Red Yeasts Research Articles

Morphological and Production Changes in Stressed Red Yeasts Monitored Using SEM and Raman Spectroscopy

Morphological and Production Changes in Stressed Red Yeasts Monitored Using SEM and Raman Spectroscopy

Proteomic analysis of Rhodotorula mucilaginosa: dealing with the issues of a non-conventional yeast.

Red yeasts ascribed to the species Rhodotorula mucilaginosa are gaining increasing attention, due to their numerous biotechnological applications, spanning carotenoid production, liquid bioremediation, heavy metal biotransformation and antifungal and plant growth-promoting actions, but also for their role as opportunistic pathogens. Nevertheless, their characterization at the 'omic' level is still scarce. Here, we applied different proteomic workflows to R. mucilaginosa with the aim of assessing their potential in generating information on proteins and functions of biotechnological interest, with a particular focus on the carotenogenic pathway. After optimization of protein extraction, we tested several gel-based (including 2D-DIGE) and gel-free sample preparation techniques, followed by tandem mass spectrometry analysis. Contextually, we evaluated different bioinformatic strategies for protein identification and interpretation of the biological significance of the dataset. When 2D-DIGE analysis was applied, not all spots returned a unambiguous identification and no carotenogenic enzymes were identified, even upon the application of different database search strategies. Then, the application of shotgun proteomic workflows with varying levels of sensitivity provided a picture of the information depth that can be reached with different analytical resources, and resulted in a plethora of information on R. mucilaginosa metabolism. However, also in these cases no proteins related to the carotenogenic pathway were identified, thus indicating that further improvements in sequence databases and functional annotations are strictly needed for increasing the outcome of proteomic analysis of this and other non-conventional yeasts. Copyright © 2016 John Wiley & Sons, Ltd.

Cloning and evaluation of different constitutive promoters in the oleaginous yeast Rhodosporidium toruloides

The oleaginous yeast Rhodosporidium toruloides is an unconventional yeast species that can accumulate a high content of lipids. Because it belongs to the basidiomycetous group of fungus, limited tools and functional elements are available for genetic engineering of R. toruloides and related red yeasts. Here we report the functional evaluation of five constitutive promoters from this yeast. We assembled a reporter gene expression cassette, consisting of a promoter, the hygromycin gene (HYG) and the nos terminator, and inserted it into the binary vector pZPK. Hygromycin-resistant transformants were obtained when R. toruloides cells were co-cultured with Agrobacterium tumefaciens AGL1 cells harbouring the engineered vector. Genomic integration of the reporter cassette was verified by successful amplification of target DNA fragments. Quantitative PCR analysis suggested that the transformant had only one copy of the reporter cassette. The strength of these promoters was demonstrated at the phenotypic level on the hygromycin-gradient plate and at the transcriptional level by real-time quantitative PCR. It was found that the strengths of these promoters varied no more than five-fold and followed a decreasing sequence of PPGI, PPGK, PFBA, PTPI, and PGPD. This study established new genetic elements for the construction of superior R. toruloides strains to produce advanced biofuels and related chemicals.

Red yeasts and carotenoid production: outlining a future for non-conventional yeasts of biotechnological interest

Carotenoids are one of the most common classes of pigments that occur in nature. Due to their biological properties, they are widely used in phytomedicine and in the chemical, pharmaceutical, cosmetic, food and feed industries. Accordingly, their global market is continuously growing, and it is expected to reach about US$1.4 billion in 2018. Carotenoids can be easily produced by chemical synthesis, although their biotechnological production is rapidly becoming an appealing alternative to the chemical route, partly due to consumer concerns against synthetic pigments. Among the yeasts, and apart from the pigmented species Phaffia rhodozyma (and its teleomorph Xanthophyllomyces dendrorhous), a handful of species of the genera Rhodosporidium, Rhodotorula, Sporobolomyces and Sporidiobolus are well known carotenoid producers. These are known as 'red yeasts', and their ability to synthesize mixtures of carotenoids from low-cost carbon sources has been broadly studied recently. Here, in agreement with the renewed interest in microbial carotenoids, the recent literature is reviewed regarding the taxonomy of the genera Rhodosporidium, Rhodotorula, Sporobolomyces and Sporidiobolus, the stress factors that influence their carotenogenesis, and the most advanced analytical tools for evaluation of carotenoid production. Moreover, a synopsis of the molecular and "-omic" tools available for elucidation of the metabolic pathways of the microbial carotenoids is reported.

