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As part of a project aimed at the selection of cold-adapted yeasts expressing biotechnologically interesting features, the extracellular enzymatic activity (EEA) of basidiomycetous yeasts isolated from glacial and subglacial waters of northwest Patagonia (Argentina) was investigated. Ninety-one basidiomycetous yeasts (belonging to the genera Cryptococcus, Leucosporidiella, Dioszegia, Mrakia, Rhodotorula, Rhodosporidium, Sporobolomyces, Sporidiobolus, Cystofilobasidium, and Udeniomyces) were screened for extracellular amylolytic, proteolytic, lipolytic, esterasic, pectinolytic, chitinolytic, and cellulolytic activities. Over 15% of the strains exhibited three or more different EEAs at 4 °C and more than 63% had at least two EEAs at the same temperature. No chitinolytic or cellulolytic activities were detected at 4 and 20 °C. Cell-free supernatants exhibited significantly higher (P < 0.01) protease and lipase activities at ≤10 °C, or even at 4 °C. In light of these findings, cold environments of Patagonia (Argentina) may be considered a potential source of cold-adapted yeasts producing industrially relevant cold-active enzymes. [ABSTRACT FROM AUTHOR]

Organisms exhibit extensive variation in ecological niche breadth, from very narrow (specialists) to very broad (generalists). Two general paradigms have been proposed to explain this variation: (i) trade-offs between performance efficiency and breadth and (ii) the joint influence of extrinsic (environmental) and intrinsic (genomic) factors. We assembled genomic, metabolic, and ecological data from nearly all known species of the ancient fungal subphylum Saccharomycotina (1154 yeast strains from 1051 species), grown in 24 different environmental conditions, to examine niche breadth evolution. We found that large differences in the breadth of carbon utilization traits between yeasts stem from intrinsic differences in genes encoding specific metabolic pathways, but we found limited evidence for trade-offs. These comprehensive data argue that intrinsic factors shape niche breadth variation in microbes.

Aureobasidium pullulans is a yeast-like fungus with remarkable phenotypic plasticity widely studied for its importance for the pharmaceutical and food industries. So far, genomic studies with strains from all over the world suggest they constitute a genetically unstructured population, with no association by habitat. However, the mechanisms by which this genome supports so many phenotypic permutations are still poorly understood. Recent works have shown the importance of sequencing yeast genomes from extreme environments to increase the repertoire of phenotypic diversity of unconventional yeasts. In this study, we present the genomic draft of A. pullulans strain from a Patagonian yeast diversity hotspot, re-evaluate its taxonomic classification based on taxogenomic approaches, and annotate its genome with high-depth transcriptomic data. Our analysis suggests this isolate could be considered a novel variant at an early stage of the speciation process. The discovery of divergent strains in a genomically homogeneous group, such as A. pullulans, can be valuable in understanding the evolution of the species. The identification and characterization of new variants will not only allow finding unique traits of biotechnological importance, but also optimize the choice of strains whose phenotypes will be characterized, providing new elements to explore questions about plasticity and adaptation., (© 2023 John Wiley & Sons Ltd.)

The Phaffia rhodozyma UCD 67-385 genome harbors a 7873 bp cluster containing DDGS , OMT, and ATPG , encoding 2-desmethy-4-deoxygadusol synthase, O-methyl transferase, and ATP-grasp ligase, respectively, of the mycosporine glutaminol (MG) biosynthesis pathway. Homozygous deletion mutants of the entire cluster, single-gene mutants, and the Δ ddgs -/- ;Δ omt -/- and Δ omt -/- ;Δ atpg -/- double-gene mutants did not produce mycosporines. However, Δ atpg -/- accumulated the intermediate 4-deoxygadusol. Heterologous expression of the DDGS and OMT or DDGS , OMT, and ATPG cDNAs in Saccharomyces cerevisiae led to 4-deoxygadusol or MG production, respectively. Genetic integration of the complete cluster into the genome of the non-mycosporine-producing CBS 6938 wild-type strain resulted in a transgenic strain (CBS 6938&#95;MYC) that produced MG and mycosporine glutaminol glucoside. These results indicate the function of DDGS , OMT, and ATPG in the mycosporine biosynthesis pathway. The transcription factor gene mutants Δ mig1 -/- , Δcyc8 -/- , and Δ opi1 -/- showed upregulation, Δ rox1 -/- and Δ skn7 -/- showed downregulation, and Δ tup6 -/- and Δ yap6 -/- showed no effect on mycosporinogenesis in glucose-containing medium. Finally, comparative analysis of the cluster sequences in several P. rhodozyma strains and the four newly described species of the genus showed the phylogenetic relationship of the P. rhodozyma strains and their differentiation from the other species of the genus Phaffia .

Species is the fundamental unit to quantify biodiversity. In recent years, the model yeast Saccharomyces cerevisiae has seen an increased number of studies related to its geographical distribution, population structure, and phenotypic diversity. However, seven additional species from the same genus have been less thoroughly studied, which has limited our understanding of the macroevolutionary events leading to the diversification of this genus over the last 20 million years. Here, we show the geographies, hosts, substrates, and phylogenetic relationships for approximately 1,800 Saccharomyces strains, covering the complete genus with unprecedented breadth and depth. We generated and analyzed complete genome sequences of 163 strains and phenotyped 128 phylogenetically diverse strains. This dataset provides insights about genetic and phenotypic diversity within and between species and populations, quantifies reticulation and incomplete lineage sorting, and demonstrates how gene flow and selection have affected traits, such as galactose metabolism. These findings elevate the genus Saccharomyces as a model to understand biodiversity and evolution in microbial eukaryotes., (© 2023. The Author(s).)

