<Emphasis Type="Italic">Nicotiana glauca</Emphasis> engineered for the production of ketocarotenoids in flowers and leaves by expressing the cyanobacterial <Emphasis Type="Italic">crtO</Emphasis> ketolase gene

Nicotiana glauca is a tobacco species that forms flowers with carotenoid-pigmented petals, sepals, pistil, ovary and nectary tissue. The carotenoids produced are lutein, ss-carotene as well as some violaxanthin and antheraxanthin. This tobacco species was genetically modified for ketocarotenoid biosynthesis by transformation with a cyanobacterial crtO ketolase gene under the 35S CaMV promoter. In the transformants, ketocarotenoids were detected in both leaves and flowers. Although astaxanthin was not detected other ketocarotenoids such as 4'-ketolutein, echinenone, 3'-hydroxyechinenone and 4-ketozeaxanthin were present. Accumulation of ketocarotenoids in leaves decreased their photosynthetic efficiency moderately. Under the green house conditions used no impairment of growth and development compared to the wild type was observed. In the crtO-transformants, an unexpected up-regulation of total carotenoid biosynthesis in leaves and especially in flower petals was observed. This led to a total ketocarotenoid concentration in leaves of 136.6 (young) or 156.1 (older) mug/g dry weight and in petals of 165 mug/g dry weight. In our engineered plants, the ketocarotenoid pathway is one step short of astaxanthin. Strategies are discussed to improve N. glauca flowers as a biological system for astaxanthin.     

Metabolic engineering of ketocarotenoid formation in higher plants

Although higher plants synthesize carotenoids, they do not possess the ability to form ketocarotenoids. In order to generate higher plants capable of synthesizing combinations of ketolated and hydroxylated carotenoids the genes responsible for the carotene 4,4' oxygenase and 3,3' hydroxylase have been transformed into tomato and tobacco. The gene products were produced as a polyprotein. Subsequent cleavage of the polyprotein, targeting of the two enzymes to the plastid and enzyme activities have been shown for both gene products. Metabolite profiling has shown the formation of ketolated carotenoids from beta-carotene and its hydroxylated intermediates in tobacco and tomato leaf. In the nectary tissues of tobacco flowers a quantitative increase (10-fold) as well as compositional changes were evident, including the presence of astaxanthin, canthaxanthin and 4-ketozeaxanthin. Interestingly, in this tissue the newly formed carotenoids resided predominantly as esters. These data are discussed in terms of metabolic engineering of carotenoids and their sequestration in higher plant tissues.     

Elucidation of the pathway to astaxanthin in the flowers of Adonis aestivalis

A few species in the genus Adonis are the only land plants known to produce the valuable red ketocarotenoid astaxanthin in abundance. Here, we ascertain the pathway that leads from the β-rings of β-carotene, a carotenoid ubiquitous in plants, to the 3-hydroxy-4-keto-β-rings of astaxanthin (3,3'-dihydroxy-β,β-carotene-4,4'-dione) in the blood-red flowers of Adonis aestivalis, an ornamental and medicinal plant commonly known as summer pheasant's eye. Two gene products were found to catalyze three distinct reactions, with the first and third reactions of the pathway catalyzed by the same enzyme. The pathway commences with the activation of the number 4 carbon of a β-ring in a reaction catalyzed by a carotenoid β-ring 4-dehydrogenase (CBFD), continues with the further dehydrogenation of this carbon to yield a carbonyl in a reaction catalyzed by a carotenoid 4-hydroxy-β-ring 4-dehydrogenase, and concludes with the addition of an hydroxyl group at the number 3 carbon in a reaction catalyzed by the erstwhile CBFD enzyme. The A. aestivalis pathway is both portable and robust, functioning efficiently in a simple bacterial host. Our elucidation of the pathway to astaxanthin in A. aestivalis provides enabling technology for development of a biological production process and reveals the evolutionary origin of this unusual plant pathway, one unrelated to and distinctly different from those used by bacteria, green algae, and fungi to synthesize astaxanthin.     

