Isolation and taxonomic study of a new canthaxanthin-containing bacterium, Gordonia jacobaea MV-1 sp. nov.

This article describes the isolation and taxonomic study of a coryneform isolate of a new Gordonia species (G. jacobaea), strain MV-1, which accumulates several carotenoids, including the ketocarotenoid trans-canthaxanthin. Identification of this new isolate by morphobiochemical methods did not allow unambiguous taxon assignment, but sequencing of the 16S rRNA gene clearly pointed to the genus Gordonia, Gordonia sputi being the closest fit. Differences in certain transversions/transitions in otherwise very well-conserved sequences of the described Gordonia species supported the proposal of this new taxon. The fact that both the best growth and best pigmentation were obtained with glucose, an inexpensive carbon source and at an industrially suitable temperature, suggests that this new bacterial strain may have good potential for the industrial production of canthaxanthin.     

Proposal of a method for the genetic transformation of Gordonia jacobaea

AIMS: Gordonia jacobaea is a recently isolated bacterial species with potential industrial application on account of its ability to store large quantities of trans-canthaxanthin. Its genetic manipulation is, however, difficult and cumbersome owing to the presence of mycolic acids in the cell wall and, especially, because of current lack of knowledge about its basic genetics. The present work describes a method for the genetic transformation of G. jacobaea. METHODS AND RESULTS: Gordonia jacobaea was grown in media supplemented with different glycine, penicillin G and isoniazid concentrations. The temperature, carbon source, growth phase and ultrasounds were analyzed for improving the method efficiency. The cells were finally transformed by electroporation. Finally, the method was applied to Brevibacteriumlactofermentum and Gordonia bronchialis. CONCLUSIONS: The growth of G. jacobaea in the presence of glycine and isoniazid is essential for obtaining electrocompetents cells. The temperature, growth phase and ultrasounds appeared as the main factors for increasing the transformation efficiency. The use of shuttle plasmids became necessary. The method described can be used with other Corynebacteria species. SIGNIFICANCE AND IMPACT OF THE STUDY: Because of the importance of the CNM group (Corynebacteria, Nocardia and Mycobacteria genera) in different areas such as industry, bioremediation improve the knowledge of their molecular mechanisms are becoming essential. The method described here improves the genetic manipulation of this group of bacteria.     

The effect of enhanced ultraviolet-B radiation on germination and seedling development of plant species occurring in a dune grassland ecosystem

The germination of seeds of seven plant species occurring in a dune grassland vegetation of the Netherlands, was studied at four levels of UV-B radiation simulating unto 45% stratospheric ozone reduction during April. With the exception of seeds of Senecio jacobaea, germination of the dune grassland species was not affected by enhanced UV-B irradiance. Although a clear UV-B fluence-response relationship was not observed, the germination rate of S. jacobaea seeds and maximal germination percentage were reduced at enhanced UV-B. Germination rate in the dark was higher than germination in the light for Oenothera biennis, Plantago lanceolata, Rumex obtusifolius and S. jacobaea. Total dry biomass accumulation of seedlings was not affected by increased UV-B radiation in any of the species tested. Clear-cut differences in UV-absorbance of methanolic extracts were observed between species. Enhanced UV-B irradiance stimulated UV-absorbance of seedling extracts of Holcus lanatus and Verbascum thapsus. A clear UV-B fluence-response relationship was observed for both species. The results indicate that germination of the studied plant species probably will not be adversely affected by the expected stratospheric ozone reduction in The Netherlands.     

Accumulation of canthaxanthin in Chlorella emersonii

A strain of Chlorella emersonii grown under high light intensity and low nitrogen degrades its chlorophyll and synthesizes canthaxanthin as the major carotenoid. Nitrogen starvation or high light alone does not induce canthaxanthin production. Norflurazon, a carotene inhibitor at the level of phytoene desaturase, inhibits production of canthaxanthin in C. emersonii with no accumulation of phytoene. Chlorella vulgaris and C. sp. 993 exposed to similar conditions do not respond in the same way as C. emersonii .     

