TY - JOUR
AU - Johnson, Eric, A.
AB - Abstract 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 (β-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 β-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. Xanthophyllomyces dendrorhous, Phaffia rhodozyma, Carotenoid, Astaxanthin, Epicoccum nigrum 1 Introduction The heterobasidiomycetous yeast Phaffia rhodozyma was initially isolated by Herman Jan Phaff and collaborators in the late 1960's from fluxes of deciduous trees rich in sugar in mountainous regions of Japan and Alaska [1]. P. rhodozyma was unusual because it was the first reported carotenoid-producing yeast capable of fermenting sugars [2]. The uniqueness of this yeast was further enhanced when Andrewes and Starr showed that the primary carotenoid in the yeast was astaxanthin [3]. Phaff and collaborators searched for the teleomorphic stage for many years but were not successful, and it was not until 1995 that Golubev reported the perfect state in certain strains and named the teleomorph Xanthophyllomyces dendrorhous[4]. Although it is commonly assumed that all strains of this yeast should be designated as X. dendrorhous, Fell and others have found that this group of yeasts is much more complex and several phylogenetic lineages probably exist [5,6]. Certain of these strains have not shown a perfect stage and the designation P. rhodozyma should be retained for these anamorphs [5,6]. The pinkish-red carotenoid astaxanthin (3,3′-dihydroxy-β,β′-carotene-4,4′-dione) is an abundant naturally occurring carotenoid that is produced by a limited number of species of microalgae, marine bacteria and fungi [7,8]. The organisms that have attracted most industrial interest are X. dendrorhous/P. rhodozyma[3] and the fresh water alga Haematococcus pluvialis[9]. These organisms are in various stages of commercial production. In the food chain, astaxanthin accumulates in animals such as salmon, crustaceans, and certain birds, imparting a vivid pink color to their flesh, carapace, or plumage [10,11]. Aquacultural production of salmonids and crustacea has become a very important industry in many countries. However, when these animals are grown in captivity, they require astaxanthin or selected precursors of carotenoids in their feed to attain their characteristic color and consumer acceptance. Astaxanthin is the most expensive feed ingredient in the aquaculture industry [7,10]. The growth of aquaculture, particularly of salmonids, and the associated requirement for astaxanthin as a feed ingredient, has created a market for pigment that has been estimated as approximately $200 million US dollars yearly [7,10]. Traditionally astaxanthin production has been achieved by chemical synthesis [12]. However, consumer and governmental concerns regarding chemical additives in foods have stimulated research in biological systems for astaxanthin production [7]. Key limitations for industrial production by biological systems are the low yields in wild-type strains and most mutant strains, and costly extraction methods [7,13,14]. Several approaches have been applied to X. dendrorhous to increase carotenoid yields, including optimization of fermentation methodologies [15,16], mutagenesis [17,18], chemical stimulants (ethanol) [19], and genetic and metabolic engineering [20]. The genes for astaxanthin biosynthesis have been elucidated, and methods are being developed to enable genetic manipulation of X. dendrorhous[17,18,21–23]. Genes involved in biosynthesis of a variety of carotenoids have been transferred to heterologous hosts such as Escherichia coli[20], but the production levels in these hosts have been low. Thus, the natural hosts have mainly been considered for industrial processes. A novel strategy that may help to enhance carotenoid yields is stimulation by extracts of other organisms in the production media, but this has not been investigated for X. dendrorhous. Previously it has been reported that an Aspergillus isolate stimulated β-carotene production in Phycomyces blakesleeanus[24]. As described in the present study, a fortuitous fungal contaminant on an agar plate of X. dendrohous markedly stimulated astaxanthin biosynthesis. 