Biogeography, Host Specificity, and Molecular Phylogeny of the Basidiomycetous Yeast Phaffia rhodozyma and Its Sexual Form, Xanthophyllomyces dendrorhous

Phaffia rhodozyma (sexual form, Xanthophyllomyces dendrorhous) is a basidiomycetous yeast that has been found in tree exudates in the Northern Hemisphere at high altitudes and latitudes. This yeast produces astaxanthin, a carotenoid pigment with biotechnological importance because it is used in aquaculture for fish pigmentation. We isolated X. dendrorhous from the Southern Hemisphere (Patagonia, Argentina), where it was associated with fruiting bodies of Cyttaria hariotii, an ascomycetous parasite of Nothofagus trees. We compared internal transcribed spacer (ITS)-based phylogenies of P. rhodozyma and its tree host (Betulaceae, Corneaceae, Fagaceae, and Nothofagaceae) and found them to be generally concordant, suggesting that different yeast lineages colonize different trees and providing an explanation for the phylogenetic distance observed between the type strains of P. rhodozyma and X. dendrorhous. We hypothesize that the association of Xanthophyllomyces with Cyttaria derives from a previous association of the yeast with Nothofagus, and the sister relationship between Nothofagaceae and Betulaceae plus Fagaceae correlates with the phylogeny of X. dendrorhous strains originating from these three plant families. The two most basal strains of X. dendrorhous are those isolated from Cornus, an ancestral genus in the phylogenetic analysis of the host trees. Thus, we question previous conclusions that P. rhodozyma and X. dendrorhous represent different species since the polymorphisms detected in the ITS and intergenic spacer sequences can be attributed to intraspecific variation associated with host specificity. Our study provides a deeper understanding of Phaffia biogeography, ecology, and molecular phylogeny. Such knowledge is essential for the comprehension of many aspects of the biology of this organism and will facilitate the study of astaxanthin production within an evolutionary and ecological framework.     

Molecular diversity and intragenomic variability in the yeast genus Xanthophyllomyces: the origin of Phaffia rhodozyma?

The teleomorphic basidiomycetous yeast Xanthophyllomyces dendrorhous is important as a commercial source of astaxanthin, which is a component of feeds for mariculture. Phaffia rhodozyma is the anamorphic state of Xanthophyllomyces; however, there are conflicting reports in the literature concerning the presence of a sexual cycle in P. rhodozyma. The current study attempted to explain this enigma. Strains were obtained from the Phaff Yeast Culture Collection (University of California, Davis) and other sources in the northern hemisphere. Molecular sequences of three nuclear rDNA regions were examined: the internal transcribed spacers (ITS), intergenic spacer (IGS1) and the D1D2 region at the 5' end of the 26S gene. Different levels of genetic variability were observed in the three regions. The D1D2 differentiated major groups of strains, while an increased variability in the ITS suggested that the ITS region could be employed as an ecological marker. The greatest variability was in the IGS1 region, where strains can be defined by the presence and location of indels. Intragenomic sequence heterogeneity in the ITS and IGS1 regions led to the hypothesis that the type strain of P. rhodozyma (CBS 5905(T), UCD 67-210(T)) was derived as a mating-deficient basidiospore from the parent teleomorphic strain CBS 9090.     

Stimulation of astaxanthin formation in the yeast Xanthophyllomyces dendrorhous by the fungus Epicoccum nigrum

A fungal contaminant on an agar plate containing colonies of Xanthophyllomyces dendrorhous markedly increased carotenoid production by yeast colonies near to the fungal growth. Spent-culture filtrate from growth of the fungus in yeast-malt medium also stimulated carotenoid production by X. dendrorhous. Four X. dendrorhous strains including the wild-type UCD 67-385 (ATCC 24230), AF-1 (albino mutant, ATCC 96816), Yan-1 (beta-carotene mutant, ATCC 96815) and CAX (astaxanthin overproducer mutant) exposed to fungal concentrate extract enhanced astaxanthin up to approximately 40% per unit dry cell weight in the wild-type strain and in CAX. Interestingly, the fungal extract restored astaxanthin biosynthesis in non-astaxanthin-producing mutants previously isolated in our laboratory, including the albino and the beta-carotene mutant. The fungus was identified as Epicoccum nigrum by morphology of sporulating cultures, and the identity confirmed by genetic characterization including rDNA sequencing analysis of the large-subunit (LSU), the internal transcribed spacer, and the D1/D2 region of the LSU. These E. nigrum rDNA sequences were deposited in GenBank under accesssion numbers AF338443, AY093413 and AY093414. Systematic rDNA homology alignments were performed to identify fungi related to E. nigrum. Stimulation of carotenogenesis by E. nigrum and potentially other fungi could provide a novel method to enhance astaxanthin formation in industrial fermentations of X. dendrorhous and Phaffia rhodozyma.     

