Effects of oxidative stress on the production of carotenoid pigments byPhaffia rhodozyma (Xanthophyllomyces dendrorhous)

The resistance to killing by free radicals of two mutants ofPhaffia rhodozyma was determined. Mutant 5–7 did not produce astaxanthin but produced β-carotene, while mutant 3–4 did not produce any carotenoid pigments. The resistance of mutant 5–7 was the same as that of the wild type but mutant 3–4 was rapidly killed. Carotenoid pigments increased the resistance to killing by free radicals. We investigated the effects of free radicals, generated by H₂O₂ and Fe²⁺ added to the medium, on wild-type cells and mutants ofP. rhodozyma. Unpigmented mutants of basidiomycetous yeasts (Rhodotorula spp. and others) are more susceptible to killing by UV-irradiation than the pigmented, wild-type strains. Therefore, we investigated the effect of free radicals on a similar basidiomycetous yeast,P. rhodozyma, a species of economic importance, in the biological production of astaxanthin.     

Astaxanthin production by <Emphasis Type="Italic">Phaffia rhodozyma</Emphasis> and <Emphasis Type="Italic">Haematococcus pluvialis</Emphasis>: a comparative study

Phaffia rhodozyma (now Xanthophyllomyces dendrorhous) and Haematococcus pluvialis are known as the major prominent microorganisms able to synthesize astaxanthin natural pigment. Important research efforts have been made to determine optimal conditions for astaxanthin synthesis. When the focus is on astaxanthin production, the maximal reported value of 9.2 mg/g cell is obtained within H. pluvialis grown on BAR medium, under continuous illumination (345 μmol photon m⁻² s⁻¹) and without aeration. Whereas fermentation by mutated R1 yeast grown on coconut milk produced 1,850 μg/g yeast. However, when looking at astaxanthin productivity, the picture is slightly different. The figures obtained with P. rhodozyma are rather similar to those of H. pluvialis. Maximal reported values are 170 μg/g yeast per day with a wild yeast strain and 370 μg/g yeast per day with mutated R1 yeast. In the case of H. pluvialis, maximal values ranged from 290 to 428 μg/g cell per day depending on the media (BG-11 or BAR), light intensity (177 μmol photon m⁻² s⁻¹), aeration, etc. The main aim of this work was to examine how astaxanthin synthesis, by P. rhodozyma and H. pluvialis, could be compared. The study is based on previous works by the authors where pigment productions have been reported.     

Biotechnological production of astaxanthin with <Emphasis Type="Italic">Phaffia rhodozyma</Emphasis>/<Emphasis Type="Italic">Xanthophyllomyces dendrorhous</Emphasis>

The oxygenated β-carotene derivative astaxanthin exhibits outstanding colouring, antioxidative and health-promoting properties and is mainly found in the marine environment. To satisfy the growing demand for this ketocarotenoid in the feed, food and cosmetics industries, there are strong efforts to develop economically viable bioprocesses alternative to the current chemical synthesis. However, up to now, natural astaxanthin from Haematococcus pluvialis, Phaffia rhodozyma or Paracoccus carotinifaciens has not been cost competitive with chemically synthesized astaxanthin, thus only serving niche applications. This review illuminates recent advances made in elucidating astaxanthin biosynthesis in P. rhodozyma. It intensely focuses on strategies to increase astaxanthin titers in the heterobasidiomycetous yeast by genetic engineering of the astaxanthin pathway, random mutagenesis and optimization of fermentation processes. This review emphasizes the potential of P. rhodozyma for the biotechnological production of astaxanthin in comparison to other natural sources such as the microalga H. pluvialis, other fungi and transgenic plants and to chemical synthesis.     

