentally friendly. PHAs can be used to replace traditional plastic to reduce environmental pollution in various fields. PHA production costs are high because PHA must be produced from a carbon substrate. The purpose of this study was to find the strain that can used the BDF by-product as the sole carbon source to produce high amounts of medium-chain-length PHA. Three isolates were evaluated for potential PHA production by using biodiesel-derived crude glycerol as the sole carbon source. Among them, Pseudomonas mosselii TO7 yielded high PHA content. The PHA produced from P. mosselii TO7 were medium-chain-length-PHAs. The PHA content of 48% cell dry weight in 48 h with a maximum PHA productivity of 13.16 mg PHAs L^?1 h^?1. The narrow polydispersity index value of 1.3 reflected the homogeneity of the polymer chain, which was conducive to industrial applications.     

The methyl migration in rosenonolactone biosynthesis

The C-10 to C-9 methyl group migration in rosane biosynthesis proceeded without loss of mevalonoid hydrogen from C-20 thus excluding a cyclopropane intermediate from the biosynthesis sequence. 10β,19-Dihydroxyrosa-15-ene was comparatively efficiently incorporated into rosenonolactone but it could not be detected in the fermentation.     

Plant growth regulators from Heracleum lanatum

The plant growth regulating substances, pimpinellin, isopimpinellin, bergapten, isobergapten, vaginidiol, sphondin, scopoletin, umbelliferone, ferulic     

Caffeine biosynthesis in Camellia sinensis

The metabolism of adenine and guanine, relating to the biosynthesis of caffeine, in excised shoot tips of tea was studied with micromolar amounts of adenine-[8-¹⁴C] or guanine-[8-¹⁴C]. Among the presumed precursors of caffeine biosynthesis, adenine was the most effective, whereas guanine was the least effective. After administration of a ‘pulse’ of adenine-[8-¹⁴C], almost all of the adenine-[¹⁴C] supplied disappeared by 30 hr, and ¹⁴C-labelled caffeine and RNA purine nucleotide (AMP and GMP) synthesis increased throughout the experimental period, whereas the radioactivities of free purine nucleotides, 7-methylxanthine and theobromine increased during the first 10 hr incubation period, followed by a steady decrease. By contrast, more than 45% of the guanine-[8-¹⁴C] supplied remained unchanged even after a 120 hr period. The main products of guanine-[8-¹⁴C] metabolism in tea shoot tips were guanine nucleotides, theobromine, caffeine and the GMP of RNA. The results support the hypothesis that the purine nucleotides are synthesized from adenine and guanine via the pathway of purine salvage. Adenylate is readily converted into other purine nucleotides, whereas the conversion rate of guanylate into other purine nucleotides is very low.The results also support the view that 7-methylxanthine and theobromine are precursors of caffeine. For the origin of the purine ring in caffeine, purine nucleotides in the nucleotide pool rather than in nucleic acids are suggested.     

Isoflavone biosynthesis in Onobrychis viciifolia: Formononetin and texasin as precursors of afrormosin

4,2′,4′-Trihydroxychalcone- [carbonyl-¹⁴C], formononetin- [Me-¹⁴C] and texasin- [Me-¹⁴C] were all good precursors of afrormosin (7-hydroxy-6,4′-dimethoxyisoflavone) in Onobrychis viciifolia seedlings, and a biosynthetic pathway involving these intermediates is proposed. 2′,4′-Dihydroxy-4-methoxychalcone- [carbonyl-¹⁴C] and daidzein-[carbonyl-¹⁴C] were poor precursors. Incorporations into formononetin were also recorded.     

