Resonance raman spectroscopy and quantum chemical modeling studies of protein-astaxanthin interactions in alpha-crustacyanin (major blue carotenoprotein complex in carapace of lobster, Homarus gammarus)

Resonance Raman spectroscopy and quantum chemical calculations were used to investigate the molecular origin of the large redshift assumed by the electronic absorption spectrum of astaxanthin in alpha-crustacyanin, the major blue carotenoprotein from the carapace of the lobster, Homarus gammarus. Resonance Raman spectra of alpha-crustacyanin reconstituted with specifically 13C-labeled astaxanthins at the positions 15, 15,15', 14,14', 13,13', 12,12', or 20,20' were recorded. This approach enabled us to obtain information about the effect of the ligand-protein interactions on the geometry of the astaxanthin chromophore in the ground electronic state. The magnitude of the downshifts of the C==C stretching modes for each labeled compound indicate that the main perturbation on the central part of the polyene chain is not homogeneous. In addition, changes in the 1250-1400 cm(-1) spectral range indicate that the geometry of the astaxanthin polyene chain is moderately changed upon binding to the protein. Semiempirical quantum chemical modeling studies (Austin method 1) show that the geometry change cannot be solely responsible for the bathochromic shift from 480 to 632 nm of protein-bound astaxanthin. The calculations are consistent with a polarization mechanism that involves the protonation or another interaction with a positive ionic species of comparable magnitude with both ketofunctionalities of the astaxanthin-chromophore and support the changes observed in the resonance Raman and visible absorption spectra. The results are in good agreement with the conclusions that were drawn on the basis of a study of the charge densities in the chromophore in alpha-crustacyanin by solid-state NMR spectroscopy. From the results the dramatic bathochromic shift can be explained not only from a change in the ground electronic state conformation but also from an interaction in the excited electronic state that significantly decreases the energy of the pi-antibonding C==O orbitals and the HOMO-LUMO gap.     

The crtS gene of Xanthophyllomyces dendrorhous encodes a novel cytochrome-P450 hydroxylase involved in the conversion of beta-carotene into astaxanthin and other xanthophylls

The conversion of beta-carotene into xanthophylls is a subject of great scientific and industrial interest. We cloned the crtS gene involved in astaxanthin biosynthesis from two astaxanthin producing strains of Xanthophyllomyces dendrorhous: VKPM Y2410, an astaxanthin overproducing strain, and the wild type ATCC 24203. In both cases, the ORF has a length of 3166 bp, including 17 introns, and codes for a protein of 62.6 kDa with similarity to cytochrome-P450 hydroxylases. crtS gene sequences from strains VKPM Y2410, ATCC 24203, ATCC 96594, and ATCC 96815 show several nucleotide changes, but none of them causes any amino acid substitution, except a G2268 insertion in the 13th exon of ATCC 96815 which causes a change in the reading frame. A G1470 --> A change in the 5' splicing region of intron 8 was also found in ATCC 96815. Both point mutations explain astaxanthin idiotrophy and beta-carotene accumulation in ATCC 96815. Mutants accumulating precursors of the astaxanthin biosynthetic pathway were selected from the parental strain VKPM Y2410 (red) showing different colors depending on the compound accumulated. Two of them were blocked in the biosynthesis of astaxanthin, M6 (orange; 1% astaxanthin, 71 times more beta-carotene) and M7 (orange; 1% astaxanthin, 58 times more beta-carotene, 135% canthaxanthin), whereas the rest produced lower levels of astaxanthin (5-66%) than the parental strain. When the crtS gene was expressed in M7, canthaxanthin accumulation disappeared and astaxanthin production was partially restored. Moreover, astaxanthin biosynthesis was restored when X. dendrorhous ATCC 96815 was transformed with the crtS gene. The crtS gene was heterologously expressed in Mucor circinelloides conferring to this fungus an improved capacity to synthesize beta-cryptoxanthin and zeaxanthin, two hydroxylated compounds from beta-carotene. These results show that the crtS gene is involved in the conversion of beta-carotene into xanthophylls, being potentially useful to engineer carotenoid pathways.     