<Emphasis Type="Italic">Phaffia rhodozyma</Emphasis>: colorful odyssey

Phaffia rhodozyma was isolated by Herman Phaff in the 1960s, during his pioneering studies of yeast ecology. Initially, the yeast was isolated from limited geographical regions, but isolates were subsequently obtained from Russia, Chile, Finland, and the United States. The biological diversity of the yeast is more extensive than originally envisioned by Phaff and his collaborators, and at least two species appear to exist, including the anamorph Phaffia rhodozyma and the teleomorph Xanthophyllomyces dendrorhous. The yeast has attracted considerable biotechnological interest because of its ability to synthesize the economically important carotenoid astaxanthin (3,3-dihydroxy-, -carotene-4,4-dione) as its major pigment. This property has stimulated research on the biology of the yeast as well as development of the yeast as an industrial microorganism for astaxanthin production by fermentation. Our laboratory has isolated several mutants of the yeast affected in carotenogenesis, giving colonies a vivid array of pigmentation. We have found that nutritional and environmental conditions regulate astaxanthin biosynthesis in the yeast, and have demonstrated that astaxanthin protects P. rhodozyma from damage by reactive oxygen species. We proposed in the 1970s that P. rhodozyma could serve as an economically important pigment source in animal diets including salmonids, lobsters, and the egg yolks of chickens and quail, in order to impart characteristic and desirable colors. Although P. rhodozyma/Xanthomyces dendrorhous has been studied by various researchers for nearly 30 years, it still attracts interest from yeast biologists and biotechnologists. There is a bright and colorful outlook for P. rhodozyma/X. dendrorhous from fundamental and applied research perspectives.     

Improvement of astaxanthin production by a newly isolated Phaffia rhodozyma mutant with low-energy ion beam implantation

Aims: Isolation, characterization and identification of Phaffia sp. ZJB 00010, and improvement of astaxanthin production with low‐energy ion beam implantation. Methods and Results: A strain of ZJB 00010, capable of producing astaxanthin, was isolated and identified as Phaffia rhodozyma, based on its physiological and biochemical characteristics as well as its internal transcribed spacer (ITS) rDNA gene sequence analysis. With low‐energy ion beam implantation, this wild‐type strain was bred for improving the yield of astaxanthin. After ion beam implantation, the best mutant, E5042, was obtained. The production of astaxanthin in E5042 was 2512 μg g^?1 (dry cell weight, DCW), while the wild‐type strain was about 1114 μg g^?1 (DCW), an increase of 125·5%. Moreover, the fermentation conditions of mutant E5042 for producing astaxanthin were optimized. The astaxanthin production under the optimized conditions was upscaled and studied in a 50‐l fermentor. Conclusions: A genetically stable mutant strain with high yield of astaxanthin was obtained using low‐energy ion beam implantation. This mutant may be a suitable candidate for the industrial‐scale production of astaxanthin. Significance and Impact of the Study: Astaxanthin production in Phaffia rhodozyma could be fficiently improved by low‐energy ion beam implantation, which is a new technology in the mutant breeding of micro‐organisms. The mutant obtained in this work could potentially be utilized in industrial production of astaxanthin.     

Production and optimization of carotenoid-enriched dried distiller’s grains with solubles by <Emphasis Type="Italic">Phaffia rhodozyma</Emphasis> and <Emphasis Type="Italic">Sporobolomyces roseus</Emphasis> fermentation of whole stillage

Whole stillage—a co-product of grain-based ethanol—is used as an animal feed in the form of dried distiller’s grain with solubles (DDGS). Since animals cannot synthesize carotenoids and animal feed is generally poor in carotenoids, about 30–120 ppm of total carotenoids are added to animal feed to improve animal health, enhance meat color and quality, and increase vitamin A levels in milk and meat. The main objective of this study was to produce carotenoid (astaxanthin and β-carotene)-enriched DDGS by submerged fermentation of whole stillage. Mono- and mixed cultures of red yeasts, Phaffia rhodozyma (ATCC 24202) and Sporobolomyces roseus (ATCC 28988), were used to produce astaxanthin and β-carotene. Media optimization was carried out in shake flasks using response surface methodology (RSM). Macro ingredients, namely whole stillage, corn steep liquor and glycerol, were fitted to a second-degree polynomial in RSM. Under optimized conditions, astaxanthin and β-carotene yields in mixed culture and P. rhodozyma monoculture were 5 and 278, 97, and 275 μg/g, respectively, while S. roseus produced 278 μg/g of β-carotene. Since the carotenoid yields are almost twice the quantity used in animal feed, the carotenoid-enriched DDGS has potential application as “value-added animal feed or feed blends.”     