The Effect of Salt Stress on the Production of Canthaxanthin and Astaxanthin by Chlorella zofingiensis Grown Under Limited Light Intensity

The fresh water green microalga Chlorella zofingiensisis known to accumulate ketocarotenoids – primarily astaxanthin but also canthaxanthin – when grown under stress conditions of high light irradiance and low nitrogen. We found that salt stress can replace light stress with respect to inducing carotenoid production: cells of C. zofingiensis grown under low light irradiance and subjected to salt and low nitrogen stress accumulated higher amounts of total secondary carotenoids than those growing under high light and low nitrogen stress. Furthermore, C. zofingiensis growing under conditions of salt stress and low light accumulated higher amounts of canthaxanthin than astaxanthin. It is suggested that for canthaxanthin accumulation under salt stress, light is not a limiting factor, but for astaxanthin accumulation high light irradiance is mandatory. These results may be applied in the future for the commercial production of canthaxanthin by C. zofingiensis in systems in which light availability is poor.     

Optimization of process parameters by response surface methodology and kinetic modeling for batch production of canthaxanthin by Dietzia maris NIT-D (accession number: HM151403)

Dietzia maris NIT-D, a canthaxanthin producer, was isolated during routine screening of pigment-producing bacteria. Response surface methodology was applied for statistical designing of process parameters for biomass and canthaxanthin production. The effects of four process parameters (considered as independent variables), namely temperature (10-30?°C), pH (4.75-5.75), shaker speed (75-135?rpm) and percentage inoculum (0.5-2.5?%) on the biomass and canthaxanthin yield (considered as dependent variables) were studied. As much as 122?mg?L(-1) of canthaxanthin was obtained when Dietzia maris NIT-D was incubated for 120?h at 25?°C and 120?rpm, initial pH and percentage inoculum being 5.5 and 2?% respectively. The pigment yield is the highest reported till date, with Dietzia maris as the test organism. The maximum biomass yield was 7.39?g?L(-1) under optimized process parameters. The predicted values were also verified by validation experiments in 5-day fermentation. Different mathematical models were used to describe growth and production, considering the effect of glucose in batch mode. The kinetic constants were calculated by fitting the experimental data to the models. Cell growth was inhibited beyond a glucose concentration of 15?g?L(-1). Andrews' model gave the best fit with a R (2) value of 0.9993. During the exponential growth phase, the specific growth rate was found to remain fairly constant with respect to time. There was no inhibitory effect due to intracellular product accumulation for all concentrations of glucose. This observation is the first of its kind, as previous studies have reported that increasing accumulation of intracellular carotenoid exerts greater degree of inhibition on growth.     

Maternal effects and heterosis influence the fitness of plant hybrids

Here we tested two possible nonexclusive explanations for the maintenance of a hybrid swarm between Senecio jacobaea and Senecio aquaticus; first, that genotype-by-environment interactions involving water and nutrient clines are involved in hybrid fitness, and second, heterosis in early hybrid generations may provide an initial hybrid advantage that contributes to hybrid persistence. In three climate chamber studies, fitness and root growth were measured for parental species and natural and artificial F1 hybrids, in order to determine whether hybrids occur in habitats where they are more fit than parental species. Natural hybrids, which are generally back-crossed to S. jacobaea, always equaled S. jacobaea in growth characteristics. Maternal effects played a role in the fitness of F1 hybrids, with offspring from S. jacobaea mothers exhibiting higher fitness than those from S. aquaticus mothers, and compared with parental species and natural hybrids. Natural hybrids are not distributed in zones where they are most fit with respect to nutrient and water regimes. Superior fitness of early generation hybrids may contribute to hybrid swarm stability.     

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.     