The brewing activity in Andean Patagonia plays a very important role in the region's economy, being microbial contamination one of the main problems in terms of quality. The presence of contaminant bacteria and wild yeasts in beer generate microbiological, physical and chemical changes that impact on its sensory attributes. However, few breweries establish criteria and policies to guarantee the quality of their products in a microbiological sense. The purpose of this work was to study for the first time the incidence of microbial contaminants in bottled craft beers from Andean Patagonia, identify the main microorganisms involved and establish relationships between contamination and the physicochemical variables of beer. We analyzed 75 beers from 37 breweries from 12 different Patagonian cities. Our results showed that 69.3% of the analyzed beer exhibited contaminant microorganism growth. Bacteria Levilactobacillus brevis and wild yeasts of Saccharomyces were the main microorganisms responsible for these contaminations. In addition, we found that microbial contamination had an impact on beer sensory profile and also that pH was correlated with the presence of lactic acid bacteria in beer, being an indicator of contamination for these bacteria. In conclusion, we observed that 8 out of 10 breweries studied showed contamination problems, highlighting the need to design prevention and control strategies in microbreweries., (Copyright © 2022 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.)

The study of natural variation can untap novel alleles with immense value for biotechnological applications. Saccharomyces eubayanus Patagonian isolates exhibit differences in the diauxic shift between glucose and maltose, representing a suitable model to study their natural genetic variation for novel strains for brewing. However, little is known about the genetic variants and chromatin regulators responsible for these differences. Here, we show how genome-wide chromatin accessibility and gene expression differences underlie distinct diauxic shift profiles in S. eubayanus. We identified two strains with a rapid diauxic shift between glucose and maltose (CL467.1 and CBS12357) and one strain with a remarkably low fermentation efficiency and longer lag phase during diauxic shift (QC18). This is associated in the QC18 strain with lower transcriptional activity and chromatin accessibility of specific genes of maltose metabolism and higher expression levels of glucose transporters. These differences are governed by the HAP complex, which differentially regulates gene expression depending on the genetic background. We found in the QC18 strain a contrasting phenotype to those phenotypes described in S. cerevisiae, where hap4Δ , hap5Δ , and cin5Δ knockouts significantly improved the QC18 growth rate in the glucose-maltose shift. The most profound effects were found between CIN5 allelic variants, suggesting that Cin5p could strongly activate a repressor of the diauxic shift in the QC18 strain but not necessarily in the other strains. The differences between strains could originate from the tree host from which the strains were obtained, which might determine the sugar source preference and the brewing potential of the strain. IMPORTANCE The diauxic shift has been studied in budding yeast under laboratory conditions; however, few studies have addressed the diauxic shift between carbon sources under fermentative conditions. Here, we study the transcriptional and chromatin structure differences that explain the natural variation in fermentative capacity and efficiency during diauxic shift of natural isolates of S. eubayanus. Our results show how natural genetic variants in transcription factors impact sugar consumption preferences between strains. These variants have different effects depending on the genetic background, with a contrasting phenotype to those phenotypes previously described in S. cerevisiae. Our study shows how relatively simple genetic/molecular modifications/editing in the lab can facilitate the study of natural variations of microorganisms for the brewing industry.

Basidiomycetous yeasts remain an almost unexplored source of enzymes with great potential in several industries. Tausonia pullulans (Tremellomycetes) is a psychrotolerant yeast with several extracellular enzymatic activities reported, although the responsible genes are not known. We performed the genomic sequencing, assembly and annotation of T. pullulans strain CRUB 1754 (Perito Moreno glacier, Argentina), a gene survey of carbohydrate-active enzymes (CAZymes), and analyzed its secretome by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) after growth in glucose (GLU) or starch (STA) as main carbon sources. T. pullulans has 7210 predicted genes, 3.6% being CAZymes. When compared to other Tremellomycetes, it contains a high number of CAZy domains, and in particular higher quantities of glucoamylases (GH15), pectinolytic enzymes (GH28) and lignocellulose decay enzymes (GH7). When the secretome of T. pullulans was analyzed experimentally after growth in starch or glucose, 98 proteins were identified. The 60% of total spectral counts belonged to GHs, oxidoreductases and to other CAZymes. A 65 kDa glucoamylase of family GH15 (TpGA1) showed the highest fold change (tenfold increase in starch). This enzyme contains a conserved active site and showed extensive N-glycosylation. This study increases the knowledge on the extracellular hydrolytic enzymes of basidiomycetous yeasts and, in particular, establishes T. pullulans as a potential source of carbohydrate-active enzymes. KEY POINTS: • Tausonia pullulans genome harbors a high number of genes coding for CAZymes. • Among CAZy domains/families, the glycoside hydrolases are the most abundant. • Secretome analysis in glucose or starch as main C sources identified 98 proteins. • A 65 kDa GH15 glucoamylase showed the highest fold increase upon culture in starch., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The diversification of beer flavor is becoming increasingly popular, especially in the field of non-alcoholic beers, where sales are growing steadily. While flavor substances of traditional beers can largely be traced back to defined secondary metabolites, the production of non-alcoholic beers with non- Saccharomyces yeasts generates novel fruity flavors, some of which cannot yet be assigned to specific flavor substances. In a recently published study, besides pear, cool mint sweets, and banana-like flavor, distinctive red berry and apple flavors were perceived in a non-alcoholic beer fermented with the yeast strain Cyberlindnera saturnus TUM 247, whose secondary metabolites were to be elucidated in this study. The trials were carried out using response surface methodology to examine the fermentation properties of the yeast strain and to optimize the beer with maximum fruitiness but minimal off-flavors and ethanol content. It turned out that a low pitching rate, a moderate fermentation temperature, and an original gravity of 10.5 °P gave the optimal parameters. Qualitative analysis of the secondary metabolites, in addition to standard analysis for traditional beers, was first performed using headspace-gas chromatography with olfactometry. ( E )- β -damascenone emerged as the decisive substance for the red berry and apple flavor and so this substance was then quantitated. Although ( E )- β -damascenone is a well-known secondary metabolite in beer and this substance is associated with apple or cooked apple- and berry-like flavors, it has not yet been reported as a main flavor component in non-alcoholic beers.