Influence of culture conditions of Gordonia jacobaea MV-26 on canthaxanthin production.

Commercial interest in the use of natural pigments isolated from microorganisms has increased in recent years; hence, molecules belonging to the polyisoprenoid group (i.e. beta-carotene, astaxanthin, and canthaxanthin) have been the focus of much attention. The bacterium Gordonia jacobaea readily synthesizes and accumulates large amounts of canthaxanthin (beta-beta'-carotene-4,4'-dione), which is widely used in the food and cosmetics industries. In the present work, the effects of different low-cost raw materials on fermentation and canthaxanthin accumulation by a hyperpigmented strain of G. jacobaea were studied. Canthaxanthin production and peak levels of accumulation varied according to the different media used.     

Carotenoid hydroxylase from Haematococcus pluvialis: cDNA sequence, regulation and functional complementation.

A cDNA homologous to beta-carotene hydroxylase from Arabidopsis thaliana was isolated from the green alga Haematococcus pluvialis. The predicted amino acid sequence for this enzyme shows homology to the three known plant beta-carotene hydroxylases from Arabidopsis thaliana and from Capsicum annuum (38% identity) and to prokaryote carotenoid hydroxylases (32-34% identities). Heterologous complementation using E. coli strains which were genetically engineered to produce carotenoids indicated that the H. pluvialis beta-carotene hydroxylase was able to catalyse not only the conversion of beta-carotene to zeaxanthin but also the conversion of canthaxanthin to astaxanthin. Furthermore, Northern blot analysis revealed increased beta-carotene hydroxylase mRNA steady state levels after induction of astaxanthin biosynthesis. In accordance with the latter results, it is proposed that the carotenoid hydroxylase characterized in the present publication is involved in the biosynthesis of astaxanthin during cyst cell formation of H. pluvialis.     

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.     

Determination of carotenoids and all-trans-retinol in fish eggs by liquid chromatography-electrospray ionization-tandem mass spectrometry

A novel method was developed for the combined determination of carotenoids and retinoids in fish eggs, which incorporates prior analyte isolation using liquid-liquid partitioning to minimize analyte degradation, and fraction analysis using high-performance liquid chromatography-electrospray (positive)-quadrupole mass spectrometry (LC-ESI(+)-MS; SIM or MRM modes). Eggs from Chinook salmon (Oncorhynchus tshawytscha) were used as the model fish egg matrix. The methodology was assessed and validated for beta-carotene, lutein, zeaxanthin, and beta-cryptoxanthin (molecular ion radicals [M](+)), canthaxanthin and astaxanthin ([M+Na](+) adducts) and all-trans-retinol ([(M+H)-H(2)O](+)). Using replicate egg samples (n=5) spiked with beta-cryptoxanthin and beta-carotene before and after extraction, matrix-sourced ESI(+) enhancement was observed as evidenced by comparable %matrix effect and %process efficiency values for beta-cryptoxanthin and beta-carotene of 114-119%. In aquaculture-raised eggs from adult Chinook salmon astaxanthin, all-trans-retinol, lutein and canthaxanthin were identified and determined at concentrations of 4.12, 1.06, 0.12 and 0.45 microg/g (egg wet weight), respectively. To our knowledge, this is the first report on a method for LC-MS determination of carotenoids and retinoids in a fish egg matrix, and the first carotenoid-specific determination in any fish egg sample.     

雨生红球藻β－胡萝卜素酮化酶（bkt）启动子功能分析

单细胞绿藻———雨生红球藻在逆境条件下积累大量的虾青素。β-胡萝卜素酮化酶(bkt)催化在β-胡萝卜素和玉米黄素的β-紫罗酮环C-4位引入酮基的反应,是虾青素合成过程中的关键酶。我们利用凝胶阻滞的方法研究雨生红球藻中bkt基因309bp(-617/-309)启动子区域的转录因子结合位点并发现在-396/-338的59bp探针存在特异的核蛋白结合位点。通过序列分析,发现此59bp区域并不包含TATA或者CAAT-box,而是存在对光、缺氧、p-香豆酸及激素反应的G-box。     