Reduction of fatty acid flux results in enhancement of astaxanthin synthesis in a mutant strain of Phaffia rhodozyma

A moderate-temperature mutant strain of the yeast Phaffia rhodozyma, termed MK19, was selected by 1-methyl-3-nitro-1-nitrosoguanidine (NTG) and Co60 mutagenesis. MK19 displayed fast cell growth and elevated astaxanthin content at 25°C, whereas optimal temperature for growth and astaxanthin synthesis of wild-type P. rhodozyma was 17–21°C. Optimized astaxanthin yield for MK19 after 4 days culture in shaking flask at 25°C, determined by response surface methodology, was 25.8 mg/l, which was 17-fold higher than that of the wild-type. MK19 was tolerant of high initial concentration of glucose (>100 g/l) in optimized medium. Total fatty acid content of MK19 was much lower than that of the wild-type. Acetyl-CoA is a common precursor of fatty acid and terpenoid biosynthesis, and it is possible that decreased fatty acid synthesis results in transfer of acetyl-CoA to the carotenoid biosynthetic pathway. Our results indicate that astaxanthin content is negatively correlated with fatty acid content in P. rhodozyma. Nutrient analysis showed that MK19 cells are enriched in lysine, vitamin E, and other rare nutrients, and have potential application as fish food without nutritional supplementation. This moderate-temperature mutant strain is a promising candidate for economical industrial-scale production.     

Use of alfalfa residual juice as a substrate for propagation of the red yeast Phaffia rhodozyma

Summary Alfalfa residual juice (ARJ) supported good growth of the yeast Phaffia rhodozyma but formation of astaxanthin was inhibited. Supplementary nutrients did not reverse the inhibition, indicating that the the juice probably contained some inhibitor of astaxanthin biosynthesis. Six strains of P. rhodozyma were tested and found to be susceptible to the inhibitory effects of the juice. Concentrations of ARJ above 1.25% (v/v) were inhibitory to pigmentation of the yeast. Above approximately 3.7%, total inhibition of astaxanthin formation was observed but some chromogenic components of the juice were adsorbed on Phaffia cells and appeared as artefacts in astaxanthin analyses. Phaffia biomass produced in ARJ showed greater susceptibility to autolysis than that produced in a peptone-glucose-salts medium. Supplementation of ARJ with glucose enhanced yield of cell mass and minimised the autolytic phenomenon, and is potentially useful for producing Phaffia biomass for use as a source of single cell protein.Unsupplemented brewer's malt wort and molasses, separately and in a suitable combination, were compared with ARJ and were found suitable for growth and pigmentation of P. rhodozyma.     

Increased astaxanthin production by a Phaffia rhodozyma mutant grown on date juice from Yucca fillifera

The wild strain and the astaxanthin-overproducing mutant strain 25–2 of Phaffia rhodozyma were analyzed in order to assess their ability to grow and synthesize astaxanthin in a minimal medium composed of g L⁻¹: KH₂PO₄ 2.0; MgSO₄ 0.5; CaCl₂ 0.1; urea 1.0 and supplemented with date juice of Yucca fillifera as a carbon source (yuca medium). The highest astaxanthin production (6170 μg L⁻¹) was obtained at 22.5 g L⁻¹ of reducing sugars. The addition of yeast extract to the yuca medium at concentrations of 0.5–3.0 g L⁻¹ inhibited astaxanthin synthesis. The yuca medium supported a higher production of astaxanthin, 2.5-fold more than that observed in the YM medium. Journal of Industrial Microbiology & Biotechnology (2000) 24, 187–190. Received 14 July 1999/ Accepted in revised form 02 December 1999     