Yeasts play a crucial role in brewing. During fermentation, besides ethanol and carbon dioxide, yeasts produce a considerable number of organic compounds, which are essential for beer flavor. In particular, Saccharomyces cerevisiae and Saccharomyces pastorianus are traditionally used in the production of ale and lager beers, respectively. Nowadays, the continuous growth of the craft beer market motivates the production of differential and innovative beers; leading specialists and brewers focus on non-conventional yeasts as tools for new product development. In this work, we describe the potential application of non-conventional yeast species such as those of the genera Brettanomyces, Torulaspora, Lachancea, Wickerhamomyces, Pichia and Mrakia in the craft brewing industry, as well as non-traditional brewing yeasts of the Saccharomyces genus. Furthermore, the fermentation conditions of these non-conventional yeasts are discussed, along with their abilities to assimilate and metabolize diverse wort components providing differential characteristics to the final product. In summary, we present a comprehensive review of the state-of-the-art of non-conventional yeasts, which is highly relevant for their application in the production of novel craft beers including flavored beers, non-alcoholic beers, low-calorie beers and functional beers., (Copyright © 2021 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.)

A set of four strains representing a novel basidiomycetous yeast species Rhodotorula sampaioana f. a., sp. nov. were isolated from two different habitats, subsurface waters of Lake Negra in Argentina, and the gut of a xylophagous termite in India. Phylogenetic analyses of LSU and ITS sequences showed that they belonged to the genus Rhodotorula of the order Sporidiobolales (subphylum Pucciniomycotina) and the closest known relative being R. kratochvilovae. The new species differed from R. kratochvilovae CBS 7436 (AF071436, AF444520) by nine nucleotide substitutions and one deletion (1.7 % sequence variation) in a 593 bp D1/D2 region, and by five nucleotide substitutions and three deletions (1.3 %) in a 592 bp ITS region, respectively. Several morphological and physiological differences were also observed between R. kratochvilovae and the strains obtained during this study. These data support the proposal of Rhodotorula sampaioana as a novel species, with CRUB 1124 as the holotype, CBS 10798 as ex-type, and NFCCI 4872 as an additional strain. The GenBank accession numbers of the LSU and ITS sequences of Rhodotorula sampaioana f. a., sp. nov. are EF595748 and MW879331. The MycoBank number is MB 838533., (© 2021. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

During a survey of Nothofagus trees and their parasitic fungi in Andean Patagonia (Argentina), genetically distinct strains of Hanseniaspora were obtained from the sugar-containing stromata of parasitic Cyttaria spp. Phylogenetic analyses based on the single-gene sequences (encoding rRNA and actin) or on conserved, single-copy, orthologous genes from genome sequence assemblies revealed that these strains represent a new species closely related to Hanseniaspora valbyensis. Additionally, delimitation of this novel species was supported by genetic distance calculations using overall genome relatedness indices (OGRI) between the novel taxon and its closest relatives. To better understand the mode of speciation in Hanseniaspora , we examined genes that were retained or lost in the novel species in comparison to its closest relatives. These analyses show that, during diversification, this novel species and its closest relatives, H. valbyensis and Hanseniaspora jakobsenii , lost mitochondrial and other genes involved in the generation of precursor metabolites and energy, which could explain their slower growth and higher ethanol yields under aerobic conditions. Similarly, Hanseniaspora mollemarum lost the ability to sporulate, along with genes that are involved in meiosis and mating. Based on these findings, a formal description of the novel yeast species Hanseniaspora smithiae sp. nov. is proposed, with CRUB 1602 H as the holotype., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Čadež, Bellora, Ulloa, Tome, Petković, Groenewald, Hittinger and Libkind.)

