Genomic content of a novel yeast species Hanseniaspora gamundiae sp. Nov. From fungal stromata (Cyttaria) associated with a unique fermented beverage in Andean Patagonia, Argentina

A novel yeast species was isolated from the sugar-rich stromata of Cyttaria hariotii collected from two different Nothofagus tree species in the Andean forests of Patagonia, Argentina. Phylogenetic analyses of the concatenated sequence of the rRNA gene sequences and the protein-coding genes for actin and translational elongation factor-1α indicated that the novel species belongs to the genus Hanseniaspora. De novo genome assembly of the strain CRUB 1928 T yielded a 10.2-Mbp genome assembly predicted to encode 4452 protein-coding genes. The genome sequence data were compared to the genomes of other Hanseniaspora species using three different methods, an alignment-free distance measure, K r , and two model-based estimations of DNA-DNA homology values, of which all provided indicative values to delineate species of Hanseniaspora. Given its potential role in a rare indigenous alcoholic beverage in which yeasts ferment sugars extracted from the stromata of Cytarria sp., we searched for the genes that may suggest adaptation of novel Hanseniaspora species to fermenting communities. The SSU1-like gene encoding a sulfite efflux pump, which, among Hanseniaspora, is present only in close relatives to the new species, was detected and analyzed, suggesting that this gene might be one factor that characterizes this novel species. We also discuss several candidate genes that likely underlie the physiological traits used for traditional taxonomic identification. Based on these results, a novel yeast species with the name Hanseniaspora gamundiae sp. nov. is proposed with CRUB 1928 T (ex-types: ZIM 2545 T = NRRL Y-63793 T = PYCC 7262 T ; MycoBank number MB 824091) as the type strain. Furthermore, we propose the transfer of the Kloeckera species, K. hatyaiensis, K. lindneri and K. taiwanica to the genus Hanseniaspora as Hanseniaspora hatyaiensis comb. nov. (MB 828569), Hanseniaspora lindneri comb. nov. (MB 828566) and Hanseniaspora taiwanica comb. nov. (MB 828567).Fil: Čadež, Neža. University of Ljubljana; EsloveniaFil: Bellora, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Biodiversidad y Biotecnología; Argentina. Universidad Nacional del Comahue. Centro Regional Universitario Bariloche; ArgentinaFil: Ulloa, José Ricardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigación y Desarrollo en Ingeniería de Procesos, Biotecnología y Energías Alternativas. Universidad Nacional del Comahue. Instituto de Investigación y Desarrollo en Ingeniería de Procesos, Biotecnología y Energías Alternativas; ArgentinaFil: Hittinger, Chris Todd. University of Wisconsin; Estados UnidosFil: Libkind Frati, Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto Andino Patagónico de Tecnologías Biológicas y Geoambientales. Universidad Nacional del Comahue. Instituto Andino Patagónico de Tecnologías Biológicas y Geoambientales; Argentina. Universidad Nacional del Comahue. Centro Regional Universitario Bariloche; Argentin

Aim18p and Aim46p are chalcone isomerase domain-containing mitochondrial hemoproteins in Saccharomyces cerevisiae

Chalcone isomerases (CHIs) have well-established roles in the biosynthesis of plant flavonoid metabolites. Saccharomyces cerevisiae possesses two predicted CHI-like proteins, Aim18p (encoded by YHR198C) and Aim46p (YHR199C), but it lacks other enzymes of the flavonoid pathway, suggesting that Aim18p and Aim46p employ the CHI fold for distinct purposes. Here, we demonstrate using proteinase K protection assays, sodium carbonate extractions, and crystallography that Aim18p and Aim46p reside on the mitochondrial inner membrane and adopt CHI folds, but they lack select active site residues and possess an extra fungal-specific loop. Consistent with these differences, Aim18p and Aim46p lack CHI activity and also the fatty acid-binding capabilities of other CHI-like proteins, but instead bind heme. We further show that diverse fungal homologs also bind heme and that Aim18p and Aim46p possess structural homology to a bacterial hemoprotein. Collectively, our work reveals a distinct function and cellular localization for two CHI-like proteins, introduces a new variation of a hemoprotein fold, and suggests that ancestral CHI-like proteins were hemoproteins

The genome sequence of Saccharomyces Eubayanus and the domestication of Lager-Brewing yeasts