Metabolic engineering of the astaxanthin-biosynthetic pathway of Xanthophyllomyces dendrorhous

This review describes the different approaches that have been used to manipulate and improve carotenoid production in Xanthophyllomyces dendrorhous. The red yeast X. dendrorhous (formerly known as Phaffia rhodozyma) is one of the microbiological production systems for natural astaxanthin. Astaxanthin is applied in food and feed industry and can be used as a nutraceutical because of its strong antioxidant properties. However, the production levels of astaxanthin in wild-type isolates are rather low. To increase the astaxanthin content in X. dendrorhous, cultivation protocols have been optimized and astaxanthin-hyperproducing mutants have been obtained by screening of classically mutagenized X. dendrorhous strains. The knowledge about the regulation of carotenogenesis in X. dendrorhous is still limited in comparison to that in other carotenogenic fungi. The X. dendrorhous carotenogenic genes have been cloned and a X. dendrorhous transformation system has been developed. These tools allowed the directed genetic modification of the astaxanthin pathway in X. dendrorhous. The crtYB gene, encoding the bifunctional enzyme phytoene synthase/lycopene cyclase, was inactivated by insertion of a vector by single and double cross-over events, indicating that it is possible to generate specific carotenoid-biosynthetic mutants. Additionally, overexpression of crtYB resulted in the accumulation of beta-carotene and echinone, which indicates that the oxygenation reactions are rate-limiting in these recombinant strains. Furthermore, overexpression of the phytoene desaturase-encoding gene (crtI) showed an increase in monocyclic carotenoids such as torulene and HDCO (3-hydroxy-3',4'-didehydro-beta,-psi-carotene-4-one) and a decrease in bicyclic carotenoids such as echinone, beta-carotene and astaxanthin.     

Production of the UVB-absorbing compound mycosporine-glutaminol-glucoside by Xanthophyllomyces dendrorhous (Phaffia rhodozyma)

The ability of the basidiomycetous yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma) to accumulate astaxanthin is responsible for the industrial use of this yeast as a microbial source of pigments for aquaculture. It is also hypothesized that astaxanthin accounts for its ability to thrive in highly oxidative and UV-exposed habitats. Here, we assessed the ability of this species to synthesize UV-absorbing compounds generally known as mycosporines, evaluated the effect of culture media in the production of these compounds and compared its UV growth resistance and tolerance with other yeasts. The 48 wild and collection strains screened were positive for mycosporines and a unique compound identified as mycosporine-glutaminol-glucoside (MGG) was detected. Thus, the ability of X. dendrorhous to produce MGG, as described here for the first time, is so far unique among the Cystofilobasidiales. The compound was synthesized constitutively, although growth under visible light and, to a greater extent, UVA radiation stimulated its production. Strains from UV-exposed habitats produced larger quantities and oligotrophic complex media seemed to favor MGG accumulation. UV tolerance and survival of X. dendrorhous was high and comparable to that of the polyextremophilic Rhodotorula mucilaginosa. The taxonomical and ecological implications of the production of MGG by X. dendrorhous are discussed.     

Increased carotenoid production in Xanthophyllomyces dendrorhous G276 using plant extracts

The red yeast Xanthophyllomyces dendrorhous (previously named Phaffia rhodozyma) produces astaxanthin pigment among many carotenoids. The mutant X. dendrorhous G276 was isolated by chemical mutagenesis. The mutant produced about 2.0 mg of carotenoid per g of yeast cell dry weight and 8.0 mg/L of carotenoid after 5 days batch culture with YM media; in comparison, the parent strain produced 0.66 mg/g of yeast cell dry weight and a carotenoid concentration of 4.5 mg/L. We characterized the utilization of carbon sources by the mutant strain and screened various edible plant extracts to enhance the carotenoid production. The addition of Perilla frutescens (final concentration, 5%) or Allium fistulosum extracts (final concentration, 1%) enhanced the pigment production to about 32 mg/L. In a batch fermentor, addition of Perilla frutescens extract reduced the cultivation time by two days compared to control (no extract), which usually required five-day incubation to fully produce astaxanthin. The results suggest that plant extracts such as Perilla frutescens can effectively enhance astaxanthin production.     