The unambiguous application of fungal names is important to communicate scientific findings. Names are critical for (clinical) diagnostics, legal compliance, and regulatory controls, such as biosafety, food security, quarantine regulations, and industrial applications. Consequently, the stability of the taxonomic system and the traceability of nomenclatural changes is crucial for a broad range of users and taxonomists. The unambiguous application of names is assured by the preservation of nomenclatural history and the physical organisms representing a name. Fungi are extremely diverse in terms of ecology, lifestyle, and methods of study. Predominantly unicellular fungi known as yeasts are usually investigated as living cultures. Methods to characterize yeasts include physiological (growth) tests and experiments to induce a sexual morph; both methods require viable cultures. Thus, the preservation and availability of viable reference cultures are important, and cultures representing reference material are cited in species descriptions. Historical surveys revealed drawbacks and inconsistencies between past practices and modern requirements as stated in the International Code of Nomenclature for Algae, Fungi, and Plants (ICNafp). Improper typification of yeasts is a common problem, resulting in a large number invalid yeast species names. With this opinion letter, we address the problem that culturable microorganisms, notably some fungi and algae, require specific provisions under the ICNafp. We use yeasts as a prominent example of fungi known from cultures. But viable type material is important not only for yeasts, but also for other cultivable Fungi that are characterized by particular morphological structures (a specific type of spores), growth properties, and secondary metabolites. We summarize potential proposals which, in our opinion, will improve the stability of fungal names, in particular by protecting those names for which the reference material can be traced back to the original isolate., (© 2021. The Author(s).)

Phaffia is an orange-colored basidiomycetous yeast genus of the order Cystofilobasidiales that contains a single species, P. rhodozyma . This species is the only fungus known to produce the economically relevant carotenoid astaxanthin. Although Phaffia was originally found in the Northern hemisphere, its diversity in the southern part of the globe has been shown to be much greater. Here we analyze the genomes of two Australasian lineages that are markedly distinct from P. rhodozyma . The two divergent lineages were investigated within a comprehensive phylogenomic study of representatives of the Cystofilobasidiales that supported the recognition of two novel Phaffia species, for which we propose the names of P. australis sp. nov. and P. tasmanica sp. nov. Comparative genomics and other analyses confirmed that the two new species have the typical Phaffia hallmark-the six genes necessary for the biosynthesis of astaxanthin could be retrieved from the draft genome sequences, and this carotenoid was detected in culture extracts. In addition, the organization of the mating-type ( MAT ) loci is similar to that of P. rhodozyma , with synteny throughout most regions. Moreover, cases of trans-specific polymorphism involving pheromone receptor genes and pheromone precursor proteins in the three Phaffia species, together with their shared homothallism, provide additional support for their classification in a single genus.

Twenty-one psychrophilic yeast isolates related to the Camptobasidiaceae family in the Microbotryomycetes class were obtained from ice collected from cold environments worldwide. A new psychrophilic species from the recently described genus Cryolevonia, Cryolevania giraudoae is proposed to accommodate 18 isolates from Patagonia (Argentina) and Antarctica (holotype CRUB 2086 T ). In addition, a new psychrophilic species in the genus Camptobasidium is described as Camptobasidium gelus sp. nov. (holotype CBS 8941 T ), based on three isolates from glacial ice in the Russel glacier (Greenland ice sheet) and Antarctica. The strict psychrophilic profile is the salient feature of both novel species.

The wild, cold-adapted parent of hybrid lager-brewing yeasts, Saccharomyces eubayanus, has a complex and understudied natural history. The exploration of this diversity can be used both to develop new brewing applications and to enlighten our understanding of the dynamics of yeast evolution in the wild. Here, we integrate whole genome sequence and phenotypic data of 200 S. eubayanus strains, the largest collection known to date. S. eubayanus has a multilayered population structure, consisting of two major populations that are further structured into six subpopulations. Four of these subpopulations are found exclusively in the Patagonian region of South America; one is found predominantly in Patagonia and sparsely in Oceania and North America; and one is specific to the Holarctic ecozone. Plant host associations differed between subpopulations and between S. eubayanus and its sister species, Saccharomyces uvarum. S. eubayanus is most abundant and genetically diverse in northern Patagonia, where some locations harbor more genetic diversity than is found outside of South America, suggesting that northern Patagonia east of the Andes was a glacial refugium for this species. All but one subpopulation shows isolation-by-distance, and gene flow between subpopulations is low. However, there are strong signals of ancient and recent outcrossing, including two admixed lineages, one that is sympatric with and one that is mostly isolated from its parental populations. Using our extensive biogeographical data, we build a robust model that predicts all known and a handful of additional regions of the globe that are climatically suitable for S. eubayanus, including Europe where host accessibility and competitive exclusion by other Saccharomyces species may explain its continued elusiveness. We conclude that this industrially relevant species has rich natural diversity with many factors contributing to its complex distribution and natural history., Competing Interests: We have read the journal's policy and the authors of this manuscript have the following competing interests: commercial use of Saccharomyces eubayanus strains requires a license from WARF or CONICET. Strains are available for academic research under a material transfer agreement.

