Global Expression Analysis of the Yeast Lachancea (Saccharomyces) kluyveri Reveals New URC Genes Involved in Pyrimidine Catabolism

Pyrimidines are important nucleic acid precursors which are constantly synthesized, degraded, and rebuilt in the cell. Four degradation pathways, two of which are found in eukaryotes, have been described. One of them, the URC pathway, has been initially discovered in our laboratory in the yeast Lachancea kluyveri. Here, we present the global changes in gene expression in L. kluyveri in response to different nitrogen sources, including uracil, uridine, dihydrouracil, and ammonia. The expression pattern of the known URC genes, URC1-6, helped to identify nine putative novel URC genes with a similar expression pattern. The microarray analysis provided evidence that both the URC and PYD genes are under nitrogen catabolite repression in L. kluyveri and are induced by uracil or dihydrouracil, respectively. We determined the function of URC8, which was found to catalyze the reduction of malonate semialdehyde to 3-hydroxypropionate, the final degradation product of the pathway. The other eight genes studied were all putative permeases. Our analysis of double deletion strains showed that the L. kluyveri Fui1p protein transported uridine, just like its homolog in Saccharomyces cerevisiae, but we demonstrated that is was not the only uridine transporter in L. kluyveri. We also showed that the L. kluyveri homologs of DUR3 and FUR4 do not have the same function that they have in S. cerevisiae, where they transport urea and uracil, respectively. In L. kluyveri, both of these deletion strains grew normally on uracil and urea.     

Mitochondrial-encoded endonucleases drive recombination of protein-coding genes in yeast

Mitochondrial recombination in yeast is well recognized, yet the underlying genetic mechanisms are not well understood. Recent progress has suggested that mobile introns in mitochondrial genomes (mitogenomes) can facilitate the recombination of their corresponding intron-containing genes through a mechanism known as intron homing. As many mitochondrial genes lack introns, there is a critical need to determine the extent of recombination and underlying mechanism of intron-lacking genes. This study leverages yeast mitogenomes to address these questions. In Saccharomyces cerevisiae, the 3′-end sequences of at least three intron-lacking mitochondrial genes exhibit elevated nucleotide diversity and recombination hotspots. Each of these 3′-end sequences is immediately adjacent to or even fused as overlapping genes with a stand-alone endonuclease. Our findings suggest that SAEs are responsible for recombination and elevated diversity of adjacent intron-lacking genes. SAEs were also evident to drive recombination of intron-lacking genes in Lachancea kluyveri, a yeast species that diverged from S. cerevisiae more than 100 million years ago. These results suggest SAEs as a common driver in recombination of intron-lacking genes during mitogenome evolution. We postulate that the linkage between intron-lacking gene and its adjacent endonuclease gene is the result of co-evolution.     

Monitoring Stress-Related Genes during the Process of Biomass Propagation of Saccharomyces cerevisiae Strains Used for Wine Making

Physiological capabilities and fermentation performance of Saccharomyces cerevisiae strains to be employed during industrial wine fermentations are critical for the quality of the final product. During the process of biomass propagation, yeast cells are dynamically exposed to a mixed and interrelated group of known stresses such as osmotic, oxidative, thermic, and/or starvation. These stressing conditions can dramatically affect the parameters of the fermentation process and the technological abilities of the yeast, e.g., the biomass yield and its fermentative capacity. Although a good knowledge exists of the behavior of S. cerevisiae under laboratory conditions, insufficient knowledge is available about yeast stress responses under the specific media and growth conditions during industrial processes. We performed growth experiments using bench-top fermentors and employed a molecular marker approach (changes in expression levels of five stress-related genes) to investigate how the cells respond to environmental changes during the process of yeast biomass production. The data show that in addition to the general stress response pathway, using the HSP12 gene as a marker, other specific stress response pathways were induced, as indicated by the changes detected in the mRNA levels of two stress-related genes, GPD1 and TRX2. These results suggest that the cells were affected by osmotic and oxidative stresses, demonstrating that these are the major causes of the stress response throughout the process of wine yeast biomass production.     