Dishwashers provide a selective extreme environment for human-opportunistic yeast-like fungi

Urban life has led to the creation of human-made environments that, from a microbiological perspective, provide extreme life conditions. Certain non-ubiquitous microorganisms such as thermophilic members of the black yeast genus Exophiala are enriched within these habitats for which no counterpart is known in nature. Dishwashers consistently accumulate a number of specific black, white and red yeasts on the rubber seals of doors and in stagnant water at the interior. Several of these yeasts are primarily known as agents of human opportunistic infections. In this review, the literature data are supported by a screening study involving 937 households in 15 cities in Turkey. Fungi were detected in 230 samples (24.5 %). Using rDNA sequencing, the prevalent species were identified as Exophiala dermatitidis (n = 116), Candida parapsilosis (n = 44), E. phaeomuriformis (n = 35), Magnusiomyces capitatus (n = 22), Rhodotorula mucilaginosa (n = 15), and C. lusitaniae (n = 14). The possible role of dishwashers in transmitting disease is discussed.

Laser Target Evaporation Fe<sub>2</sub>O<sub>3</sub> Nanoparticles for Water-Based Ferrofluids for Biomedical Applications

Maghemite spherical magnetic nanoparticles (MNPs) were prepared by laser target evaporation. X-ray diffraction, transmission electron microscopy, specific surface area, and dynamic light scattering studies were performed. For water-based suspensions prepared on the basis of obtained MNPs, the zeta potential was measured. Magnetic and microwave measurements were performed both for MNPs and ferrofluids. To estimate the inductive magnetic heating of electrostatically self-stabilized or electrostatically stabilized by adsorbed citrate ions ferrofluids, magneto-inductive heating experiments were performed that showed heating efficiency. For the study of cytotoxicity and maghemite MNPs accumulation process, two non-pathogenic Exophiala nigrum (black) and its mutant strain (red) yeasts were studied. In both cases, no significant alterations of cell morphology were observed.

Two yeast species Cystobasidium psychroaquaticum f.a. sp. nov. and Cystobasidium rietchieii f.a. sp. nov. isolated from natural environments, and the transfer of Rhodotorula minuta clade members to the genus Cystobasidium.

Many species of dimorphic basidiomycetes are known only in their asexual phase and typically those pigmented in different hues of red have been classified in the large polyphyletic genus Rhodotorula. These yeasts are ubiquitous and include a few species of some clinical relevance. The phylogenetic distribution of Rhodotorula spans three classes: Microbotryomycetes, Cystobasidiomycetes and Exobasidiomycetes. Here, the presented multi-gene analyses resolved phylogenetic relationships between the second largest group of Rhodotorula and the mycoparasite Cystobasidium fimetarium (Cystobasidiales, Cystobasidiomycetes, Pucciniomycotina). Based on the results, we propose the transfer of nine species belonging to the Rhodotorula minuta clade into the genus Cystobasidium. As a result, the clinically relevant species R. minuta will be renamed Cystobasidium minutum. This proposal follows ongoing reassessments of the anamorphic genus Rhodotorula reducing the polyphyly of this genus. The delimitation of the R. minuta clade from Rhodotorula species comprised in Sporidiobolales including the type species Rhodotorula glutinis is an important step to overcome obsolete generic placements of asexual basidiomycetous yeasts. Our proposal will also help to distinguish most common red yeasts from clinical samples such as members of Sporidiobolales and Cystobasidiales. The diagnosis of the genus Cystobasidium is amended by including additional characteristics known for the related group of species. The taxonomic change enables us to classify two novel species with the phylogenetically related members of the R. minuta clade in Cystobasidium. The recently from natural environments isolated species are described here as Cystobasidium psychroaquaticum f.a. sp. nov. (K-833(T)=KBP 3881(T)=VKPM Y-3653(T)=CBS11769(T)=MUCL 52875(T)=DSM 27713(T)) and Cystobasidium rietchiei f.a. sp. nov. (K-780(T)=KBP 4220(T)=VKPM Y-3658(T)=CBS12324(T)=MUCL 53589(T)=DSM 27155(T)). The new species were registered in MycoBank under MB 809336 and MB 809337, respectively.