The most common fermented beverage, lager beer, is produced by interspecies hybrids of the brewing yeast Saccharomyces cerevisiae and its wild relative S. eubayanus. Lager-brewing yeasts are not the only example of hybrid vigour or heterosis in yeasts, but the full breadth of interspecies hybrids associated with human fermentations has received less attention. Here we present a comprehensive genomic analysis of 122 Saccharomyces hybrids and introgressed strains. These strains arose from hybridization events between two to four species. Hybrids with S. cerevisiae contributions originated from three lineages of domesticated S. cerevisiae, including the major wine-making lineage and two distinct brewing lineages. In contrast, the undomesticated parents of these interspecies hybrids were all from wild Holarctic or European lineages. Most hybrids have inherited a mitochondrial genome from a parent other than S. cerevisiae, which recent functional studies suggest could confer adaptation to colder temperatures. A subset of hybrids associated with crisp flavour profiles, including both lineages of lager-brewing yeasts, have inherited inactivated S. cerevisiae alleles of critical phenolic off-flavour genes and/or lost functional copies from the wild parent through multiple genetic mechanisms. These complex hybrids shed light on the convergent and divergent evolutionary trajectories of interspecies hybrids and their impact on innovation in lager brewing and other diverse fermentation industries.

Among the main beer components, fusel alcohols are important because of their influence on the flavor of the final product, and therefore on its quality. During the production process, these compounds are generated by yeasts through the metabolism of amino acids. The yeasts, fermentation conditions and wort composition affect fusel alcohols profiles and their concentrations. In this review, we provide detailed information about the enzymes involved in fusel alcohols formation and their regulation. Moreover, we describe how the type of yeast used, the fermentation temperature and the composition of carbohydrates and nitrogen source in wort, among other fermentation parameters, affect the biosynthesis of these alcohols. Knowing how fusel alcohol levels vary during beer production provides a relevant tool for brewers to achieve the desired characteristics in the final product and at the same time highlights the aspects still unknown to science., (Copyright © 2018 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.)

During brewing process yeast produce more than 500 chemical compounds that can negatively and positively impact beer at the organoleptic level. In recent years, and particularly thanks to the advancement of molecular biology and genomics, there has been considerable progress in our understanding about the molecular and cellular basis of the synthesis and regulation of many of these flavor compounds. This article focuses on esters, responsible for the floral and fruity beer flavor. Its formation depends on various enzymes and factors such as the concentration of wort nutrients, the amount of dissolved oxygen and carbon dioxide, fermentation temperature and mainly the genetics of the yeast used. We provide information about how the esters originate and how is the impact of different fermentative parameters on the final concentrations of these compounds and the quality of the end product., (Copyright © 2018 The Authors. Publicado por Elsevier España, S.L.U. All rights reserved.)

Astaxanthin is a xanthophyll pigment of high economic value for its use as a feeding component in aquaculture. Phaffia rhodozyma (Xanthophyllomyces dendrorhous) is a basidiomycetous fungi able to synthesize astaxanthin as its major carotenoid, the only known yeast species bearing the capability to produce this type of carotenoid and the only tremellomycetes with biotechnological application. Recently, the habitat and intraspecific variability of this species have been found to be wider than previously expected, encouraging the search for new wild strains with potential biotechnological applications. Here we describe effective procedures for isolation of P. rhodozyma from environmental samples, accurate identification of the strains, analysis of their astaxanthin content, and proper conservation of the isolates.

Exposure to ultraviolet radiation (UVR) is harmful to living organisms, causing damage to macromolecules such as DNA, RNA, proteins and lipids. Depending on the wavelength, the injury could be direct or indirect through reactive oxygen intermediates, so it is desirable to find compounds that can reduce both. Many organic chemicals used in commercial sunscreen possess estrogenic activity in vivo. In this report we analyzed recent patents related to UV sunscreens of microbial origin, in particular mycosporines (MYC) and mycosporine-like aminoacids (MAA). Both are promising natural alternatives for both direct (UV-absorption) and indirect (antioxidant) protection, given they show strong photostability and absence of cytotoxicity. It becomes clear that although the search for natural photoprotective molecules is relatively recent, efforts have been invested mainly in marine environments, remaining still many potential photoprotective molecules to find in other type of habitats. Furthermore, unicellular microorganisms have several advantages for the production of metabolites of interest, since they improve the production costs due to its simplicity of culture and easy genetic manipulation. The knowledge of the biosynthesis pathway of MYC and MAA is essential to improve rationally their expression levels. Currently, only the MAA pathway in bacteria has been reported, remaining the MYC pathway unclear. Future perspectives include the heterologous expression of MYC and/or MAA in industrially friendly microorganisms (bacteria and yeast) in order to co-produce different UV-protective molecules and thus cover a broader UV spectrum and simplify the production process.

