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.     

Mode of Action of the Massively Accumulated beta-Carotene of Dunaliella bardawil in Protecting the Alga against Damage by Excess Irradiation

When grown under defined conditions Dunaliella bardawil accumulates a high concentration of β-carotene, which is composed primarily of two isomers, all-trans and 9-cis β-carotene. The high β-carotene alga is substantially resistant to photoinhibition of photosynthetic oxygen evolution when compared with low β-carotene D. bardawil or with Dunaliella salina which is incapable of accumulating β-carotene. Protection against photoinhibition in the high β-carotene D. bardawil is very strong when blue light is used as the photoinhibitory agent, intermediate with white light, and nonexistent with red light. These observations suggest that the massively accumulated β-carotene in D. bardawil protects the alga against damage by high irradiation by screening through absorption of the blue region of the spectrum. Irradiation of D. bardawil by high intensity blue light results in the following temporal sequence of events: photoinhibition of oxygen evolution, photodestruction of 9-cis β-carotene, photodestruction of all-trans β-carotene, photodestruction of chlorophyll and cell death.     

Protection of Photosynthesis against Ultraviolet-B Radiation by Carotenoids in Transformants of the Cyanobacterium Synechococcus PCC7942

The cyanobacterium Synechococcus PCC7942 was transformed with various carotenogenic genes, and the resulting transformants either accumulated higher amounts of β-carotene and zeaxanthin or showed a shift in the carotenoid pattern toward the formation of zeaxanthin. These transformants were exposed to ultraviolet-B (UV-B) radiation, and the degradation of phycobilins, the inactivation of photosynthetic oxygen evolution, and the activity of photosystem II were determined. In the genetically modified cells, the influence on destruction of phycobilins was negligible. However, protection of photosynthetic reactions against UV-B damage was observed and was dependent on the carotenoid concentrations in the different transformants. Furthermore, it was shown that endogenous zeaxanthin is more effective than β-carotene. Our results suggest that carotenoids exert their protective function as antioxidants to inactivate UV-B-induced radicals in the photosynthetic membrane.     

Novel Carotenoid Oxidase Involved in Biosynthesis of 4,4′-Diapolycopene Dialdehyde

Biosynthesis of C₃₀ carotenoids is relatively restricted in nature but has been described in Staphylococcus and in methylotrophic bacteria. We report here identification of a novel gene (crtNb) involved in conversion of 4,4′-diapolycopene to 4,4′-diapolycopene aldehyde. An aldehyde dehydrogenase gene (ald) responsible for the subsequent oxidation of 4,4′-diapolycopene aldehyde to 4,4′-diapolycopene acid was also identified in Methylomonas. CrtNb has significant sequence homology with diapophytoene desaturases (CrtN). However, data from knockout of crtNb and expression of crtNb in Escherichia coli indicated that CrtNb is not a desaturase but rather a novel carotenoid oxidase catalyzing oxidation of the terminal methyl group(s) of 4,4′-diaponeurosporene and 4,4′-diapolycopene to the corresponding terminal aldehyde. It has moderate to low activity on neurosporene and lycopene and no activity on β-carotene or ζ-carotene. Using a combination of C₃₀ carotenoid synthesis genes from Staphylococcus and Methylomonas, 4,4′-diapolycopene dialdehyde was produced in E. coli as the predominant carotenoid. This C₃₀ dialdehyde is a dark-reddish purple pigment that may have potential uses in foods and cosmetics.     

β-Carotene Attenuates Angiotensin II-Induced Aortic Aneurysm by Alleviating Macrophage Recruitment in Apoe?/? Mice