Selection of Astaxanthin-Overproducing Mutants of Phaffia rhodozyma with β-Ionone

β-Ionone, an end ring analog of β-carotene, inhibits astaxanthin production in the red yeast Phaffia rhodozyma. Astaxanthin-overproducing mutants of this yeast are easily spotted on β-ionone-containing yeast malt agar plates. β-Ionone appears to block astaxanthin synthesis at the β-carotene level.     

Relationship between astaxanthin production and the intensity of anabolic processes in the yeast <Emphasis Type="Italic">Phaffia rhodozyma</Emphasis>

The astaxanthin synthesis in the yeast Phaffia rhodozyma was shown to depend on the rate of growth occurring in the first two days of cultivation. The growth rate of the yeast culture studied was preset by the cultivation conditions, among which the C : N ratio was decisive. The intense anabolic processes coupled with active culture growth during the first 24 h significantly inhibited the synthesis of the key enzymes involved in astaxanthin synthesis, which led to a marked decrease in the carotenoid production. It was demonstrated that, for the maximum yield of astaxanthin to be obtained from 1 l of nutrient medium, it is necessary to carry out cultivation, beginning with the first day, at a growth rate significantly lower than µ_max. The optimum budding rate of the mutant strain Ph. rhodozyma VKPM Y-2409 consistent with the maximum astaxanthin synthesis was determined. The specific astaxanthin productivity of the strain studied was about 7.0 mg/g of dry biomass at a budding rate of <0.5.Translated from Mikrobiologiya, Vol. 73, No. 6, 2004, pp. 751–757.Original Russian Text Copyright © 2004 by Vustin, Belykh, Kishilova.     

High concentrations of biotechnologically produced astaxanthin by lowering pH in a <Emphasis Type="Italic">Phaffia rhodozyma</Emphasis> bioprocess

Astaxanthin additions to animal diets predominantly serve as colorization aid to satisfy consumer expectations and desire for a consistent product with familiar coloration, e.g. the characteristic pink colorization of the flesh of species being produced by aquaculture. The heterobasidiomycetous yeast Phaffia rhodozyma (Xanthophyllomyces dendrorhous) can be used as natural feed source of astaxanthin. However, currently, the majority of astaxanthin used for the feed market is produced by chemical synthesis. We present a further step in direction of a competitive production of natural astaxanthin in an optimized bioprocess with non-genetically modified Phaffia rhodozyma. After medium optimization AXJ-20, a mutant strain of P. rhodozyma wild-type strain ATCC 96594, was able to grow to a cell dry weight concentration of over 114 g per kg of culture broth in a fed-batch process. In this bioprocess, where pH was lowered from 5.5 to 3.5 during the maturation phase, AXJ-20 produced the highest value reported for astaxanthin production with P. rhodozyma up to now: 0.7 g astaxanthin per kg of culture broth with a space-time-yield of 3.3 mg astaxanthin per kg of culture broth per hour. Lowering the pH during the bioprocess and increasing trace element and vitamin concentrations prevented loss of cell dry weight concentration in the maturation phase and proved to be critical for astaxanthin concentration and purity.     