Xanthophyllomyces dendrorhous (Phaffia rhodozyma) yeasts are biotechnologically exploited as a natural source of astaxanthin for aquaculture. Based on results of recent studies, it has become clear that this species possesses a greater genetic variability generating the necessity to uncover it and assess its potential for the astaxanthin industry. However, difficulties for the isolation of the X. dendrorhous hinder extensive environmental surveys which need to be carried out to better understand the habitat, distribution and genetic diversity of this species. We extensively searched for distinctive physiological traits of X. dendrorhours by testing phenotypic properties simultaneously with a panel of common sympatric fungi. As a result we obtained a new and innovative strategy for improving X. dendrorhous recovery rate and identification from environmental samples. This strategy involved the use of trehalose-based media, and a rapid X. dendrorhous identification method based on the simultaneous spectrophotometric detection of astaxanthin and UV-absorbing compounds (mycosporines). The proposed procedures proved effective in field trials conducted in natural environments of Patagonia (Argentina) and thus represent an important tool for the discovery of new astaxanthin-producing strains of X. dendrorhous useful for the aquaculture industry., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Astaxanthin is a xanthophyll pigment of high economic value for its use as a feeding component in aquaculture. Xanthophyllomyces dendrorhous is a basidiomycetous fungi able to synthesize astaxanthin as its major carotenoid, and the only known yeast species bearing the capability to produce this type of carotenoid. Recently, the habitat and intraspecific variability of this species have been found to be wider than previously expected, encouraging the search for new wild strains with potential biotechnological applications. Here we describe effective procedures for isolation of X. dendrorhous from environmental samples, accurate identification of the strains, analysis of their astaxanthin content, and proper conservation of the isolates.

Yeasts of the genera Rhodotorula are able to synthesize different pigments of high economic value like β-carotene, torulene, and torularhodin, and therefore represent a biotechnologically interesting group of yeasts. However, the low production rate of pigment in these microorganisms limits its industrial application. Here we describe some strategies to obtain hyperpigmented mutants of Rhodotorula mucilaginosa by means of ultraviolet-B radiation, the procedures for total carotenoids extraction and quantification, and a method for identification of each pigment.

Soil microorganisms play an important role in soil quality and they interact closely with vegetation. Little is known about yeast diversity and function in forest soil ecosystems and their interactions with other biotic soil components, particularly in the mycorrhizosphere. We studied the diversity of yeasts inhabiting the bulk-soil, rhizosphere and ectomycorrhizosphere of a Nothofagus pumilio forest in Nahuel Huapi National Park (Bariloche, Argentina). Ectomycorrhizal infection was observed in all N. pumilio trees studied. A total of 126 yeast isolates were obtained, including 18 known and three possibly new species. Basidiomycetous yeasts were predominant in all soil fractions, and the most frequently isolated species was Cryptococcus podzolicus. Diversity indices and multivariate analyses were used to study and compare yeast communities in the bulk-soil, rhizosphere and ectomycorrhizosphere. Yeasts able to ferment glucose were found associated with the rhizosphere. Many of the recovered yeast species were associated with lignocelluloses compound degradation, which suggest that yeast plays an important role as a decomposer in these forest soils. Each soil fraction has a distinct yeast assemblage related to their physiologic capacities and soil nutrient availability., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Domestication of plants and animals promoted humanity's transition from nomadic to sedentary lifestyles, demographic expansion, and the emergence of civilizations. In contrast to the well-documented successes of crop and livestock breeding, processes of microbe domestication remain obscure, despite the importance of microbes to the production of food, beverages, and biofuels. Lager-beer, first brewed in the 15th century, employs an allotetraploid hybrid yeast, Saccharomyces pastorianus (syn. Saccharomyces carlsbergensis), a domesticated species created by the fusion of a Saccharomyces cerevisiae ale-yeast with an unknown cryotolerant Saccharomyces species. We report the isolation of that species and designate it Saccharomyces eubayanus sp. nov. because of its resemblance to Saccharomyces bayanus (a complex hybrid of S. eubayanus, Saccharomyces uvarum, and S. cerevisiae found only in the brewing environment). Individuals from populations of S. eubayanus and its sister species, S. uvarum, exist in apparent sympatry in Nothofagus (Southern beech) forests in Patagonia, but are isolated genetically through intrinsic postzygotic barriers, and ecologically through host-preference. The draft genome sequence of S. eubayanus is 99.5% identical to the non-S. cerevisiae portion of the S. pastorianus genome sequence and suggests specific changes in sugar and sulfite metabolism that were crucial for domestication in the lager-brewing environment. This study shows that combining microbial ecology with comparative genomics facilitates the discovery and preservation of wild genetic stocks of domesticated microbes to trace their history, identify genetic changes, and suggest paths to further industrial improvement.

The occurrence and distribution of Xanthophyllomyces dendrorhous associated with Cyttaria hariotii parasitizing three Nothofagus species (N. dombeyi, N. antarctica and N. pumilio) in northwestern Patagonia (Argentina), as well as the factors that may affect this distribution were herein studied. Between 2000 and 2007, samples were obtained from 18 different locations. Based on physiological tests and morphological characteristics of sexual structures, 72 isolates were identified as X. dendrorhous. Representative strains were studied by MSP-PCR fingerprinting and sequence analysis of the ITS region. MSP-PCR fingerprints were similar for the newly isolated strains, and were also identical to the profiles of the strains previously found in this region. Patagonian strains appear to be a genetically uniform and distinct population, supporting the hypothesis that the association with different host species has determined genetically distinct X. dendrorhous populations worldwide. X. dendrorhous was recovered from N. dombeyi and N. antarctica. Approximately half the sampling sites and samples were positive for X. dendrorhous, but the isolation recovery rate was low. X. dendrorhous was absent in the early stages of ascostromata maturation, becoming more abundant in later stages. The present work represents a step forward in the understanding of the natural distribution and ecology of this biotechnologically relevant yeast.