Abdominal aortic aneurysm (AAA) is a common chronic degenerative disease characterized by progressive aortic dilation and rupture. The mechanisms underlying the role of α-tocopherol and β-carotene on AAA have not been comprehensively assessed. We investigated if α-tocopherol and β-carotene supplementation could attenuate AAA, and studied the underlying mechanisms utilized by the antioxidants to alleviate AAA. Four-months-old Apoe^−/− mice were used in the induction of aneurysm by infusion of angiotensin II (Ang II), and were orally administered with α-tocopherol and β-carotene enriched diet for 60 days. Significant increase of LDL, cholesterol, triglycerides and circulating inflammatory cells was observed in the Ang II-treated animals, and gene expression studies showed that ICAM-1, VCAM-1, MCP-1, M-CSF, MMP-2, MMP-9 and MMP-12 were upregulated in the aorta of aneurysm-induced mice. Extensive plaques, aneurysm and diffusion of inflammatory cells into the tunica intima were also noticed. The size of aorta was significantly (P = 0.0002) increased (2.24±0.20 mm) in the aneurysm-induced animals as compared to control mice (1.17±0.06 mm). Interestingly, β-carotene dramatically controlled the diffusion of macrophages into the aortic tunica intima, and circulation. It also dissolved the formation of atheromatous plaque. Further, β-carotene significantly decreased the aortic diameter (1.33±0.12 mm) in the aneurysm-induced mice (β-carotene, P = 0.0002). It also downregulated ICAM-1, VCAM-1, MCP-1, M-CSF, MMP-2, MMP-9, MMP-12, PPAR-α and PPAR-γ following treatment. Hence, dietary supplementation of β-carotene may have a protective function against Ang II-induced AAA by ameliorating macrophage recruitment in Apoe^−/− mice.     

Substrate Specificity of Purified Recombinant Human β-Carotene 15,15′-Oxygenase (BCO1)

Humans cannot synthesize vitamin A and thus must obtain it from their diet. β-Carotene 15,15′-oxygenase (BCO1) catalyzes the oxidative cleavage of provitamin A carotenoids at the central 15–15′ double bond to yield retinal (vitamin A). In this work, we quantitatively describe the substrate specificity of purified recombinant human BCO1 in terms of catalytic efficiency values (k_cat/K_m). The full-length open reading frame of human BCO1 was cloned into the pET-28b expression vector with a C-terminal polyhistidine tag, and the protein was expressed in the Escherichia coli strain BL21-Gold(DE3). The enzyme was purified using cobalt ion affinity chromatography. The purified enzyme preparation catalyzed the oxidative cleavage of β-carotene with a V_max = 197.2 nmol retinal/mg BCO1 × h, K_m = 17.2 μm and catalytic efficiency k_cat/K_m = 6098 m⁻¹ min⁻¹. The enzyme also catalyzed the oxidative cleavage of α-carotene, β-cryptoxanthin, and β-apo-8′-carotenal to yield retinal. The catalytic efficiency values of these substrates are lower than that of β-carotene. Surprisingly, BCO1 catalyzed the oxidative cleavage of lycopene to yield acycloretinal with a catalytic efficiency similar to that of β-carotene. The shorter β-apocarotenals (β-apo-10′-carotenal, β-apo-12′-carotenal, β-apo-14′-carotenal) do not show Michaelis-Menten behavior under the conditions tested. We did not detect any activity with lutein, zeaxanthin, and 9-cis-β-carotene. Our results show that BCO1 favors full-length provitamin A carotenoids as substrates, with the notable exception of lycopene. Lycopene has previously been reported to be unreactive with BCO1, and our findings warrant a fresh look at acycloretinal and its alcohol and acid forms as metabolites of lycopene in future studies.     

Conversion of carotenoids into vitamins A(1) and A(2) in two species of freshwater fish

1. Examination of two zooplankton species predominating in fish ponds, Daphnia magna and Chironomus larvae, revealed the presence of α- and β-carotene, echinenone, canthaxanthin and 3-hydroxy-4-oxo-β-carotene in Daphnia, and β-carotene and cryptoxanthin ester in Chironomus. No specific provitamins A₂ (containing a 3,4-dehydro-β-ionone ring) were detected. 2. Guppies (Lebistes reticulatus) and platies (Xiphophorus variatus) were found to form vitamin A from β-carotene and from its oxygen-containing derivatives isozeaxanthin, canthaxanthin and astaxanthin. Slight conversion into vitamin A₂ seemed to occur simultaneously. 3,4-Dehydro-3′-hydroxy-β-carotene formed little vitamin A, and the latter was mainly of the A₂ type. Lutein was devoid of provitamin A properties. 3. In addition to vitamin A, β-carotene was detected in fish receiving the 4-oxo- and 4-hydroxy-carotenoids. A reaction scheme for the conversion of carotenoids into retinal and and 3,4-dehydroretinal is presented. 4. It is concluded that natural 4-oxo derivatives of β-carotene may play a significant role as vitamin A precursors for fish.     

