Preparation of the red yeast,Xanthophyllomyces dendrorhous,as feed additive with increased availability of astaxanthin

Xanthophyllomyces dendrorhous (Phaffia rhodozyma) is used as a colorant for aquaculture, egg yolks, and crustaceans but its carotenoids can only be absorbed by animals when its cell wall is degraded. Conditions that degraded the cell wall of X. dendrorhous were developed. To measure the degrees of cell wall degradation, the carotenoid extractability (extracted carotenoid by acetone/total carotenoid) unit was used. Treatment with HCl (0.2 M, 9 h, 90 °C) followed by neutralization to pH 3 by NaOH and spray-drying increased carotenoid extractability to 100% with minimal decomposition.     

ATP-citrate lyase activity and carotenoid production in batch cultures of Phaffia rhodozyma under nitrogen-limited and nonlimited conditions

ATP-citrate lyase (ACL) is the key cytoplasmic enzyme which supplies acetyl-CoA for fatty acids in oleaginous yeast. Although it has been suggested that fatty acid and carotenoid biosynthesis may have a common source of acetyl-CoA in Phaffia rhodozyma, the source for carotenoids is currently unknown. The purpose of this work was to analyze the development of ACL activity during batch cultures of P. rhodozyma under ammonium-limited and nonammonium-limited conditions and study its possible relationship with carotenoid synthesis. Every experiment showed carotenoid accumulation linked to an increasing ACL activity. Moreover, the ACL activity increased with dissolved oxygen (DO), i.e., ACL responded to DO in a similar way as carotenoid synthesis. Additionally, in the ammonium-limited culture, ACL activity increased upon ammonium depletion. However, the contribution to carotenoid accumulation in that case was negligible. This suggests that P. rhodozyma has developed two components of ACL, each one responsive to a different environmental stimulus, i.e., DO and ammonium depletion. The role of each component is still unknown; however, considering that the former responds to DO and the known role of carotenoids as antioxidants, it may be a provider of acetyl-CoA for carotenoid synthesis.     

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.     

Production of carotenoids byPhaffia rhodozyma grown on media composed of corn wet-milling co-products

Summary Natural isolates of the carotenoid-producing yeastPhaffia rhodozyma were analyzed for their ability to grow and to produce carotenoids in culture media composed exclusively of co-products of corn wet-milling for fuel ethanol production. FiveP. rhodozyma strains were tested for biomass produced (dry weight) and carotenoid yield. Six co-products were examined, ranging in cost from approximately $0.02 per kg to $0.11 per kg, all less expensive than conventional or agricultural growth substrates previously tested. The three co-products allowing the greatest accumulation of biomass and carotenoids byP. rhodozyma were thin stillage (TS), corn condensed distiller's solubles (CCDS) and corn gluten feed (CGF). Of the medium compositions tested, 10–15% CGF, 70% TS and 6–8% CCDS generally allowed maximum carotenoid production. Cultures grown in these three media produced up to 65%. 148% and 104% of the carotenoid yield per ml of yeast extract/malt extract (YM) cultures, respectively. Under the conditions tested, this was at an approximate medium cost of $0.67 per g carotenoids for CCDS and $0.73 per g for CGF as compared to $385.00 per g for YM. These results indicate that certain co-products of corn wet-milling can serve, at the appropriate concentration, as efficient, economical substrates for growth and carotenoid production byPhaffia rhodozyma.The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned.     

An unusual Xanthophyllomyces strain from leaves of Eucalyptus globulus in Chile

Xanthophyllomyces sp. was isolated as an epiphytic red yeast from leaves of Eucalyptus glo-bulus in Concepción, Chile. Sexual reproduction was by basidiospores produced from one or rarely two metabasidia arising from a yeast cell without preceding paedogamy. The main carotenoid pigment was astaxanthin. This isolate did not cluster with the X. dendrorhous complex (including Phaffia rhodozyma) in ITS and 26S rDNA-based phylogenetic analyses. The phylloplane may be a further habitat for Xanthophyllomyces, in addition to the well-known spring sap-flows of deciduous trees and the recently-characterised ascostromata of Cyttaria hariotii.     

