Effect of CPTA and cycocel on the biosynthesis of carotenoids by Phycomyces blakesleeanus mutants

CPTA and cycocel cause accumulation of lycopene and γ-carotene, simultaneously inhibiting the formation of β-carotene and β-zeacarotene in Phycomyces blakesleeanus mutant strain C115. Phytoene synthesis is enhanced. CPTA is more effective than cycocel. Kinetic studies show that with increasing concentrations of CPTA, lycopene and γ-carotene increase with the concomitant decrease in β-carotene, the total of these three carotenes being almost equal to β-carotene present in the control. When CPTA-treated mycelium is washed free of the chemical and resuspended in phosphate buffer solution containing 2·5% glucose (pH 5·6), β-carotene is formed at the expense of both γ-carotene and lycopene. β-Zeacarotene, which is not present in the mycelium, reappears upon resuspension. These results indicate that CPTA is inhibiting the enzymes causing cyclization both at neurosporene and lycopene levels. Studies on the effect of CPTA on the high lycopene mutant strain C9 reveal that with increasing concentrations of the compound, lycopene increases slightly and both β-carotene and γ-carotene decrease. Phytoene synthesis is stimulated up to a certain level of CPTA and then becomes steady. In the albino mutant strain C5, there is a slight increase in phytoene formation on the addition of CPTA to the medium. No other carotenoid is formed, suggesting that CPTA cannot remove the block caused by genetic mutation and exerts its influence in an already existing biosynthetic pathway.     

Alternative pathways of carotene cyclisation in Phycomyces blakesleeanus

Three carotene substrates, lycopene-[¹⁴C], γ-carotene-[¹⁴C] and neurosporene-[¹⁴C] were each incorporated into β-carotene using a cell extract of the C115 car-42 mad-107(?) mutant of P. blakesleeanus. Neurosporene-[¹4C] was also converted into β-zeacarotene and lycopene. Addition of either unlabelled lycopene or β-zeacarotene with neurosporene-[¹⁴C] to the cell extract caused approximately equal reductions of incorporation into β-carotene, indicating that both cyclisation pathways are of equivalent quantitative importance.     

The effect of CPTA analogs and other nitrogenous compounds on the biosynthesis of carotenoids in Phycomyces blakesleeanus mutants

The inhibitory effect of a series of analogs of CPTA, 2-(4-chlorophenylthio)-triethylamine-HCl, and ammonia derivatives on carotenoid biosynthesis in Phycomyces blakesleeanus mutants was studied. The types of inhibition exhibited allowed no firm conclusions about the biosynthetic route to β-carotene from either β-zeacarotene or lycopene. However, the evidence suggests at present that both pathways are operative. It was found that a slight change in structure of inhibitor resulted in a different type of action. Conclusions based on a single inhibitor could be cited as “evidence” for a certain pathway.     

Carotenogenesis in Phycomyces

Time course studies of carotenoid production and of mycelial growth in liquid cultures of Phycomyces blakesleeanus wild type [NRRL 1555 (?)], red mutants C9, C10 and C13 and the heterokaryon C2 * C9 are reported. The ratios of the concentrations of lycopene, γ-carotene and β-carotene in the red mutant C13 and in the heterokaryon C2 * C9 during the growth periods were measured. In these strains the concentration of lycopene is close to its final value after 2 days of growth, at a time at which β-carotene is just beginning to be produced. It is suggested that the β-carotene produced late is possibly synthesized via β-zeacarotene.     

<Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 CruA (sll0147) encodes lycopene cyclase and requires bound chlorophyll <Emphasis Type="Italic">a</Emphasis> for activity

The genome of the model cyanobacterium, Synechococcus sp. PCC 7002, encodes two paralogs of CruA-type lycopene cyclases, SynPCC7002_A2153 and SynPCC7002_A0043, which are denoted cruA and cruP, respectively. Unlike the wild-type strain, a cruA deletion mutant is light-sensitive, grows slowly, and accumulates lycopene, γ-carotene, and 1-OH-lycopene; however, this strain still produces β-carotene and other carotenoids derived from it. Expression of cruA from Synechocystis sp. PCC 6803 (cruA ₆₈₀₃) in Escherichia coli strains that synthesize either lycopene or γ-carotene did not lead to the synthesis of either γ-carotene or β-carotene, respectively. However, expression of this orthologous cruA ₆₈₀₃ gene (sll0147) in the Synechococcus sp. PCC 7002 cruA deletion mutant produced strains with phenotypic properties identical to the wild type. CruA₆₈₀₃ was purified from Synechococcus sp. PCC 7002 by affinity chromatography, and the purified protein was pale yellow-green due to the presence of bound chlorophyll (Chl) a and β-carotene. Native polyacrylamide gel electrophoresis of the partly purified protein in the presence of lithium dodecylsulfate at 4 °C confirmed that the protein was yellow-green in color. When purified CruA₆₈₀₃ was assayed in vitro with either lycopene or γ-carotene as substrate, β-carotene was synthesized. These data establish that CruA₆₈₀₃ is a lycopene cyclase and that it requires a bound Chl a molecule for activity. Possible binding sites for Chl a and the potential regulatory role of the Chl a in coordination of Chl and carotenoid biosynthesis are discussed.     