2 Materials and methods 2.1 Yeast and fungal strains X. dendrorhous strains were obtained from EAJ's collection at the Department of Food Microbiology and Toxicology, University of Wisconsin-Madison. Most of the wild-types originated from Phaff's original collection at the University of California-Davis (UCD), while the mutants were mainly generated in our laboratory. X. dendrorhous UCD 67-385 (ATCC 24230), AF-1 (albino mutant, ATCC 96816), Yan-1 (β-carotene mutant, ATCC 96815) and CAX (astaxanthin overproducer mutant) were used in this work. The strains were maintained at −70°C in yeast–malt (YM) broth (Difco Laboratories, Detroit, MI, USA)+30% glycerol. The fungal isolate was identified as Epicoccum nigrum by cultural methods and ribosomal DNA (rDNA) sequencing as described below. 2.2 Chemicals and media YM broth and agar were obtained from Difco or were prepared in our laboratory. The components of YM are 10 g glucose, 5 g bactopeptone, 3 g yeast extract, 3 g malt extract, and 20 g agar (for plates) in 1 l distilled water. E. nigrum was cultured on YM agar, and on sporulation agar (2% agar in distilled water) to stimulate spore formation. All media were sterilized by autoclaving for 20 min at 121°C. Astaxanthin and β-carotene standards, as well as chromatographic high-performance liquid chromatography (HPLC)-grade solvents were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). 2.3 E. nigrum sporulation and visualization E. nigrum was incubated on a microscope slide coated with water agar and kept moist in a Petri dish for about a week at 21°C until black spots (conidia) appeared. Photos were obtained using an Olympus BH2-RFCA microscope with an attached Olympus C-35AD-4 camera. 2.4 X. dendrorhous growth and pigment extraction X. dendrorhous liquid cultures were grown for 120 h at 21°C in 50 ml YM broth media with continuous shaking (150 rpm) in dark conditions. Cells were pelleted by centrifuging for 3 min at 6000 rpm and washed with distilled water. Yeast cells were resuspended in 4 ml PBS buffer (0.1 M phosphate buffer, 0.1 M NaCl, pH 7.4), kept on ice for at least 10 min and broken in an AMINCO French press (American Instrument Company, Silver Springs, MD, USA), generally using three passes. The French press was set at 1500 psi, and cell disruption was checked microscopically. To extract carotenoids, a solvent mixture of acetone and hexane (1:2) was added to each sample, and after vigorous shaking for 2 min the organic phase was separated from the aqueous phase by centrifugation at 6000 rpm for 6 min. The upper organic phase was transferred to a 10-ml glass tube wrapped with aluminum foil to exclude light. This process was repeated until the pellet was colorless, usually requiring three extractions. The organic phase was dried under N2 and stored at −20°C. The same methodology was used for isolating carotenoids from E. nigrum. 2.5 Analysis of carotenoids Carotenoids were quantitatively analyzed on an HPLC (HPLX Rainin Instrument Co., Emeryville, CA, USA) using a reverse phase Alltech Econosphere C18 column (250×4.6 mm, 5 μm particle size) and an Alltech Alltima C18 5-μm guard column (Alltech Associates, Inc., Deerfield, IL, USA). The mobile phase, solvent A, consisted of 85% acetonitrile, 15% methanol, while solvent B was 100% dichloromethane. The flow rate was 1 ml min−1 and runs were 18 min in duration. A solvent gradient was run as follows: (a) 100% solvent A for 1 min; (b) linear gradient over 1.5 min to 32% solvent B; (c) isocratic with 32% solvent B for 11 min; (c) decreasing linear gradient of solvent B to 100% solvent A for 30 s; (b) and a final system reequilibration with 100% solvent A for 4 min. Astaxanthin and β-carotene HPLC peaks were monitored at 474 nm with a Dynamax UV-1 variable wavelength UV/visible absorbance detector (Rainin Instrument Co., Woburn, MA, USA). HPLC fractions eluting at 3.85 min (astaxanthin) and 10.9 min (β-carotene) were collected and evaporated to dryness under nitrogen gas. 2.6 Preparation of E. nigrum extracts for addition to X. dendrorhous cultures Inocula for the fungal fermentations were prepared by growing the E. nigrum isolate in 5 ml YM broth in a roller drum at 20°C for 3 days, and the broth containing the mycelial balls were inoculated to the production flask at 0.5% v/v. The fungal isolate was grown in 500 ml YM broth in a 1-l flask for 5 days in dark conditions with continuous shaking at 150 rpm. Following culture, the broth containing the mycelial pellets was filtered using a 0.20-μm filter apparatus (Nalge Nunc International, Rochester, NY, USA) yielding a sterile supernatant. The supernatant was concentrated by centrifuging at 2000×g using a Millipore Biomax 10 K concentrator (Millipore Corporate, Bedford, MA, USA), which gave a five-fold concentration of the initial filtrate. The final filtrate was mixed into YM to obtain 1, 5, 10, 15 or 20% v/v and X. dendrorhous was cultured in these supplemented media. 