Riboswitch-mediated inducible expression of an astaxanthin biosynthetic operon in plastids

The high-value carotenoid astaxanthin (3,3′-dihydroxy-β,β-carotene-4,4′-dione) is one of the most potent antioxidants in nature. In addition to its large-scale use in fish farming, the pigment has applications as a food supplement and an active ingredient in cosmetics and in pharmaceuticals for the treatment of diseases linked to reactive oxygen species. The biochemical pathway for astaxanthin synthesis has been introduced into seed plants, which do not naturally synthesize this pigment, by nuclear and plastid engineering. The highest accumulation rates have been achieved in transplastomic plants, but massive production of astaxanthin has resulted in severe growth retardation. What limits astaxanthin accumulation levels and what causes the mutant phenotype is unknown. Here, we addressed these questions by making astaxanthin synthesis in tobacco (Nicotiana tabacum) plastids inducible by a synthetic riboswitch. We show that, already in the uninduced state, astaxanthin accumulates to similarly high levels as in transplastomic plants expressing the pathway constitutively. Importantly, the inducible plants displayed wild-type–like growth properties and riboswitch induction resulted in a further increase in astaxanthin accumulation. Our data suggest that the mutant phenotype associated with constitutive astaxanthin synthesis is due to massive metabolite turnover, and indicate that astaxanthin accumulation is limited by the sequestration capacity of the plastid.

Inducible expression of a synthetic astaxanthin operon in plastids alleviates the growth phenotype of constitutive pathway expression and provides insights into carotenoid biosynthesis bottlenecks.     

Biosynthesis of astaxanthin in tobacco leaves by transplastomic engineering

The natural pigment astaxanthin has attracted much attention because of its beneficial effects on human health, despite its expensive market price. In order to produce astaxanthin, transgenic plants have so far been generated through conventional genetic engineering of Agrobacterium -mediated gene transfer. The results of trials have revealed that the method is far from practicable because of low yields, i.e. instead of astaxanthin, large quantities of the astaxanthin intermediates, including ketocarotenoids, accumulated in the transgenic plants. In the present study, we have overcome this problem, and have succeeded in producing more than 0.5% (dry weight) astaxanthin (more than 70% of total caroteniods) in tobacco leaves, which turns their green color to reddish brown, by expressing both genes encoding CrtW (β-carotene ketolase) and CrtZ (β-carotene hydroxylase) from a marine bacterium Brevundimonas sp., strain SD212, in the chloroplasts. Moreover, the total carotenoid content in the transplastomic tobacco plants was 2.1-fold higher than that of wild-type tobacco. The tobacco transformants also synthesized a novel carotenoid 4-ketoantheraxanthin. There was no significant difference in the size of the aerial part of the plant between the transformants and wild-type plants at the final stage of their growth. The photosynthesis rate of the transformants was also found to be similar to that of wild-type plants under ambient CO₂ concentrations of 1500 μmol photons m⁻² s⁻¹ light intensity.     

Metabolic engineering of ketocarotenoid biosynthesis in leaves and flowers of tobacco species

Ketocarotenoids and especially astaxanthin are high-valued pigments used as feed additives. Conventionally, they are provided by chemical synthesis. Their biological production is a promising alternative. For the development of a plant production system, Nicotiana glauca, a species with carotenoid-containing yellow pigmented flower petals, was transformed with a cyanobacterial ketolase gene. The resulting plants accumulated 4-ketozeaxantin (adinoxanthin), which is the first ketocarotenoid synthesized in flower petals by genetic modification. Due to the very late flowering in this tobacco species, N. tabacum was used to optimize the yield and ketocarotenoid product pattern by metabolic engineering of the ketolation steps of carotenogenesis. The highly carotenogenic nectary tissue in the flowers represents a model of a flower chromoplast system. By expression of a ketolase gene, it was possible to engineer the biosynthetic pathway towards the formation of 3'-hydroxyechinenone, 3-hydroxyechinenone, 4-ketozeaxanthin, 4-ketozeaxanthin esters, 4-ketolutein and 4-ketolutein esters. Some of these ketocarotenoids were also formed in the leaves of the trangenic plants. In particular, by co-expression of the ketolase gene in combination with a hydroxylase gene under an ubiquitous promoter, the formation of total carotenoids in nectaries increased by more than 2.5-fold. In the nectaries of this type of transformants, more than 50% of the accumulating carotenoids were keto derivatives. In addition, the levels of ketocarotenoid esters were much lower and a higher percentage of the free ketocarotenoids accumulated. These results open new promising perspectives for a successful metabolic engineering of keto-hydroxy carotenoid production in carotenogenic flowers.     