Separation of astaxanthin from cells of Phaffia rhodozyma using colloidal gas aphrons in a flotation column

The aim of this study is to investigate the separation of astaxanthin from the cells of Phaffia rhodozyma using colloidal gas aphrons (CGA), which are surfactant stabilized microbubbles, in a flotation column. It was reported in previous studies that optimum recoveries are achieved at conditions that favor electrostatic interactions. Therefore, in this study, CGA generated from the cationic surfactant hexadecyl trimethyl ammonium bromide (CTAB) were applied to suspensions of cells pretreated with NaOH. The different operation modes (batch or continuous) and the effect of volumetric ratio of CGA to feed, initial concentration of feed, operating height, and flow rate of CGA on the separation of astaxanthin were investigated. The volumetric ratio was found to have a significant effect on the separation of astaxanthin for both batch and continuous experiments. Additionally, the effect of homogenization of the cells on the purity of the recovered fractions was investigated, showing that the homogenization resulted in increased purity. Moreover, different concentrations of surfactant were used for the generation of CGA for the recovery of astaxanthin on batch mode; it was found that recoveries up to 98% could be achieved using CGA generated from a CTAB solution 0.8 mM, which is below the CTAB critical micellar concentration (CMC). These results offer important information for the scale‐up of the separation of astaxanthin from the cells of P. rhodozyma using CGA. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Hydrogen peroxide-induced astaxanthin biosynthesis and catalase activity in Xanthophyllomyces dendrorhous

Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma) in shake-flask cultures was exposed to 10–20 mmol/L H₂O₂ at various culture stages, and the astaxanthin production was significantly increased by H₂O₂ fed at 0 or 24 h (exponential phase), but only slightly at 48 h (near stationary phase). The astaxanthin production was enhanced most significantly with double feeding of 10 mmol/L H₂O₂ at 0 and 24 h, reaching a cellular content of 1.30 mg/g cell and a volumetric yield of 10.4 mg/L, which were 83 and 65% higher, respectively, than those of the control (0.71 mg/g cell and 6.3 mg/L). The intracellular catalase (CAT) activity was also increased after H₂O₂ treatment. The increases in CAT and astaxanthin of cells could be detected within 4 h of H₂O₂ treatment. The increase in the astaxanthin content of cells was concomitant with a notable decrease in the β-carotene content. The older yeast cells at late culture stage (120 h), due perhaps in part to their higher astaxanthin contents, were more tolerant to H₂O₂ toxicity than the younger cells (24 h). No enhancement of the astaxanthin biosynthesis was attained when H₂O₂ was added to the yeast culture together with a sufficient amount of exogenous CAT. The results suggest that astaxanthin biosynthesis in X. dendrorhous can be stimulated by H₂O₂ as an antioxidative response.     

Influence of high-pressure homogenization,ultrasonication, and supercritical fluid on free astaxanthin extraction from β-glucanase-treated Phaffia rhodozyma cells

In this study astaxanthin production by Phaffia rhodozyma was enhanced by chemical mutation using ethyl methane sulfonate. The mutant produces a higher amount of astaxanthin than the wild yeast strain. In comparison to supercritical fluid technique, high-pressure homogenization is better for extracting astaxanthin from yeast cells. Ultrasonication of dimethyl sulfoxide, hexane, and acetone-treated cells yielded less astaxanthin than β-glucanase enzyme-treated cells. The combination of ultrasonication with β-glucanase enzyme is found to be the most efficient method of extraction among all the tested physical and chemical extraction methods. It gives a maximum yield of 435.71 ± 6.55 µg free astaxanthin per gram of yeast cell mass.     

Carotenoids of Phaffia rhodozyma, a red-pigmented fermenting yeast

The red-pigmented fermenting yeast Phaffia rhodozyma contains astaxanthin as the principal carotenoid pigment. Echinenone, 3-hydroxyechinenone and phoenicoxanthin were also isolated and identified; isocryptoxanthin and canthaxanthin were absent. Evidence is presented for a new carotenoid, 3-hydroxy-3′4′-didehydro-β,ψ-caroten-4-one. A possible biosynthetic scheme for the formation of astaxanthin in P. rhodozyma is suggested.     

