Isolation of a β-Carotene Over-Producing Soil Bacterium, Sphingomonas sp.

A carotenoid-accumulating bacterium isolated from soil, identified as a Sphingomonas sp., grew at 0.18 h(-1) and produced 1.7 mg carotenoids g(-1) dry cell, among which beta-carotene (29% of total carotenoids) and nostoxanthin (36%). A mutant strain, obtained by treatment with ethyl methanesulfonate, accumulated up to 3.5 mg carotenoids g(-1) dry cell. Accumulation of beta-carotene by this strain depended on the oxygenation of the growth medium, with maximal accumulation (89%) occurring under limiting conditions. Beta-carotene accumulation could be further enhanced by incubating the cells in the presence of glycerol (either not or only slowly assimilated) and yeast extract resulting in an accumulation of 5.7 mg beta-carotene g(-1) dry cell wt. The strain used lactose as carbon source with similar biomass and carotenoid production, providing a viable alternative use for cheese whey ultra-filtrate.     

2,2'-beta-hydroxylase (CrtG) is involved in carotenogenesis of both nostoxanthin and 2-hydroxymyxol 2'-fucoside in Thermosynechococcus elongatus strain BP-1

We identified the molecular structures, including the stereochemistry, of all carotenoids in Thermosynechococcus elongatus strain BP-1. The major carotenoid was beta-carotene, and its hydroxyl derivatives of (3R)-beta-cryptoxanthin, (3R,3'R)-zeaxanthin, (2R,3R,3'R)-caloxanthin and (2R,3R,2'R,3'R)-nostoxanthin were also identified. The myxol glycosides were identified as (3R,2'S)-myxol 2'-fucoside and (2R,3R,2'S)-2-hydroxymyxol 2'-fucoside. 2-Hydroxymyxol 2'-fucoside is a novel carotenoid, and similar carotenoids of 4-hydroxymyxol glycosides were previously named aphanizophyll. Ketocarotenoids, such as echinenone and 4-ketomyxol, which are unique carotenoids in cyanobacteria, were absent, and genes coding for both beta-carotene ketolases, crtO and crtW, were absent in the genome. From a homology search, the Tlr1917 amino acid sequence was found to be 41% identical to 2,2'- beta-hydroxylase (CrtG) from Brevundimonas sp. SD212, which produces nostoxanthin from zeaxanthin. In the crtG disruptant mutant, 2-hydroxymyxol 2'-fucoside, caloxanthin and nostoxanthin were absent, and the levels of both myxol 2'-fucoside and zeaxanthin were higher. Therefore, the gene has a CrtG function for both myxol to 2-hydroxymyxol and zeaxanthin to nostoxanthin. This is the first functional identification of CrtG in cyanobacteria. We also investigated the distribution of crtG-like genes, and 2-hydroxymyxol and/or nostoxanthin, in cyanobacteria. Based on the identification of the carotenoids and the completion of the entire nucleotide sequence of the genome in T. elongatus, we propose a biosynthetic pathway of the carotenoids and the corresponding genes and enzymes.     

Identification of carotenoids in Erwinia herbicola and in a transformed Escherichia coli strain

The yellow pigments of Erwinia herbicola Eho 10 and of a transformed Escherichia coli LE392 pPL376 have been identified as carotenoids. HPLC separation, spectra and in some cases mass spectroscopy demonstrated the presence of phytoene (15-cis isomer), beta-carotene (all-trans, 9-cis and 15-cis), beta-cryptoxanthin ( = 3-hydroxy beta-carotene), zeaxanthin (3,3'-dihydroxy beta-carotene) and corresponding carotene glycosides. In addition, lycopene and gamma-carotene accumulated in the presence of the inhibitor 2-(4-chlorophenylthio)-triethylamine.HCl. Carotenoid content in the transformed E. coli was two-fold higher than in E. herbicola. The pattern of the carotenoids was similar in the two organisms. Inactivation of the katF gene in E. coli resulted in an 85% lowering of carotenoid formation, as did the addition of 0.5% glucose to the medium. Suppression of carotenoid formation by inactivation of the katF gene lowered, but did not abolish, the protection offered by carotenoids against inactivation by alpha-terthienyl plus near-ultraviolet light (320-400 nm).     

Carotenoid Pigments of Mycobacterium kansasii

Partitioned between aqueous methanol and petroleum ether, the unsaponifiable pigments of Mycobacterium kansasii were all epiphasic. Thin-layer chromatography of these carotenoids showed that M. kansasii formed at least nine pigments. These pigments were identified by their chromatographic properties and spectral characteristics as phytoene, zeta-carotene, neurosporene, lycopene, leprotene, gamma-carotene, delta-carotene, alpha-carotene, and beta-carotene. Three additional pigmented spots on thin-layer chromatography found in trace amounts were possibly degradation products of the major carotenoids.     