Characterization of cyanobacterial carotenoid ketolase CrtW and hydroxylase CrtR by complementation analysis in Escherichia coli

The pathway from beta-carotene to astaxanthin is a crucial step in the synthesis of astaxanthin, a red antioxidative ketocarotenoid that confers beneficial effects on human health. Two enzymes, a beta-carotene ketolase (carotenoid 4,4'-oxygenase) and a beta-carotene hydroxylase (carotenoid 3,3'-hydroxylase), are involved in this pathway. Cyanobacteria are known to utilize the carotenoid ketolase CrtW and/or CrtO, and the carotenoid hydroxylase CrtR. Here, we compared the catalytic functions of CrtW ketolases, which originated from Gloeobacter violaceus PCC 7421, Anabaena (also known as Nostoc) sp. PCC 7120 and Nostoc punctiforme PCC 73102, and CrtR from Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120 and Anabaena variabilis ATCC 29413 by complementation analysis using recombinant Escherichia coli cells that synthesized various carotenoid substrates. The results demonstrated that the CrtW proteins derived from Anabaena sp. PCC 7120 as well as N. punctiforme PCC 73102 (CrtW148) can convert not only beta-carotene but also zeaxanthin into their 4,4'-ketolated products, canthaxanthin and astaxanthin, respectively. In contrast, the Anabaena CrtR enzymes were very poor in accepting either beta-carotene or canthaxanthin as substrates. By comparison, the Synechocystis sp. PCC 6803 CrtR converted beta-carotene into zeaxanthin efficiently. We could assign the catalytic functions of the gene products involved in ketocarotenoid biosynthetic pathways in Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120 and N. punctiforme PCC 73102, based on the present and previous findings. This explains why these cyanobacteria cannot produce astaxanthin and why only Synechocystis sp. PCC 6803 can produce zeaxanthin.     

Functional characterization of various algal carotenoid ketolases reveals that ketolating zeaxanthin efficiently is essential for high production of astaxanthin in transgenic Arabidopsis

Extending the carotenoid pathway to astaxanthin in plants is of scientific and industrial interest. However, expression of a microbial β-carotene ketolase (BKT) that catalyses the formation of ketocarotenoids in transgenic plants typically results in low levels of astaxanthin. The low efficiency of BKTs in ketolating zeaxanthin to astaxanthin is proposed to be the major limitation for astaxanthin accumulation in engineered plants. To verify this hypothesis, several algal BKTs were functionally characterized using an Escherichia coli system and three BKTs were identified, with high (up to 85%), moderate (～38%), and low (～1%) conversion rate from zeaxanthin to astaxanthin from Chlamydomonas reinhardtii (CrBKT), Chlorella zofingiensis (CzBKT), and Haematococcus pluvialis (HpBKT3), respectively. Transgenic Arabidopsis thaliana expressing the CrBKT developed orange leaves which accumulated astaxanthin up to 2 mg g(-1) dry weight with a 1.8-fold increase in total carotenoids. In contrast, the expression of CzBKT resulted in much lower astaxanthin content (0.24 mg g(-1) dry weight), whereas HpBKT3 was unable to mediate synthesis of astaxanthin in A. thaliana. The none-native astaxanthin was found mostly in a free form integrated into the light-harvesting complexes of photosystem II in young leaves but in esterified forms in senescent leaves. The alteration of carotenoids did not affect chlorophyll content, plant growth, or development significantly. The astaxanthin-producing plants were more tolerant to high light as shown by reduced lipid peroxidation. This study advances a decisive step towards the utilization of plants for the production of high-value astaxanthin.     

Flower color alteration in <Emphasis Type="Italic">Lotus japonicus</Emphasis> by modification of the carotenoid biosynthetic pathway

To establish a model system for alteration of flower color by carotenoid pigments, we modified the carotenoid biosynthesis pathway of Lotus japonicus using overexpression of the crtW gene isolated from marine bacteria Agrobacterium aurantiacum and encoding β-carotene ketolase (4,4′-β-oxygenase) for the production of pink to red color ketocarotenoids. The crtW gene with the transit peptide sequence of the pea Rubisco small subunit under the regulation of the CaMV35S promoter was introduced to L. japonicus. In most of the resulting transgenic plants, the color of flower petals changed from original light yellow to deep yellow or orange while otherwise exhibiting normal phenotype. HPLC and TLC analyses revealed that leaves and flower petals of these plants accumulated novel carotenoids, believed to be ketocarotenoids consisting of including astaxanthin, adonixanthin, canthaxanthin and echinenone. Results indicated that modification of the carotenoid biosynthesis pathway is a means of altering flower color in ornamental crops.     