Alternative Splicing and Subfunctionalization Generates Functional Diversity in Fungal Proteomes

Alternative splicing is commonly used by the Metazoa to generate more than one protein from a gene. However, such diversification of the proteome by alternative splicing is much rarer in fungi. We describe here an ancient fungal alternative splicing event in which these two proteins are generated from a single alternatively spliced ancestral SKI7/HBS1 gene retained in many species in both the Ascomycota and Basidiomycota. While the ability to express two proteins from a single SKI7/HBS1 gene is conserved in many fungi, the exact mechanism by which they achieve this varies. The alternative splicing was lost in Saccharomyces cerevisiae following the whole-genome duplication event as these two genes subfunctionalized into the present functionally distinct HBS1 and SKI7 genes. When expressed in yeast, the single gene from Lachancea kluyveri generates two functionally distinct proteins. Expression of one of these proteins complements hbs1, but not ski7 mutations, while the other protein complements ski7, but not hbs1. This is the first known case of subfunctionalization by loss of alternative splicing in yeast. By coincidence, the ancestral alternatively spliced gene was also duplicated in Schizosaccharomyces pombe with subsequent subfunctionalization and loss of splicing. Similar subfunctionalization by loss of alternative splicing in fungi also explains the presence of two PTC7 genes in the budding yeast Tetrapisispora blattae, suggesting that this is a common mechanism to preserve duplicate alternatively spliced genes.     

Horizontal gene transfer promoted evolution of the ability to propagate under anaerobic conditions in yeasts

The ability to propagate under anaerobic conditions is an essential and unique trait of brewers or bakers yeast (Saccharomyces cervisiae). To understand the evolution of facultative anaerobiosis we studied the dependence of de novo pyrimidine biosynthesis, more precisely the fourth enzymic activity catalysed by dihydroorotate dehydrogenase (DHODase), on the enzymes of the respiratory chain in several yeast species. While the majority of yeasts possess a mitochondrial DHODase, Saccharomyces cerevisiae has a cytoplasmatic enzyme, whose activity is independent of the presence of oxygen. From the phylogenetic point of view, this enzyme is closely related to a bacterial DHODase from Lactococcus lactis. Here we show that S. kluyveri, which separated from the S. cerevisiae lineage more than 100 million years ago, represents an evolutionary intermediate, having both cytoplasmic and mitochondrial DHODases. We show that these two S. kluyveri enzymes, and their coding genes, differ in their dependence on the presence of oxygen. Only the cytoplasmic DHODase promotes growth in the absence of oxygen. Apparently a Saccharomyces yeast progenitor which had a eukaryotic-like mitochondrial DHODase acquired a bacterial gene for DHODase, which subsequently allowed cell growth gradually to become independent of oxygen.Communicated by C. P. Hollenberg     

Induction of Stress Genes in Saccharomyces cerevisiae during Starvation

In order to analyze the response of Saccharomyces cerevisiae to starvation on a gene expression level, microarray experiments were performed using a yeast whole genome array. It is well known that under stress conditions like heat, high salt concentrations, pressure or the presence of toxins, special stress response genes are induced in Saccharomyces cerevisiae. This includes the genes encoding the typical heat shock proteins as well as numerous genes concerning cell membrane composition, central carbon metabolism or cell cycle. In this contribution, the Saccharomyces cerevisiae starvation‐stress response is analyzed. Starvation is a living condition often experienced by yeast in natural surroundings. As Saccharomyces cerevisiae is an eukaryote, many results from the gene expression analysis are valid for mammalians as well. The understanding of response of the yeast to the absence of a nutrient is also important for the development of feeding strategies in cultivations. Therefore, knowledge about the gene expression during starvation is important for both research and industrial applications. The regulation of 233 genes, which are involved in the stress response according to the literature, was examined via microarray experiments. In addition, a screening was carried out identifying 115 genes, which are hitherto not known to be comprised in the stress response, but which were significantly up‐regulated during starvation.     