Effect of polar and non-polar carotenoids on Xanthophylomyces dendrorhous membranes by EPR

The red yeast Xanthophyllomyces dendrorhous is one of the microbiological production systems for natural carotenoids. High-performance liquid chromatography (HPLC) and electron paramagnetic resonance spectroscopy (EPR) experiments were performed on X. dendrorhous membranes in order to study the effect of incorporation rates of different type of carotenoids. In the case of fluid-phase membranes, it was found that polar carotenoids, such as astaxanthin and cis-astaxanthin, increased the EPR order parameter and decreased the motional freedom and phase-transition temperature. In contrast the non-polar carotenoids beta-cryptoxanthin and beta-carotene decreased the EPR order parameter and increased motional freedom and phase-transition temperature. A noteworthy coherence was observed between the polarities of the strains and the phase-transition temperatures.     

Carotenoids of an Antarctic psychrotolerant bacterium, Sphingobacterium antarcticus, and a mesophilic bacterium, Sphingobacterium multivorum

The major carotenoid pigments of an Antarctic psychrotolerant bacterium, Sphingobacterium antarcticus, and a mesophilic bacterium, Sphingobacterium multivorum, were identified as zeaxanthin, beta-cryptoxanthin, and beta-carotene. Analysis was based on ultraviolet-visible spectroscopy, mass spectroscopy, and reversed-phase HPLC. Photoacoustic spectroscopy of intact bacterial cells revealed that the bulk of the pigments in S. antarcticus and S. multivorum was associated with the cell membrane. In vitro studies with synthetic membranes of phosphatidylcholine demonstrated that the major pigment was bound to the membranes and decreased their fluidity. The relative amounts of polar pigments were higher in cells grown at 5 degrees C than in cells grown at 25 degrees C. In the mesophilic strain, the synthesis of polar carotenoids was quantitatively less than that of the psychrotolerant strain.     

Peridinin as the major biological carotenoid quencher of singlet oxygen in marine algae Gonyaulax polyedra

Carotenoids in light-harvesting proteins and reaction centers increase the overall efficiency of photosynthesis by transferring absorbed light energy to chlorophylls. Peridinin and beta-carotene were isolated from Gonyaulax polyedra in a one-step purification protocol using the preparative circular chromatography (Chromatotron), performed on silica gel under N(2) atmosphere and n-hexane/acetone 8:2 as mobile phase and characterized by extensive (1)H NMR, infrared, and electrospray ionization mass spectrometry analyses. The quenching of singlet molecular oxygen [O(2) ((1)Delta(g))] was evaluated by NIR-emission assays using singlet oxygen generated by sensitization of either perinaphthenone or methylene blue. The NIR-emission assay showed that peridinin quench as singlet oxygen (k(q) = 9.5 x 10(8) M(-1) s(-1)) 5-fold less efficiently than beta-carotene (52 x 10(8) M(-1) s(-1)). A method, based on the use of high-performance liquid chromatography with UV-VIS detection, was then developed for the sensitive quantification of peridinin (55% of total carotenoids) and beta-carotene (4.1% of total carotenoids). Thus, since peridinin is 10-fold more abundant than beta-carotene, it is expected to be the major protector against the deleterious effects of O(2) ((1)Delta(g)) in Gonyaulax polyedra.     

Isolation and characterization of marine pigmented bacteria from Norwegian coastal waters and screening for carotenoids with UVA-blue light absorbing properties

Microbial culture collections are important resources for isolation of natural compounds with novel properties. In this study, a culture collection of around 1,500 pigmented heterotrophic bacteria was established. The bacteria were isolated from the sea surface microlayer at different sampling sites along the mid-part of the Norwegian coast. The bacterial isolates produced pigments of various coloration (e.g. golden, yellow, red, pink and orange). Methanol extracts of sixteen isolates were characterized with LC-Diodearray-TOF mass spectrometry analysis. The number of pigments per isolate varied considerably, and a tentative identification of the pigments was performed based on UV-absorbance profile and molecular formula assignation based on the accurate mass determination. The LC-MS analyses revealed that most of the pigments probably were carotenoids. Furthermore, we developed a high throughput LC-MS method for characterization and screening of a larger sub-fraction (300 isolates) of the culture collection. The aim was to screen and identify bacterial isolates producing carotenoids that absorb light in the UVA-Blue light. Six of the bacterial strains were selected for detailed investigation, including 16s rRNA sequencing, preparative HPLC for purification of major carotenoids and subsequent structural elucidation with NMR. Among the identified carotenoids were zeaxanthin, nostoxanthin and sarcinaxanthin, some with novel glycosylation patterns.     