Isolation and characterization ofPhaffia rhodozyma mutants

Mutagenic treatment with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) inPhaffia rhodozyma generated 15 mutants with a wide diversity of color variants ranging from white to dark red. Characterization of the mutants by absorption spectra, TLC and HPLC was performed. Two categories could be distinguished: astaxanthin hyperproducing and astaxanthin hypoproducing mutants. Hyperproducing mutants exhibited considerable increases in astaxanthin content whereas hypoproducing mutants showed higher β-carotene contents than the wild-type strain. The characterization of carotenoid mutants inP. rhodozyma could contribute to the knowledge of the biosynthetic pathway of astaxanthin production of this microorganism.     

Engineering of geranylgeranyl pyrophosphate synthase levels and physiological conditions for enhanced carotenoid and astaxanthin synthesis in <Emphasis Type="Italic">Xanthophyllomyces dendrorhous</Emphasis>

The basidiomycetous yeast, Xanthophyllomyces dendrorhous, is one of the very few organisms which can be used for biological production of the carotenoid astaxanthin. crtE cDNA has been cloned from this fungus for engineering of the terpenoid pathway. The function of its gene product as a geranylgeranyl pyrophosphate synthase was established. X. dendrorhous was transformed with the crtE cDNA to divert metabolite flow from the sterol pathway towards carotenoid biosynthesis. Transformants were obtained with increased levels of geranylgeranyl pyrophosphate synthase leading to higher carotenoid levels including astaxanthin. Physiological conditions for maximum carotenoid synthesis for wild type and the CrtE transformant were dim light and extra air supply of the shaking culture. These conditions and the transformation with crtE had additive effects and resulted in an 8-fold higher astaxanthin formation as compared to the initial wild type culture without illumination and extra air supply yielding 451 μg/g dry wt within 4 days of growth.     

Metabolic engineering of astaxanthin biosynthesis in maize endosperm and characterization of a prototype high oil hybrid

Maize was genetically engineered for the biosynthesis of the high value carotenoid astaxanthin in the kernel endosperm. Introduction of a β-carotene hydroxylase and a β-carotene ketolase into a white maize genetic background extended the carotenoid pathway to astaxanthin. Simultaneously, phytoene synthase, the controlling enzyme of carotenogenesis, was over-expressed for enhanced carotenoid production and lycopene ε-cyclase was knocked-down to direct more precursors into the β-branch of the extended ketocarotenoid pathway which ends with astaxanthin. This astaxanthin-accumulating transgenic line was crossed into a high oil- maize genotype in order to increase the storage capacity for lipophilic astaxanthin. The high oil astaxanthin hybrid was compared to its astaxanthin producing parent. We report an in depth metabolomic and proteomic analysis which revealed major up- or down- regulation of genes involved in primary metabolism. Specifically, amino acid biosynthesis and the citric acid cycle which compete with the synthesis or utilization of pyruvate and glyceraldehyde 3-phosphate, the precursors for carotenogenesis, were down-regulated. Nevertheless, principal component analysis demonstrated that this compositional change is within the range of the two wild type parents used to generate the high oil producing astaxanthin hybrid.     

Metabolic engineering of the astaxanthin-biosynthetic pathway of Xanthophyllomyces dendrorhous

This review describes the different approaches that have been used to manipulate and improve carotenoid production in Xanthophyllomyces dendrorhous. The red yeast X. dendrorhous (formerly known as Phaffia rhodozyma) is one of the microbiological production systems for natural astaxanthin. Astaxanthin is applied in food and feed industry and can be used as a nutraceutical because of its strong antioxidant properties. However, the production levels of astaxanthin in wild-type isolates are rather low. To increase the astaxanthin content in X. dendrorhous, cultivation protocols have been optimized and astaxanthin-hyperproducing mutants have been obtained by screening of classically mutagenized X. dendrorhous strains. The knowledge about the regulation of carotenogenesis in X. dendrorhous is still limited in comparison to that in other carotenogenic fungi. The X. dendrorhous carotenogenic genes have been cloned and a X. dendrorhous transformation system has been developed. These tools allowed the directed genetic modification of the astaxanthin pathway in X. dendrorhous. The crtYB gene, encoding the bifunctional enzyme phytoene synthase/lycopene cyclase, was inactivated by insertion of a vector by single and double cross-over events, indicating that it is possible to generate specific carotenoid-biosynthetic mutants. Additionally, overexpression of crtYB resulted in the accumulation of beta-carotene and echinone, which indicates that the oxygenation reactions are rate-limiting in these recombinant strains. Furthermore, overexpression of the phytoene desaturase-encoding gene (crtI) showed an increase in monocyclic carotenoids such as torulene and HDCO (3-hydroxy-3',4'-didehydro-beta,-psi-carotene-4-one) and a decrease in bicyclic carotenoids such as echinone, beta-carotene and astaxanthin.     