Pyruvate decarboxylases from the petite-negative yeast<Emphasis Type="Italic"> Saccharomyces kluyveri</Emphasis>

Saccharomyces kluyveri is a petite-negative yeast, which is less prone to form ethanol under aerobic conditions than is S. cerevisiae. The first reaction on the route from pyruvate to ethanol is catalysed by pyruvate decarboxylase, and the differences observed between S. kluyveri and S. cerevisiae with respect to ethanol formation under aerobic conditions could be caused by differences in the regulation of this enzyme activity. We have identified and cloned three genes encoding functional pyruvate decarboxylase enzymes ( PDC genes) from the type strain of S. kluyveri (Sk-PDC11, Sk-PDC12 and Sk-PDC13). The regulation of pyruvate decarboxylase in S. kluyveri was studied by measuring the total level of Sk-PDC mRNA and the overall enzyme activity under various growth conditions. It was found that the level of Sk-PDC mRNA was enhanced by glucose and oxygen limitation, and that the level of enzyme activity was controlled by variations in the amount of mRNA. The mRNA level and the pyruvate decarboxylase activity responded to anaerobiosis and growth on different carbon sources in essentially the same fashion as in S. cerevisiae. This indicates that the difference in ethanol formation between these two yeasts is not due to differences in the regulation of pyruvate decarboxylase(s), but rather to differences in the regulation of the TCA cycle and the respiratory machinery. However, the PDC genes of Saccharomyces/Kluyveromyces yeasts differ in their genetic organization and phylogenetic origin. While S. cerevisiae and S. kluyveri each have three PDC genes, these have apparently arisen by independent duplications and specializations in each of the two yeast lineages.Communicated by C. P. Hollenberg     

The Sir2-Sum1 Complex Represses Transcription Using Both Promoter-Specific and Long-Range Mechanisms to Regulate Cell Identity and Sexual Cycle in the Yeast Kluyveromyces lactis

Deacetylases of the Sir2 family regulate lifespan and response to stress. We have examined the evolutionary history of Sir2 and Hst1, which arose by gene duplication in budding yeast and which participate in distinct mechanisms of gene repression. In Saccharomyces cerevisiae, Sir2 interacts with the SIR complex to generate long-range silenced chromatin at the cryptic mating-type loci, HMLα and HMR a. Hst1 interacts with the SUM1 complex to repress sporulation genes through a promoter-specific mechanism. We examined the functions of the non-duplicated Sir2 and its partners, Sir4 and Sum1, in the yeast Kluyveromyces lactis, a species that diverged from Saccharomyces prior to the duplication of Sir2 and Hst1. KlSir2 interacts with both KlSir4 and KlSum1 and represses the same sets of target genes as ScSir2 and ScHst1, indicating that Sir2 and Hst1 subfunctionalized after duplication. However, the KlSir4-KlSir2 and KlSum1-KlSir2 complexes do not function as the analogous complexes do in S. cerevisiae. KlSir4 contributes to an extended repressive chromatin only at HMLα and not at HMR a. In contrast, the role of KlSum1 is broader. It employs both long-range and promoter-specific mechanisms to repress cryptic mating-type loci, cell-type–specific genes, and sporulation genes and represents an important regulator of cell identity and the sexual cycle. This study reveals that a single repressive complex can act through two distinct mechanisms to regulate gene expression and illustrates how mechanisms by which regulatory proteins act can change over evolutionary time.     

Yeast species associated with spontaneous wine fermentation of Cabernet Sauvignon from Ningxia,China

The eastern base of the Helan Mountains in Ningxia is a fast developing wine production area in China. Of urgent necessity to the Ningxia wine industry is to be able to produce wines with typical regional characteristics. It is well known that autochthonous yeast species and strains play an important role in introducing local character or terroir into the winemaking practice. The aim of this study was to investigate indigenous yeast species diversity and preselect desirable S. cerevisiae strains in the Ningxia region. Four hundred wine-related yeast colonies were isolated from Cabernet Sauvignon musts in three vineyards at the beginning, middle and final stages of spontaneous fermentations. Yeast species were first classified according to colony morphologies on Wallerstein Laboratory Nutrient Agar (WL) and confirmed by sequencing of the D1/D2 domain of the 26S rRNA gene. Nine unique colony morphology types were profiled on WL agar and three new types were found. After sequence analysis of representative colonies from each WL group, nine yeast species were identified, namely H. uvarum, H. occidentalis, M. pulcherrima, C. zemplinina, H. vineae, I. orientalis, Z. bailii, P. kluyveri and S. cerevisiae. The non-Saccharomyces yeasts appeared mainly in the early stage of the fermentation while H. uvarum, I. orientalis and P. kluyveri were able to remain in the final stage. Fourty-six S. cerevisiae isolates were preselected according to physiological characteristics and twelve strains with valuable fermentation properties were obtained.     