Chlorophyll and carotenoid degradation mediated by thylakoid-associated peroxidative activity in olives (Olea europaea) cv. hojiblanca

A peroxidative activity was found in solubilized thylakoid membranes of olives (Olea europaea) cv. hojiblanca that catalyses degradation of chloroplast pigments in the presence of H2O2 and 2,4-dichlorophenol (DCP). The intermediate products of this degradation were analyzed using HPLC with diode array detection and the results indicated that 13(2)-OH-chlorophyll a and 13(2)-OH-chlorophyll b were the primary catabolites. The peroxidative activity assosiated with the thylakoid membranes affected, not only chlorophyll a and chlorophyll b, but also other accessory pigments in the photosynthetic process, such as the carotenoids. Quantitatively, the progressive decrease of the ratios Chl a/b and total Chls a+b/carotenoids indicated a more rapid disappearance of Chl a than of Chl b and a faster degradation of Chls a+b than of carotenoids.     

Improvement of carotenoid-synthesizing yeast Rhodotorula rubra by chemical mutagenesis

A mutant Rhodotorula rubra with enhanced carotenoid-synthesizing activity for synthesizing total carotenoids and beta-carotene was obtained by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. When co-cultivated with yogurt starter bacteria (Lactobacillus bulgaricus + Streptococcus thermophilus) in whey ultrafiltrate it produced 15.7 mg total carotenoids l(-1) culture fluid or 946 microg total carotenoids g(-1) dry cells of which 71% was beta-carotene. Grown as a monoculture in glucose substrate, the mutant shown 1.4 times lower carotenoid-synthesizing activity, and the relative share of beta-carotene in the total carotenoids was lower (63%). The individual pigments torulene and torularhodin were identified, whose mass fractions were (29% and 7%) and (24% and 4%), respectively, for the mutant grown as a monoculture and as a mixed culture with the yogurt bacteria.     

Thermotropic phase behaviour of alpha-dipalmitoylphosphatidylcholine multibilayers is influenced to various extents by carotenoids containing different structural features--evidence from differential scanning calorimetry

Carotenoids are the effective modulators of physical properties of model and natural membranes. To demonstrate the relationship between the structure of carotenoids and their effect on the molecular dynamics of membranes, we have investigated the influence of five structurally different carotenoids: beta-carotene, lycopene, lutein, violaxanthin, zeaxanthin and additionally carotane--a fully saturated derivative of beta-carotene, on thermotropic phase behaviour of dipalmitoylphosphatidylcholine (DPPC) multilamellar vesicles by means of differential scanning calorimetry (DSC). The results obtained indicate that the carotenoids used modulated the thermotropic properties of multibilayers to various extents, broadening the pretransition and the main phase transition peaks and shifting them to lower temperatures. Pronounced decrease of pretransition enthalpy (DeltaH(p)) proves that carotenoids very strongly alter the membrane properties in its gel phase. Comparison of the influence of several carotenoids shows that a rigid, polyisoprenoid chain plays a basic role in altering the thermotropic properties of such membranes and the presence of rings without oxygen-containing groups has a minor significance for the observed interactions. Carotenoids containing epoxy and/or hydroxy groups attached to their rings modify the thermotropic phase behaviour of DPPC multilamellar vesicles stronger than carotenes--a result of their orientation in the DPPC bilayer.     

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 profiles of yeasts belonging to the genera Rhodotorula, Rhodosporidium, Sporobolomyces, and Sporidiobolus

Eighteen yeast species of the genera Rhodotorula, Rhodosporidium, Sporobolomyces, and Sporidiobolus, each one represented by its type strain, were investigated with the objective of evaluating their carotenoid composition. The pigments were extracted from yeast cells, quantified by high pressure liquid chromatography diode array detector and the main compounds were confirmed by atmospheric pressure chemical ionization quadrupole mass spectrometry. Significant (P < 0.01) differences among several species and (or) genera were observed. Thirteen strains were seen to be able to produce carotenoids, from 16.4 to 184 microg/g cell dry mass and from 6.0 to 1993.4 microg/L culture. The main carotenoids produced were identified as torularhodin, torulene, gamma-carotene, and beta-carotene. The correlation matrix calculated on the basis of the carotenoid composition data matrix indicated significant (P < 0.01) relationships between torulene and torularhodin (r = 0.81), gamma-carotene and torulene (r = 0.49), beta-carotene and torulene (r = -0.72), as well as beta-carotene and gamma-carotene (r = 0.64). These significant correlation coefficients may suggest that species belonging to the genera Rhodosporidium, Sporobolomyces, and Sporidiobolus possess a carotenoid biosynthetic pathway analogous to that elsewhere postulated for Rhodotorula species.     

