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.     

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     

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.     

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.     

Multiple improvement of astaxanthin biosynthesis in Xanthophyllomyces dendrorhous by a combination of conventional mutagenesis and metabolic pathway engineering

Xanthophyllomyces dendrorhous (Phaffia rhodozyma) is the only yeast or fungus that synthesizes the commercially attractive carotenoid astaxanthin. For a suitable bioprocess, the wild type has to be modified for increasing biomass content. To achieve this, a two step strategy has been followed. At first, random mutagenesis was applied leading to colonies with substantially higher astaxanthin content. Then, the resulting strain was genetically engineered by targeting limiting reactions for further enhancement of astaxanthin biosynthesis. This combinatorial approach together with selection of an appropriate growth medium resulted in highest astaxanthin biomass contents reported to date for X. dendrorhous. In a fermenter culture, its maximum content was 9.7 mg/g dry weight.     

<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.     

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.     

Global distribution,diversity hot spots and niche transitions of an astaxanthin‐producing eukaryotic microbe

Microbes establish very diverse but still poorly understood associations with other microscopic or macroscopic organisms that do not follow the more conventional modes of competition or mutualism. Phaffia rhodozyma, an orange‐coloured yeast that produces the biotechnologically relevant carotenoid astaxanthin, exhibits a Holarctic association with birch trees in temperate forests that contrasts with the more recent finding of a South American population associated with Nothofagus (southern beech) and with stromata of its biotrophic fungal parasite Cyttaria spp. We investigated whether the association of Phaffia with Nothofagus–Cyttaria could be expanded to Australasia, the other region of the world where Nothofagus are endemic, studied the genetic structure of populations representing the known worldwide distribution of Phaffia and analysed the evolution of the association with tree hosts. The phylogenetic analysis revealed that Phaffia diversity in Australasia is much higher than in other regions of the globe and that two endemic and markedly divergent lineages seem to represent new species. The observed genetic diversity correlates with host tree genera rather than with geography, which suggests that adaptation to the different niches is driving population structure in this yeast. The high genetic diversity and endemism in Australasia indicate that the genus evolved in this region and that the association with Nothofagus is the ancestral tree association. Estimates of the divergence times of Phaffia lineages point to splits that are much more recent than the break‐up of Gondwana, supporting that long‐distance dispersal rather than vicariance is responsible for observed distribution of P. rhodozyma.     

Sterol regulatory element-binding protein Sre1 regulates carotenogenesis in the red yeast Xanthophyllomyces dendrorhous

Xanthophyllomyces dendrorhous is a basidiomycete yeast that produces carotenoids, mainly astaxanthin. Astaxanthin is an organic pigment of commercial interest due to its antioxidant and coloring properties. X. dendrorhous has a functional SREBP pathway, and the Sre1 protein is the SREBP homolog in this yeast. However, how sterol regulatory element (Sre)1 promotes the biosynthesis of sterols and carotenoids in X. dendrorhous is unknown. In this work, comparative RNA-sequencing analysis between modified X. dendrorhous strains that have an active Sre1 protein and the WT was performed to identify Sre1-dependent genes. In addition, Sre1 direct target genes were identified through ChIP combined with lambda exonuclease digestion (ChIP-exo) assays. SRE motifs were detected in the promoter regions of several Sre1 direct target genes and were consistent with the SREs described in other yeast species. Sre1 directly regulates genes related to ergosterol biosynthesis as well as genes related to the mevalonate (MVA) pathway, which synthesizes the building blocks of isoprenoids, including carotenoids. Two carotenogenic genes, crtE and crtR, were also identified as Sre1 direct target genes. Thus, carotenogenesis in X. dendrorhous is regulated by Sre1 through the regulation of the MVA pathway and the regulation of the crtE and crtR genes. As the crtR gene encodes a cytochrome P450 reductase, Sre1 regulates pathways that include cytochrome P450 enzymes, such as the biosynthesis of carotenoids and sterols. These results demonstrate that Sre1 is a sterol master regulator that is conserved in X. dendrorhous.     