Tomato QM-Like Protein Protects Saccharomyces cerevisiae Cells against Oxidative Stress by Regulating Intracellular Proline Levels

Exogenous proline can protect cells of Saccharomyces cerevisiae from oxidative stress. We altered intracellular proline levels by overexpressing the proline dehydrogenase gene (PUT1) of S. cerevisiae. Put1p performs the first enzymatic step of proline degradation in S. cerevisiae. Overexpression of Put1p results in low proline levels and hypersensitivity to oxidants, such as hydrogen peroxide and paraquat. A put1-disrupted yeast mutant deficient in Put1p activity has increased protection from oxidative stress and increased proline levels. Following a conditional life/death screen in yeast, we identified a tomato (Lycopersicon esculentum) gene encoding a QM-like protein (tQM) and found that stable expression of tQM in the Put1p-overexpressing strain conferred protection against oxidative damage from H₂O₂, paraquat, and heat. This protection was correlated with reactive oxygen species (ROS) reduction and increased proline accumulation. A yeast two-hybrid system assay was used to show that tQM physically interacts with Put1p in yeast, suggesting that tQM is directly involved in modulating proline levels. tQM also can rescue yeast from the lethality mediated by the mammalian proapoptotic protein Bax, through the inhibition of ROS generation. Our results suggest that tQM is a component of various stress response pathways and may function in proline-mediated stress tolerance in plants.     

Additions, Losses, and Rearrangements on the Evolutionary Route from a Reconstructed Ancestor to the Modern Saccharomyces cerevisiae Genome

Comparative genomics can be used to infer the history of genomic rearrangements that occurred during the evolution of a species. We used the principle of parsimony, applied to aligned synteny blocks from 11 yeast species, to infer the gene content and gene order that existed in the genome of an extinct ancestral yeast about 100 Mya, immediately before it underwent whole-genome duplication (WGD). The reconstructed ancestral genome contains 4,703 ordered loci on eight chromosomes. The reconstruction is complete except for the subtelomeric regions. We then inferred the series of rearrangement steps that led from this ancestor to the current Saccharomyces cerevisiae genome; relative to the ancestral genome we observe 73 inversions, 66 reciprocal translocations, and five translocations involving telomeres. Some fragile chromosomal sites were reused as evolutionary breakpoints multiple times. We identified 124 genes that have been gained by S. cerevisiae in the time since the WGD, including one that is derived from a hAT family transposon, and 88 ancestral loci at which S. cerevisiae did not retain either of the gene copies that were formed by WGD. Sites of gene gain and evolutionary breakpoints both tend to be associated with tRNA genes and, to a lesser extent, with origins of replication. Many of the gained genes in S. cerevisiae have functions associated with ethanol production, growth in hypoxic environments, or the uptake of alternative nutrient sources.     

Yeast Diversity Isolated from Grape Musts During Spontaneous Fermentation from a Brazilian Winery

Saccharomyces and non-Saccharomyces yeast species from a winery located in Brazil were identified by ribosomal gene-sequencing analysis. A total of 130 yeast strains were isolated from grape surfaces and musts during alcoholic fermentation from Isabel, Bordeaux, and Cabernet Sauvignon varieties. Samples were submitted to PCR–RFLP analysis and genomic sequencing. Thirteen species were identified: Candida quercitrusa, Candida stellata, Cryptococcus flavescens, Cryptococcus laurentii, Hanseniaspora uvarum, Issatchenkia occidentalis, Issatchenkia orientalis, Issatchenkia terricola, Pichia kluyveri, Pichia guilliermondii, Pichia sp., Saccharomyces cerevisiae, and Sporidiobolus pararoseus. A sequential substitution of species during the different stages of fermentation, with a dominance of non-Saccharomyces yeasts at the beginning, and a successive replacement of species by S. cerevisiae strains at the final steps were observed. This is the first report about the yeast distribution present throughout the alcoholic fermentation in a Brazilian winery, providing supportive information for future studies on their contribution to wine quality.     

