Echinenone production of a dark red-coloured strain of Botryococcus braunii

Echinenone has been used as an edible orange pigment, antioxidant and provitamin A. An echinenone-accumulating strain, BOT-20, of Botryococcus braunii was isolated from freshwater environments in Japan. The B. braunii BOT-20 strain is different from other strains of B. braunii, as it appeared dark red during its growth in the laboratory culture as opposed to green. The biomass of the strain was 1.9?g?L^?1 at 1?month after cultivation. The n-hexane/acetone (3:1, v/v) extract of the strain was 45.5% of the dry biomass weight and consisted of carotenoids (92%, of which 73% was echinenone) and hydrocarbons (8%). The echinenone content was 30.5% of the dry biomass weight, and production was 630?mg?L^?1. Hydrocarbons comprised only 3.7% of the total dry biomass weight. The main component of hydrocarbon was an analogue of botryococene by ¹H and ¹³C NMR. With high values of echinenone content and production, the B. braunii strain BOT-20 is expected to be a new bioresource for the commercial production of echinenone.     

Nature and Distribution of Carotenoid Pigments in Astasia ocellata

SYNOPSIS. The pigments synthesized by Astasia ocellata include α- and ε-carotene, 4-keto-β-carotene (echinenone), and 4,4'-diketo-β-carotene (canthaxanthin); 4-keto-α-carotene, accounting for about half the pigment in the cells, was tentatively identified; a strongly adsorbed keto-carotenoid, accounting for 25% of the pigments and bearing some similarities to astacin, polytomaxanthin and phoenicoxanthin, was also found.     

THE CAROTENOIDS OF FOUR BLUE-GREEN ALGAE1

The carotenoids of 4 species of blue-green algae, Anabaena variabilis, Phormidium persicinum, P. ectocarpi, and P. fragile, were investigated. In each, ft-carotene was a major pigment and the only carotene detected. The xanthophylls present in Anabaena variabilis were echinenone, canthaxanthin, and myxo-xanthophyll. Each of the Phormidium species contained zeaxanthin as the major xanthophyll. In each, this was accompanied by trace amounts of echinenone and isocryptoxanthin. In addition, 2 new xanthophylls, spectrally resembling ^-carotene, were found in Phormidium persicinum and P. ectocarpi, while another, with a spectrum similar to that of myxoxanthophyll, was found in P. fragile.     

Influence of zeaxanthin and echinenone binding on the activity of the Orange Carotenoid Protein

In most cyanobacteria high irradiance induces a photoprotective mechanism that downregulates photosynthesis by increasing thermal dissipation of the energy absorbed by the phycobilisome, the water-soluble antenna. The light activation of a soluble carotenoid protein, the Orange-Carotenoid-Protein (OCP), binding hydroxyechinenone, a keto carotenoid, is the key inducer of this mechanism. Light causes structural changes within the carotenoid and the protein, leading to the conversion of a dark orange form into a red active form. Here, we tested whether echinenone or zeaxanthin can replace hydroxyechinenone in a study in which the nature of the carotenoid bound to the OCP was genetically changed. In a mutant lacking hydroxyechinenone and echinenone, the OCP was found to bind zeaxanthin but the stability of the binding appeared to be lower and light was unable to photoconvert the dark form into a red active form. Moreover, in the strains containing zeaxanthin-OCP, blue-green light did not induce the photoprotective mechanism. In contrast, in mutants in which echinenone is bound to the OCP, the protein is photoactivated and photoprotection is induced. Our results strongly suggest that the presence of the carotenoid carbonyl group that distinguishes echinenone and hydroxyechinenone from zeaxanthin is essential for the OCP activity.     

Metabolic engineering of <Emphasis Type="Italic">Lilium</Emphasis> × <Emphasis Type="Italic">formolongi</Emphasis> using multiple genes of the carotenoid biosynthesis pathway