2.7 DNA isolation from E. nigrum E. nigrum was grown in tubes containing 10 ml YM broth on a roller drum at 35 rpm and 21°C for 96 h. Mycelia were collected by filtering culture broth through No. 2 Whatman filter paper (Whatman International Ltd., Maidstone, England) and washed using phosphate buffer. The mycelia were resuspended in 2 ml predigestion buffer (0.8 M KCl, 0.1 M sodium citrate, pH 5, with 7.5%β-mercaptoethanol) and incubated for 30 min with shaking in a water bath at 37°C. Predigestion buffer was removed after centrifugation, and the mycelia washed twice in 5 ml prelysis buffer (0.8 M KCl, 0.1 M sodium citrate, pH 5). Mycelia were resuspended in 1 ml lysis buffer containing 5 mg ml−1β-d-glucanase (InterSpec Products, Inc, San Mateo, CA, USA). RNase (Qiagen, Valencia, CA, USA) was added at 0.1 mg ml−1, and the mycelia incubated for 3 h at 25°C with shaking. Spheroplast formation was monitored by light microscopy. Lysis buffer was removed by centrifugation of the cell pellet at 4000 rpm for 5 min. The spheroplasts were washed twice in lysis buffer. Spheroplasts were bursted by incubation for 1 h at 50°C with gentle shaking in 1 ml TE buffer (50 mM Tris–HCl, 1 mM EDTA, pH 7.4) in which proteinase K and sodium dodecyl sulfate were present at 1 mg ml−1 and 1% (v/v), respectively. DNA was isolated in the aqueous phase after standard phenolic extraction (phenol/chloroform/isoamyl alcohol 25/24/1, pH 8). DNA was precipitated in cold 100% ethanol, and incubated overnight in 70% ethanol. DNA quality and concentration were evaluated by agarose gel electrophoresis and the 280/260 nm ratio. 2.8 Polymerase chain reaction (PCR) amplification, sequencing, and genetic analysis of E. nigrum rDNA DNA fragments encoding rDNA internal transcribed spacer (ITS) and D1/D2 sequences were amplified from the E. nigrum isolate using PCR. Yeast-specific primer designs were obtained from the literature [6] and checked for their utility in E. nigrum. PCR was performed using DNA polymerase from Eppendorf MasterMix (Eppendorf, Hamburg, Germany) following the manufacturer's instructions in a PCR system 9700 (Applied BioSystems, Foster City, CA). PCR primers were purchased from Integrated DNA Technologies, Inc. (Coralville, IA, USA). The primers selected for E. nigrum ITS amplifications were ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) [6] and the primers used for D1/D2 amplification were D1/D2-F63 (5′-GCATATCAATAAGCGGAGGAAAAG-3′) and D1/D2-LR3 (5′-GGTCCGTGTTTCAAGACGG-3′) [6]. Amplifications were performed using the following program: 94°C for 4 min followed by 10 cycles consisting of 94°C for 45 s, 50°C for 60 s, and 72°C for 45 s, followed by 20 cycles consisting of 94°C for 30 s, 55°C for 45 s, and 72°C for 45 s with a final extension of 72°C for 7 min. The PCR products were visualized in a 1% agarose gel stained with ethidium bromide. Next, PCR products were purified by enzymatic digestion with ExoSap-IT (USB Corporation, Cleveland, OH, USA) for sequencing. The nucleotide sequences were determined on both strands from at least two different PCR experiments. If the nucleotide sequences differed, more PCR products were sequenced to obtain a consensus sequence. Sequencing was performed using an ABI Prism BigDye Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA, USA) at the University of Wisconsin Biotechnology Center. The nucleotide sequences were aligned and analyzed with sequence analysis software MacVector and AssemblyLIGN (Genetics Computer Group, Madison, WI, USA) and with the DNASTAR software package (DNASTAR, Inc., Madison, WI, USA). Homology searches were performed using the web-based BLAST service provided by the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). Additionally, the E. nigrum large-subunit (LSU) rDNA region was amplified and sequenced using Applied Biosystem's MicroSeq D2 LSU rDNA Fungal Sequencing Kit. This provided approximately 300 bp of sequence data from a variable D2 region of the LSU rRNA gene. 2.9 Nucleotide sequence accession numbers The sequences determined in this paper were deposited in GenBank with the following accession numbers: AF338443 (LSU region rDNA), AY093413 (ITS region rDNA), and AY093414 (D1/D2 region of the LSU rDNA). 