Production of the Carotenoids Lycopene, β-Carotene, and Astaxanthin in the Food Yeast Candida utilis

The food-grade yeast Candida utilis has been engineered to confer a novel biosynthetic pathway for the production of carotenoids such as lycopene, β-carotene, and astaxanthin. The exogenous carotenoid biosynthesis genes were derived from the epiphytic bacterium Erwinia uredovora and the marine bacterium Agrobacterium aurantiacum. The carotenoid biosynthesis genes were individually modified based on the codon usage of the C. utilis glyceraldehyde 3-phosphate dehydrogenase gene and expressed in C. utilis under the control of the constitutive promoters and terminators derived from C. utilis. The resultant yeast strains accumulated lycopene, β-carotene, and astaxanthin in the cells at 1.1, 0.4, and 0.4 mg per g (dry weight) of cells, respectively. This was considered to be a result of the carbon flow into ergosterol biosynthesis being partially redirected to the nonendogenous pathway for carotenoid production.     

Mutants and Intersexual Heterokaryons of Blakeslea trispora for Production of β-Carotene and Lycopene

The industrial production of β-carotene with the zygomycete Blakeslea trispora involves the joint cultivation of mycelia of opposite sex in the presence of β-ionone and other chemical activators. We have obtained improved strains by mutation and heterokaryosis. We chose wild strains on the basis of their growth and carotene content in single and mated cultures. Following exposure of their spores to N-methyl-N′-nitro-N-nitrosoguanidine, we obtained high-carotene mutants, which were more productive than their parents but similar to them in having β-carotene as the main product. Further increases in carotene content were obtained after a new round of mutagenesis in one of the mutants. The production was shifted to lycopene in cultures incubated in the presence of nicotine and in lycopene-rich mutants derived from the wild strains. The highest production levels were achieved in intersexual heterokaryons, which contained mutant nuclei of opposite sex. These contained up to 39 mg of β-carotene or 15 mg of lycopene per g (dry mass) under standard laboratory conditions in which the original wild strains contained about 0.3 mg of β-carotene per g (dry mass). β-Ionone did not increase the carotene content of these strains. Not all wild strains lent themselves to these improvements, either because they produced few mutants or because they did not increase their carotene production in mated cultures.     

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.     

Enhancement of β-Carotene Synthesis by Citrus Products

β-Ionone, a stimulatory compound in the microbiological production of β-carotene by mated cultures of Blakeslea trispora, could be replaced with low-cost agricultural by-products (citrus oils, citrus pulp, or citrus molasses) with as good or better carotene yields. Peak yields (81 to 129 mg of carotene per g of dry solids) were achieved in 5 days. The various citrus products tested did not change the pigments produced; all trans-β-carotene remained the pre-dominant pigment. The acid-hydrolyzed soybean meal and corn used in previous production media could be replaced with unhydrolyzed cottonseed embryo meal and corn in a medium that also contained a natural lipid, deodorized kerosene, nonionic detergent, and a precursor.     

Metabolic Engineering of the Carotenoid Biosynthetic Pathway in the Yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma)

The crtYB locus was used as an integrative platform for the construction of specific carotenoid biosynthetic mutants in the astaxanthin-producing yeast Xanthophyllomyces dendrorhous. The crtYB gene of X. dendrorhous, encoding a chimeric carotenoid biosynthetic enzyme, could be inactivated by both single and double crossover events, resulting in non-carotenoid-producing transformants. In addition, the crtYB gene, linked to either its homologous or a glyceraldehyde-3-phosphate dehydrogenase promoter, was overexpressed in the wild type and a β-carotene-accumulating mutant of X. dendrorhous. In several transformants containing multiple copies of the crtYB gene, the total carotenoid content was higher than in the control strain. This increase was mainly due to an increase of the β-carotene and echinone content, whereas the total content of astaxanthin was unaffected or even lower. Overexpression of the phytoene synthase-encoding gene (crtI) had a large impact on the ratio between mono- and bicyclic carotenoids. Furthermore, we showed that in metabolic engineered X. dendrorhous strains, the competition between the enzymes phytoene desaturase and lycopene cyclase for lycopene governs the metabolic flux either via β-carotene to astaxanthin or via 3,4-didehydrolycopene to 3-hydroxy-3′-4′-didehydro-β-ψ-caroten-4-one (HDCO). The monocylic carotenoid torulene and HDCO, normally produced as minority carotenoids, were the main carotenoids produced in these strains.     