Differential effects of carotenoids on lipid peroxidation due to membrane interactions: X-ray diffraction analysis

The biological benefits of certain carotenoids may be due to their potent antioxidant properties attributed to specific physico-chemical interactions with membranes. To test this hypothesis, we measured the effects of various carotenoids on rates of lipid peroxidation and correlated these findings with their membrane interactions, as determined by small angle X-ray diffraction approaches. The effects of the homochiral carotenoids (astaxanthin, zeaxanthin, lutein, beta-carotene, lycopene) on lipid hydroperoxide (LOOH) generation were evaluated in membranes enriched with polyunsaturated fatty acids. Apolar carotenoids, such as lycopene and beta-carotene, disordered the membrane bilayer and showed a potent pro-oxidant effect (>85% increase in LOOH levels) while astaxanthin preserved membrane structure and exhibited significant antioxidant activity (40% decrease in LOOH levels). These findings indicate distinct effects of carotenoids on lipid peroxidation due to membrane structure changes. These contrasting effects of carotenoids on lipid peroxidation may explain differences in their biological activity.     

The impact of coral bleaching on the pigment profile of the symbiotic alga, Symbiodinium

Bleaching of corals by loss of symbiotic dinoflagellate algae and/or photosynthetic pigments is commonly triggered by elevated temperatures coupled with high irradiance, and is a first-order threat to coral reef communities. In this study, a high-resolution high-performance liquid chromatography method integrated with mass spectrometry was applied to obtain the first definitive identification of chlorophyll and carotenoid pigments of three clades of symbiotic dinoflagellate algae (Symbiodinium) in corals, and their response to experimentally elevated temperature and irradiance. The carotenoids peridinin, dinoxanthin, diadinoxanthin (Dn), diatoxanthin (Dt) and beta-carotene were detected, together with chlorophylls a and c2, and phaeophytin a, in all three algal clades in unstressed corals. On exposure to elevated temperature and irradiance, three coral species (Montastrea franksi and Favia fragum with clade B algae, and Montastrea cavernosa with clade C) bleached by loss of 50-80% of their algal cells, with no significant impact to chlorophyll a or c2, or peridinin in retained algal cells. One species (Agaricia sp. with clade C) showed no significant reduction in algal cells at elevated temperature and irradiance, but lost substantial amounts of chlorophyll a and carotenoid pigments, presumably through photo-oxidative processes. Two coral species (Porites astreoides and Porites porites both bearing clade A algae) did not bleach. The impact of elevated temperature and irradiance on the levels of the photoprotective xanthophylls (Dn + Dt) and beta-carotene varied among the corals, both in pool size and xanthophyll cycling, and was not correlated to coral bleaching resistance.     

Relationship between mycobacterial species and their carotenoid pigments

A study of the relationship between mycobacterial species and their carotenoid pigments was carried out. According to the carotenoid pigments contained, the mycobacterial species tested were divided into four groups: the first group of Mycobacterium kansasii and M. marinum, which formed principally only beta-carotene; the second group of M. gordonae, M. scrofulaceum, M. szulgai, M. xenopi, M. flavescens, M. phlei, M. rhodesiae, M. neoaurum, and M. aichiense, which formed beta-carotene and a zeaxanthin-like substance; the third group of M. aurum and M. obuense, which formed beta-carotene and an eschscholtzxanthin-like substance; and the fourth group of M. chubuense and M. tokaiense, which formed beta-carotene and zeaxanthin- and eschscholtzxanthin-like substances. The common carotenoid pigment throughout the genus Mycobacterium was beta-carotene and the hypophasic carotenoids differed according to the species.     

Pigments and ultrastructures of pigment cells in xanthic sailfin mollies (Poecilia latipinna).