Production of carotenoids, ergosterol and other lipidic compounds by red yeasts cultivated on lignocellulose waste substrates

Production of carotenoids, ergosterol and other lipidic compounds by red yeasts cultivated on lignocellulose waste substrates

ISOLATION OF Phaffia rhodozyma YEASTS MUTANTS UNDER INCREASED CAROTENOID CONTENT

49 Natural carotenoid pegments play the important role in the life of the animals and humans as biostimulators, antioxydants, vitamin A substitutes, coloring and tumor inhibiting compounds. Red yeasts of the genus Phaffia are known to produce the different carotenoids presented by torulene, torularhodin and astaxanthin. The low carotenoid content in the biomass limited these yeasts in industrial exploitation. Carotenoid production by microorganisms can be increased using mutagenic treatment of the cells. Natural pigments carotenoids in most cases are presented by tetraterpenoids synthesized from eight isoprene pyrophosphate units [1]. Many red yeasts of the genera Rhodotorula, Rhodospiridium, Sporobolomyces and Phaffia are known to produce different carotenoids in natural conditions [2 4]. Carotenoids play the important role in the life of animals and humans as biostimulators, antioxidants, vitamin A substitutes, coloring and tumor inhibiting pigments. Representatives of the genera Rhodotorula and Phaffia synthesize different commercially important carotenoids (beta-carotene, torulene, toriularhodin and astaxanthin) and therefore belong to potential industrial microorganisms. Industrial exploitation of these yeasts is limited by the low carotenoid content in their biomass. The aim of the paper was the selection of the improved strains of Phaffia rhodozyma using chemical mutagenesis and the identification of individual carotenoids synthesized by isolated more pigmented mutants.

Phaeohyphomycosis caused by Microsphaeropsis arundinis: a case report

A 68-year-old woman presented with a four-year history of rheumatoid arthritis and a four-month history of erythematous plaque on the left forearm. Dermatological examination revealed a dark erythematous, ill- defined plaque measuring 5 cmx 10 cm over the left dorsal hand, wrist, and forearm, with purpura and a small amount of purulent discharge. Direct microscopic examination of the pus in 10% potassium hydroxide showed pale brown, branched and septate hyphae. Culture of pus and lesional tissue grew dark gray, velvety, confined colony on Sabouraud dextrose agar. Slide cultures on potato dextrose agar resulted in the formation of dark septate hyphae, but no spores were observed. Histopathological examination of the lesional tissue revealed infectious granuloma, and periodic acid-Schiff staining showed violet to red yeasts and pseudohyphae. Sequencing analysis of the internal transcribed spacer （ITS） region revealed a 99% consistence between the clinical isolate and reference strain of Microsphaeropsis arundini~ Based on the above findings, the patient was diagnosed with subcutaneous phaeohyphomycosis caused by M. arnndinis. She was given itraconazole 400 mg once daily orally as well as thermotherapy. Four months later, the lesion completely resolved. No recurrence was observed during one-year follow-up. Key words: Phaeohyphomycosis; Coelomycetes; Microsphaeropsis arundinis; Arthritis, rheumatoid

Response Surface Methodology for Ultrasound-Assisted Extraction of Astaxanthin from Haematococcus pluvialis

Astaxanthin is a novel carotenoid nutraceutical occurring in many crustaceans and red yeasts. It has exhibited various biological activities including prevention or amelioration of cardiovascular disease, gastric ulcer, hypertension, and diabetic nephropathy. In this study, ultrasound-assisted extraction was developed for the effective extraction of astaxanthin from Haematococcus pluvialis. Some parameters such as extraction solvent, liquid-to-solid ratio, extraction temperature, and extraction time were optimized by single-factor experiment and response surface methodology. The optimal extraction conditions were 48.0% ethanol in ethyl acetate, the liquid-to-solid ratio was 20:1 (mL/g), and extraction for 16.0 min at 41.1 °C under ultrasound irradiation of 200 W. Under optimal conditions, the yield of astaxanthin was 27.58 ± 0.40 mg/g. The results obtained are beneficial for the full utilization of Haematococcus pluvialis, which also indicated that ultrasound-assisted extraction is a very useful method for extracting astaxanthin from marine life.

Production of biomass, carotenoid and other lipid metabolites by several red yeast strains cultivated on waste glycerol from biofuel production – a comparative screening study