The dramatic phenotypic changes that occur in organisms during domestication leave indelible imprints on their genomes. Although many domesticated plants and animals have been systematically compared with their wild genetic stocks, the molecular and genomic processes underlying fungal domestication have received less attention. Here, we present a nearly complete genome assembly for the recently described yeast species Saccharomyces eubayanus and compare it to the genomes of multiple domesticated alloploid hybrids of S. eubayanus × S. cerevisiae (S. pastorianus syn. S. carlsbergensis), which are used to brew lager-style beers. We find that the S. eubayanus subgenomes of lager-brewing yeasts have experienced increased rates of evolution since hybridization, and that certain genes involved in metabolism may have been particularly affected. Interestingly, the S. eubayanus subgenome underwent an especially strong shift in selection regimes, consistent with more extensive domestication of the S. cerevisiae parent prior to hybridization. In contrast to recent proposals that lager-brewing yeasts were domesticated following a single hybridization event, the radically different neutral site divergences between the subgenomes of the two major lager yeast lineages strongly favor at least two independent origins for the S. cerevisiae × S. eubayanus hybrids that brew lager beers. Our findings demonstrate how this industrially important hybrid has been domesticated along similar evolutionary trajectories on multiple occasions.Fil: Baker, Emily Clare. University Of Wisconsin; Estados UnidosFil: Wang, Bing. University Of Wisconsin; Estados UnidosFil: Bellora, Nicolás. Universidad Nacional del Comahue. Centro Regional Universidad de Bariloche. Departamento de Biologia. Laboratorio de Microbiologia Aplicada y Biotecnologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Patagonia Norte. Instituto de Investigación en Biodiversidad y Medioambiente; ArgentinaFil: Peris, David. University Of Wisconsin; Estados UnidosFil: Hulfachor, Amanda Beth. University Of Wisconsin; Estados UnidosFil: Koshalek, Justin A.. University Of Wisconsin; Estados UnidosFil: Adams, Marie. University Of Wisconsin; Estados UnidosFil: Libkind Frati, Diego. Universidad Nacional del Comahue. Centro Regional Universidad de Bariloche. Departamento de Biologia. Laboratorio de Microbiologia Aplicada y Biotecnologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Patagonia Norte. Instituto de Investigación en Biodiversidad y Medioambiente; ArgentinaFil: Hittinger, Chris Todd. University Of Wisconsin; Estados Unido

The Awesome Power of Yeast Evolutionary Genetics: New Genome Sequences and Strain Resources for the Saccharomyces sensu stricto Genus

High-quality, well-annotated genome sequences and standardized laboratory strains fuel experimental and evolutionary research. We present improved genome sequences of three species of Saccharomyces sensu stricto yeasts: S. bayanus var. uvarum (CBS 7001), S. kudriavzevii (IFO 1802T and ZP 591), and S. mikatae (IFO 1815T), and describe their comparison to the genomes of S. cerevisiae and S. paradoxus. The new sequences, derived by assembling millions of short DNA sequence reads together with previously published Sanger shotgun reads, have vastly greater long-range continuity and far fewer gaps than the previously available genome sequences. New gene predictions defined a set of 5261 protein-coding orthologs across the five most commonly studied Saccharomyces yeasts, enabling a re-examination of the tempo and mode of yeast gene evolution and improved inferences of species-specific gains and losses. To facilitate experimental investigations, we generated genetically marked, stable haploid strains for all three of these Saccharomyces species. These nearly complete genome sequences and the collection of genetically marked strains provide a valuable toolset for comparative studies of gene function, metabolism, and evolution, and render Saccharomyces sensu stricto the most experimentally tractable model genus. These resources are freely available and accessible through www.SaccharomycesSensuStricto.org