Electron micrographs of skin from xanthic (gold) sailfin mollies revealed numerous xanthophores, as well as scattered melanophores. The melanophores were seen to contain premelanosomes in various stages of development. This is consistent with the fact that xanthic mollies have been shown to be tyrosinase positive. Melanosomes in xanthic mollies appear to develop by one of two pathways: 1) from an endoplasmic reticulum-derived vesicle which develops an internal lamellar framework, and 2) by fusion of multiple Golgi-derived vesicles which lack an internal lamellar framework. Analysis of the pigments in the skin of the xanthic mollies identified four colorless pteridine pigments (xanthopterin, isoxanthopterin, neopterin, and pterin) and a carotenoid with an absorbance spectrum similar to beta-carotene. It appears that, unlike some other poeciliid fishes, sailfin mollies do not use pteridine pigments for orange coloration. Rather, they appear to rely primarily on carotenoids.     

The solubilisation pattern of lutein, zeaxanthin, canthaxanthin and beta-carotene differ characteristically in liposomes, liver microsomes and retinal epithelial cells

The incorporation efficiencies of lutein, zeaxanthin, canthaxanthin and beta-carotene into Retinal Pigment Epithelial (RPE) cells (the human RPE cell line D 407), liver microsomes and EYPC liposomes are investigated. In RPE cells the efficiency ratio of lutein and zeaxanthin compared to canthaxanthin and beta-carotene is higher than in the other membranes. The preferential interactions of lutein and zeaxanthin with RPE cells are discussed considering special protein binding properties. Incorporation yields were obtained from the UV-Vis spectra of the carotenoids. Membrane modulating effects of the carotenoids were obtained from the fluorescence spectra of co-incorporated Laurdan (6-dodecanoyl-2-dimethylaminonaphtalene). The Laurdan fluorescence quenching efficiencies of the membrane bound carotenoids offer an access to direct determinations of membrane carotenoid concentrations. Fetal calf serum as carrier for carotenoid incorporation appears superior to tetrahydrofuran.     

Myxol and 4-ketomyxol 2'-fucosides, not rhamnosides, from Anabaena sp. PCC 7120 and Nostoc punctiforme PCC 73102, and proposal for the biosynthetic pathway of carotenoids

We identified the molecular structures of carotenoids in some Anabaena and Nostoc species. The myxoxanthophyll and ketomyxoxanthophyll in Anabaena (also known as Nostoc) sp. PCC 7120, Anabaena variabilis IAM M-3, Nostoc punctiforme PCC 73102 and Nostoc sp. HK-01 were (3R,2'S)-myxol 2'-fucoside and (3S,2'S)-4-ketomyxol 2'-fucoside, respectively. The glycoside moiety of the pigments was fucose, not rhamnose. The major carotenoids were beta-carotene and echinenone, and the minor ones were beta-cryptoxanthin, zeaxanthin, canthaxanthin and 3'-hydroxyechinenone. Based on the identification of the carotenoids and the completion of the entire nucleotide sequence of the genome in Anabaena sp. PCC 7120 and N. punctiforme PCC 73102, we proposed a biosynthetic pathway for the carotenoids and the corresponding genes and enzymes. Since only zeta-carotene desaturase (CrtQ) from Anabaena sp. PCC 7120 and beta-carotene ketolase (CrtW) from N. punctiforme PCC 73102 have been functionally identified, the other genes were searched by sequence homology only from the functionally confirmed genes. Finally, we investigated the phylogenetic relationships among some Anabaena and Nostoc species, including some newly isolated species.     

Carotenoids from the aerobic photosynthetic bacterium,Erythrobacter longus: β-Carotene and its hydroxyl derivatives

About 20 different carotenoids were found in a strictly aerobic photosynthetic bacterium, Erythrobacter longus. All the carotenoids except the highly polar ones were identified as C₄₀-skeletal carotenoids, which could be devided into three groups: (1) bicyclic carotenoids: -carotene and its hydroxyl derivatives; -cryptoxanthin, zeaxanthin, caloxanthin and nostoxanthin, (2) monocyclic carotenoids: rubixanthin, bacteriorubixanthin and bacteriorubixanthinal, which was a unique cross-conjugated carotenal, and (3) acyclic carotenoids: anhydrorhodovibrin and spirilloxanthin. Bacteriorubixanthinal and zeaxanthin were the major components. (3R)-3-Hydroxy--ionone has rarely been found in carotenoids of purple photosynthetic bacteria, while the acyclic carotenoids have been found exclusively in photosynthetic bacteria. Thus, this bacterium is interesting in its composition of carotenoids.Abbreviations DPA diphenylamine - HPLC high-performance liquid chromatography - HP-TLC high-performance thin layer chromatography - FD-MS field desorption mass spectrometry - ¹HNMR proton nuclear magnetic resonance - CD circular dichroism