This study was focused on a comparison of growth and production properties of seven red yeast strains of the genus Rhodotorula, Sporobolomyces and Cystofilobasidium cultivated on glycerol substrate. Production of enriched yeast biomas and specific yeast metabolites (carotenoids, ergosterol, lipids) was evaluated on medium with glucose, pure technical glycerol and/or waste glycerol from biofuel production (40 g/L) and mixture of glycerol and glucose (1:3, 1:1, 3:1; C/N ratio 57 in all cultivations). All tested strains were able to utilize glycerol as the only carbon source. Production of biomass on waste glycerol was in most strains higher than in control as well as in medium with pure technical glycerol and reached 15.97–21.76 g/L. Production of carotenoids and ergosterol was better in glucose medium than in medium with glycerol only. Nevertheless, using glycerol medium with addition of glucose, higher yields of total carotenoids, beta-carotene and ergosterol were obtained than in control. The highest yields of total pigments were reached by Sporobolomyces roseus (3.60 mg/g cell dry weight (CDW); glycerol:glucose 1:3), Sporobolomyces salmonicolor (2.85 mg/g CDW; glycerol:glucose 1:3) and Rhodotorula glutinis (2.80 mg/g CDW; glycerol:glucose 3:1) In glucose medium, most tested strains except Cystofilobasidium capitatum (22.6 %) produced neutral lipids in the range of 11–15 %. Production of triacylglycerols in all strains was in 10–30 % better in glycerol medium, in which Rhodotorula aurantiaca and Sporobolomyces shibatanus also reached intracellular triacylglycerol concentrations up to 20 % of biomass. This study has shown that oleaginous red yeasts could have great potential for converting crude glycerol to valuable lipids and carotenoids in respect of efficient bioresources utilization.

Utilization of hydrothermally pretreated wheat straw for production of bioethanol and carotene-enriched biomass

In this work hydrothermally pretreated wheat straw was used for production of bioethanol by Saccharomyces cerevisiae and carotene-enriched biomass by red yeasts Rhodotorula glutinis, Cystofilobasidium capitatum and Sporobolomyces roseus. To evaluate the convertibility of pretreated wheat straw into ethanol, simultaneous saccharification and fermentation of S. cerevisiae was performed under semi-anaerobic conditions. The highest ethanol production efficiency of 65–66% was obtained following pretreatment at 200°C without the catalytic action of acetic acid, and at 195 and 200°C respectively in the presence of catalyst. Red yeast strain S. roseus produced 1.73–2.22mgg−1 of ergosterol on the filter cake, 1.15–4.17mgg−1 of ergosterol and 1.23–1.56mgg−1 of β-carotene on pretreated wheat straw hydrolysates and also the highest amount of carotenoids and ergosterol on untreated wheat straw (1.70 and 4.17mgg−1, respectively).

Effect of selenium on lipid alternations in pigment-forming yeasts

The work deals with lipid modifications of pigment-forming yeasts Rhodotorula and Sporobolomyces growing under presence of selenium. This metal in the medium significantly prolonged lag-phase of all cultures and enlarged yeast cells. Total, neutral, and membrane yeast lipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol) consisted of predominantly palmitic, palmitoleic, stearic, oleic, linoleic, and linolenic acids. Selenium activated fatty acid unsaturation mainly in phosphatidylcholine due to elevated levels of linoleic and linolenic acids. Because biosynthesis of C18 unsaturated fatty acids in Rhodotorula and Sporobolomyces species may be associated with phosphatidylcholine moieties, selenium might be involved to the induction of membranebound fatty acid Δ12 and Δ15 desaturases in red yeasts. Oppositely, neutral lipids (primarily triacylglycerols) did not show such intensive changes in fatty acid composition as their polar counterparts. These observations could be applied for preparation of selenized red yeasts containing carotenoid pigments with enhanced accumulation of linoleic and linolenic acids.

From crude glycerol to carotenoids by using a Rhodotorula glutinis mutant

In this work eighteen red yeasts were screened for carotenoids production on glycerol containing medium. Strain C2.5t1 of Rhodotorula glutinis, that showed the highest productivity, was UV mutagenized. Mutant 400A15, that exhibited a 280% increase in β-carotene production in respect to the parental strain, was selected. A central composite design was applied to 400A15 to optimize carotenoids and biomass productions. Regression analyses of the quadratic polynomial equations obtained (R(2)=0.87 and 0.94, for carotenoids and biomass, respectively) suggest that the models are reliable and significant (P<0.0001) in the prediction of carotenoids and biomass productions on the basis of the concentrations of crude glycerol, yeast extract and peptone. Accordingly, total carotenoids production achieved (14.07±1.45mgl(-1)) under optimized growth conditions was not statistically different from the maximal predicted (14.64±1.57mgl(-1)) (P<0.05), and it was about 100% higher than that obtained under un-optimized conditions. Therefore mutant 400A15 may represent a biocatalyst of choice for the bioconversion of crude glycerol into value-added metabolites, and a tool for the valorization of this by-product of the biodiesel industry.