Flexible responses to visual and olfactory stimuli by foraging Manduca sexta: larval nutrition affects adult behaviour

Here, we show that the consequences of deficient micronutrient (β-carotene) intake during larval stages of Manduca sexta are carried across metamorphosis, affecting adult behaviour. Our manipulation of larval diet allowed us to examine how developmental plasticity impacts the interplay between visual and olfactory inputs on adult foraging behaviour. Larvae of M. sexta were reared on natural (Nicotiana tabacum) and artificial laboratory diets containing different concentrations of β-carotene (standard diet, low β-carotene, high β-carotene and cornmeal). This vitamin-A precursor has been shown to be crucial for photoreception sensitivity in the retina of M. sexta. After completing development, post-metamorphosis, starved adults were presented with artificial feeders that could be either scented or unscented. Regardless of their larval diet, adult moths fed with relatively high probabilities on scented feeders. When feeders were unscented, moths reared on tobacco were more responsive than moths reared on β-carotene-deficient artificial diets. Strikingly, moths reared on artificial diets supplemented with increasing amounts of β-carotene (low β and high β) showed increasing probabilities of response to scentless feeders. We discuss these results in relationship to the use of complex, multi-modal sensory information by foraging animals.     

On the Factors Which Determine Massive beta-Carotene Accumulation in the Halotolerant Alga Dunaliella bardawil

Dunaliella bardawil, a β-carotene-accumulating halotolerant alga, has been analyzed for the effect of various growth conditions on its pigment content, and compared with Dunaliella salina, a β-carotene nonaccumulating species. In D. bardawil, increasing light intensity and light period or inhibiting growth by various stress conditions such as nutrient deficiency or high salt concentration caused a decrease in the content of chlorophyll per cell and an increase in the amount of β-carotene per cell. As a result, the β-carotene-to-chlorophyll ratio increased from about 0.4 to 13 grams per gram and the alga changed its visual appearance from green to deep orange. D. salina grown similarly decreased in content of both chlorophyll and β-carotene per cell and the culture turned from green to yellowish. Low chlorophyll-containing cells of D. bardawil or D. salina exhibit very high photosynthetic rates when expressed on a chlorophyll basis (~600 micromoles O₂ evolved per milligram chlorophyll per hour).

Variation of pigment content in D. bardawil by a large variety of environmental agents has been correlated with the integral irradiance received by the algal culture during a division cycle. The higher the integral irradiance per division cycle, the lower the chlorophyll content per cell; the higher the β-carotene content per cell, and therefore the higher the β-carotene-to-chlorophyll ratio. The results are interpreted as indicating a protecting effect of β-carotene against injury by high irradiance under conditions of impairment in chlorophyll content per cell.

     

Development of β-Carotene Rich Maize Hybrids through Marker-Assisted Introgression of β-carotene hydroxylase Allele

Development of vitamin A-rich cereals can help in alleviating the widespread problem of vitamin A deficiency. We report here significant enhancement of kernel β-carotene in elite maize genotypes through accelerated marker-assisted backcross breeding. A favourable allele (543 bp) of the β-carotene hydroxylase (crtRB1) gene was introgressed in the seven elite inbred parents, which were low (1.4 µg/g) in kernel β-carotene, by using a crtRB1-specific DNA marker for foreground selection. About 90% of the recurrent parent genome was recovered in the selected progenies within two backcross generations. Concentration of β-carotene among the crtRB1-introgressed inbreds varied from 8.6 to 17.5 µg/g - a maximum increase up to 12.6-fold over recurrent parent. The reconstituted hybrids developed from improved parental inbreds also showed enhanced kernel β-carotene as high as 21.7 µg/g, compared to 2.6 µg/g in the original hybrid. The reconstituted hybrids evaluated at two locations possessed similar grain yield to that of original hybrids. These β-carotene enriched high yielding hybrids can be effectively utilized in the maize biofortification programs across the globe.     

Metagenomic Identification of a Novel Salt Tolerance Gene from the Human Gut Microbiome Which Encodes a Membrane Protein with Homology to a brp/blh-Family β-Carotene 15,15′-Monooxygenase

The human gut microbiome consists of at least 3 million non-redundant genes, 150 times that of the core human genome. Herein, we report the identification and characterisation of a novel stress tolerance gene from the human gut metagenome. The locus, assigned brpA, encodes a membrane protein with homology to a brp/blh-family β-carotene monooxygenase. Cloning and heterologous expression of brpA in Escherichia coli confers a significant salt tolerance phenotype. Furthermore, when cultured in the presence of exogenous β-carotene, cell pellets adopt a red/orange pigmentation indicating the incorporation of carotenoids in the cell membrane.     