Lilium × formolongi was genetically engineered by Agrobacterium-mediated transformation with the plasmid pCrtZW-N8idi-crtEBIY, which contains seven enzyme genes under the regulation of the CaMV 35S promoter. In the transformants, ketocarotenoids were detected in both calli and leaves, which showed a strong orange color. In transgenic calli, the total amount of carotenoids [133.3 μg/g fresh weight (FW)] was 26.1-fold higher than in wild-type calli. The chlorophyll content and photosynthetic efficiency in transgenic orange plantlets were significantly lowered; however, after several months of subculture, they had turned into plantlets with green leaves that showed significant increases in chlorophyll and photosynthetic efficiency. The total carotenoid contents in leaves of transgenic orange and green plantlets were quantified at 102.9 and 135.2 μg/g FW, respectively, corresponding to 5.6- and 7.4-fold increases over the levels in the wild-type. Ketocarotenoids such as echinenone, canthaxanthin, 3′-hydroxyechinenone, 3-hydroxyechinenone, and astaxanthin were detected in both transgenic calli and orange leaves. A significant change in the type and composition of ketocarotenoids was observed during the transition from orange transgenic plantlets to green plantlets. Although 3′-hydroxyechinenone, 3-hydroxyechinenone, astaxanthin, and adonirubin were absent, and echinenone and canthaxanthin were present at lower levels, interestingly, the upregulation of carotenoid biosynthesis led to an increase in the total carotenoid concentration (+31.4%) in leaves of the transgenic green plantlets.     

Carotenoids in the sea urchin Paracentrotus lividus: occurrence of 9'-cis-echinenone as the dominant carotenoid in gonad colour determination

Regular sampling of wild Paracentrotus lividus was carried out over a 12-month period to examine seasonal effects on the pigment profile and content of the gonads, especially in comparison to gonad colour. The major pigments detected in the gut wall were breakdown products of fucoxanthin, namely fucoxanthinol and amarouciaxanthin A. Lower levels of other dietary carotenoids (lutein and β-carotene) together with some carotenoids not found in the diet, namely isozeaxanthin and echinenone ( 20% total carotenoid) were also detected in the gut wall. The presence of echinenone in the gut wall demonstrates that this organ acts as a major site of carotenoid metabolism. Echinenone is the dominant carotenoid in the gonads, accounting for approx. 50–60% of the total pigment. Both all-trans and 9′-cis forms of echinenone were detected in both the gut wall and in the gonad, with levels of the 9′-cis form typically 10-fold greater than the all-trans form in the gonad. The detection of large levels of 9′-cis-echinenone in wild sea urchins is unexpected due to the absence of 9- or 9′-cis forms of carotenoids in the natural, algal, diet. Whilst echinenone clearly contributes towards gonad pigmentation, levels of this carotenoid, cannot be directly linked to a qualitative assessment of gonad colour in terms of market acceptability. Indeed, unacceptable gonad colouration can be seen with both very low and high levels of echinenone and total carotenoid. The presence of 9′-cis-echinenone as the major carotenoid contributing to the pigmentation/colour of the gonad is an important observation in terms of developing artificial diets for urchin cultivation.     

Step of Dichlorvos Inhibition in the Pathway of Aflatoxin Biosynthesis

Dichlorvos (dimethyl 2,2-dichlorovinyl phosphate) inhibits the biosynthesis of aflatoxin by Aspergillus parasiticus. Cultures treated with dichlorvos excrete an orange pigment which can be converted into aflatoxin B(1) by the untreated mycelia. The orange pigment was partially identified as an acetyl derivative of versiconol-type compound. In the presence of dichlorvos, sterigmatocystin is converted into aflatoxin B(1) without being interfered, but averufin is converted into the orange pigment instead of aflatoxin B(1). Therefore, dichlorvos appears to block an enzymatic step in the aflatoxin biosynthetic pathway, which lies beyond averufin but before sterigmatocystin, at the formation of the orange pigment.     

Reinvestigation of Brevibacterium sp. Strain KY-4313 as a Source of Canthaxanthin

The hydrocarbon-utilizing Brevibacterium sp. strain KY-4313 was reevaluated for its potential to produce canthaxanthin, a carotenoid pigment of strong commercial interest. Three approaches were used to optimize the canthaxanthin yield from this organism, i.e., the preparation of mutants, the addition of supposedly carotenogenic chemicals to the growth medium, and growth promotion. Following treatment of the parent strain with N-nitrosomethylurea, a presumed mutant was isolated which showed a 32% increase in cellular canthaxanthin content. No effective carotenogenic chemicals were found in connection with hydrocarbon fermentations, in which mainly growth promotion through periodic medium renewal proved conducive to enhanced pigment production. Carotenogenesis could be stimulated in brain heart infusion broth by adding alcohols or retinol. Improved growth in this medium was generally not associated with higher canthaxanthin yields. Both superior growth and pigment levels were obtained in a newly designed medium based on fumaric acid-molasses. The maximum yields of canthaxanthin in shake flasks were (in milligrams per liter) 4.2 (brain heart infusion broth plus propanol-zinc sulfate), 3.6 (hydrocarbon medium), and 9.3 (fumaric acid-molasses), which represent a significant improvement over the originally reported optimal result (1 mg/liter). The corresponding yields of echinenone, the direct precursor of canthaxanthin, were 1.2, 1.6, and 2.3 mg/liter, respectively. Two-liter hydrocarbon batch fermentations involving medium renewal maximally produced 7.2 mg of canthaxanthin and 3.7 mg of echinenone per liter.     