Several yeast species are able to synthesize and accumulate UV-radiation-absorbing mycosporine metabolites that are of unclear physiological function. In this work we analyzed the relationship between mycosporine-glutaminol glucoside (MGG) production, cell survival after UVB irradiation, and formation of cyclobutane pyrimidine dimers (CPDs). We also assessed the photostability and singlet oxygen quenching activity of MGG. A set of nine isolates of the basidiomycetous yeast Cryptococcus steppossus cultured in both dark and light conditions was used for the studies. Survival of the UVB-irradiated isolates and MGG concentration had a linear relationship when the concentration was over 2.5 mg g(-1). CPD accumulation and MGG accumulation were inversely related. MGG in aqueous solution was photostable with a photodecomposition quantum yield of 1.16 × 10(-5). MGG quenching of singlet oxygen was also observed, and the rate constant for the process in D(2)O was 5.9 × 10(7) M(-1) s(-1). Our results support the idea that MGG plays an important role as a UVB photoprotective metabolite in yeasts by protecting against direct damage on DNA and probably against indirect damage by singlet oxygen quenching.

In this paper we report the relationship between carotenoids and ergosterol and cell UV-B resistance in different strains of the yeast Rhodotorula mucilaginosa. Cell survival was studied using a set of 13 strains; additionally, two mutants (a hyper-producing one and a colourless one) in combination with diphenylamine (DPA), a carotenogenesis inhibitor, were used. A positive correlation between total carotenoids and survival to UV-B radiation was found. However, when individual carotenoid concentrations were tested, only torularhodin was found to be significantly related to UV-B survival. On the contrary, ergosterol did not affect survival. The hyper-pigmented strain showed an enhanced survival (up to 250%) compared to the parental strain, while the survival of the albino mutant was similar to that experienced by the parental strain; however, observed changes in survival were dose dependent. The cyclobutane pyrimidine dimers (CPDs), one of the major forms of DNA damage caused by UV exposure, appears as unrelated to the accumulation of carotenoids and cell survival. These results indicate that bearing higher torularhodin concentrations enhances UV-B survival in yeasts and, thus, the accumulation of this pigment constitutes an important mechanism that improves the resistance of yeasts to UV-B.

In this work, the photoprotective role of carotenoids in yeasts was analysed by contrasting the responses to UV-B of pigmented and naturally occurring albino strains of Sporobolomyces ruberrimus and Cystofilobasidium capitatum in different conditions. Albino and pigmented strains were confirmed to be conspecific by PCR fingerprinting and rDNA sequencing. Experimental exposure to UV-B conducted with both yeast species showed that the pigmented strains were more tolerant to UV-B than the albino strains and that the increment in carotenoid contents during the stationary growth phase enhance survivorship. These results indicated that carotenoid pigments afford UV-B protection in yeasts.

Here, we report on two novel sexual basidiomycetous red yeast species of the genus Cystofilobasidium. Cystofilobasidium lacus-mascardii sp. nov. is based on sexually compatible strains isolated from Lake Mascardi, an ultraoligotrophic lake in north-western Patagonia, Argentina. Following the discovery of the first isolate of this species, additional (sexually compatible) strains were isolated using a selective medium containing erythritol as the sole source of carbon. The second novel species corresponds to the sexual state of Cryptococcus macerans. In spite of accounts over the last 20 years of sexually compatible strains of this species, the complete life has never been observed. We provide evidence of a Cystofilobasidium-like basidial stage with teliospores and slender holobasidia, based on the study of self-fertile (homothallic) and self-sterile (heterothallic) isolates of Cryptococcus macerans. A revised molecular phylogeny of the genus Cystofilobasidium is presented and the most salient features of Cystofilobasidium lacus-mascardii sp. nov. (type strain CBS 10642(T) =PYCC 5819(T) =CRUB 1046(T)) and Cystofilobasidium macerans sp. nov. (type strain CBS 10757(T)) are discussed and compared with those of the remaining species in the genus. Information on additional Patagonian isolates belonging to the Cystofilobasidiales is also included in this report.

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.

In the Iberian Pyrite Belt (IPB), acid rock drainage gives rise to aquatic habitats with low pH and high concentrations of heavy metals, a situation that causes important environmental problems. We investigated the occurrence and diversity of yeasts in two localities of the IPB: São Domingos (Portugal) and Rio Tinto (Spain). Yeast isolation was performed on conventional culture media (MYP), acidified (pH 3) media (MYP3), and on media prepared with water from the study sites (MYPw). The main goal of the study was to determine the structure of the yeast community; a combination of molecular methods was used for accurate species identifications. Our results showed that the largest fraction of the yeast community was recovered on MYPw rather than on MYP and MYP3. Twenty-seven yeast species were detected, 48% of which might represent undescribed taxa. Among these, an undescribed species of the genus Cryptococcus required low pH for growth, a property that has not been observed before in yeasts. The communities of S. Domingos and R. Tinto showed a considerable resemblance, and eight yeast species were simultaneously found in both localities. Taking into consideration the physicochemical parameters studied, we propose a hierarchic organization of the yeast community in terms of high-, intermediate-, or low-stress conditions of the environment. According to this ranking, the acidophile yeast Cryptococcus sp. 5 is considered the most tolerant species, followed by Cryptococcus sp. 3 and Lecytophora sp. Species occurring in situations of intermediate environmental stress were Candida fluviatilis, Rhodosporidium toruloides, Williopsis californica, and three unidentified yeasts belonging to Rhodotorula and Cryptococcus.