Ethanol increases carotenoid production in Phaffia rhodozyma

Addition of ethanol (0.2%) to cultures of the yeast Phaffia rhodozyma increased the specific rate of carotenoid production [(carotenoid)(cell mass)⁻¹(time)⁻¹]. The incremental increase in carotenoid synthesis with ethanol was highest in carotenoid-hyperproducing strains. Ethanol increased carotenoid production when it was added at various points during the lag and active growth phases. Ethanol increased alcohol dehydrogenase and hydroxy-methyl-glutaryl-CoA (HMG-CoA) reductase activities. Our results indicate that increased carotenoid production by ethanol is associated with induction of HMG-CoA reductase and possibly activation of oxidative metabolism. Received 24 December 1996/ Accepted in revised form 27 May 1997     

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.     

<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.     

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.     

Effect of the carbon source on the carotenoid profiles of Phaffia rhodozyma strains

Phaffia rhodozyma strains ATCC 24202, ATCC 24203, ATCC 24228, ATCC 24229, ATCC 24261, NRRL Y-10921, NRRL Y-10922 and NRRL Y-17268 were grown on culture media containing glucose, sucrose or xylose as carbon sources. Carotenoids were extracted from biomass and analyzed by HPLC with diode-array detection. The carotenoid profiles depended on both the strain considered and the carbon source employed. Astaxanthin, the main pigment found in P. rhodozyma, accounted for 42–91% of total carotenoids. Other carotenoids such as canthaxanthin, echinenone, 3-hydroxyechinenone, lycopene, 4-hydroxy-3′, 4′-didehydro-β-ψ-carotene and phoenicoxanthin were detected. The highest volumetric carotenoid concentration (3.60 mg L⁻¹) was obtained with strain NRRL Y-17268 growing on xylose. In this case, astaxanthin accounted for 82% of total carotenoids. Received 29 May 1997/ Accepted in revised form 08 August 1997     

Improved growth of the red yeast, Phaffia rhodozyma (Xanthophyllomyces dendrorhous), in the presence of tricarboxylic acid cycle intermediates

Catabolites related to tricarboxylic acid cycle affected growth and carotenogenesis in Phaffia rhodozyma. Glutamate, glutamine, aspartate, asparagine and proline at 75 mM of N increased biomass from 2 g l^–1 to 2.9–4.7 g l^–1 but decreased carotenoid from 420 g g^–1 yeast to 200–260 g g^–1 yeast in strain 67-385. However, simple nitrogen sources did not decrease carotenoid formation. Tricarboxylic acid intermediates repressed carotenogenesis to a less degree than the corresponding amino acids. Carotenoid hyper-producing mutants were impaired in nitrogen utilization. These results indicated that nitrogen assimilation and the concentrations of tricarboxylic acid cycle intermediates are involved in regulation of carotenoid biosynthesis.     

Production of carotenoids by β-ionone-resistant mutant of <Emphasis Type="Italic">Xanthophyllomyces dendrorhous</Emphasis> using various carbon sources

Batch culture kinetics of the red yeast, Xanthophyllomyces dendrorhous SKKU 0107, revealed reduction in biomass with glucose and lower intracellular carotenoid content with fructose. Figures were different when compared to sucrose, which is a disaccharide of glucose and fructose. In contrast, specific growth rate constant stayed between 0.094~0.098 h⁻¹, irrespective of the carbon sources employed. Although the uptake rate of glucose was found to be 2.9-fold faster than that of fructose, sucrose was found to be a more suitable carbon source for the production of carotenoids by the studied strain. When sugar cane molasses was used, both the specific growth rate constant and the intracellular carotenoid content decreased by 27 and 17%, respectively. Compared with the batch culture using 28 g/L sugar cane molasses, fed-batch culture with the same strain resulted in a 1.45-fold higher cell yield together with a similar level of carotenoid content in X. dendrorhous SKKU 0107.     