Functional Characterization of the Xanthophyllomyces dendrorhous Farnesyl Pyrophosphate Synthase and Geranylgeranyl Pyrophosphate Synthase Encoding Genes That Are Involved in the Synthesis of Isoprenoid Precursors

The yeast Xanthophyllomyces dendrorhous synthesizes the carotenoid astaxanthin, which has applications in biotechnology because of its antioxidant and pigmentation properties. However, wild-type strains produce too low amounts of carotenoids to be industrially competitive. Considering this background, it is indispensable to understand how the synthesis of astaxanthin is controlled and regulated in this yeast. In this work, the steps leading to the synthesis of the carotenoid precursor geranylgeranyl pyrophosphate (GGPP, C₂₀) in X. dendrorhous from isopentenyl pyrophosphate (IPP, C₅) and dimethylallyl pyrophosphate (DMAPP, C₅) was characterized. Two prenyl transferase encoding genes, FPS and crtE, were expressed in E. coli. The enzymatic assays using recombinant E. coli protein extracts demonstrated that FPS and crtE encode a farnesyl pyrophosphate (FPP, C₁₅) synthase and a GGPP-synthase, respectively. X. dendrorhous FPP-synthase produces geranyl pyrophosphate (GPP, C₁₀) from IPP and DMAPP and FPP from IPP and GPP, while the X. dendrorhous GGPP-synthase utilizes only FPP and IPP as substrates to produce GGPP. Additionally, the FPS and crtE genes were over-expressed in X. dendrorhous, resulting in an increase of the total carotenoid production. Because the parental strain is diploid, the deletion of one of the alleles of these genes did not affect the total carotenoid production, but the composition was significantly altered. These results suggest that the over-expression of these genes might provoke a higher carbon flux towards carotenogenesis, most likely involving an earlier formation of a carotenogenic enzyme complex. Conversely, the lower carbon flux towards carotenogenesis in the deletion mutants might delay or lead to a partial formation of a carotenogenic enzyme complex, which could explain the accumulation of astaxanthin carotenoid precursors in these mutants. In conclusion, the FPS and the crtE genes represent good candidates to manipulate to favor carotenoid biosynthesis in X. dendrorhous.     

The inter-generic fungicidal activity of <Emphasis Type="Italic">Xanthophyllomyces dendrorhous</Emphasis>

In this study, the existence of intra-specific and inter-generic fungicidal activity in Xanthophyllomyces dendrorhous and Phaffia rhodozyma strains isolated from different regions of the earth was examined. Assays were performed under several culture conditions, showing that all the analyzed X. dendrorhous and P. rhodozyma strains have killing activity against Kloeckera apiculata, Rhodotorula sloffiae, and R. minuta. This activity was greater in rich media at a pH from 4.6 to 5.0. Extracellular protein extracts with fungicidal activity were obtained from cultures of all strains, and their characterization suggested that a protein of 33 kDa is the antifungal factor. According to peptide mass fingerprinting and an analysis of the results with the MASCOT search engine, this protein was identified as an aspartic protease. Additionally, extrachromosomal double-stranded DNA elements (dsDNAs) were observed in all X. dendrorhous and P. rhodozyma strains. Although there is a high variability, two dsDNAs of 5.4 and 6.8 kb are present in all strains.     

Evaluation and screening of efficient promoters to improve astaxanthin production in Xanthophyllomyces dendrorhous

Astaxanthin is a valuable carotenoid that is widely used in the aquaculture, food, pharmaceutical, and cosmetic industries. Xanthophyllomyces dendrorhous is a carotenoid-synthesizing yeast strain that produces astaxanthin as its main pigment. Although metabolic engineering using gene manipulation is a valuable way to improve astaxanthin production, a gene expression system for X. dendrorhous has been poorly developed. In this study, three known promoters of X. dendrorhous, glycerol-3-phosphate dehydrogenase (gpd) promoter (Pgpd), glucose dehydrogenase (gdh) promoter (Pgdh), and actin (act) promoter (Pact), were evaluated for use in the overexpression of target proteins using green fluorescence protein (GFP) as an expression level indicator protein. The actin promoter, Pact, showed the highest expression level of GFP when compared with Pgpd and Pgdh. Additionally, to obtain new promoters for higher expression of target protein in X. dendrorhous, intracellular GFP intensity was evaluated for 13 candidate promoters. An alcohol dehydrogenase promoter, Padh4, showed more efficient expression of GFP rather than Pact. Overexpression of crtE gene encoding rate-limiting enzyme of carotenoid synthesis under the adh4 promoter yielded an increase in intracellular astaxanthin content of about 1.7-fold compared with the control strain. The promoters identified in this study must be useful for improving carotenoids production in X. dendrorhous.     