Ketocarotenoid Production in Soybean Seeds through Metabolic Engineering

The pink or red ketocarotenoids, canthaxanthin and astaxanthin, are used as feed additives in the poultry and aquaculture industries as a source of egg yolk and flesh pigmentation, as farmed animals do not have access to the carotenoid sources of their wild counterparts. Because soybean is already an important component in animal feed, production of these carotenoids in soybean could be a cost-effective means of delivery. In order to characterize the ability of soybean seed to produce carotenoids, soybean cv. Jack was transformed with the crtB gene from Pantoea ananatis, which codes for phytoene synthase, an enzyme which catalyzes the first committed step in the carotenoid pathway. The crtB gene was engineered together in combinations with ketolase genes (crtW from Brevundimonas sp. strain SD212 and bkt1 from Haematococcus pluvialis) to produce ketocarotenoids; all genes were placed under the control of seed-specific promoters. HPLC results showed that canthaxanthin is present in the transgenic seeds at levels up to 52 μg/g dry weight. Transgenic seeds also accumulated other compounds in the carotenoid pathway, such as astaxanthin, lutein, β-carotene, phytoene, α-carotene, lycopene, and β-cryptoxanthin, whereas lutein was the only one of these detected in non-transgenic seeds. The accumulation of astaxanthin, which requires a β-carotene hydroxylase in addition to a β-carotene ketolase, in the transgenic seeds suggests that an endogenous soybean enzyme is able to work in combination with the ketolase transgene. Soybean seeds that accumulate ketocarotenoids could potentially be used in animal feed to reduce or eliminate the need for the costly addition of these compounds.     

Production of the Carotenoids Lycopene, β-Carotene,and Astaxanthin in the Food Yeast Candida utilis

The food-grade yeast Candida utilis has been engineered to confer a novel biosynthetic pathway for the production of carotenoids such as lycopene, β-carotene, and astaxanthin. The exogenous carotenoid biosynthesis genes were derived from the epiphytic bacterium Erwinia uredovora and the marine bacterium Agrobacterium aurantiacum. The carotenoid biosynthesis genes were individually modified based on the codon usage of the C. utilis glyceraldehyde 3-phosphate dehydrogenase gene and expressed in C. utilis under the control of the constitutive promoters and terminators derived from C. utilis. The resultant yeast strains accumulated lycopene, β-carotene, and astaxanthin in the cells at 1.1, 0.4, and 0.4 mg per g (dry weight) of cells, respectively. This was considered to be a result of the carbon flow into ergosterol biosynthesis being partially redirected to the nonendogenous pathway for carotenoid production.Carotenoids are yellow, orange, and red pigments which are widely distributed in nature (3). Industrially, carotenoid pigments such as β-carotene are utilized as food or feed supplements. β-Carotene is also a precursor of vitamin A in mammals (11). Recently, carotenoids have attracted greater attention, due to their beneficial effect on human health: e.g., the functions of lycopene and astaxanthin include strong quenching of singlet oxygen (12), involvement in cancer prevention (2), and enhancement of immune responses (6). Astaxanthin has also been exploited for industrial use, principally as an agent for pigmenting cultured fish and shellfish.The genes responsible for the synthesis of carotenoids such as lycopene, β-carotene, and astaxanthin have been isolated from the epiphytic Erwinia species or the marine bacteria Agrobacterium aurantiacum and Alcaligenes sp. strain PC-1, and their functions have been elucidated (13, 14). The first substrate of the encoded enzymes for carotenoid synthesis is farnesyl pyrophosphate (diphosphate) (FPP), which is the common precursor for the biosynthesis of numerous isoprenoid compounds such as sterols, hopanols, dolicols, and quinones. The ubiquitous nature of FPP among yeasts has been utilized in the microbial production of lycopene and β-carotene by the yeast Saccharomyces cerevisiae carrying the Erwinia uredovora carotenogenic genes (19). However, the amount of carotenoids produced in these hosts was only 0.1 mg of lycopene and 0.1 mg of β-carotene per g (dry weight) of cells, respectively.The edible yeast Candida utilis is generally recognized as a safe substance by the Food and Drug Administration. Large-scale production of the yeast cells has been developed with cheap biomass-derived sugars as the carbon source for the production of single-cell protein and several chemicals such as glutathione and RNA (1, 4). This yeast was also found to accumulate a large amount of ergosterol in the cell during stationary phase (6 to 13 mg/g [dry weight] of cells) (17). Thus, C. utilis has the potential to produce a large amount of carotenoids by redirecting the carbon flux for the ergosterol biosynthesis into the nonendogenous pathway for carotenoid synthesis via FPP. Previously, a C. utilis strain was made to produce lycopene (0.8 mg/g [dry weight]) by expressing the three nonmodified genes crtE, crtB, and crtI derived from E. uredovora (15).In this paper, the de novo biosynthesis of lycopene, β-carotene, and astaxanthin has been performed in C. utilis by using six carotenogenic genes, which were synthesized according to the codon usage of the C. utilis glyceraldehyde-3-phosphate dehydrogenase (GAP) gene, which is expressed at high levels. By this approach, increased carotenoid production in C. utilis was achieved.     