基于动力学模型的法夫酵母发酵生产虾青素的补料策略优化

对法夫酵母的不同补料发酵方式进行了研究.基于底物抑制模型,提出了一种优化的两阶段补料策略,用于法夫酵母产虾青素的高密度发酵.在发酵的延迟期和对数生长期早期,糖浓度控制在25 g/L左右,在此条件下,生物量可以达到最大,且时间缩短.在对数生长期后期及稳定期,糖浓度控制在5 g/L,虾青素的合成时间可以有效延长.与传统的补料方式相比,采用此补料策略取得了较好的发酵效果.发酵终点细胞干重达到23.8g/L,虾青素产量达到29.05 mg/L,分别比分批发酵提高了52.8%和109%.     

Homothallic life cycle in the diploid red yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma)

Sexual activity was induced in the basidiomyceteous Phaffia rhodozyma (Xanthophyllomyces dendrorhous) by depletion of nitrogen from the culture medium. This activity involved both mating between two yeast cells and the formation of basidiospores. Mating is possibly started by a G1 phase arrest of the cell cycle, as in other yeasts. The life cycle exhibited homothallic features. Crosses between genetically marked strains, and pulse-field gel electrophoresis of the chromosomal DNA of cells derived from individual spores revealed evidence of karyogamy, meiosis and even recombination. The segregation ratio in tetrads pointed to diploid vegetative cells, which formed tetraploid zygotes and the immediate meiosis then gave rise to diploid progenies again. Apart from the type strain Phaffia rhodozyma CBS 5905, all the examined strains were able to sporulate.     

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.     

Efficient screening for astaxanthin-overproducing mutants of the yeast Xanthophyllomyces dendrorhous by flow cytometry

Astaxanthin possesses higher antioxidant activity than other carotenoids and, for this and other reasons, has great commercial potential for use in the aquaculture, pharmaceutical, and food industries. The basidiomycetous yeast Xanthophyllomyces dendrorhous is one of the best natural producers of astaxanthin, but wild-type cells accumulate only a small amount of astaxanthin. In this study, we developed an efficient flow cytometry method to screen for astaxanthin-overproducing mutants of X. dendrorhous. We first examined the relationship between cellular astaxanthin content and the intensity of fluorescence emitted from the cell. Although the fluorescence emission maximum of astaxanthin dissolved in acetone occurred at 570 nm, intracellular astaxanthin content correlated better with emission at around 675 nm in different X. dendrorhous strains. Using this emission wavelength, we screened cells mutagenized with ethyl methanesulfonate and successfully isolated mutants that produced 1.5-3.8-fold more astaxanthin than parent cells. This method enabled us to obtain overproducers five times more efficient than conventional screening from plate culture.     

Heterologous expression of pentalenene synthase (PSS) from Streptomyces UC5319 in Xanthophyllomyces dendrorhous

For the first time, the pentalenene synthase (PSS) gene from Streptomyces UC5319 was expressed in Xanthophyllomyces dendrorhous, a native producer of astaxanthin. For the expression of the gene and the concurrent knock out of the native crtE or crtYB genes, two new vectors were engineered and used for the transformation of the wild-type strain of X. dendrorhous. The transformations resulted in white colonies, showing a complete shutdown of the carotenoid production. Furthermore, an additional vector was constructed for the insertion of the PSS gene in the rDNA of the yeast. All the mutant strains produce the sesquiterpene pentalenene and show no difference in growth when compared to the wild-type strain. In this report, we demonstrate that X. dendrorhous is a suitable host for the expression of heterologous terpene cyclases and for the production of foreign terpene compounds.     