Thermotropic phase behaviour of lipid bilayers containing carotenoid pigment canthaxanthin: a differential scanning calorimetry study

In this study we address the problem of the effect of canthaxanthin on the thermotropic properties of lipid membranes formed with lipids which differ in the thickness of their hydrophobic core, size of polar heads or presence of the ester carbonyl group. For all the lipids a decrease in main transition enthalpy has been observed, indicating that canthaxanthin alters the membrane properties in its gel phase. The strongest influence of canthaxanthin on main phase transition and pretransition has been observed for the lipid having the thinnest hydrophobic region. Component analysis indicates a distinct cooperativity change, which most probably colligates with the formation of new thermotropic phases. The effect of canthaxanthin has been almost negligible in the case of phosphatidylethanolamines. The absence of the ester carbonyl group results in different thermotropic behavior, especially for low canthaxanthin concentrations. The effect of canthaxanthin is explained in terms of its organization within the membrane.     

Interactions between canthaxanthin and lipid membranes — possible mechanisms of canthaxanthin toxicity

Canthaxanthin (β, β-carotene 4, 4′ dione) is used widely as a drug or as a food and cosmetic colorant, but it may have some undesirable effects on human health, mainly caused by the formation of crystals in the macula lutea membranes of the retina. This condition is called canthaxanthin retinopathy. It has been shown that this type of dysfunction of the eye is strongly connected with damage to the blood vessels around the place of crystal deposition. This paper is a review of the experimental data supporting the hypothesis that the interactions of canthaxanthin with the lipid membranes and the aggregation of this pigment may be the factors enhancing canthaxanthin toxicity towards the macula vascular system. All the results of the experiments that have been done on model systems such as monolayers of pure canthaxanthin and mixtures of canthaxanthin and lipids, oriented bilayers or liposomes indicate a very strong effect of canthaxanthin on the physical properties of lipid membranes, which may explain its toxic action, which leads to the further development of canthaxanthin retinopathy.     

Phylogeny reconstruction in the gap between too little and too much divergence: the closest relatives of Senecio jacobaea (Asteraceae) according to DNA sequences and AFLPs

The species composition and molecular phylogeny of Senecio sect. Jacobaea (Asteraceae; Senecioneae) were studied to identify the closest relatives of Senecio jacobaea. Maximum parsimony and Bayesian inference analyses of DNA sequence data of the plastid (the trnT-L igs, the trnL intron, two parts of the trnK intron, and the psbA-trnH igs) and nuclear genome (ITS1, 5.8S, and ITS2) showed these markers to be suitable to assess the species composition of sect. Jacobaea, identifying 24 species as members of this section. Of these, nine species were not previously assigned to the section. The selected DNA sequence regions, however, showed too little sequence divergence to be optimal for phylogenetic inference within sect. Jacobaea. In contrast, AFLPs proved to be too variable to be used to study relationships between the basal lineages in sect. Jacobaea. Nonetheless, these markers are very useful to study the phylogeny of S. jacobaea and its closest relatives. The combined use of DNA sequence data and AFLPs allowed us to take a major step towards resolving phylogenetic relationships in sect. Jacobaea, identifying Senecio alpinus, Senecio pancicii and Senecio subalpinus (using DNA sequence data) or Senecio chrysanthemoides (using AFLPs) as the closest relatives of S. jacobaea.     

Response of serum carotenoid levels to dietary astaxanthin and canthaxanthin in immature rainbow trout Oncorhynchus mykiss

Rainbow trout were fed a diet supplemented with astaxanthin (89 mg/kg) or canthaxanthin (116 mg/kg) in two different experiments: experiment 1 was designed to measure the kinetics of the appearance and disappearance of carotenoids in the serum; experiment 2 was undertaken to establish the serum dose-response to synthetic astaxanthin and canthaxanthin for immature rainbow trout. The serum carotenoid concentrations of immature rainbow trout increased when fish were fed carotenoid supplemented feed and then reached a plateau after 1 day of intake for astaxanthin and after 2 days for canthaxanthin. Circulating astaxanthin represented a value 2.3 times that of canthaxanthin. After dietary supplementation was discontinued, the serum carotenoid concentrations decreased within 3 days for both carotenoids. The average decreasing slopes for the two carotenoid pigments were parallel, indicating a similarity in the rate of which astaxanthin and canthaxanthin are utilized by rainbow trout. The serum dose-response of trout that received dietary keto-carotenoids increased with increasing pigment levels. The hypothesis that absorption of dietary carotenoids in 12.5–200 mg/kg range of concentration across the gut wall may be by passive diffusion is proposed.     