The carotenoids of wild and blue disease affected farmed tiger shrimp (Penaeus monodon, Fabricus)

1. The main carotenoids in wild Penaeus monodon exoskeleton were astaxanthin di- and mono-esters, astaxanthin, and beta-carotene. 2. Wild P. monodon exoskeleton contained on average 26.3 ppm total carotenoid; normally pigmented farmed shrimp had a similar concentration (25.3 ppm). 3. Exoskeletons of farmed "blue" P. monodon (i.e. blue-coloured, as opposed to the normally red-blue/black banded shrimp) contained significantly less total carotenoid (4.3-7 ppm). The only major carotenoid being astaxanthin. 4. Commercially available diets contained only trace quantities of canthaxanthin. 5. Nutritional deficiency with respect to carotenoids is suggested as the cause of blue disease in farmed P. monodon.     

Cloning of two carotenoid ketolase genes from Nostoc punctiforme for the heterologous production of canthaxanthin and astaxanthin

For the heterologous synthesis of keto-carotenoids such as astaxanthin, two carotenoid ketolase genes crtW38 and crtW148, were cloned from the cyanobacterium, Nostoc punctiforme PCC 73102 and functionally characterized. Upon expression in Escherichia coli, both genes mediated the conversion of beta-carotene to canthaxanthin. However in a zeaxanthin-producing E. coli, only the gene product of crtW148 introduced 4-keto groups into the 3,3'-dihydroxy carotenoid zeaxanthin yielding astaxanthin. The gene product of crtW38 was unable to catalyze this reaction. Both ketolases differ in their interaction with a hydroxylase in the biosynthetic pathway from beta-carotene to astaxanthin.     

Raman and surface enhanced Raman spectroscopy of 2,2,5,5-tetramethyl-3-pyrrolin-1-yloxy-3-carboxamide labeled proteins: bovine serum albumin and cytochrome c

2,2,5,5-Tetramethyl-3-pyrrolin-1-yloxy-3-carboxamide (tempyo) labeled bovine serum albumin and cytochrome c at different pH values were prepared and investigated using Raman-resonance Raman (RR) spectroscopy and surface enhanced Raman scattering (SERS) spectroscopy. The Raman spectra of tempyo labeled proteins in the pH 6.7-11 range were compared to those of the corresponding free species. The SERS spectra were interpreted in terms of the structural changes of the tempyo labeled proteins adsorbed on the silver colloidal surface. The tempyo spin label was found to be inactive in the Raman-RR and SERS spectra of the proteins. The alpha-helix conformation was concluded to be more favorable as the SERS binding site of bovine serum albumin. In the cytochrome c the enhancement of the bands assigned to the porphyrin macrocycle stretching mode allowed the supposition of the N-adsorption onto the colloidal surface.     

Identification by HPLC-MS of carotenoids of the Thraustochytrium CHN-1 strain isolated from the Seto Inland Sea

The orange-pigmented Thraustochytrium, CHN-1 strain was found to contain astaxanthin as the main carotenoid pigment. Echinenone, canthaxanthin, phoenicoxanthin and beta-carotene were also identified by high-performance liquid chromatography (HPLC) and HPLC-mass spectrometry. The total extractable carotenoid level was found to increase with culture age.     

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.     

Isolation and Identification of Canthaxanthin from Micrococcus roseus

Cooney, J. J. (University of Dayton, Dayton, Ohio), H. W. Marks, Jr., and Anne M. Smith. Isolation and identification of canthaxanthin from Micrococcus roseus. J. Bacteriol. 92:342-345. 1966.-The principal colored carotenoid of Micrococcus roseus was purified by solvent partitioning followed by column and thin-layer chromatography. Absorption spectra, partition coefficients, and infrared spectra suggested that the pigment was a diketo derivative of beta-carotene. The pigment was subjected to reduction, and the reduced pigment was subsequently dehydrated. Spectral data and partition coefficients of these derivatives indicated that the original pigment was canthaxanthin (4',4'-diketo-beta-carotene). The pigment was an all-trans isomer; it does not exist as an ester in M. roseus. Canthaxanthin has not previously been identified as a bacterial pigment.     