Totally chlorine-free bleaching of flax pulp

Invertase and urease are enzyme entities highly associated with the cells of the astaxanthin-producer yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma) during any stage of its cell growth cycle. In this study cellobiose was a more efficient carbon source than sucrose or its hexose counterparts for invertase expression. Extensive ultrasonication or abrasion with glass pearls were required in order to promote enzyme release. In contrast to the yeast whose growth declines above 27 degrees C, the released enzymes displayed a higher optimum temperature range when assayed in vitro. Isoforms from both enzymes could be resolved either by FPLC on DEAE-Sepharose or by an affinity approach on immobilized Concanavalin. The zymogram for invertase showed a pI somewhat less acidic than that of the similar enzyme from S. cerevisiae.     

Characterization of a novel South American population of the astaxanthin producing yeast <Emphasis Type="Italic">Xanthophyllomyces dendrorhous</Emphasis> (<Emphasis Type="Italic">Phaffia rhodozyma</Emphasis>)

A novel population of the biotechnologically important yeast Xanthophyllomyces dendrorhous, the sexual stage of Phaffia rhodozyma, has been recently isolated for the first time in the southern Hemisphere (Patagonia, Argentina). The aim of the present work was to phenotypically and genotypically characterize two representative strains of this new population, and assess such strains as a potential biotechnological source of astaxanthin, fatty acids and extracellular enzymes. Minor variations were found in physiological tests. PCR fingerprinting studies (MSP-PCR) showed the main differences between X. dendrorhous Patagonian and Type strains. Patagonian strains accumulated a xanthophyll-like pigment, which was identified as astaxanthin. These strains showed low fatty acids content (mainly polyunsaturated fatty acids) and, of a total of six extracellular enzymes tested, only produced amylase. Genetic differences between Patagonian and collection X. dendrorhous strains could be explained by geographic isolation and habitat specificity.     

Co-production of carotenoids and xylitol by Xanthophyllomyces dendrorhous (Phaffia rhodozyma)

Carotenoid production by Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma) when growing on xylose depended on the initial substrate concentration. Astaxanthin was the main carotenoid, others were echinenone, 3-hydroxyechinenone and canthaxanthin. Xylitol (an intermediate of xylose metabolism, with application as sweetener) accumulates in the fermentation media. Xylitol concentrations up to 22 g/l were obtained in experiments carried out with an initial xylose concentration of 42 g/l. Xylitol was assimilated after xylose depletion.     

Separation of strains of the yeasts Xanthophyllomyces dendrorhousand Phaffia rhodozyma based on rDNA IGS and ITS sequence analysis

The type strains of the anamorph Phaffia rhodozyma (CBS 5905) and the teleomorph Xanthophyllomyces dendrorhous (VKM Y-2786) were analyzed by nucleotide sequence analysis and compared to the sequences found in three additional strains (ATCC 24228, ATCC 24230 and CBS 6938). The results of ribosomal DNA Internal transcribed spacer (ITS) and Intergenic spacer (IGS) region analyses indicate that P. rhodozyma, which was isolated from a beech tree, is a distinct species from the other four strains. The latter that were collected from birch trees are considered to be strains of X. dendrorhous. These individual strains of X. dendrorhous, which have geographically distinct isolation sources, can be distinguished by nucleotide substitutions and deletion/insertion gaps in sub-repeat regions of the Intergenic spacer. The conclusions demonstrate that differences in the IGS region provide molecular markers for denoting strains that may differ in their biochemical and physiological capabilities. The hypothesis is presented that strain differences in the IGS region may be useful to demonstrate geographic and host specificity. Received 28 January 1999/ Accepted in revised form 17 April 1999     

Metabolic engineering of the carotenoid biosynthetic pathway in the yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma)

The crtYB locus was used as an integrative platform for the construction of specific carotenoid biosynthetic mutants in the astaxanthin-producing yeast Xanthophyllomyces dendrorhous. The crtYB gene of X. dendrorhous, encoding a chimeric carotenoid biosynthetic enzyme, could be inactivated by both single and double crossover events, resulting in non-carotenoid-producing transformants. In addition, the crtYB gene, linked to either its homologous or a glyceraldehyde-3-phosphate dehydrogenase promoter, was overexpressed in the wild type and a beta-carotene-accumulating mutant of X. dendrorhous. In several transformants containing multiple copies of the crtYB gene, the total carotenoid content was higher than in the control strain. This increase was mainly due to an increase of the beta-carotene and echinone content, whereas the total content of astaxanthin was unaffected or even lower. Overexpression of the phytoene synthase-encoding gene (crtI) had a large impact on the ratio between mono- and bicyclic carotenoids. Furthermore, we showed that in metabolic engineered X. dendrorhous strains, the competition between the enzymes phytoene desaturase and lycopene cyclase for lycopene governs the metabolic flux either via beta-carotene to astaxanthin or via 3,4-didehydrolycopene to 3-hydroxy-3'-4'-didehydro-beta-psi-caroten-4-one (HDCO). The monocylic carotenoid torulene and HDCO, normally produced as minority carotenoids, were the main carotenoids produced in these strains.     