Phosphatidylglycerol Depletion Induces an Increase in Myxoxanthophyll Biosynthetic Activity in Synechocystis PCC6803 Cells

Phosphatidylglycerol (PG) depletion suppressed the oxygen-evolvingactivity of Synechocystis PCC6803 pgsA mutant cells. Shortageof PG led to decreased photosynthetic activity, which, similarto the effect of high light exposure, is likely to generatethe production of reactive oxygen species (ROS) or free radicals.Protection of the PG-depleted cells against light-induced damageincreased the echinenone and myxoxanthophyll content of thecells. The increased carotenoid content was localized in a solublefraction of the cells as well as in isolated thylakoid and cytoplasmicmembranes. The soluble carotenoid fraction contained carotenederivatives, which may bind to proteins. These carotene–proteincomplexes are similar to orange carotenoid protein that is involvedin yielding protection against free radicals and ROS. An increasein the content of myxoxanthophyll and echinenone upon PG depletionsuggests that PG depletion regulates the biosynthetic pathwayof specific carotenoids.     

Pigmentation and Antibacterial Activity of Fast Neutron- and X-Ray-induced Strains of Monascus purpureus Went

Seven new strains of Monascus purpureus Went were induced by neutron and x-ray irradiation. The quantity and quality of pigments produced by these strains differed. Strains N4S and N11S produced twice as much pigment as normal, while another strain, N14S, was albino. An unknown orange pigment was found in young colonies of the N11S strain. This orange pigment reacted with alcohols and malt extract medium to form red pigments. Strains N4S, N11S, X2P, and wild type inhibited the growth of certain bacteria, especially the Bacillus species. Strain N11S had more antibacterial activity than wild type. A major active compound was isolated with an ultraviolet absorption spectrum that was related to those of the red pigments found in this fungus. The active compound(s) was named monascidin.     

Growth and pigment accumulation in nutrient-depleted Isochrysis aff. galbana T-ISO

The effect of three different nutrient depletions (nitrogen, sulphur and magnesium) on the growth and pigment accumulation of the haptophyte Isochrysis aff. galbana (clone T-ISO) has been studied. Pigments were quantified based on RP-UHPLC-PDA-MSⁿ analysis. All nutrient depletions led to reduced maximal biomass concentrations. Besides, all nutrient-depleted cultures accumulated 3-hydroxyechinenone. To our knowledge, this is the first time that 3-hydroxyechinenone has been found in I. aff. galbana T-ISO. Most 3-hydroxyechinenone, as well as the most echinenone and diatoxanthin, were found in the nitrogen-limited culture in which a more severe limitation resulted in higher cellular contents. Similar to accumulation of diatoxanthin, accumulation of 3-hydroxyechinenone and echinenone may be part of a global (stress) response mechanism to oversaturating light conditions.     

Effect of pH and nitrogen source on pigment production by Monascus purpureus

The effect of pH and nitrogen source on pigment production by Monascus purpureus 192F using glucose as the carbon and energy source, was studied in pH-controlled, batch fermentor cultures using HPLC analysis to determine individual pigment concentrations. A maximum of four pigments were detected in fungal extracts. These were the yellow pigments monascin and ankaflavin, the orange rubropunctatin and the red pigment monascorubramine. Monascorubramine was present as the major product in all instances. Fungal growth and ankaflavin synthesis were favoured at low pH (pH 4.0), whereas production of the other pigments was relatively independent of pH. The nature of the nitrogen source affected fungal growth and pigment production, independent of pH. Ammonium and peptone as nitrogen sources gave superior growth and pigment concentrations compared to nitrate. Ankaflavin was not detected in nitrate cultures. The highest red pigment production was obtained using a glucose-peptone medium at pH 6.5, due to the secretion of red pigments into the medium under these conditions. Correspondence to: M. R. Johns     