3 Results 3.1 E. nigrum stimulation of carotenoid biosynthesis in wild-type and mutant strains of X. dendrorhous During routine agar plating of the β-carotene mutant X. dendrorhous Yan-1, it was observed that a fungal contaminant on a 4-day-old plate increased colony pigmentation in the vicinity of the contaminant (Fig. 1). The normal yellow colonies of Yan-1 mutant assumed a pink-red coloration near to the fungal mycelium. The fungus was isolated in pure culture on YM agar from the contaminated plate. 1 Open in new tabDownload slide Representative photomicrographs of X. dendrorhous and E. nigrum, in co-culture of the two fungi. a: X. dendrorhous (UCD 67-385) cells observed at 400× magnification; b: X. dendrorhousβ-carotene mutant Yan-1 co-cultured with E. nigrum for 120 h at 21°C on YM agar; c: E. nigrum mycelia and conidia after growing on water agar for one week at 21°C. Photographs (a) and (c) were taken using an Olympus BH2-RFCA microscope with an attached Olympus C-35AD-4 camera, while photo (b) was taken with a Nikon Coolpix digital camera. 1 Open in new tabDownload slide Representative photomicrographs of X. dendrorhous and E. nigrum, in co-culture of the two fungi. a: X. dendrorhous (UCD 67-385) cells observed at 400× magnification; b: X. dendrorhousβ-carotene mutant Yan-1 co-cultured with E. nigrum for 120 h at 21°C on YM agar; c: E. nigrum mycelia and conidia after growing on water agar for one week at 21°C. Photographs (a) and (c) were taken using an Olympus BH2-RFCA microscope with an attached Olympus C-35AD-4 camera, while photo (b) was taken with a Nikon Coolpix digital camera. Changes in carotenoid pigmentation were also observed when other X. dendrorhous strains were exposed to the fungal isolate on agar plates (data not shown). To further investigate the yeast–fungus interaction on carotenoid formation, 5× concentrated culture filtrates of the fungus grown in YM were prepared. These filtrates were added to YM media at levels of 0, 1, 5, 10, 15 and 20% (v/v), and the flasks inoculated with various X. dendrorhous strains including the wild-type strain UCD 67-385, the albino mutant AF-1, the β-carotene mutant Yan-1, and the astaxanthin overproducer mutant CAX. These were grown for 5 days at 20°C. The growth rates and final cell yields of the cultures in the presence of the extract did not differ from the controls lacking filtrate. The X. dendrorhous yeasts were analyzed for specific quantity of total carotenoids (μg per gram cell dry weight (gCDW) yeast) as well as for the levels of individual pigments (Figs. 2 and 3). Astaxanthin yields of UCD 67-385 and CAX strains were markedly enhanced when grown with the fungal extracts (Fig. 2). The yields were increased in these two strains by 38% and 33%, respectively, in the cultures containing 20% (v/v) fungal extract, and to a lesser extent in cultures containing less extract (Fig. 2). The major pigments of X. dendrorhous grown in the presence of the fungal extract were astaxanthin, β-carotene, and lycopene (Fig. 3). Extracts heated for 20 min at 121°C did not stimulate carotenoid formation. 2 Open in new tabDownload slide Enhancement of astaxanthin production in X. dendrorhous by E. nigrum extracts. X. dendrorhous was grown in dark conditions at 21°C in YM containing the extracts for 120 h with continuous shaking at 50 rpm, and cells harvested, carotenoids extracted and analyzed as described in Section 2. 2 Open in new tabDownload slide Enhancement of astaxanthin production in X. dendrorhous by E. nigrum extracts. X. dendrorhous was grown in dark conditions at 21°C in YM containing the extracts for 120 h with continuous shaking at 50 rpm, and cells harvested, carotenoids extracted and analyzed as described in Section 2. 3 Open in new tabDownload slide HPLC chromatograms comparing carotenoid profiles of X. dendrorhous strains grown in YM broth (upper chromatograms) or in YM+20% (v/v) E. nigrum extract (bottom chromatograms). Astaxanthin (peak a) elutes at 3.85 min, lycopene (peak b) at 10.9 min, and β-carotene (peak c) at 14.69 min. 3 Open in new tabDownload slide HPLC chromatograms comparing carotenoid profiles of X. dendrorhous strains grown in YM broth (upper chromatograms) or in YM+20% (v/v) E. nigrum extract (bottom chromatograms). Astaxanthin (peak a) elutes at 3.85 min, lycopene (peak b) at 10.9 min, and β-carotene (peak c) at 14.69 min. Surprisingly, we observed that the β-carotene mutant (Yan-1), which under normal conditions produces no astaxanthin, synthesized low levels of astaxanthin in the presence of the fungal filtrate (Figs. 2 and 3). The Yan-1 mutant formed up to 160 μg astaxanthin gCDW−1 yeast in 20% (v/v) fungal extract, but none in media lacking extract. It was also surprising to find that the albino mutant AF-1 