Gender differences in the immune system activities of sea urchin Paracentrotus lividus

In the immune system of vertebrates, gender-specific differences in individual immune competence are well known. In general, females possess more powerful immune response than males. In invertebrates, the situation is much less clear. For this purpose we have chosen to study the immune response of the two sexes of the echinoderm Paracentrotus lividus in pre- and post-spawning phases. The coelomic fluid from the echinoderms contains several coelomocyte types and molecules involved in innate immune defenses. In this article we report that the degree of immune responses in the P. lividus differs according to sex in both pre- and post-spawning phases. We found in all tests that females were more active than males. The results indicate that females possess a significant higher number of immunocytes consisting of phagocytes and uncolored spherulocytes. Since the immunological activity is mainly based on immunocytes, it was not surprising that females possessed the highest values of cytotoxicity and hemolysis activity and showed a greater ability to uptake neutral red and phagocyte yeasts cells, while the average number of ingested particles per active phagocyte was not significantly different. Furthermore, agglutinating activity was more evident in the coelomocyte lysate and coelomic fluid of females than in those of males. Finally we found that the acidic extract of female gonads possessed greater antimicrobial activity than that of male gonads. These results make it very likely that gender differences in the immune response are not restricted to vertebrates; rather, they are a general evolutionary phenomenon.

Overcoming recalcitrant transformation and gene manipulation in Pucciniomycotina yeasts

The red yeasts of the Pucciniomycotina have rarely been transformed with DNA molecules. Transformation methods were recently developed for a species of Sporobolomyces, based on selection using uracil auxotrophs and plasmids carrying the wild-type copies of the URA3 and URA5 genes. However, these plasmids were ineffective in the transformation of closely related species. Using the genome-sequenced strain of Rhodotorula graminis as a starting point, the URA3 and URA5 genes were cloned and tested for the transformation ability into different Pucciniomycotina species by biolistic and Agrobacterium-mediated transformations. Transformation success depended on the red yeast species and the origin of the URA3 or URA5 genes, which may be related to the high G + C DNA content found in several species. A new vector was generated to confer resistance to nourseothricin, using a native promoter from R. graminis and the naturally high G + C nourseothricin acetyltransferease gene. This provides a second selectable marker in these species. Targeted gene disruption was tested in Sporobolomyces sp. IAM 13481 using different lengths of homologous DNA with biolistic and Agrobacterium transformation methods. Both DNA delivery methods were effective for targeted replacement of a gene required for carotenoid pigment biosynthesis. The constructs also triggered transgene silencing. These developments open the way to identify and manipulate gene functions in a large group of basidiomycete fungi.

Highly‐efficient colony PCR method for red yeasts and its application to identify mutations within two leucine auxotroph mutants

Red yeasts hold great promise in the production of microbial lipids and carotenoids. Genetic study of red yeasts has attracted much attention; however, rapid amplification of genes from red yeast samples remains technically challenging. Here a highly efficient method for the preparation of genomic DNA (gDNA) template, which could be directly used for PCR, was developed. Cells from colonies or liquid cultures were collected and sequentially treated by microwave, plMAN5C, proteinase K and boiling (MMPB) in a single tube to give cell lysates that were qualified as PCR templates. Single-copied gDNA fragments o up to 2.8 kb were successfully amplified. We also demonstrated successful application of this method for species in the Ascomycetes and Basidiomycetes and identification of two leucine auxotroph mutants of Rhodotorula glutinis. This method could be widely employed for the screening and genetic engineering of various yeasts.

Production of carotenoids and ergosterol from wheat straw employing red yeasts

Production of carotenoids and ergosterol from wheat straw employing red yeasts

Black microcolonial fungi as deteriogens of two famous marble statues in Florence, Italy

Blackened areas on outdoor marble statues are a significant esthetic problem due to the presence of deteriorating agents. Microcolonial black fungi, which have their natural ecological niche on rocks, play an important role in deterioration of stones used in monuments, such as marble and limestone. Black fungi were isolated from two very valuable statues exposed to the outdoor environment in Florence, Italy, the “Ratto delle Sabine” and the “Copia del David,” and these fungi were demonstrated to be responsible for the blackening areas on the statues. The black strains showed many features common to members of rock-inhabitants dematiaceous fungi. Morphological and molecular characterization, including phylogenetic analysis, indicated that the strains isolated from both statues and in different times belong to the same species and can be assigned to the rock-inhabitant genera Sarcinomyces and Phaeococcomyces. Red yeasts growing in close proximity to the black ones, with no visible effect on the statues, were also characterized on the morphological and molecular level and identified as Sporobolomyces yunnanensis.