Carotenoid and lipid content in muscle of Atlantic salmon, Salmo salar, transferred to seawater as 0+ or 1+ smolts

Accumulation of lipids and carotenoids, including 4'-hydroxyechinenone (4'-hydroxy-beta,beta-carotene-4-one), growth and condition factor were investigated in Atlantic salmon (Salmo salar) transferred to seawater as 0+ and 1+ smolts. Salmon were fed a diet with 30 mg/kg astaxanthin (3,3'-dihydroxy-beta,beta-carotene-4,4'-dione) and 30 mg/kg canthaxanthin (beta,beta-carotene-4,4'-dione) for 35 weeks. The 0+ smolt contained more carotenoids than the 1+ smolt when mass differences were corrected for (P<0.0001), a difference also reflected by the tristimulus colour measurements (C1E a*- and b*-values). Astaxanthin and canthaxanthin comprised more than 93% of the total carotenoids, but small differences were observed in carotenoid composition. The condition factor was significantly higher in 0+ than 1+ smolts after correction for mass differences (P<0.01). There was a high correlation between ln-transformed muscle lipid (%) and ln-transformed body mass for 0+(R2=0.94) and 1+smolts (R2=0.97). The canthaxanthin metabolite 4'-hydroxyechinenone was isolated from muscle of Atlantic salmon fed a diet supplemented with canthaxanthin. It was characterised and identified by its absorption maximum (lambda(max)=458 nm in n-hexane), mass spectrometry (M+=566) and co-chromatography with authentic standard obtained by NaBH4-reduction of canthaxanthin on thin-layer chromatography and HPLC. HPLC of the camphanates of 4'-hydroxyechinenone revealed a stereoselective transformation in favour of the (4'S)-isomer, the (4'S) and (4'R)-isomers comprising approximately 81 and 19% of the total 4'-hydroxyechinenone, respectively. The percentage of 4'-hydroxyechineone of total carotenoids ranged from 1.3 to 3.1% and declined with fish size (P<0.001). We conclude that effects of time of seawater transfer of Atlantic salmon smolts have significant effect on carotenoid accumulation and other quality traits. The detailed biochemical and physiological basis for these differences require further elucidation.     

Host-plant choice and larval growth in the cinnabar moth: do pyrrolizidine alkaloids play a role?

Witte et al. (1992) described two distinct chemotypes of Senecio jacobaea L. Asteraceae, a chemotype with jacobine as one of the major pyrrolizidine alkaloids (PAs) and a chemotype with erucifoline as one of the major PAs. We hypothesized that the presence of erucifoline might be the factor responsible for the lack of success of the cinnabar moth on Senecio erucifolius L. Asteraceae and the S. jacobaea erucifoline chemotype. We performed a survey of the distribution of the two chemotypes in the Netherlands and compared this with the distribution map of Tyria jacobaeae L. Lepidoptera, Arctiidae. The distribution of the two chemotypes in the Netherlands is poorly correlated with the distribution of the cinnabar moth. The jacobine chemotype occurs along the coast and the erucifoline chemotype predominantly inward.An oviposition experiment showed that the cinnabar moth did not discriminate between the two chemotypes of S. jacobaea and S. erucifolius. Larval performance did not differ between the two chemotypes and species. Although the distribution of S. jacobaea jacobine chemotype is loosely associated with the abundance of the cinnabar moth the oviposition and growth experiments indicate that other factors than the presence of erucifoline play a role in this association.The absence of recordings of S. erucifolius as a foodplant for the cinnabar moth might be explained by the phenology of the foodplant. Ovipositing females of the univoltine cinnabar moth prefer flowering plants for oviposition. S. erucifolius starts flowering about 1–2 month later than S. jacobaea just after the peak density of moths.     