Pigmentation and Acriflavine Resistance in Serratia marcescens

Stable, orange, acriflavine-resistant variants were selected by treatment of a wild-type, red, acriflavine-sensitive strain of Serratia marcescens with acriflavine. Visible, ultraviolet, infrared, and nuclear magnetic resonance spectra of purified pigment from the red strain were identical to those of the pigment from the orange strain, and the orange mutant was not due to a mutation affecting the structure of the pigment, prodigiosin. The color of the red strain was not affected by variations in pH between 5.0 and 8.0, whereas the color of the orange mutant changed from pink to orange over the same pH range. This variation was mimicked by the pH-induced variation in color of prodigiosin purified from either the red, wild-type or the orange, mutant strains. Density-gradient centrifugation of cell fragments after ultrasonic disintegration resulted in characteristic pigmented bands. Biochemical characterization of these pigmented bands showed that they contained pigment and a protein component, but no lipids, polysaccharides, sugars, glucosamine, or phosphates were detected. Further fractionation of these pigmented bands by zone electrophoresis on a sucrose density gradient indicated that some pigment in S. marcescens was specifically attached to protein components.     

Chlorophylle und Carotinoide der Chaetophorineae (Chlorophyceae; Ulotrichales)

In air-supplied inorganic liquid cultures, the highly differentiated green alga Fritschiella tuberosa forms only branched filaments with long slender cells. In nitrogen-deficient medium and with ageing of the cultures these cells become much shorter in length by subsequent formation of transverse walls. The chloroplasts of the slender cells contain the typical pigments of green algae. Together with the morphogenetic change to “short cells” secondary carotenoids are synthesized and stored in lipid droplets. Besides traces of lutein, violaxanthin and neoxanthin and reduced amounts of β-carotene, the following pigments have been demonstrated: Esters of astaxanthin (main pigment) and adonixanthin, canthaxanthin, echinenone and a Keto-α-carotinoid, which presumably is identical with α-doradexanthin, (3,3′-Dihydroxy-4-keto-α-Carotene) a pigment not known in plants until now. In nitrogen deficient cultures the chlorophylls are totally decomposed, the total-lipids increase by about 500%. By supplying nitrogen-deficient cultures with nitrogen and subsequent illumination regreening of the cells starts already 16 hrs later.     

Expression of a costly, plastic secondary sexual trait is correlated with age and condition in a damselfly with two male morphs

Males of the damselfly Mnais costalis Selys (Odonata: Calopterygidae) are morphologically and behaviourally polymorphic, typically existing as clear-winged non-territorial ‘sneaks’ and orange-winged territorial ‘fighters’. The amount of orange pigment in the wing, as measured with a chromameter, varied between individuals, and decreased as the reproductive season progressed. Young individuals maintained in the laboratory on high or low nutrient diets differed in the amount of pigment that developed in the wing. Males in the high nutrient group developed darker wings faster than those in the low nutrient group. Young adults of both sexes and morphs were fed ¹⁴C-radiolabelled tryptophan or tyrosine (precursors of the pigments ommochrome and melanin, respectively). Ommochrome was restricted to the pseudopterostigma of the males of both morphs and was not present in females. The presence of tyrosine in the wing cells of orange males, but not of clear males, indicated that the orange pigment is at least partly constituted from melanin. These data show that at least some pigment levels must be maintained continuously in the wings of orange males, and that maintenance is costly as it is compromised at low nutrient levels.     

Factors influencing the variability of pigments in the surface sediments of mountain lakes

1. Surface sediment biofilm samples from 82 Pyrenean lakes were analysed for marker pigment composition using high performance liquid chromatography (HPLC). 2. Variability in the pigment composition among lakes was investigated by multivariate statistical analyses using a large data set of factors describing lake chemical, physical, morphological and catchment characteristics. 3. Due to the widely varying light penetration in the lakes, the most significant gradient of pigment composition extended from a benthic to a planktonic signal. The most important pigments in the gradient were alloxanthin (cryptophytes marker pigment, planktonic signal) and diatoxanthin (diatoms marker pigment, benthic signal). The molar ratio between these two marker pigments was positively correlated with lake depth. 4. Chlorophyll‐a preservation was found to be positively related to light penetration and the development of an autothrophic biofilm on the surface sediment and negatively related to decreasing pH and the percentage of alpine meadows in the lake catchments. 5. Zooplankton marker pigments in the surface sediment, including grazing by‐products (e.g. phaeophorbides) and carotenoids (astaxanthin, canthaxanthin, echinenone) incorporated into their tissues, were correlated with the areal abundance of zooplankton. 6. Marker pigments for photosynthetic bacteria, BChl‐e and okenone, were found mainly in relatively shallow lakes with large catchments that are forested, probably because of their higher loading of allochthonous organic matter. 7. The evaluation of a preservation index (Chl‐a expressed as a percentage of a‐phorbins) and the alloxanthin/diatoxanthin ratios throughout the sediment record of mountain lakes can provide evidence of historical changes in the relative importance of planktonic versus benthic primary production and might ultimately be interpreted in terms of climatic or environmental changes.     