Carotene biosynthesis by cell extracts of mutants of Phycomyces blakesleeanus

Cell extracts capable of converting MVA-[2-¹⁴C] into isoprenoids were obtained from the yellow C115-mad-107(−) and red C9-carR21(−) mutants of Phycomyces blakesleeanus. Neither air nor light was essential for carotene biosynthesis. The specific activities of the terpenoid-synthesizing enzymes varied with the age of the cultures although the formation of lycopene (ψ,ψ-carotene) in the C9 and of β-carotene (β,β-carotene) in the C115 'mutants. respectively, followed the increase in the dry weight yield of the cultures. The significance of these results to the biosynthesis of carotenes and to the classification of these compounds as secondary metabolites is discussed.     

Biosynthesis of Carotenoids in Brevibacterium sp. KY-4313

The biosynthesis of 4-keto and 4,4′-diketo carotenoids in Brevibacterium sp. KY-4313 was studied. Echinenone and canthaxanthin were isolated from the cultures grown on a medium containing several n-alkanes. When glutathione was added to the bacterial cultures, the formation of canthaxanthin was inhibited while β-carotene and its hydroxy derivatives accumulated. It is suggested that these 4-hydroxy compounds, isocryptoxanthin, isozeaxanthin, and 4-hydroxy-4′-keto-β-carotene, are intermediates in the biosynthesis of canthaxanthin. In the presence of 2-(4-chlorophenylthio)-triethylamine hydrochloride or nicotine, lycopene and neurosporene accumulated. The β-carotene level decreased slightly but β-zeacarotene remained unchanged. β-carotene and its derivatives were resynthesized upon removal of the inhibitors. It was concluded that cyclization can take place at either the neurosporene or lycopene level in Brevibacterium sp. KY-4313.     

Production of β-carotene and acetate in recombinant Escherichia coli with or without mevalonate pathway at different culture temperature or pH

Natural β-carotene has received much attention as consumers have become more health conscious. Its production by various microorganisms including metabolically engineered Escherichia coli or Saccharomyces cerevisiae has been attempted. We successfully created a recombinant E. coli with an engineered whole mevalonate pathway in addition to β-carotene biosynthetic genes and evaluated the engineered cells from the aspects of metabolic balance between central metabolism and β-carotene production by comparison with conventional β-carotene producing recombinant E. coli (control) utilizing a native methylerythritol phosphate (MEP) pathway using bioreactor cultures generated at different temperatures or pHs. Better production of β-carotene was obtained in E. coli cultured at 37°C than at 25°C. A two-fold higher titer and 2.9-fold higher volumetric productivity were obtained in engineered cells compared with control cells. Notably, a marginal amount of acetate was produced in actively growing engineered cells, whereas more than 8 g/L of acetate was produced in control cells with reduced cell growth at 37°C. The data indicated that the artificial operon of the whole mevalonate pathway operated efficiently in redirecting acetyl-CoA into isopentenyl pyrophosphate (IPP), thereby improving production of β-carotene, whereas the native MEP pathway did not convert a sufficient amount of pyruvate into IPP due to endogenous feedback regulation. Engineered cells also produced lycopene with a reduced amount of β-carotene in weak alkaline cultures, consistent with the inhibition of lycopene cyclase.     