The biosynthesis of kaurenolide diterpenoids by gibberella fujikuroi

The biosynthesis of 7β-hydroxy- and 7β,18-dihydroxy-kaurenolides from ent-kaur-16-en-19-oic acid has been investigated by incubating unlabelled     

Biosynthetic intermediates in the conversion of fucosterol to oogoniol

When grown in the presence of [CD₃]-methionine Achlya heterosexual produces oogoniols containing two deuterium atoms which are located at C-28     

Tetrapyrrole biosynthesis in greening etiolated barley seedlings

Allyl isopropylacetamide (AIA) does not stimulate porphyrin biosynthesis in greening barley; AIA inhibits the synthesis of 5-aminolaevulinate (ALA) in plants and does not overcome the repression of ALA-synthetase. This indicates that the ALA synthesis system of green plants is regulated differently from ALA synthetase of mammalian systems. Laevulinic acid (LA) inhibited the biosynthesis of tetrapyrrole pigments in greening barley and diminished the insertion of ⁵⁵Fe into extractable protohaem, confirming that haem was synthesized at a time of little net increase in protohaem. ALA feeding increased iron incorporation into protohaem without increasing either extractable protohaem or cytochromes b and f. Since ALA feeding greatly increased the protochlorophyllide content of darkgrown plants and subsequent chlorophyll levels in the light, the regulation of haem pigment synthesis in plants occurs after protoporphyrin and protohaem synthesis and is likely to involve the turnover of protohaem produced in excess of haem protein requirements.     

Methionine biosynthesis in isolated Pisum sativum mitochondria

Homocysteine-dependent transmethylases utilizing 5-methyltetrahydropteroylglutamic acid and S-adenosylmethionine as methyl donors have been examined using ammonium sulphate fractions prepared from isolated mitochondria of pea cotyledons. Substantial levels of a 5-rnethyltetrahydropteroylglutamate transmethylase were detected, the catalytic properties of this enzyme being found similar to those of a previously reported enzyme present in cotyledon extracts. The mitochondrial 5-CH₃-H₄PteGlu transmethylase had an apparent K_m of 25 μM for the methyl donor, was saturated with homocysteine at 1 mM and was inhibited 50% by l-methionine at 2.5 mM. At similar concentrations of methyl donor the mitochondrial S-adenosylmethionine methyltransferase was not saturated. Mitochondrial preparations were found capable of synthesizing substantial amounts of S-adenosylmethionine but lacked ability to form S-methylmethionine. Significant levels of β-cystathionase, cystathionine-γ-synthase, l-homoserine transacetylase and l-homoserine transsuccinylase were detected in the isolated mitochondria. The activity of the enzymes of homocysteine biosynthesis was not affected by l-methionine in vitro. It is concluded that pea mitochondria have ability to catalyze the synthesis of methionine de novo.     

Alkaloid biosynthesis in Croton linearis

The ability of Croton linearis to form crotonosine from linearisine has been demonstrated; the extent of this conversion appears to vary according to the sex of the plant.     

Sterol biosynthesis in heterotrophic plant parasites

Cycloartenol derivatives are present in the non-photosynthetic parasitic plants Cuscuta europaea (dodder), Cuscuta epithymum and Orobanche lutea (broomrape). C.europaea and O.lutea are capable of biosynthesizing their own sterols. There is therefore no direct link, in a chlorophyll-containing phylum, between the cycloartenol pathway to sterols and photosynthesis.     

Glucofructosan biosynthesis in Fusarium oxysporum

Low MW glucofructosans have been detected in the medium of Fusarium oxysporum. A 53-fold purification of fructosyl transferase has been achieved by ethanol precipitation, DEAE-cellulose and Sephadex G-100 column chromatography. Maximum fructosyl transferase activity coincided with maximum glucofructosan concentration in the medium. Invertase showed greatest activity in the later stages of growth when glucofructosans were absent. Fructosyl transferase and invertase have been separated by DEAE-cellulose column chromatography. On the basis of kinetic studies and effect of nucleotides on fructosyl transferase in the presence and absence of MgCl₂, a two site active centre linked through a nucleotide bridge is proposed. Fructosyl transferase and invertase are highly phosphorylated.     

N-Coronafacoyl-L-isoleucine and N-coronafacoyl-L-alloisoleucine,potential biosynthetic intermediates of the phytotoxin coronatine

Two new naturally-occurring analogues of the phytotoxin coronatine have been isolated from liquid cultures of Pseudomonas syringae pv. glycinea. These have been identified as N-coronafacoyl-L-isoleucine and N-coronafacoyl-L-alloisoleucine by mass spectrometry and by studies of the products of acid hydrolysis of the two compounds. The compounds were purified as a mixture of ca 2:1 composition, but the two parent components were not preparatively separated. The possible significance of the two compounds, to the biosynthesis of coronatine, is discussed.     