Selection and evaluation of astaxanthin-overproducing mutants of Phaffia rhodozyma

Mutagenesis of Phaffia rhodozyma with NTG yielded a mutant with an astaxanthin content of 1688 g (g dry biomass)^-1, a cell yield coefficient of 0.47 on glucose and a maximum specific growth rate of 0.12 h^-1. Re-mutation of the mutant decreased the cell yield and maximum specific growth rate but increased the astaxanthin content. The use of mannitol or succinate as carbon sources enhanced pigmentation, yielding astaxanthin contents of 1973 g g^-1 and 1926 g g^-1, respectively. The use of valine as sole nitrogen source also increased astaxanthin production, but severely decreased the maximum specific growth rate and cell yield coefficient. The optimum pH for growth of P. rhodozyma was between pH 4.5 and 5.5, whereas the astaxanthin content remained constant above pH 3.     

Inhibition of the red yeast Phaffia rhodozyma by saponin

Summary Production of cell mass and astaxanthin by the yeast Phaffia rhodozyma was inhibited by saponin. The inhibition was partially reversed by oleic and linoleic acids and by ergosterol and cholesterol, thereby suggesting a possible interference of saponin with the yeast cell membrane.     

Improvement of astaxanthin production by Phaffia rhodozyma through mutation and optimization of culture conditions

Phaffia rhodozyma strains were treated with the mutagenic agent NTG several times and plated onto yeast-malt agar containing β-ionone as a selective medium. One of the NTG-treated strains (NCHU-FS301) produced considerably more astaxanthin than the parent CBS-6938 (strain NCHU-FS301 produced 1515.63 μg/g and CBS-6938 565.08 μg/l). When the kinetic parameters of the specific growth rate (μ) and specific astaxanthin productivity (q_p) were used to judge the association between growth behavior and product formation, NCHU-FS301 was shown to be a more positive growth-associated fermentation type than the parent strain. A study of the effects of the carbon source on red pigment formation revealed that glucose could support the highest total astaxanthin production (7809.3 μg/l). Yeast extract was the best nitrogen source in supporting the highest total astaxanthin formation (8637.5 μg/l). When mixed nitrogen sources were used, a mixture of yeast extract, beef extract, and potassium nitrate (1:1:1) supported more pigmentation (8052.6 μg/l) than the other mixtures tested. Astaxanthin-overproducing mutants could be useful in providing a natural source of astaxanthin for the aquacultural industry.     

Carotenoids protectPhaffia rhodozyma against singlet oxygen damage

Summary The only known habitat of the astaxanthin-containingPhaffia rhodozyma is in slime fluxes of deciduous trees at high altitudes. In this habitat, the function of carotenoids inP. rhodozyma is probably to provide protection against photogenerated antifungal substances in the tree flux such as singlet oxygen (¹O₂). To investigate the role of carotenoids inP. rhodozyma, genetic selections were employed to determine if carotenogenic yeast strains ofP. rhodozyma have enhanced ability to quench¹O₂. Singlet oxygen was generated in liquid culture by the interaction of visible light (-550 nm) with the photosensitizer rose bengal or by the activation of -terthienyl with ultraviolet light (=366 nm). In each case the treatments selected for growth of pigmented strains ofP. rhodozyma. Albino (carotenoid-less) or yellow (-carotene producing) strains grew less well in media containing¹O₂. Addition of the¹O₂ quencher sodium azide to the medium with -terthienyl allowed growth of non-pigmented strains. Since the ecological niche ofP. rhodozyma is highly specific, we investigated whether extracts of birch trees (Betula), the original source ofP. rhodozyma, contained a compound that would select for pigmented populations of the yeast. WhenP. rhodozyma strains were exposed to ethyl acetate extracts ofBetula papyrifera excited with 366 nm ultraviolet light, only pigmented cells were able to grow. These results suggest that carotenogenesis developed inP. rhodozyma in response to the presence of photoactivatable antifungal compounds produced by the host tree.This paper is dedicated to Professor Herman Jan Phaff in honor of his 50 years of active research which still continues.