produced light pink color colonies on agar plates, and formed up to 47 μg astaxanthin gCDW−1 yeast when grown in 20% (v/v) extract (Figs. 2 and 3). Prior to this observation, Yan-1 or AF-1 have never been observed by our laboratory to produce astaxanthin during years of culture of these strains under various conditions. E. nigrum mycelia were colorless when grown on YM agar, but when stored on YM plates for 3–5 days at a low temperature (4°C), developed a light yellow-red color. Analysis of the cold-incubated mycelium by HPLC revealed that it contained low yields of β-carotene (45 μg gCDW−1), but did not have a trace of astaxanthin (data not shown). A polar brown pigment was also detected in the extract by chromatographic separation, but this pigment was not a carotenoid based on chromatographic, solubility, and spectroscopic properties (data not shown). 3.2 Phylogenetic analysis of the E. nigrum isolate Because of the interesting property of stimulating astaxanthin biosynthesis in X. dendrorhous, we investigated the identity of the fungus and its phylogenetic relationship to other fungi. Black spots containing numerous conidia were observed when the fungal isolate was grown for approximately one week on water agar at 21°C. The morphology of the fungal mycelium and the characteristics of the spores determined by microscopic analysis of fungal conidia suggested the identity of the fungus as E. nigrum. We investigated the phylogeny of E. nigrum by sequencing rDNA in order to confirm species identity, and its phylogenetic relationship to other fungi. The ITS region within the 5.8S rDNA locus was sequenced (GenBank accession number AY093413) and BLAST analysis was performed using GenBank to identify related fungi (see Table 1 and Fig. 4). PCR amplification of the ITS region resulted in a 496-nucleotide sequence that exactly matched several submitted sequences of Epicoccum sp. isolated from different ecosystems in Africa, America and Europe [25], while an isolate from Spain showed 97.2% identity. Additionally, the ITS sequence of Phoma epicoccina had 100% identity with that of the E. nigrum isolate, supporting the view that they may be the same biological species [25]. Other species also showed a high degree of similarity to E. nigrum, including Cerebella andropogonis (97.4%), Microsphaeropsis amaranthi (94.3%), and Phoma glomerata (93.5%). 1 Identification of fungi related to E. nigrum by ITS, LSU and D1/D2 rDNA sequence homology Open in new tab 1 Identification of fungi related to E. nigrum by ITS, LSU and D1/D2 rDNA sequence homology Open in new tab 4 Open in new tabDownload slide Phylogenetic analysis of E. nigrum compared to other fungi based on nucleotide sequences of LSU, ITS, and D1/D2 regions (see Section 2). The nucleotide sequences were aligned and analyzed by AssemblyLIGN (MacVector) and the trees were constructed using DNASTAR. The E. nigrum isolate from this study is marked with *. 4 Open in new tabDownload slide Phylogenetic analysis of E. nigrum compared to other fungi based on nucleotide sequences of LSU, ITS, and D1/D2 regions (see Section 2). The nucleotide sequences were aligned and analyzed by AssemblyLIGN (MacVector) and the trees were constructed using DNASTAR. The E. nigrum isolate from this study is marked with *. Approximately 300 nucleotides of the LSU rDNA starting at position 3334 of the D2 LSU region in the 28S rDNA gene of E. nigrum was PCR amplified and sequenced. The LSU E. nigrum rDNA sequence was registered in GenBank database under the accession number AF338443. Based on the LSU sequence, the most closely related fungal species was Mycosphaerella mycopappi followed by Ophiobolus fulgidus, Spilocaea oleaginea, and Trematosphaeria heterospora (see Table 1 and Fig. 4). The analogous sequences from P. epicoccina, P. glomerata, C. andropogonis, and M. amaranthi were not available for comparison. Finally, a 580-nucleotide-long segment of the 28S D1/D2 region was amplified by PCR. As shown in Fig. 4 and Table 1, Leptophaeria doliolum, Cochliobolus heliconeae, and Clathrospora diplospora also seem to be closely related to E. nigrum. 