Responses of Saccharomyces cerevisiae Strains from Different Origins to Elevated Iron Concentrations

Iron is an essential micronutrient for all eukaryotic organisms. However, the low solubility of ferric iron has tremendously increased the prevalence of iron deficiency anemia, especially in women and children, with dramatic consequences. Baker''s yeast Saccharomyces cerevisiae is used as a model eukaryotic organism, a fermentative microorganism, and a feed supplement. In this report, we explore the genetic diversity of 123 wild and domestic strains of S. cerevisiae isolated from different geographical origins and sources to characterize how yeast cells respond to elevated iron concentrations in the environment. By using two different forms of iron, we selected and characterized both iron-sensitive and iron-resistant yeast strains. We observed that when the iron concentration in the medium increases, iron-sensitive strains accumulate iron more rapidly than iron-resistant isolates. We observed that, consistent with excess iron leading to oxidative stress, the redox state of iron-sensitive strains was more oxidized than that of iron-resistant strains. Growth assays in the presence of different oxidative reagents ruled out that this phenotype was due to alterations in the general oxidative stress protection machinery. It was noteworthy that iron-resistant strains were more sensitive to iron deficiency conditions than iron-sensitive strains, which suggests that adaptation to either high or low iron is detrimental for the opposite condition. An initial gene expression analysis suggested that alterations in iron homeostasis genes could contribute to the different responses of distant iron-sensitive and iron-resistant yeast strains to elevated environmental iron levels.     

Molecular genetic differentiation of yeast α-glucosidases: Maltase and isomaltase

The review is dedicated to the molecular genetics of yeast ??-glucosidases: the maltase and isomaltase isozymes. Comparative analysis of the genome sequence of the yeast Saccharomyces cerevisiae S288C using the isomaltase gene of Saccharomyces cerevisiae ATCC56960 revealed a new family of polymeric isomaltase genes IMA1-IMA5 located in the telomeric regions of chromosomes VII, XV, IX, X, and X, respectively. The isomaltase overexpression and substrate specificity are discussed.     

Genetically Engineered Saccharomyces Yeast Capable of Effective Cofermentation of Glucose and Xylose

Xylose is one of the major fermentable sugars present in cellulosic biomass, second only to glucose. However, Saccharomyces spp., the best sugar-fermenting microorganisms, are not able to metabolize xylose. We developed recombinant plasmids that can transform Saccharomyces spp. into xylose-fermenting yeasts. These plasmids, designated pLNH31, -32, -33, and -34, are 2μm-based high-copy-number yeast-E. coli shuttle plasmids. In addition to the geneticin resistance and ampicillin resistance genes that serve as dominant selectable markers, these plasmids also contain three xylose-metabolizing genes, a xylose reductase gene, a xylitol dehydrogenase gene (both from Pichia stipitis), and a xylulokinase gene (from Saccharomyces cerevisiae). These xylose-metabolizing genes were also fused to signals controlling gene expression from S. cerevisiae glycolytic genes. Transformation of Saccharomyces sp. strain 1400 with each of these plasmids resulted in the conversion of strain 1400 from a non-xylose-metabolizing yeast to a xylose-metabolizing yeast that can effectively ferment xylose to ethanol and also effectively utilizes xylose for aerobic growth. Furthermore, the resulting recombinant yeasts also have additional extraordinary properties. For example, the synthesis of the xylose-metabolizing enzymes directed by the cloned genes in these recombinant yeasts does not require the presence of xylose for induction, nor is the synthesis repressed by the presence of glucose in the medium. These properties make the recombinant yeasts able to efficiently ferment xylose to ethanol and also able to efficiently coferment glucose and xylose present in the same medium to ethanol simultaneously.