Engineering a beta-carotene ketolase for astaxanthin production

A new beta-carotene ketolase gene (crtW) was cloned from an environmental isolate Sphingomonas sp. DC18. A robust and reliable color screen was developed for protein engineering to improve its activity on hydroxylated carotenoids for astaxanthin production. Localized random mutagenesis was performed on the crtW gene including the upstream ribosomal binding site (RBS). Six mutations (H96L, R203W, A205V, A208V, F213L and A215T) in the crtW gene were isolated multiple times that showed improved astaxanthin production. These mutations were localized near the conserved histidine motifs, which were proposed for binding iron required for enzymatic activity. Combination of two of the mutations (R203W/F213L) further improved astaxanthin production. One mutation at the RBS (a438t) was shown to have additional effect on improving astaxanthin production. Most of the mutants still retained high activity on beta-carotene, however, the F213L single mutant and the R203W/F213L double mutant that yielded the highest improvement for astaxanthin production showed decreased activity for canthaxanthin production.     

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.     

Adaptative role of carotenoids and carotenoproteins in Cyclops kolensis Lilljeborg (Crustacea: Copepoda) specimens to extremely eutrophical conditions

The authors, using column, thin-layer, and ion-exchange chromatography, investigated carotenoid and carotenoprotein complex content in Cyclops kolensis specimens from an extremely eutrophic pond. The following carotenoids were found to be present: beta-carotene, beta-cryptoxanthin, lutein epoxide, crustaxanthin, 4'-hydroxyechinenone, canthaxanthin, and astaxanthin. Carotenoprotein complex containing astaxanthin as the prosthetic group name gamma-crustacyanine was purified from Cyclops kolensis individuals examined. The authors justify the adaptative role of these pigments in Cyclops kolensis specimens in extremely eutrophical conditions.     

Carotenoid content of chlorophycean microalgae: factors determining lutein accumulation in Muriellopsis sp. (Chlorophyta)

Fifteen strains of chlorophycean microalgae have been investigated with regard to their carotenoid profile. Lutein, beta-carotene and violaxanthin were present in virtually all of the strains, lutein, in general, being the most abundant carotenoid, whereas canthaxanthin and astaxanthin were found in some strains only. Chlorella fusca SAG 211-8b, Chlorococcum citriforme SAG 62.80, Muriellopsis sp., Neospongiococcum gelatinosum SAG B 64.80 and Chlorella zofingiensis CCAP 211/14 exhibited high lutein levels, the latter strain containing in addition substantial amounts of astaxanthin. Muriellopsis sp. was further characterized, since besides a high lutein content (up to 35 mg l(-1) culture), it had the highest growth rate (up to 0.17-0.23 h(-1)) and maximal standing cell density (up to 8 x 10(10) cells l(-1) culture). These levels of lutein are in the range of those reported for astaxanthin in Haematococcus and for beta-carotene in Dunaliella, microalgae of recognized interest for the production of these carotenoids. Lutein content of Muriellopsis sp. increased during the exponential phase of growth, with the highest value being recorded in the early stationary phase. Maximum levels of lutein in Muriellopsis sp. cultures were recorded at 20-40 mM NaNO3, 2-100 mM NaCl, 460 micromol photon m(-2) s(-1), pH 6.5 and 28 degrees C, conditions which were, in general, also optimal for cell growth. Growth-limiting conditions, such as pH values of 6 or 9 and a temperature of 33 degrees C, were found to stimulate carotenogenesis in Muriellopsis sp. This strain represents a potential source of lutein, a commercially interesting carotenoid of application in aquaculture and poultry farming, as well as in the prevention of cancer and diseases related to retinal degeneration.     

Astaxanthin and canthaxanthin are potent antioxidants in a membrane model.