Interference in Carotenogenesis as a Mechanism of Action of the Pyridazinone Herbicide Sandoz 6706: Accumulation of C-40 Carotenoid Precursors Inhibition of beta-Carotene Synthesis and Enhancement of Phytoene Epoxidation

The herbicide Sandoz 6706 (4-chloro-5-(dimethylamino)-2-α,α,α, (trifluoro-m-tolyl)-3(2H)-pyridazinone), when applied as a preplant soil treatment at a concentration of 0.05 μg/g reduced the content of β-carotene and chlorophylls in 21-day-old wheat seedlings (Triticum aestivum L.) by 55% and 29%, respectively, without affecting the fresh or dry matter of the seedlings. At 0.8 μg/g, the herbicide reduced the content of β-carotene and chlorophyll by as much as 98%, while the fresh weight of the albino seedlings was reduced by only 24%. The effect of the herbicide on chlorophyll b was much stronger than on chlorophyll a. Time course studies of pigment synthesis in Sandoz 6706-treated seedlings showed that chlorophyll, β-carotene, cyclic xanthophylls, phytoene, phytofluene, and ζ-carotene were accumulating during the first 7 days after sowing. Later on, there was a sharp decline in the content of chlorophyll and β-carotene and a gradual reduction in the content of phytofluene, ζ-carotene, and cyclic xanthophylls; the content of phytoene remained essentially unchanged. Coinciding with the drop in content of β-carotene and chlorophyll, there was a remarkable increase in the content of epoxy phytoene. It is suggested that Sandoz 6706 might act as an inhibitor of the cyclization reaction in the biosynthetic pathway of carotenoids and that other effects, such as the bleaching of chlorophyll, are a consequence of this inhibition.     

Isorenieratene biosynthesis in green sulfur bacteria requires the cooperative actions of two carotenoid cyclases

The cyclization of lycopene to γ- or β-carotene is a major branch point in the biosynthesis of carotenoids in photosynthetic bacteria. Four families of carotenoid cyclases are known, and each family includes both mono- and dicyclases, which catalyze the formation of γ- and β-carotene, respectively. Green sulfur bacteria (GSB) synthesize aromatic carotenoids, of which the most commonly occurring types are the monocyclic chlorobactene and the dicyclic isorenieratene. Recently, the cruA gene, encoding a conserved hypothetical protein found in the genomes of all GSB and some cyanobacteria, was identified as a lycopene cyclase. Further genomic analyses have found that all available fully sequenced genomes of GSB encode an ortholog of cruA. Additionally, the genomes of all isorenieratene-producing species of GSB encode a cruA paralog, now named cruB. The cruA gene from the chlorobactene-producing GSB species Chlorobaculum tepidum and both cruA and cruB from the brown-colored, isorenieratene-producing GSB species Chlorobium phaeobacteroides strain DSM 266^T were heterologously expressed in lycopene- and neurosporene-producing strains of Escherichia coli, and the cruB gene of Chlorobium clathratiforme strain DSM 5477^T was also heterologously expressed in C. tepidum by inserting the gene at the bchU locus. The results show that CruA is probably a lycopene monocyclase in all GSB and that CruB is a γ-carotene cyclase in isorenieratene-producing species. Consequently, the branch point for the synthesis of mono- and dicyclic carotenoids in GSB seems to be the modification of γ-carotene, rather than the cyclization of lycopene as occurs in cyanobacteria.     

Carotene Hydroxylase Activity Determines the Levels of Both α-Carotene and Total Carotenoids in Orange Carrots

The typically intense carotenoid accumulation in cultivated orange-rooted carrots (Daucus carota) is determined by a high protein abundance of the rate-limiting enzyme for carotenoid biosynthesis, phytoene synthase (PSY), as compared with white-rooted cultivars. However, in contrast to other carotenoid accumulating systems, orange carrots are characterized by unusually high levels of α-carotene in addition to β-carotene. We found similarly increased α-carotene levels in leaves of orange carrots compared with white-rooted cultivars. This has also been observed in the Arabidopsis thaliana lut5 mutant carrying a defective carotene hydroxylase CYP97A3 gene. In fact, overexpression of CYP97A3 in orange carrots restored leaf carotenoid patterns almost to those found in white-rooted cultivars and strongly reduced α-carotene levels in the roots. Unexpectedly, this was accompanied by a 30 to 50% reduction in total root carotenoids and correlated with reduced PSY protein levels while PSY expression was unchanged. This suggests a negative feedback emerging from carotenoid metabolites determining PSY protein levels and, thus, total carotenoid flux. Furthermore, we identified a deficient CYP97A3 allele containing a frame-shift insertion in orange carrots. Association mapping analysis using a large carrot population revealed a significant association of this polymorphism with both α-carotene content and the α-/β-carotene ratio and explained a large proportion of the observed variation in carrots.     