Astaxanthin Synthesis by Yeast Xanthophyllomyces dendrorhous and its Mutants on Media Based on Plant Extracts

The study evaluated the effect of media based on plant extracts: potato, carrot and barley malt broth, on growth and astaxanthin synthesis by yeast Xanthophyllomyces dendrorhous DSM 5626 and its mutants. The carrot medium promoted carotenogenesis most effectively. In cultures on this medium the highest volumetric and cellular concentrations of astaxanthin were recorded for four out of five tested strains. Also the share of astaxanthin in the total carotenoids produced by the tested strains was the highest.     

Xanthophyllomyces dendrorhous for the industrial production of astaxanthin

Astaxanthin is a red xanthophyll (oxygenated carotenoid) with large importance in the aquaculture, pharmaceutical, and food industries. The green alga Haematococcus pluvialis and the heterobasidiomycetous yeast Xanthophyllomyces dendrorhous are currently known as the main microorganisms useful for astaxanthin production at the industrial scale. The improvement of astaxanthin titer by microbial fermentation is a requirement to be competitive with the synthetic manufacture by chemical procedures, which at present is the major source in the market. In this review, we show how the isolation of new strains of X. dendrorhous from the environment, the selection of mutants by the classical methods of random mutation and screening, and the rational metabolic engineering, have provided improved strains with higher astaxanthin productivity. To reduce production costs and enhance competitiveness from an industrial point of view, low-cost raw materials from industrial and agricultural origin have been adopted to get the maximal astaxanthin productivity. Finally, fermentation parameters have been studied in depth, both at flask and fermenter scales, to get maximal astaxanthin titers of 4.7 mg/g dry cell matter (420 mg/l) when X. dendrorhous was fermented under continuous white light. The industrial scale-up of this biotechnological process will provide a cost-effective method, alternative to synthetic astaxanthin, for the commercial exploitation of the expensive astaxanthin (about $2,500 per kilogram of pure astaxanthin).     

Crystal macropatterns in leaves of Fagaceae and Nothofagaceae: a comparative study

Summary Crystal macropatterns in Fagaceae and Nothofagaceae were examined from cleared herbarium leaves (eight genera, 122 spp.) by polarizing microscopy. Prisms and druses dominate, but previously undescribed intermediate forms occur in 25 species of Fagaceae. Sixty-two species had only prisms associated with vein orders 1–4, but smallest orders 5 and 6 had few or no crystals. In mesophyll, 98 species had small druses in spongy parenchyma and larger but fewer druses in palisade parenchyma. Subfamily Fagoideae conformed closely to this macropattern, Castaneoideae less so. Six Castaneoideae species lacked vein crystals. Two Fagoideae species and seven Nothofagus species lacked mesophyll crystals. Druses with prominent cores occur throughout Fagaceae but none were seen in Nothofagaceae. Tabular presentation of results shows significant although overlapping trends from Fagoideae to Castaneoideae to Nothofagaceae, with the latter taxon deviating most from Fagaceae.     

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.     

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.     

Enhancing astaxanthin accumulation in Xanthophyllomyces dendrorhous by a phytohormone: metabolomic and gene expression profiles

Xanthophyllomyces dendrorhous is a promising source of natural astaxanthin due to its ability to accumulate high amounts of astaxanthin. This study showed that 6-benzylaminopurine (6-BAP) is an effective substrate that enhances cell biomass and astaxanthin accumulation in X. dendrorhous. In the current study, the biomass and astaxanthin content in X. dendrorhous were determined to be improved by 21.98% and 24.20%, respectively, induced by 6-BAP treatments. To further understand the metabolic responses of X. dendrorhous to 6-BAP, time-course metabolomics and gene expression levels of X. dendrorhous cultures with and without 6-BAP feeding were investigated. Metabolome analysis revealed that 6-BAP facilitated glucose consumption, promoted the glycolysis, suppressed the TCA cycle, drove carbon flux of acetyl-CoA into fatty acid and mevalonate biosynthesis, and finally facilitated the formation of astaxanthin. ROS analysis suggested that the antioxidant mechanism in X. dendrorhous can be induced by 6-BAP. Additionally, the process of 6-BAP significantly upregulated the expression of six key genes involved in pathways related to astaxanthin biosynthesis. This research demonstrates the metabolomic mechanism of phytohormone stimulation of astaxanthin production iNn X. dendrorhous and presents a new strategy to improve astaxanthin production to prevent the dilemma of choosing between accumulation of astaxanthin and cell biomass.