Effect of the ratio of (+) and (−) mating type of Blakeslea trispora on carotene production from cheese whey in submerged fermentation

The effect of the ratio of (+) and (?) mating type of Blakeslea trispora on carotene production from deproteinized hydrolysed whey in shake flask culture was investigated. Also, the inoculum ratio of the two mating types on the morphology of the microorganism and the relationship between morphological changes of the fungus and product formation were studied. The concentration of carotenes was significantly affected by the ratio of (+) and (?) mating type of B. trispora. A ratio of 1:10 up to 1:100 of (+) and (?) type was found to achieve the highest carotene yields. The optimum ratio of the (+) and (?) mating types for the maximum pigment production (175.0 mg/g dry biomass at 8 days of fermentation) was found to be 1:10. The carotene content in B. trispora consisted of β-carotene, γ-carotene, and lycopene. At the maximum concentration of carotenes the proportion of β-carotene, γ-carotene, and lycopene (as percent of total carotenes) was 80, 12, and 8%, respectively. B.trispora growing in submerged fermentation is able to develop complex morphologies which have been classified into three major groups: freely dispersed hyphae, clumps and pellets. These parameters are strongly influence the production of carotenes.     

Effect of light intensity on the formation of carotenoids in normal and mutant maize leaves

Carotenoid composition in leaves of normal, lycopenic and ζ-carotenic mutants of Zea mays were investigated. In lycopenic leaves, in addition to lycopene, phytoene, phytofluene, δ- and γ-carotene, trace amounts of α- and β-carotene and antheraxanthin were identified. Low light promoted accumulation of α- and β-carotene; high light brought about an increase in antheraxanthin content. In the leaves of the ζ-carotenic mutant, phytoene, phytofluene and ζ-carotene were synthesized. Illumination of low intensity stimulated carotenoid synthesis to a slight extent. Relative amounts of carotenoid components were essentially the same as in etiolated material, except for a small increase in cis-ζ-carotene. Under high intensity illumination, carotenoids were rapidly destroyed.     

Effect of Inhibitors on the Formation of Stereoisomers in the Biosynthesis of {beta}-Carotene in Dunaliella bardawil

An assay system was developed in which the effects of inhibitorsof ß-carotene biosynthesis in Dunaliella bardawilcould be tested. Since D. bardawil can be induced to accumulateover 10% of its dry weight as ß-carotene, it is particularlysuitable for such studies. Norflurazon a desaturation inhibitor,caused the accumulation of phytoene, or of phytoene and phytofluene,depending on the concentration employed. J-334, a substituted6-methylpyrimidine which also inhibits desaturation, causedthe accumulation of ß-zeacarotene, -carotene and phytoenein different proportions, depending on the concentration employed.The cyclization inhibitors, nicotine, CPTA and MPTA, severelyaffected the growth and survival of the alga, and their effectscould therefore not be studied directly. However, their actionwas observed indirectly by following the transformation of phytoenein norflurazon-pretreated phytoene-rich algae. Under these conditions,presence of the cyclase inhibitors caused the transformationof phytoene to lycopene, rather than to ß-carotene.The accumulated ß-carotene or the intermediates ß-zeacarotene,lycopene, -carotene, phytofluene and phytoene in D. bardawilwere all composed of two stereoisomers, tentatively assignedas the all-trans stereoisomer (55%) and the 9-cis stereoisomer(45%). This suggests that the isomerization reaction which leadsto the production of the presumed 9-cis isomers occurs earlyin the pathway of carotene biosynthesis, at or before phytoene,with no isomerization during the further transformations leadingto ß-carotene. (Received January 29, 1990; Accepted May 9, 1990)     

Strong and weak plasma response to dietary carotenoids identified by cluster analysis and linked to beta-carotene 15,15'-monooxygenase 1 single nucleotide polymorphisms

The mechanisms as well the genetics underlying the bioavailability and metabolism of carotenoids in humans remain unclear. To begin to address these questions, we used cluster analysis to examine individual temporal responses of plasma carotenoids from a controlled-diet study of subjects who consumed carotenoid-rich beverages. Treatments, given daily for 3 weeks, were watermelon juice at two levels (20-mg lycopene, 2.5-mg β-carotene, n=23 and 40-mg lycopene, 5-mg β-carotene, n=12) and tomato juice (18-mg lycopene, 0.6-mg β-carotene, n=10). Cluster analysis revealed distinct groups of subjects differing in the temporal response of plasma carotenoids and provided the basis for classifying subjects as strong responders or weak responders for β-carotene, lycopene, phytoene and phytofluene. Individuals who were strong or weak responders for one carotenoid were not necessarily strong or weak responders for another carotenoid. Furthermore, individual responsiveness was associated with genetic variants of the carotenoid metabolizing enzyme β-carotene 15,15'-monooxygenase 1. These results support the concept that individuals absorb or metabolize carotenoids differently across time and suggest that bioavailability of carotenoids may involve specific genetic variants of β-carotene 15,15'-monooxygenase 1.     