Mitochondrial genome diversity across the subphylum Saccharomycotina

IntroductionEukaryotic life depends on the functional elements encoded by both the nuclear genome and organellar genomes, such as those contained within the mitochondria. The content, size, and structure of the mitochondrial genome varies across organisms with potentially large implications for phenotypic variance and resulting evolutionary trajectories. Among yeasts in the subphylum Saccharomycotina, extensive differences have been observed in various species relative to the model yeast Saccharomyces cerevisiae, but mitochondrial genome sampling across many groups has been scarce, even as hundreds of nuclear genomes have become available.MethodsBy extracting mitochondrial assemblies from existing short-read genome sequence datasets, we have greatly expanded both the number of available genomes and the coverage across sparsely sampled clades.ResultsComparison of 353 yeast mitochondrial genomes revealed that, while size and GC content were fairly consistent across species, those in the genera Metschnikowia and Saccharomyces trended larger, while several species in the order Saccharomycetales, which includes S. cerevisiae, exhibited lower GC content. Extreme examples for both size and GC content were scattered throughout the subphylum. All mitochondrial genomes shared a core set of protein-coding genes for Complexes III, IV, and V, but they varied in the presence or absence of mitochondrially-encoded canonical Complex I genes. We traced the loss of Complex I genes to a major event in the ancestor of the orders Saccharomycetales and Saccharomycodales, but we also observed several independent losses in the orders Phaffomycetales, Pichiales, and Dipodascales. In contrast to prior hypotheses based on smaller-scale datasets, comparison of evolutionary rates in protein-coding genes showed no bias towards elevated rates among aerobically fermenting (Crabtree/Warburg-positive) yeasts. Mitochondrial introns were widely distributed, but they were highly enriched in some groups. The majority of mitochondrial introns were poorly conserved within groups, but several were shared within groups, between groups, and even across taxonomic orders, which is consistent with horizontal gene transfer, likely involving homing endonucleases acting as selfish elements.DiscussionAs the number of available fungal nuclear genomes continues to expand, the methods described here to retrieve mitochondrial genome sequences from these datasets will prove invaluable to ensuring that studies of fungal mitochondrial genomes keep pace with their nuclear counterparts

Extensive loss of cell-cycle and DNA repair genes in an ancient lineage of bipolar budding yeasts

Cell-cycle checkpoints and DNA repair processes protect organisms from potentially lethal mutational damage. Compared to other budding yeasts in the subphylum Saccharomycotina, we noticed that a lineage in the genus Hanseniaspora exhibited very high evolutionary rates, low Guanine–Cytosine (GC) content, small genome sizes, and lower gene numbers. To better understand Hanseniaspora evolution, we analyzed 25 genomes, including 11 newly sequenced, representing 18/21 known species in the genus. Our phylogenomic analyses identify two Hanseniaspora lineages, a faster-evolving lineage (FEL), which began diversifying approximately 87 million years ago (mya), and a slower-evolving lineage (SEL), which began diversifying approximately 54 mya. Remarkably, both lineages lost genes associated with the cell cycle and genome integrity, but these losses were greater in the FEL. E.g., all species lost the cell-cycle regulator WHIskey 5 (WHI5), and the FEL lost components of the spindle checkpoint pathway (e.g., Mitotic Arrest-Deficient 1 [MAD1], Mitotic Arrest-Deficient 2 [MAD2]) and DNA-damage–checkpoint pathway (e.g., Mitosis Entry Checkpoint 3 [MEC3], RADiation sensitive 9 [RAD9]). Similarly, both lineages lost genes involved in DNA repair pathways, including the DNA glycosylase gene 3-MethylAdenine DNA Glycosylase 1 (MAG1), which is part of the base-excision repair pathway, and the DNA photolyase gene PHotoreactivation Repair deficient 1 (PHR1), which is involved in pyrimidine dimer repair. Strikingly, the FEL lost 33 additional genes, including polymerases (i.e., POLymerase 4 [POL4] and POL32) and telomere-associated genes (e.g., Repressor/ activator site binding protein-Interacting Factor 1 [RIF1], Replication Factor A 3 [RFA3], Cell Division Cycle 13 [CDC13], Pbp1p Binding Protein [PBP2]). Echoing these losses, molecular evolutionary analyses reveal that, compared to the SEL, the FEL stem lineage underwent a burst of accelerated evolution, which resulted in greater mutational loads, homopolymer instabilities, and higher fractions of mutations associated with the common endogenously damaged base, 8-oxoguanine. We conclude that Hanseniaspora is an ancient lineage that has diversified and thrived, despite lacking many otherwise highly conserved cell-cycle and genome integrity genes and pathways, and may represent a novel, to our knowledge, system for studying cellular life without them.Fil: Steenwyk, Jacob L.. Vanderbilt University; Estados UnidosFil: Opulente, Dana A.. University of Wisconsin; Estados UnidosFil: Kominek, Jacek. University of Wisconsin; Estados UnidosFil: Shen, Xing-Xing. Vanderbilt University; Estados UnidosFil: Zhou, Xiaofan. South China Agricultural University; ChinaFil: Labella, Abigail L.. Vanderbilt University; Estados UnidosFil: Bradley, Noah P.. Vanderbilt University; Estados UnidosFil: Eichman, Brandt F.. Vanderbilt University; Estados UnidosFil: Cadez, Neza. University of Ljubljana; EsloveniaFil: Libkind Frati, Diego. Universidad Nacional del Comahue. Centro Regional Universitario Bariloche; ArgentinaFil: DeVirgilio, Jeremy. United States Department of Agriculture. Agricultural Research Service; ArgentinaFil: Hulfachor, Amanda Beth. University of Wisconsin; Estados UnidosFil: Kurtzman, Cletus P.. United States Department of Agriculture. Agricultural Research Service; ArgentinaFil: Hittinger, Chris Todd. University of Wisconsin; Estados UnidosFil: Rokas, Antonis. Vanderbilt University; Estados Unido