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

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

Influence of mixed culture conditions on yeast-wall hydrolytic activity of Bacillus circulans WL-12 and on extractability of astaxanthin from the yeast Phaffia rhodozyma

O kagbue , R.N. & L ewis , M.J. 1985. Influence of mixed culture conditions on yeast-wall hydrolytic activity of Bacillus circulans WL-12 and on extractability of astaxanthin from the yeast Phaffia rhodozyma. Journal of Applied Bacteriology 59 , 243–255.
In mixed culture Bacillus circulans WL-12 hydrolysed cell walls of Phaffia rhodozyma and rendered astaxanthin extractable from the yeast. pH control was critical to survival and lytic activity of the bacillus; the optimum range was 6.2–6.8. The optimum range of temperature was 20–24C. Glucose (1–2%) was efficient in minimizing catabolite repression of the lytic enzyme complex of the bacillus. Slow-feeding of glucose improved ultimate yields of lytic enzyme but did not acclerate yeast cell wall modification. A relatively high inoculum level of B. circulans accelerated modification of P. rhodozyma in the mixed culture: when the bacterial inoculum was four times that of the yeast, over 80% of total astaxanthin was extractable in 48 h. High bacterial inoculum size also stimulated yeast autolysis and necessitated early harvest of the mixed culture. Results obtained in shake flasks were duplicated in 5-litre fermentors and suggest that the mixed culture has potential industrial value for producing a biomass containing biologically-available astaxanthin. Extractability of astaxanthin was also achieved when mixed culture filtrate was incubated with pure cultured Phaffia cells. When suitably fortified with nutrients, the filtrate also supported simultaneous yeast growth and modification of the yeast cell walls. A scheme incorporating mixed culture with B. circulans WL-12 and re-use of culture filtrate has been proposed for enzymatic processing of Phaffia rhodozyma for inclusion in animal diets.     

Simple method for the isolation of astaxanthin from the basidiomycetous yeast Phaffia rhodozyma.

A method is described for the quantitative and, possibly, large-scale extraction of astaxanthin from the yeast Phaffia rhodozyma. The method utilizes extracellular enzymes produced by the bacterium Bacillus circulans WL-12, which partially digests the yeast cell wall and renders the carotenoid pigments extractable by acetone or ethanol. Complete recovery of astaxanthin from heat-killed P. rhodozyma cells was obtained after growing B. circulans WL-12 on these yeast cells for 26 h and then extracting the yeast-bacterium mixture with acetone. A bacteria-free lytic system, which gave quantitative extraction of astaxanthin from P. rhodozyma, was obtained by concentrating the culture broth from the growth of B. circulans WL-12 on P. rhodozyma cells. Hydrolytic enzyme activities detected in this concentrate included beta-(1 leads to 3)-glucanase, beta-(1 leads to 6)-glucanase, alpha-(1 leads to 3)-glucanase, xylanase, and chitinase. The lytic system was found to work most efficiently at pH 6.5 and with low concentrations of yeast.     

烷化剂NTG诱变虾青素产生菌红法夫酵母的研究

虾青素是一种很有效的生物抗氧化剂和某些生物的天然着色剂,应用前景广阔。红法夫酵母是 生产虾青素的一个来源,优点颇多。天然菌株虾青素产量较少,缺少实用价值。实验采用烷化剂ＮＴＧ 诱变红法夫酵母,筛选出类胡萝卜素产量高的诱变株。用薄层层析对红法夫酵母产生的色素及其皂 化产物进行分析,并对各个成分的扫描光谱进行了比较。认为红法夫酵母产生的类胡萝卜素成分主 要是虾青素及虾青素二酯,还有一部分β－胡萝卜素。同时,还对虾青素产生的时相和ＢＨＴ对虾青素 光分解的保护作用进行了初步研究。