4 Discussion In this study it was found that a fungal contaminant on a plate of X. dendrorhous markedly affected carotenogenesis in various strains of the yeast. The fungus was isolated in pure culture and tested by various approaches for its influence on astaxanthin biosynthesis. In the presence of fungal extract astaxanthin biosynthesis was increased by nearly 40% in the wild-type UCD 67-385 and in the hyperproducer (CAX). Most interestingly, culture filtrates from growth of the fungus also stimulated astaxanthin biosynthesis in a β-carotene mutant and in an albino mutant, AF-1. These mutants had originally been isolated in our laboratory [17,18], and were believed to contain mutation(s) in the structural genes of carotenoid biosynthesis. However, the data obtained in this paper raise the intriguing possibility that mutations may lie within regulatory genes in these strains. Thus, caution must be exercised when interpreting data obtained during manipulation of these mutants and perhaps other carotenoid yeast mutants [23]. The mechanism by which E. nigrum extracts stimulated carotenoid biosynthesis in X. dendrorhous is unknown, but the elucidation of the yeast/fungus interaction could contribute to our understanding of the function of astaxanthin in the yeast and its role in X. dendrorhous/P. rhodozyma ecology. E. nigrum is a plant pathogen and is known to synthesize secondary metabolites including isoprenoids, and certain of these compounds or intermediates could affect carotenoid formation [26–28]. E. nigrum also produces pigments including flavonoids [26] and carotenoids including β-carotene, γ-carotene, rhodoxanthin and torularhodin [27,28], which share early stages of common biosynthetic pathways. Previous studies have shown that carotene precursors or substrates such as mevalonic acid enhanced astaxanthin yields in P. rhodozyma[29]. Additionally, it was reported that 0.2% (v/v) ethanol stimulated astaxanthin production in P. rhodozyma[19]. Our laboratory has demonstrated that reactive oxygen species (ROS) including singlet oxygen and peroxyl radicals (1O2, H2O2, etc.) induce astaxanthin biosynthesis in P. rhodozyma[30]. It was demonstrated that astaxanthin protected P. rhodozyma against ROS in vitro, and it was postulated that carotenoids afford protection of the yeast in its native habitats at high altitudes and associated ROS and UV exposure [31]. The present study suggests a novel function for astaxanthin, in that it may protect X. dendrorhous against antagonistic compounds produced by plant pathogens such as E. nigrum. It would be of interest to determine if fungi closely related to E. nigrum also affect carotenoid biosynthesis. Consequently, we conducted a phylogenetic analysis to identify potential candidate fungi. It is intriguing that the identified related fungi are also plant pathogens and produce secondary metabolites and oxidative enzymes. These oxidizing enzymes degrade plant cell walls, allowing access to plant nutrients such as cellulose and lignin. These degradation processes generate ROS including H2O2 and 1O2, which enhance astaxanthin yields in P. rhodozyma[30,31]. It is conceivable that oxidizing agents produced from the metabolic activities of E. nigrum and other wood-rotting fungi could stimulate astaxanthin production in X. dendrorhous/P. rhodozyma. The results presented in this study suggest the interesting possibility that fungal extracts could be used as a stimulant in industrial fermentations for production of astaxanthin or other economically valuable carotenoids, including β-carotene, lycopene, canthaxanthin, zeaxanthin, and lutein [7]. Intriguingly, it has been demonstrated that an Aspergillus sp. stimulated β-carotene production in P. blakesleeanus[24]. Similarly, it has been reported that an arbuscular mycorrhizal fungus induced the non-mevalonate pathway of isoprenoid biosynthesis in plants, with corresponding increased carotene yields [32]. In conclusion, stimulation by fungi may be a more common regulatory mechanism for carotenogenesis than has previously been recognized. Acknowledgements The authors would like to thank mycologist Dr. Harold Burdsall from the Forest Product Laboratory of USDA Forest Service for helping to characterize E. nigrum morphology. We also thank Byron Brehm-Stecher in our laboratory for his advice and fruitful collaboration. This research was supported by the USDA, sponsors of the Food Research Institute, and the College of Agriculture and Life Sciences, University of Wisconsin. 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TI - Stimulation of astaxanthin formation in the yeast Xanthophyllomyces dendrorhous by the fungus Epicoccum nigrum
JF - FEMS Yeast Research
DO - 10.1016/S1567-1356(03)00177-6
DA - 2004-01-01
UR - https://www.deepdyve.com/lp/oxford-university-press/stimulation-of-astaxanthin-formation-in-the-yeast-xanthophyllomyces-oeNruXkb5T
SP - 511
EP - 519
VL - 4
IS - 4-5
DP - DeepDyve
ER -