Cyclization of lycopene in the biosynthesis of β-carotene

Mycobacterium marinum produces carotenoids when exposed to light or when antimycin A is added. Although the major pigment synthesized is β-carotene, lycopene is accumulated when the induced bacteria are incubated in the presence of nicotine (5 mM) or 2-(4-chlorophenylthio)-triethylamine hydrochloride (CPTA) (50 μM). Both of these compounds inhibit β-carotene synthesis by blocking the cyclization of lycopene. When nicotine is removed by washing the cells, the accumulated lycopene is cyclized to form β-carotene. The cyclization of lycopene is not an energy-requiring reaction and, furthermore, does not require oxygen or any other electron acceptor. Chloramphenicol addition also does not inhibit the conversion of lycopene to β-carotene indicating that no de novo protein synthesis is involved. Nicotine appears to act by inhibiting the activity of the enzyme required for the cyclization of lycopene.Although the mode of action of CPTA is similar to nicotine, it cannot be removed by washing once the cells have been incubated in its presence, suggesting that the molecule is tightly bound to the enzyme. The possible active molecular sites of nicotine and CPTA are discussed.     

The Carotenoid Hydrocarbons of Euglena gracilis and Derived Mutants

An examination of the carotene fractions extracted from Euglena gracilis Z and pressure-bleached Euglena mutants PR-1, PR-2, PR-3, and PR-4 revealed phytoene in mutants PR-1, PR-2, and PR-3. Photosynthetic E. gracilis Z cultured at different light intensities showed no detectable phytoene, nor was phytoene found in mutant PR-4. However, dark-cultured E. gracilis Z yielded readily assayable amounts of phytoene. With the exception of PR-4, in which no C₄₀ carotenoids were detected, the following carotenes were identified in all from their mass spectra: phytoene, phytofluene, ζ-carotene, β-zeacarotene, and β-carotene. Of these, phytoene and β-zeacarotene had not previously been unequivocally identified in Euglena.     

In vitro and in vivo biosynthesis of xanthophylls by the cyanobacterium Aphanocapsa

Of the six carotenoids identified in the cyanobacterium Aphanocapsa, β-carotene, zeaxanthin, echinenone and myxoxanthophyll are the major pigments, whilst β-cryptoxanthin and 3-hydroxy-4-keto-β-carotene are present only in trace amounts. With the exception of zeaxanthin, the other xanthophylls could be formed in vitro from [¹⁴C]phytoene in high yields, especially β-cryptoxanthin and 3-hydroxy-4-keto-β-carotene. In a time course experiment of xanthopyll biosynthesis the flow of radioactivity from [¹⁴C]phytoene was followed through the pools of phytofluene, lycopene, and β-carotene. The reaction sequence from phytoene to xanthophylls is sensitive in vitro to both difunone, an inhibitor of carotene desaturation, and CPTA, an inhibitor of cyclization.     

Chemical induction of β-carotene biosynthesis

Marsh white seedless grapefruit were treated with the 2-diethylaminoethanol esters of the following acids: benzoic, phenylacetic, hydrocinnamic, 4-phenylbutyric, 5-phenylvaleric, valeric, hexanoic, heptanoic, octanoic, nonanoic, 5-chlorovaleric, cyclohexanecarboxylic, phenoxyacetic, p-chlorophenoxyacetic, 3-phenoxypropionic, cinnamic and p-chlorocinnamic. Several of these esters, in particular the hexanoate, 4-phenylbutyrate and cinnamate, caused the accumulation of large amounts of β-carotene. The effects of the hexanoate and of 2-phenoxytriethylamine, which causes only lycopene accumulation, were studied as functions of time. The hexanoate caused the rapid accumulation of lycopene during the first day. The amount of lycopene then began to decrease and that of β-carotene increased until, after 14 days, β-carotene was the major pigment. 2-Phenoxytriethylamine caused rapid lycopene accumulation during the first day and a slow steady increase afterwards. Thus, the mode of action of the β-carotene inducers may be similar to that of the lycopene inducers except that the former are probably rapidly hydrolysed by the esterase(s) in the flavedo, so that they no longer inhibit the cyclase(s), and β-carotene is accumulated at the expanse of lycopene.     