Construction of transgenic Ipomoea obscura that exhibits new reddish leaf and flower colors due to introduction of β-carotene ketolase and hydroxylase genes

Ipomoea obscura, small white morning glory, is an ornamental plant belonging to the family Convolvulaceae, and cultivated worldwide. I. obscura generates white petals including a pale-yellow colored star-shaped center (flower vein). Its fully opened flowers were known to accumulate trace amounts of carotenoids such as β-carotene. In the present study, the embryogenic calli of I. obscura, were successfully produced through its immature embryo culture, and co-cultured with Agrobacterium tumefaciens carrying the β-carotene 4,4′-ketolase (crtW) and β-carotene 3,3′-hydroxylase (crtZ) genes for astaxanthin biosynthesis in addition to the isopentenyl diphosphate isomerase (idi) and hygromycin resistance genes. Transgenic plants, in which these four genes were introduced, were regenerated from the infected calli. They generated bronze (reddish green) leaves and novel petals that exhibited a color change from pale-yellow to pale-orange in the star-shaped center part. Especially, the color of their withered leaves changed drastically. HPLC-PDA-MS analysis showed that the expanded leaves of a transgenic line (T₀) produced astaxanthin (5.2% of total carotenoids), adonirubin (3.9%), canthaxanthin (3.8%), and 3-hydroxyechinenone (3.6%), which indicated that these ketocarotenoids corresponded to 16.5% of the total carotenoids produced there (530 µg g⁻¹ fresh weight). Furthermore, the altered traits of the transgenic plants were found to be inherited to their progenies by self-crossing.     

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.     

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.     

Role of carotenoid β-cryptoxanthin in bone homeostasis

Bone homeostasis is maintained through a balance between osteoblastic bone formation and osteoclastic bone resorption. Aging induces bone loss due to decreased osteoblastic bone formation and increased osteoclastic bone resorption. Osteoporosis with its accompanying decrease in bone mass is widely recognized as a major public health problem. Nutritional factors may play a role in the prevention of bone loss with aging. Among various carotenoids (carotene and xanthophylls including beta (β)-cryptoxanthin, lutein, lycopene, β-carotene, astaxanthin, and rutin), β-cryptoxanthin, which is abundant in Satsuma mandarin orange (Citrus unshiu MARC.), has been found to have a stimulatory effect on bone calcification in vitro. β-cryptoxanthin has stimulatory effects on osteoblastic bone formation and inhibitory effects on osteoclastic bone resorption in vitro, thereby increasing bone mass. β-cryptoxanthin has an effect on the gene expression of various proteins that are related osteoblastic bone formation and osteoclastic bone resororption in vitro. The intake of β-cryptoxanthin may have a preventive effect on bone loss in animal models for osteoporosis and in healthy human or postmenopausal women. Epidemiological studies suggest a potential role of β-cryptoxanthin as a sustainable nutritional approach to improving bone health of human subjects. β-Cryptoxanthin may be an osteogenic factor in preventing osteoporosis in human subjects.     

Microbial Conversion of α-Ionone, α-Methylionone, and α-Isomethylionone

α-Ionone, α-methylionone, and α-isomethylionone were converted by Aspergillus niger JTS 191. The individual bioconversion products from α-ionone were isolated and identified by spectrometry and organic synthesis. The major products were cis-3-hydroxy-α-ionone, trans-3-hydroxy-α-ionone, and 3-oxo-α-ionone. 2,3-Dehydro-α-ionone, 3,4-dehydro-β-ionone, and 1-(6,6-dimethyl-2-methylene-3-cyclohexenyl)-buten-3-one were also identified. Analogous bioconversion products from α-methylionone and α-isomethylionone were also identified. From results of gas-liquid chromatographic analysis during the fermentation, we propose a metabolic pathway for α-ionones and elucidation of stereochemical features of the bioconversion.     

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.     

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.     

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.     

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.     

Evaluation of Structurally Different Carotenoids in Escherichia coli Transformants as Protectants against UV-B Radiation

Escherichia coli cells transformed with several carotenogenic genes to mediate the formation of ζ-carotene, neurosporene, lycopene, β-carotene, and zeaxanthin were exposed to UV-B radiation. Short-term kinetics revealed that endogenous levels of neurosporene and β-carotene protected E. coli against irradiation with UV-B. Zeaxanthin protected against only the photosensitized UV-B treatment. All other carotenoids were ineffective.