角黄素生产技术

角黄素是类胡萝卜素的一种,可作为食品氧化剂和着色剂,在食品工业和医药中应用前景广。对角黄素的生产技术进展、应用前景作了介绍,为我国角黄素的开发研究提供参考。     

Effects of Potassium Chloride‐Induced Stress on the Carotenoids Canthaxanthin,Astaxanthin, and Lipid Accumulations in the Green Chlorococcal Microalga Strain TISTR 9500

Microalgae are a diverse group of photosynthetic eukaryotic organisms that are widely distributed globally. They are prolific sources of highly valuable compounds with fascinating chemical structures. Due to their balanced nutritional compositions and health benefits, they are increasingly being used as functional food ingredients. Carotenoid‐based pigments and polyunsaturated fatty acids (PUFAs) are examples of high‐value nutrients that can be accumulated abundantly in microalgae. Here, the effects of potassium chloride‐induced stress on the productions of lipids and carotenoids in the green microalga of the Chlorococcaceae family were investigated. Under normal BG11 medium, this green microalga strain TISTR 9500 accumulated high levels of PUFA and primary carotenoid lutein. Stress tests revealed that KCl enhanced and modulated lipid and carotenoid accumulation levels. The liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) analysis revealed that secondary carotenoids astaxanthin and canthaxanthin were robustly produced under KCl stress with the similar content of lutein. Further, this stress led to a significant increase in the total FA amount with the higher proportion of unsaturated FA than saturated FA. Thus, this green microalga could be an attractive and alternative natural biosource for canthaxanthin and astaxanthin, as well as for functional lipids.     

Carotenoids reverse multidrug resistance in cancer cells by interfering with ABC-transporters

Proteins of the ATP-binding cassette superfamily, mainly P-glycoprotein (P-gp; MDR1), play an important role in the development of multidrug resistance (MDR) in cancer cells and thus in the potential failure of chemotherapy. A selection of carotenoids (β-carotene, crocin, retinoic acid, canthaxanthin, and fucoxanthin) was investigated whether they are substrates of P-gp, and if they can reverse MDR in resistant Caco-2 and CEM/ADR5000 cells as compared to the sensitive parent cell line CCRF-CEM. The activity of ABC transporter was determined in resistant and sensitive cells by spectrofluorometry and flow cytometry using the substrates doxorubicin, rhodamine 123, and calcein as fluorescent probes. The carotenoids increased accumulation of these P-gp substrates in a dose-dependent manner indicating that they themselves also function as substrates. Fucoxanthin and canthaxanthin (50-100μM) produced a 3-5-fold higher retention of the fluorescent probes than the known competitive inhibitor verapamil. Carotenoids showed a low cytotoxicity in cells with MDR with IC(50) values between 100 and 200μM. The combination of carotenoids with eight structurally different cytotoxic agents synergistically enhanced their cytotoxicity in Caco-2 cells, probably by inhibiting the function of the ABC transporters. For example, fucoxanthin synergistically enhanced the cytotoxicity of 5-FU 53.37-fold, of vinblastine 51.01-fold, and of etoposide 12.47-fold. RT-PCR was applied to evaluate the mRNA levels of P-gp in Caco-2 cells after treatment with carotenoids. Fucoxanthin and canthaxanthin significantly decreased P-gp levels to 12% and 24%, respectively as compared to untreated control levels (p<0.001). This study implies that carotenoids may be utilised as chemosensitisers, especially as adjuvants in chemotherapy.     