Carotenoids of hemipteran insects,from the perspective of chemo-systematic and chemical ecological studies

Carotenoids of 47 species of insects belonging to Hemiptera, including 16 species of Sternorrhyncha (aphids and a whitefly), 11 species of Auchenorrhyncha (planthoppers, leafhoppers, and cicadas), and 20 species of Heteroptera (stink bugs, assassin bugs, water striders, water scorpions, water bugs, and backswimmers), were investigated from the viewpoints of chemo-systematic and chemical ecology. In aphids, carotenoids belonging to the torulene biosynthetic pathway such as β-zeacarotene, β,ψ-carotene, and torulene, and carotenoids with a γ-end group such as β,γ-carotene and γ,γ-carotene were identified. Carotenoids belonging the torulene biosynthetic pathway and with a γ-end group were also present in water striders. On the other hand, β-carotene, β-cryptoxanthin, and lutein, which originated from dietary plants, were present in both stink bugs and leafhoppers. Assassin bugs also accumulated carotenoids from dietary insects. Trace amounts of carotenoids were detected in cicadas. Carotenoids of insects belonging to Hemiptera well-reflect their ecological life histories.     

Lipid-dissolved γ-carotene, β-carotene,and lycopene in globular chromoplasts of peach palm (Bactris gasipaes Kunth) fruits

Main conclusion

High levels of β-carotene, lycopene, and the rare γ-carotene occur predominantly lipid-dissolved in the chromoplasts of peach palm fruits. First proof of their absorption from these fruits is reported. The structural diversity, the physical deposition state in planta, and the human bioavailability of carotenoids from the edible fruits of diverse orange and yellow-colored peach palm (Bactris gasipaes Kunth) varieties were investigated. HPLC–PDA–MSⁿ revealed a broad range of carotenes, reaching total carotenoid levels from 0.7 to 13.9 mg/100 g FW. Besides the predominant (all-E)-β-carotene (0.4–5.4 mg/100 g FW), two (Z)-isomers of γ-carotene (0.1–3.9 mg/100 g FW), and one (Z)-lycopene isomer (0.04–0.83 mg/100 g FW) prevailed. Approximately 89–94 % of total carotenoid content pertained to provitamin A carotenoids with retinol activity equivalents ranging from 37 to 609 µg/100 g FW. The physical deposition state of these carotenoids in planta was investigated using light, transmission electron, and scanning electron microscopy. The plastids found in both orange and yellow-colored fruit mesocarps were amylo-chromoplasts of the globular type, containing carotenoids predominantly in a lipid-dissolved form. The hypothesis of lipid-dissolved carotenoids was supported by simple solubility estimations based on carotenoid and lipid contents of the fruit mesocarp. In our study, we report first results on the human bioavailability of γ-carotene, β-carotene, and lycopene from peach palm fruit, particularly proving the post-prandial absorption of the rarely occurring γ-carotene. Since the physical state of carotenoid deposition has been shown to be decisive for carotenoid bioavailability, lipid-dissolved carotenoids in peach palm fruits are expected to be highly bioavailable, however, further studies are required.     

Antioxidant Activity of Supramolecular Water-Soluble Fullerenes Evaluated by β-Carotene Bleaching Assay

We investigated the antioxidant activity of supramolecular water-soluble fullerenes, polyvinylpyrrolidone (PVP)-entrapped C₆₀, and γ-cyclodextrin (CD)-bicapped C₆₀, based on comparable β-carotene bleaching assay. Antioxidant activity against reactive oxygen species (ROS) generated by three different methods, (i) autoxidation of linoleic acid, (ii) hydrogen peroxide promoter, and (iii) photoirradiation, was evaluated as percent of inhibition relative to a control experiment in view of the bleaching rate constant (k _obs) as well as the persistent absorbancy of β-carotene. Water-soluble fullerenes exhibit significant inhibitory effects on the oxidative discoloration of β-carotene in any system.     

Effects of CPTA upon carotenogenesis and lipoidal constituents in Rhodotorula species

The effects of 2-(4-chlorophenyl)-triethylamine (CPTA) upon carotenogenesis in Rhodotorula glutinis, and upon various lipoidal constituents of R. rubra were studied. CPTA was found to cause the accumulation of lycopene and γ-carotene and to inhibit the formation of fatty acids and ergosterol. Upon removal of the inhibitor lycopene was metabolized to the cyclic carotenes and the levels of ergosterol increased. It was proposed that CPTA inhibits the cyclase enzyme of carotenogenesis, inhibits the formation of ergosterol, and causes the build-up of intermediates of these compounds.