Elicitor induction of furanocoumarin biosynthetic pathway in cell cultures ofRuta graveolens

Treatment with an autoclaved culture homogenate of the yeastRhodotorula rubra induces rapid accumulation of acridone epoxides, furoquinolines and furanocoumarins in cell cultures ofRuta graveolens (L). The increased accumulation is preceeded by an induction of enzymes of the biosynthetic pathways. In the case of furanocoumarins induction was shown for phenylalanine ammonia-lyase (PAL), 4-coumarate: CoA ligase (4-CL) and S-adenosyl-l-methionine: xanthotoxol O-methyltransferase (XOMT). For PAL and 4-CL time courses of induced activity showed an early maximum, 8–12 h after treatment, whereas XOMT was found to reach its maximum later, about 36–42 h after treatment. The elicitor dose-response curve showed saturation at an elicitor concentration of 1%. At any time during the whole culturing period cells responded to elicitiation but the maximum enzyme activities induced were lower at the late stages. Experiments with different suspension culture strains, a shoot teratoma culture and hydroponically grown sterile photomixotrophic plants were performed to assess the influence of differentiation on constitutive activities of these enzymes and their inducibility by elicitation. Constitutive furanocoumarin accumulation was positively correlated with the level of differentiation. Although induction of PAL, 4-CL and XOMT activity always accompanied induced furanocoumarin accumulation no absolute correlation existed between induced enzyme activities and the induced product level or relative product increase.Abbreviations 4-CL 4-coumarate:CoA ligase - COMT S-adenosyl-l-methionine:caffeic acid 3-O-methyltransferase - PAL phenylalanine:ammonia-lyase - XOMT S-adenosyl-l-methionine:xanthotoxol O-methyltransferase     

Oxygen incorporation in the biosynthesis of p-coumaric acid in seedlings of Zea mays

The results of experiments in which seedlings of Zea mays were grown in the light in an atmosphere enriched with oxygen-18 indicated that the hydroxyl oxygen atom in p-coumaric acid is derived from molecular oxygen.     

1,2 Hydrogen-shifts in the biosynthesis of the thujane skeleton

Degradation of (+)-isothujone (trans-thujan-3-one) biosynthesized in Tanacetum vulgare from (3RS)-mevalonic acid (MVA)-[2-¹⁴C, 2-³H₂] showed that one hydrogen from C-2 of the precursor was specifically incorporated at C-4 of product whereas the other was lost. Feeding of α-terpineol-[9-¹⁴C, 4-³H₁, 10-³H₃] (p-menth-1-en-8-ol) yielded isothujone with the same isotope ratios as in precursor. These results indicate 1,2 hydrogen-shifts at two locations in the construction of the the thujane skeleton from α-terpineol or its biogenetic equivalent, and are consistent with a mechanism involving direct cyclization of the latter to a product that by-passes the formation of the biogenetic equivalent of terpinen-4-ol (p-menth-1-en-4-ol) as an intermediate. (3R)-MVA-[¹⁴C, ³H] was more effectively incorporated (up to 1.5 %) into (+)-isothujone in vivo during autumn or winter than in summer (up to 0.02%).     

Metabolism of phenylpropanoids in Hydrangea serrata var. thunbergii and the biosynthesis of phyllodulcin

DL-Phenylalanine-[3-¹⁴C] and cinnamic acid-[3-¹⁴C] were fed to this plant and the label from cinnamic acid was incorporated into gallic acid, phyllodulcin and quercetin. By feeding p- coumaric acid-[U-³H], caffeic acid-[U-³H] and hydrangea glucoside A-[U-³H], it was possible to show that hydroxylation at C-3′in phyllodulcin occurs after the ring closure of dihydroisocoumarin. The biosynthetic pathway of phyllodulcin in this plant is thus: phenylalanine → cinnamic acid → p- coumaric acid → hydrangenol → phyllodulcin.