The Inhibition of Macrophage Foam Cell Formation by 9-Cis β-Carotene Is Driven by BCMO1 Activity

Atherosclerosis is a major cause of morbidity and mortality in developed societies, and begins when activated endothelial cells recruit monocytes and T-cells from the bloodstream into the arterial wall. Macrophages that accumulate cholesterol and other fatty materials are transformed into foam cells. Several epidemiological studies have demonstrated that a diet rich in carotenoids is associated with a reduced risk of heart disease; while previous work in our laboratory has shown that the 9-cis β-carotene rich alga Dunaliella inhibits atherogenesis in mice. The effect of 9-cis β-carotene on macrophage foam cell formation has not yet been investigated. In the present work, we sought to study whether the 9-cis β-carotene isomer, isolated from the alga Dunaliella, can inhibit macrophage foam cell formation upon its conversion to retinoids. The 9-cis β-carotene and Dunaliella lipid extract inhibited foam cell formation in the RAW264.7 cell line, similar to 9-cis retinoic acid. Furthermore, dietary enrichment with the algal powder in mice resulted in carotenoid accumulation in the peritoneal macrophages and in the inhibition of foam cell formation ex-vivo and in-vivo. We also found that the β-carotene cleavage enzyme β-carotene 15,15’-monooxygenase (BCMO1) is expressed and active in macrophages. Finally, 9-cis β-carotene, as well as the Dunaliella extract, activated the nuclear receptor RXR in hepa1-6 cells. These results indicate that dietary carotenoids, such as 9-cis β-carotene, accumulate in macrophages and can be locally cleaved by endogenous BCMO1 to form 9-cis retinoic acid and other retinoids. Subsequently, these retinoids activate the nuclear receptor RXR that, along with additional nuclear receptors, can affect various metabolic pathways, including those involved in foam cell formation and atherosclerosis.     

Selection of Astaxanthin-Overproducing Mutants of Phaffia rhodozyma with β-Ionone

β-Ionone, an end ring analog of β-carotene, inhibits astaxanthin production in the red yeast Phaffia rhodozyma. Astaxanthin-overproducing mutants of this yeast are easily spotted on β-ionone-containing yeast malt agar plates. β-Ionone appears to block astaxanthin synthesis at the β-carotene level.     

CAROTENOID CLEAVAGE DIOXYGENASE4 Is a Negative Regulator of β-Carotene Content in Arabidopsis Seeds

Experimental approaches targeting carotenoid biosynthetic enzymes have successfully increased the seed β-carotene content of crops. However, linkage analysis of seed carotenoids in Arabidopsis thaliana recombinant inbred populations showed that only 21% of quantitative trait loci, including those for β-carotene, encode carotenoid biosynthetic enzymes in their intervals. Thus, numerous loci remain uncharacterized and underutilized in biofortification approaches. Linkage mapping and genome-wide association studies of Arabidopsis seed carotenoids identified CAROTENOID CLEAVAGE DIOXYGENASE4 (CCD4) as a major negative regulator of seed carotenoid content, especially β-carotene. Loss of CCD4 function did not affect carotenoid homeostasis during seed development but greatly reduced carotenoid degradation during seed desiccation, increasing β-carotene content 8.4-fold relative to the wild type. Allelic complementation of a ccd4 null mutant demonstrated that single-nucleotide polymorphisms and insertions and deletions at the locus affect dry seed carotenoid content, due at least partly to differences in CCD4 expression. CCD4 also plays a major role in carotenoid turnover during dark-induced leaf senescence, with β-carotene accumulation again most strongly affected in the ccd4 mutant. These results demonstrate that CCD4 plays a major role in β-carotene degradation in drying seeds and senescing leaves and suggest that CCD4 orthologs would be promising targets for stabilizing and increasing the level of provitamin A carotenoids in seeds of major food crops.