Macroevolutionary diversity of traits and genomes in the model yeast genus Saccharomyces

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.Some computations were performed on Tirant III of the Spanish Supercomputing Network (“Servei d’Informàtica de la Universitat de València”) under the project BCV-2021-1-0001 granted to DP, while others were performed at the Wisconsin Energy Institute and the Center for High-Throughput Computing of the University of Wisconsin–Madison. During a portion of this project, DP was a researcher funded by the European Union’s Horizon 2020 research and innovation program Marie Sklodowska-Curie, grant agreement No. 747775, the Research Council of Norway (RCN) grant Nos. RCN 324253 and 274337, and the Generalitat Valenciana plan GenT grant No. CIDEGENT/2021/039. D.P. is a recipient of an Illumina Grant for Illumina Sequencing Saccharomyces strains in this study. Q.K.L. was supported by the National Science Foundation under Grant No. DGE-1256259 (Graduate Research Fellowship) and the Predoctoral Training Program in Genetics, funded by the National Institutes of Health (5T32GM007133). This material is based upon work supported in part by the Great Lakes Bioenergy Research Center, Office of Science, Office of Biological and Environmental Research under Award Numbers DE-SC0018409 and DE-FC02-07ER64494; the National Science Foundation under Grant Nos. DEB-1253634, DEB−1442148, and DEB-2110403; and the USDA National Institute of Food and Agriculture Hatch Project Number 1020204. C.T.H. is an H. I. Romnes Faculty Fellow, supported by the Office of the Vice Chancellor for Research and Graduate Education with funding from the Wisconsin Alumni Research Foundation. QMW was supported by the National Natural Science Foundation of China (NSFC) under Grant Nos. 31770018 and 31961133020. C.R.L. holds the Canada Research Chair in Cellular Systems and Synthetic Biology, and his research on wild yeast is supported by an NSERC Discovery Grant.Peer reviewe

Hybridization and adaptive evolution of diverse Saccharomyces species for cellulosic biofuel production

Additional file 15. Summary of whole genome sequencing statistics

Evolution of a novel chimeric maltotriose transporter in Saccharomyces eubayanus from parent proteins unable to perform this function.

At the molecular level, the evolution of new traits can be broadly divided between changes in gene expression and changes in protein-coding sequence. For proteins, the evolution of novel functions is generally thought to proceed through sequential point mutations or recombination of whole functional units. In Saccharomyces, the uptake of the sugar maltotriose into the cell is the primary limiting factor in its utilization, but maltotriose transporters are relatively rare, except in brewing strains. No known wild strains of Saccharomyces eubayanus, the cold-tolerant parent of hybrid lager-brewing yeasts (Saccharomyces cerevisiae x S. eubayanus), are able to consume maltotriose, which limits their ability to fully ferment malt extract. In one strain of S. eubayanus, we found a gene closely related to a known maltotriose transporter and were able to confer maltotriose consumption by overexpressing this gene or by passaging the strain on maltose. Even so, most wild strains of S. eubayanus lack native maltotriose transporters. To determine how this rare trait could evolve in naive genetic backgrounds, we performed an adaptive evolution experiment for maltotriose consumption, which yielded a single strain of S. eubayanus able to grow on maltotriose. We mapped the causative locus to a gene encoding a novel chimeric transporter that was formed by an ectopic recombination event between two genes encoding transporters that are unable to import maltotriose. In contrast to classic models of the evolution of novel protein functions, the recombination breakpoints occurred within a single functional domain. Thus, the ability of the new protein to carry maltotriose was likely acquired through epistatic interactions between independently evolved substitutions. By acquiring multiple mutations at once, the transporter rapidly gained a novel function, while bypassing potentially deleterious intermediate steps. This study provides an illuminating example of how recombination between paralogs can establish novel interactions among substitutions to create adaptive functions