Structure and function of the genes involved in the biosynthesis of carotenoids in the mucorales

Carotenoids are widely distributed natural pigments which are in an increasing demand by the market, due to their applications in the human food, animal feed, cosmetics, and pharmaceutical industries. Although more than 600 carotenoids have been identified in nature, only a few are industrially important (β-carotene, astaxanthin, lutein or lycopene). To date chemical processes manufacture most of the carotenoid production, but the interest for carotenoids of biological origin is growing since there is an increased public concern over the safety of artificial food colorants. Although much interest and effort has been devoted to the use of biological sources for industrially important carotenoids, only the production of biological β-carotene and astaxanthin has been reported. Among fungi, several Mucorales strains, particularlyBlakeslea trispora, have been used to develop fermentation process for the production of β-carotene on almost competitive cost-price levels. Similarly, the basidiomycetous yeastXanthophyllomyces dendrorhous (the perfect state ofPhaffia rhodozyma), has been proposed as a promising source of astaxanthin. This paper focuses on recent findings on the fungal pathways for carotenoid production, especially the structure and function of the genes involved in the biosynthesis of carotenoids in the Mucorales. An outlook of the possibilities of an increased industrial production of carotenoids, based on metabolic engineering of fungi for carotenoid content and composition, is also discussed.     

Extraction and quantitation of astaxanthin from Phaffia rhodozyma

Summary The rapid, quantitative release of astaxanthin and other carotenoids from the yeast Phaffia rhodozyma is described. Hashed cells are ruptured with dimethylsulfoxide (DMSO) and carotenoids extracted into an organic solvent. Extraction and spectrophotometric quantitation of total carotenoids is rapid, reproducible and only small volumes (0.1–2 ml) of culture are required. HPLC analysis in normal phase silica gel column indicates that astaxanthin comprises 65–95% of the total pigmented carotenoids of P. rhodozyma.     

Magnetic field as a trigger of carotenoid production by Phaffia rhodozyma

This study aimed to evaluate the influence of magnetic fields (MF) on inoculum cultivation and carotenoid production by Phaffia rhodozyma. The application of MF in the inoculum culture was evaluated (0 m T – control and 30 m T). Cellular concentration increased by 12.8 % after 24 h-culture with MF application compared to the control assay, and this was the best alternative for the preparation of inoculum. Different intervals of MF application were evaluated over 168 h. The highest volumetric carotenoids concentration was achieved by applying MF throughout cultivation, with values of 1146.39 ± 26.18 μg L⁻¹ and carotenoid productivity of 11.94 ± 1.11 μg L⁻¹ h⁻¹ in 96 h. As a result, carotenoid production increased by 59.4 % and carotenoid productivity by 99.3 %. This study is one of the first to consider MF application in carotenoid production using P. rhodozyma as a viable and low-cost alternative for carotenoid production in a shorter cultivation time.     

Comparative study of the endemic freshwater fauna of Lake Baikal-VII. Carotenoid composition of the deep-water amphipod crustaceanAcanthogammarus (Brachyuropus)grewingkii

The carotenoid composition of the deep-water gammaride is reported. Astaxanthin and their derivatives were determined to be major components of the carotenoids (58.4%). Astaxanthin-glycoside-esters comprised 21.6% of total carotenoids. A new carotenoid glycoside ester (CGE) was isolated from the deep-water gammarideAcanthogammarus (Brachyuropus)grewingkii and its structure was elucidated. The structures were determined from spectra (¹H-NMR,¹³C-NMR, MS, IR) after their isolation and quantification by means of semipreparative RP-HPLC and capillary GC-MS.     