When the conjugated keto-carotenoids, either astaxanthin or canthaxanthin, are added to rat liver microsomes undergoing radical-initiated lipid peroxidation under air, they are as effective as alpha-tocopherol in inhibiting this process. This contrasts with the effect of beta-carotene, which is a much less potent antioxidant when added in this system, without the addition of other antioxidants.     

Comparative studies of carotenoids in the fauna of the Gullmar Fjord (Bohuslan,Sweden) III. Echinodermata

The author investigated the presence of various carotenoids in the Echinodermata from Gullmar Fjord (Bohuslan, Sweden) by means of columnar and thin-layer chromatography. The investigations revealed the presence of the following:

- inHenricia sanguinolenta:β-carotene, echinenone, canthaxanthin, guraxanthin, lutein-5, 6-epoxide and astaxanthin.

- inAmphiura filiformis: canthaxanthin, cryptoxanthin, lutein, lutein-5, 6-epoxide, isozeaxanthin, zeaxanthin, astaxanthin and 4-hydroxy-4-keto-β-carotene.

- inAmphipholis squamata:β-carotene, cryptoxanthin, lutein, lutein-5, 6-epoxide, astaxanthin, astaxanthin ester, asterin-acid and rubixanthin derivative.

- inOphiopholis aculeata: canthaxanthin, cryptoxanthin, isozeaxanthin, astaxanthin, astaxanthin ester, asterinacid, 4-hydroxy-4-keto-β-carotene, hydroxy rubixanthin and gazaniaxanthin-like substances.

- inOphiothrix fragilis: canthaxanthin, lutein-5, 6-epoxide, isozeaxanthin, astaxanthin, astaxanthin ester, 4-hydroxy-4-keto-β-carotene, and hydroxy rubixanthin.

- inAntedon petatus:canthaxanthin, guaraxanthin, isozeaxan-thin, zeaxanthin, astaxanthin, astaxanthin ester and 4-keto-4-ethoxy-β-carotene.

- inEchinocardium cordatum:β-carotene,γ-carotene, canthaxanthin, lutein, isozeaxanthin, zeaxanthin, astaxanthin and astaxanthin ester.

- inSpatangus purpureus: isozeaxanthin, astaxanthin, astaxanthin ester and 4-hydroxy-4-keto-β-carotene.

     

Protection by beta-carotene and related compounds against oxygen-mediated cytotoxicity and genotoxicity: implications for carcinogenesis and anticarcinogenesis.

beta-Carotene protects against photooxidative dermatitis in porphyric humans and mice by quenching of photoactivated species. Other actions of beta-carotene in vivo are explained on the basis of its ability to scavenge free radicals in vitro. For example, in guinea pigs treated with CCl4, beta-carotene decreases pentane and ethane production. Epidemiological studies link low serum beta-carotene levels to elevated risk of lung and other cancers, and in intervention trials, beta-carotene diminishes preneoplastic lesions. However, the dose/response relationships are not well established, and antineoplastic mechanisms await clarification. Given a radical quenching mechanism, beta-carotene should block tumor promotion, but more typically the site of action is progression and an even later role in invasion has not been ruled out. Some antineoplastic actions of carotenoids (such as increased rejection of fibrosarcomas in mice) are attributed to immunoenhancement; others may reflect conversion to retinoids and subsequent gene regulation. Carotenoids other than beta-carotene may act at an earlier stage of carcinogenesis or be more effective as anticarcinogens at certain target sites. As scavengers of hydroxyl radicals, canthaxanthin and astaxanthin are more effective than beta-carotene. Canthaxanthin is sometimes more effective than beta-carotene in chemoprevention, but it is sometimes completely ineffective. Lycopene quenches singlet oxygen more than twice as effectively as beta-carotene. However, the antineoplastic actions of lycopene or astaxanthin remain untested. Explorations of the interactions of carotenoids with other nutrients are just beginning. Dietary fat increases absorption of carotene but decreases antineoplastic effectiveness. Research is hampered by technical problems, including the unavailability of rigorous controls, the instability of carotenoids, and the heterogeneous phase structure induced by hydrophobic compounds in aqueous media. Areas of current controversy and promising approaches for future research are identified.