A comparative study on carbohydrate reserves and ethanolic fermentation in the roots of two wetland and non‐wetland species after commencement of hypoxia

The problem of availability of storage carbohydrates (fructans and starch) in oxygen‐deficient roots was investigated in wetland species ( Senecio aquaticus Hill., Myosotis palustris [L.] L. Em. Rchb.) and compared with related non‐wetland species ( Senecio jacobaea L., Myosotis arvensis [L.] Hill.) with respect to ethanolic fermentation (PDC activity, ethanol production).
In response to 24 h of hypoxic treatment, the pyruvate decarboxylase (PDC) activity in roots increased 4‐fold in M. arvensis and S. jacobaea , 2‐fold in S. aquaticus and only slightly in M. palustris . The rise in PDC activity was accompanied by an increase in ethanol content in the roots. The increase in ethanolic fermentation in roots of intact plants was associated with a slight increase in fructose and glucose and in a clear rise in sucrose content during the first 24‐48 h after commencement of the hypoxic treatment. Following 24 h of hypoxia, the content of fructans started to increase significantly for the duration of the experiment (9 days) in the four species. Since starch content changed only slightly during this period, the fructan:starch ratio increased under low energy availability. In the roots of flooding‐tolerant Senecio aquaticus , the ratio shifted most clearly from 2:1 in normoxia to 9:1 in hypoxia. For the roots of the two wetland species investigated, the results indicate a stronger accumulation of carbohydrates accompanied by a lower increase in PDC activity under root hypoxia, when compared with the related non‐wetland species respectively.     

Selective absorption of carotenoids in the common green iguana (Iguana iguana)

In the animal kingdom, species-specific differences with regard to the absorption of intact carotenoids are observed. The causes of these differences are not entirely understood. To investigate the absorption of selected carotenoids, 20 juvenile green iguanas (Iguana iguana) were fed a carotenoid deficient basal diet for 56 days. Thereafter, the iguanas were assigned to receive a basal diet supplemented with different carotenoids (80 mg/kg diet) such as beta-carotene, canthaxanthin and apo-8'-carotenoic acid ethyl ester for 28 days. Changes in plasma carotinoid concentrations associated with the individual diets were used as indicators of carotenoid absorption. In both the experimental and control groups, only the oxygenated carotenoids (xanthophylls), lutein, zeaxanthin and canthaxanthin, were found in the plasma. Canthaxanthin and apo-8'-carotenoic acid ethyl ester were readily absorbed and recovered from the plasma. However, the supplementation of beta-carotene caused no increase in plasma beta-carotene concentration. Additionally, beta-carotene, canthaxanthin or apo-8'-carotenoic acid ethyl ester did not affect the concentrations of retinol and alpha-tocopherol in plasma. In conclusion, the study demonstrates that iguanas appear to be selective accumulators of polar xanthophylls. The iguana might, therefore, be a valuable model to investigate the selectiveness of carotenoid absorption as well as the function of xanthophylls in animals.     

Improvement of a CrtO-type of beta-carotene ketolase for canthaxanthin production in Methylomonas sp

Two types of non-homologous beta-carotene ketolases (CrtW and CrtO) were previously described. We report improvement of a CrtO-type of beta-carotene ketolase for canthaxanthin production in a methylotrophic bacterium, Methylomonas sp. 16a, which could use the C1 substrate (methane or methanol) as sole carbon and energy source. The crtO gene from Rhodococcus erythropolis was improved for canthaxanthin production in an E. coli strain engineered to produce high titer carotenoids by error-prone PCR mutagenesis followed by in vitro recombination. The best mutants from protein engineering could produce approximately 90% of total carotenoids as canthaxanthin in the high titer E. coli strain compared to approximately 20% canthaxanthin produced by the starting gene. Canthaxanthin production in Methylomonas was also significantly improved to approximately 50% of total carotenoids by the mutant genes. Further improvement of canthaxanthin production to approximately 93% in Methylomonas was achieved by increased expression of the best mutant gene. Some mutations were found in many of the improved genes, suggesting that these sites, and possibly the regions around these sites, were important for improving the crtO's activity for canthaxanthin production.