Synthesis of carotenoids by<Emphasis Type="Italic"> Rhodotorula rubra</Emphasis> GED8 co-cultured with yogurt starter cultures in whey ultrafiltrate

Two cultures, a yeast (Rhodorula rubra GED8) and a yogurt starter (Lactobacillus bulgaricus 2–11+Streptococcus thermophilus 15HA), were selected for associated growth in whey ultrafiltrate (WU) and active synthesis of carotenoids. In associated cultivation with the yogurt culture L bulgaricus 2–11+S. thermophilus 15HA under intensive aeration (1.3 l^–1min^–1 air-flow rate) in WU (45 g lactose l^–1), initial pH 5.5, 30 °C, the lactose-negative strain R. rubra GED8 synthesized large amounts of carotenoids (13.09 mg l^–1 culture fluid). The carotenoid yield was approximately two-fold higher in association with a mixed yogurt culture than in association with pure yogurt bacteria. The major carotenoid pigments comprising the total carotenoids were -carotene (50%), torulene (12.3%) and torularhodin (35.2%). Carotenoids with a high -carotene content were produced by the microbial association 36 h earlier than by Rhodotorula yeast species. No significant differences were notd in the ratio between the pigments synthesized by R. rubra GED8+L. bulgaricus 2–11, R. rubra GED8+S. thermophilus 15HA, and R.rubra GED8+yogurt culture, despite the fact that the total carotenoid concentrations were lower in the mixed cultures with pure yogurt bacteria.     

Effect of squalene on astaxanthin production by a mutant of Phaffia rhodozyma

The effect of different sesquiterpenes on carotenoid synthesis in Phaffia rhodozyma was studied. Addition of squalene to the culture medium resulted in a decrease in the echinenone and trans-astaxanthin concentrations, whereas -carotene remained unchanged. The role of squalene as an inhibitor of ketocarotenoid synthesis in Ph. rhodozyma is discussed.     

beta-Carotene production in sugarcane molasses by a Rhodotorula glutinis mutant

Several wild strains and mutants of Rhodotorula spp. were screened for growth, carotenoid production and the proportion of -carotene produced in sugarcane molasses. A better producer, Rhodotorula glutinis mutant 32, was optimized for carotenoid production with respect to total reducing sugar (TRS) concentration and pH. In shake flasks, when molasses was used as the sole nutrient medium with 40 g l⁻¹ TRS, at pH 6, the carotenoid yield was 14 mg l⁻¹ and -carotene accounted for 70% of the total carotenoids. In a 14-l stirred tank fermenter, a 20% increase in torulene content was observed in plain molasses medium. However, by addition of yeast extract, this effect was reversed and a 31% increase in -carotene content was observed. Dissolved oxygen (DO) stat fed-batch cultivation of mutant 32 in plain molasses medium yielded 71 and 185 mg l⁻¹ total carotenoids in double- and triple-strength medium, respectively. When supplemented with yeast extract, the yields were 97 and 183 mg l⁻¹ total carotenoid with a 30% increase in -carotene and a simultaneous 40% decrease in torulene proportion. Higher cell mass was also achieved by double- and triple-strength fed-batch fermentation. Journal of Industrial Microbiology & Biotechnology (2001) 26, 327–332. Received 18 September 2000/ Accepted in revised form 02 March 2001     

The leaves of the common box,Buxus sempervirens (Buxaceae), become red as the level of a red carotenoid,anhydroeschscholtzxanthin, increases

Carotenoids from the leaves of the common box,Buxus sempervirens (Buxaceae), which turn red in late autumn to winter, were analyzed by reversed-phase HPLC. A novel carotenoid, monoanhydroeschscholtzxanthin (3), was isolated from the red-colored leaves. UV-VIS, MS,¹H-NMR and CD spectral data showed that the structure of 3 was (3S)-2′, 3′, 4′, 5′-tetradehydro-4, 5′-retro-β, β-caroten-3-ol. As well as anhydroeschscholtzxanthin (2), the major red carotenoid in the leaves, eschscholtzxanthin (4) was identified. Very small amounts of yellow carotenoids (neoxanthin, violaxanthin, lutein and β-carotene), which are major components of green leaves, were present in the red-colored leaves. The amounts of chlorophylla andb in the leaves decreased markedly during coloration, even at the early stages, whereas those of the yellow carotenoids decreased gradually. In contrast, the content of 2, a red carotenoid, increased steadily during coloration. The biosynthetic pathway of 2 inB. sempervirens was deduced tentatively on the basis of the individual carotenoid contents during autumnal coloration.