Reconstitution of gloeobacter rhodopsin with echinenone: role of the 4-keto group

In previous work, we reconstituted salinixanthin, the C(40)-carotenoid acyl glycoside that serves as a light-harvesting antenna to the light-driven proton pump xanthorhodopsin, into a different protein, gloeobacter rhodopsin expressed in Escherichia coli, and demonstrated that it transfers energy to the retinal chromophore [Imasheva, E. S., et al. (2009) Biochemistry 48, 10948]. The key to binding of salinixanthin was the accommodation of its ring near the retinal β-ionone ring. Here we examine two questions. Do any of the native Gloeobacter carotenoids bind to gloeobacter rhodopsin, and does the 4-keto group of the ring play a role in binding? There is no salinixanthin in Gloeobacter violaceous, but a simpler carotenoid, echinenone, also with a 4-keto group but lacking the acyl glycoside, is present in addition to β-carotene and oscillol. We show that β-carotene does not bind to gloeobacter rhodopsin, but its 4-keto derivative, echinenone, does and functions as a light-harvesting antenna. This indicates that the 4-keto group is critical for carotenoid binding. Further evidence of this is the fact that salinixanthol, an analogue of salinixanthin in which the 4-keto group is reduced to hydroxyl, does not bind and is not engaged in energy transfer. According to the crystal structure of xanthorhodopsin, the ring of salinixanthin in the binding site is turned out of the plane of the polyene conjugated chain. A similar conformation is expected for echinenone in the gloeobacter rhodopsin. We suggest that the 4-keto group in salinixanthin and echinenone allows for the twisted conformation of the ring around the C6-C7 bond and probably is engaged in an interaction that locks the carotenoid in the binding site.     

PIGMENT AND PHOTOSYNTHETIC RESPONSES OF OSCILLATORIA AGARDHII (CYANOPHYTA) TO PHOTON FLUX DENSITY AND SPECTRAL QUALITY1

The effects of photon flux density (PFD) and spectral quality on biomass, pigment content and composition, and the photosynthetic activity of Oscillatoria agardhii Gomont were investigated in steady-state populations. For alterations of PFD, chemostat populations were exposed to 50, 130 and 230 μmol photons·m^?2·s^?1 of photosynthetic active radiation (PAR). Decreases in biomass, chlorophyll a (Chl a) and c-phycocyanin (CPC) contents, and CPC: Chl a and CPC: carotenoid content was not altered. Increases in the relative abundances of myxoxanthophyll and zeaxanthin and deceases in the relative abundances of echinenone and β-carotene within the carotenoid pigments coincided with increasing PFD. Increases in Chl a-specific photosynthetic rates and maxima and decreases in biomass-specific photosynthetic rates and maxima with increasing PFD were attributed to increased light harvesting by carotenoids per unit Chl a and reduction in total pigment content, respectively. Responses to spectral quality were tested by exposing chemostat populations to a gradient of spectral transmissions at 50 μmol photons·m^?2·s^?1 PAR. Biomass differences among populations were likely attributable to the distinct absorption of the PAR spectrum by Chl a, CPC, and carotenoids. Although pigment contents were not altered by spectral quality, relative abundances of zeaxanthin and echinenone in the carotenoid pigments increased in populations exposed to high-wavelength PAR. The population adapted to green light possessed a greater photosynthetic maximum than populations adapted to other spectral qualities.     

一株产槲皮素类色素的银杏内生真菌的研究

从银杏树根中分离出70余株内生真菌.将它们置于马铃薯葡萄糖(PD)液体培养基中培养,发现其中一株(Gh01)能产生橙黄色色素.经过化学反应及HPLC检测证明该橙黄色色素为槲皮素类糖苷.这是关于内生真菌产槲皮素类糖苷的首次报道.本文深入探讨了碳源,氮源,金属离子,初始PH及培养温度对色素产量的影响.PD液体培养基的最适培养温度和初始PH分别为28℃和7.0.正交设计结果显示:最适碳源和氮源分别为20g/L葡萄糖和5g/L蛋白胨.增加1g/L的氯化锌可提高色素产量.在最适培养条件下连续培养120h色素的产量可达到27.515g/L.