Mycosporine-like amino-acids (MAAs) are found in aquatic bacteria, algae, and animals. A related compound, the mycosporine-glutaminol-glucoside (myc-glu-glu), has recently been reported in freshwater yeasts. Although animals depend on other organisms as their source of MAAs, they can efficiently accumulate them in their tissues. In this work we assessed the potential transfer of the yeast mycosporine myc-glu-glu from the diet into the copepod Boeckella antiqua and the ciliate Paramecium bursaria. For this purpose, we performed experiments to study the feeding of B. antiqua and P. bursaria on the yeast Rhodotorula minuta and their ability to bioaccumulate myc-glu-glu. Bioaccumulation of myc-glu-glu in B. antiqua was assessed through long-term factorial experiments manipulating the diet (Chlamydomonas reinhardii and C. reinhardii + yeasts) and radiation exposure (PAR and PAR + UVR). Shorter term experiments were designed in the case of P. bursaria. The composition and concentration of MAAs in the diet and in the consumers were determined by HPLC analyses. Our results showed that even though both consumers ingested yeast cells, they were unable to accumulate myc-glu-glu. Moreover, when exposed to conditions that stimulated the accumulation of photoprotective compounds (i.e. UVR exposure), an increase in MAAs concentration occurred in copepods fed C. reinhardii plus yeasts as well as in those fed only C. reinhardii. This suggests that the copepods were able to modify their tissue concentrations of MAAs in response to environmental clues but also that the contribution of yeast mycosporines to total MAAs concentration was negligible.

Twelve yeasts isolated from lakes of Northwestern Patagonia, Argentina, belonging to eight genera (Sporobolomyces, Sporidiobolus, Rhodotorula, Rhodosporidium, Cystofilobasidium, Cryptococcus, Torulaspora, and Candida) were analysed for their ability to produce photoprotective compounds. For this purpose, three laboratory experiments were performed to study the effect of photosynthetically active radiation (PAR) and PAR in combination with UV radiation (PAR + UVR) on the production of carotenoids and mycosporines. The synthesis of carotenoid compounds was clearly stimulated in six out of nine red yeast strains tested upon exposure to PAR or PAR + UVR; however, the latter conditions produced a stronger response than PAR alone. The increase in carotenoids in the red strains under PAR + UVR irradiation showed a negative exponential relationship with their basal carotenoid content, suggesting that cells with higher constitutive levels of carotenoids are less responsive to induction by PAR + UVR. Three red yeasts, Rhodotorula minuta, Rh. pinicola, and Rhodotorula sp., and the colourless Cryptococcus laurentii produced a UV-absorbing compound when exposed to PAR or PAR + UVR. This compound showed an absorption maximum at 309-310 nm and was identified as mycosporine-glutaminol-glucoside (myc-glu-glu). In these strains, exposure to PAR or PAR + UVR resulted in elevated concentrations of both carotenoids and myc-glu-glu. This is the first report on the production of mycosporines by yeasts. All strains that developed under PAR + UVR were able to synthesise carotenoids either constitutively or in response to PAR exposure, and a few of them also produced myc-glu-glu when exposed to PAR. Collectively, our results suggest that the presence of carotenoids, either alone or in combination with mycosporines, are required for sustaining growth under exposure to PAR + UVR in the freshwater yeast strains studied.

Fifteen aquatic environments (lakes, lagoons and rivers) of glacial origin in the northern Andean Patagonia (Argentina) were surveyed for the occurrence of red yeasts. Subsurface water samples were filtered and used for colony counting and yeast isolation. A preliminary quantitative analysis indicated that total yeast counts ranged between 0 and 250 cells l(-1). A polyphasic approach including physiological and molecular methods was used for the identification of 64 carotenogenic yeast strains. The molecular characterisation of the isolates was based on the mini/microsatellite-primed PCR technique (MSP-PCR) employing the (GTG)5 and the M13 primers. Comparison of representative fingerprints of each group with those of the type strains of pigmented yeasts allowed the expeditious identification of 87.5% isolates. The sequence analysis of the D1/D2 domains of the 26S rDNA was employed to confirm identifications and in the characterization of the unidentified MSP-PCR groups. Teleomorphic yeast species were detected by performing sexual compatibility assays. The isolates corresponded to 6 genera and 15 yeast species, including four new yeast species of the genera Cryptococcus (1), Rhodotorula (1) and Sporobolomyces (2). Rhodotorula mucilaginosa was found in the majority of the samples and represented ca. 50% of the total number of isolates. However, this yeast was not detected in aquatic environments with very low anthropic influence. Other frequent yeast isolates were teleomorphic yeast species of Rhodosporidium babjevae, R. kratochvilovae and Sporidiobolus salmonicolor. This study represents the first report on red yeast occurrence and biodiversity in northwestern Patagonia.