A study in scarlet: enzymes of ketocarotenoid biosynthesis in the flowers of Adonis aestivalis

The red ketocarotenoid astaxanthin (3,3'-dihydroxy-4,4'-diketo-beta,beta-carotene) is widely used as an additive in feed for the pigmentation of fish and crustaceans and is frequently included in human nutritional supplements as well. There is considerable interest in developing a plant-based biological production process for this valuable carotenoid. Adonis aestivalis (Ranunculaceae) is unusual among plants in synthesizing and accumulating large amounts of astaxanthin and other ketocarotenoids. The formation of astaxanthin requires only the addition of a carbonyl at the number 4 carbon of each beta-ring of zeaxanthin (3,3'-dihydroxy-beta,beta-carotene), a carotenoid typically present in the green tissues of higher plants. We screened an A. aestivalis flower library to identify cDNAs that might encode the enzyme that catalyzes the addition of the carbonyls. Two closely related cDNAs selected in this screen were found to specify polypeptides similar in sequence to plant beta-carotene 3-hydroxylases, enzymes that convert beta-carotene (beta,beta-carotene) into zeaxanthin. The Adonis enzymes, however, exhibited neither 4-ketolase nor 3-hydroxylase activity when presented with beta-carotene as the substrate in Escherichia coli. Instead, the products of the Adonis cDNAs were found to modify beta-rings in two distinctly different ways: desaturation at the 3,4 position and hydroxylation of the number 4 carbon. The 4-hydroxylated carotenoids formed in E. coli were slowly metabolized to yield compounds with ketocarotenoid-like absorption spectra. It is proposed that a 3,4-desaturation subsequent to 4-hydroxylation of the beta-ring leads to the formation of a 4-keto-beta-ring via an indirect and unexpected route: a keto-enol tautomerization.     

Regulation of carotenoid biosynthesis in plants: evidence for a key role of hydroxymethylbutenyl diphosphate reductase in controlling the supply of plastidial isoprenoid precursors

Carotenoids are isoprenoid pigments that function as photoprotectors, precursors of the hormone abscisic acid (ABA), colorants and nutraceuticals. A major problem for the metabolic engineering of high carotenoid levels in plants is the limited supply of their isoprenoid precursor geranylgeranyl diphosphate (GGPP), formed by condensation of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) units usually synthesized by the methylerythritol phosphate (MEP) pathway in plastids. Our earlier work with three of the seven MEP pathway enzymes suggested that the first reaction of the pathway catalyzed by deoxyxylulose 5-phosphate synthase (DXS) is limiting for carotenoid biosynthesis during tomato (Lycopersicon esculentum) fruit ripening. Here we investigate the contribution of the enzyme hydroxymethylbutenyl diphosphate reductase (HDR), which simultaneously synthesizes IPP and DMAPP in the last step of the pathway. A strong upregulation of HDR gene expression was observed in correlation with carotenoid production during both tomato fruit ripening and Arabidopsis thaliana seedling deetiolation. Constitutive overexpression of the tomato cDNA encoding HDR in Arabidopsis did not increase carotenoid levels in etioplasts. By contrast, light-grown transgenic plants showed higher carotenoid levels and an enhanced seed dormancy phenotype suggestive of increased ABA levels. The analysis of double transgenic Arabidopsis plants overproducing both the enzyme taxadiene synthase (which catalyzes the production of the non-native isoprenoid taxadiene from GGPP) and either HDR or DXS showed a twofold stronger effect of HDR in increasing taxadiene levels. Together, the data support a major role for HDR in controlling the production of MEP-derived precursors for plastid isoprenoid biosynthesis.     

Growth and carotenoid production of Thraustochytrium sp. CHN-1 cultured under superbright red and blue light-emitting diodes

Isomers of astaxanthin produced by Thraustochytrium sp. CHN-1 are identified as (3S,3S')-trans-astaxanthin, (3R,3R')-trans-astaxanthin and (3S,3S')-cis-astaxanthin by chirality column HPLC, and 1H and 13C NMR. We studied the effects of light generated by superbright blue, red and near-red LEDs on the growth and carotenoid production of Thraustochytrium sp. CHN-1. Thraustochytrium sp. CHN-1 responded to blue LEDs light: It produced carotenoid pigments (astaxanthin)