Aneuploidy influences the gene expression profiles in Saccharomyces pastorianus group I and II strains during fermentation.

The lager yeasts, Saccharomyces pastorianus, are hybrids of Saccharomyces cerevisiae and Saccharomyces eubayanus and are divided into two broad groups, Group I and II. The two groups evolved from at least one common hybridisation event but have subsequently diverged with Group I strains losing many S. cerevisiae chromosomes while the Group II strains retain both sub-genomes. The complex genomes, containing orthologous alleles from the parental chromosomes, pose interesting questions regarding gene regulation and its impact on the fermentation properties of the strains. Superimposed on the presence of orthologous alleles are complexities of gene dosage due to the aneuploid nature of the genomes. We examined the contribution of the S. cerevisiae and S. eubayanus alleles to the gene expression patterns of representative Group I and II strains during fermentation. We show that the relative expression of S. cerevisiae and S. eubayanus orthologues is positively correlated with gene copy number. Despite the reduced S. cerevisiae content in the Group I strain, S. cerevisiae orthologues contribute to biochemical pathways upregulated during fermentation which may explain the retention of specific chromosomes in the strain. Conversely, S. eubayanus genes are significantly overrepresented in the upregulated gene pool in the Group II strain. Comparison of the transcription profiles of the strains during fermentation identified both common and unique gene expression patterns, with gene copy number being a dominant contributory factor. Thus, the aneuploid genomes create complex patterns of gene expression during fermentation with gene dosage playing a crucial role both within and between strains.

Population genomics of the pathogenic yeast Candida tropicalis identifies hybrid isolates in environmental samples.

Candida tropicalis is a human pathogen that primarily infects the immunocompromised. Whereas the genome of one isolate, C. tropicalis MYA-3404, was originally sequenced in 2009, there have been no large-scale, multi-isolate studies of the genetic and phenotypic diversity of this species. Here, we used whole genome sequencing and phenotyping to characterize 77 isolates of C. tropicalis from clinical and environmental sources from a variety of locations. We show that most C. tropicalis isolates are diploids with approximately 2-6 heterozygous variants per kilobase. The genomes are relatively stable, with few aneuploidies. However, we identified one highly homozygous isolate and six isolates of C. tropicalis with much higher heterozygosity levels ranging from 36-49 heterozygous variants per kilobase. Our analyses show that the heterozygous isolates represent two different hybrid lineages, where the hybrids share one parent (A) with most other C. tropicalis isolates, but the second parent (B or C) differs by at least 4% at the genome level. Four of the sequenced isolates descend from an AB hybridization, and two from an AC hybridization. The hybrids are MTLa/α heterozygotes. Hybridization, or mating, between different parents is therefore common in the evolutionary history of C. tropicalis. The new hybrids were predominantly found in environmental niches, including from soil. Hybridization is therefore unlikely to be associated with virulence. In addition, we used genotype-phenotype correlation and CRISPR-Cas9 editing to identify a genome variant that results in the inability of one isolate to utilize certain branched-chain amino acids as a sole nitrogen source.

Transcriptional Profile of the Industrial Hybrid Saccharomyces pastorianus Reveals Temperature-Dependent Allele Expression Bias and Preferential Orthologous Protein Assemblies.

Saccharomyces pastorianus is a natural yeast evolved from different hybridization events between the mesophilic S. cerevisiae and the cold-tolerant S. eubayanus. This complex aneuploid hybrid carries multiple copies of the parental alleles alongside specific hybrid genes and encodes for multiple protein isoforms which impart novel phenotypes, such as the strong ability to ferment at low temperature. These characteristics lead to agonistic competition for substrates and a plethora of biochemical activities, resulting in a unique cellular metabolism. Here, we investigated the transcriptional signature of the different orthologous alleles in S. pastorianus during temperature shifts. We identified temperature-dependent media-independent genes and showed that 35% has their regulation dependent on extracellular leucine uptake, suggesting an interplay between leucine metabolism and temperature response. The analysis of the expression of ortholog parental alleles unveiled that the majority of the genes expresses preferentially one parental allele over the other and that S. eubayanus-like alleles are significantly over-represented among the genes involved in the cold acclimatization. The presence of functionally redundant parental alleles may impact on the nature of protein complexes established in the hybrid, where both parental alleles are competing. Our expression data indicate that the majority of the protein complexes investigated in the hybrid are likely to be either exclusively chimeric or unispecific and that the redundancy is discouraged, a scenario that fits well with the gene balance hypothesis. This study offers the first overview of the transcriptional pattern of S. pastorianus and provides a rationalization for its unique industrial traits at the expression level.

Enhanced flavour profiles through radicicol induced genomic variation in the lager yeasts, Saccharomyces pastorianus.

The yeasts, Saccharomyces pastorianus, are hybrids of Saccharomyces cerevisiae and Saccharomyces eubayanus and have acquired traits from the combined parental genomes such as ability to ferment a range of sugars at low temperatures and to produce aromatic flavour compounds, allowing for the production of lager beers with crisp, clean flavours. The polyploid strains are sterile and have reached an evolutionary bottleneck for genetic variation. Here we describe an accelerated evolution approach to obtain lager yeasts with enhanced flavour profiles. As the relative expression of orthologous alleles is a significant contributor to the transcriptome during fermentation, we aimed to induce genetic variation by altering the S. cerevisiae to S. eubayanus chromosome ratio. Aneuploidy was induced through the temporary inhibition of the cell's stress response and strains with increased production of aromatic amino acids via the Shikimate pathway were selected by resistance to amino acid analogues. Genomic changes such as gross chromosomal rearrangements, chromosome loss and chromosome gain were detected in the characterised mutants, as were single-nucleotide polymorphisms in ARO4, encoding for DAHP synthase, the catalytic enzyme in the first step of the Shikimate pathway. Transcriptome analysis confirmed the upregulation of genes encoding enzymes in the Ehrlich pathway and the concomitant increase in the production of higher alcohols and esters such as 2-phenylethanol, 2-phenylethyl acetate, tryptophol, and tyrosol. We propose that the polyploid nature of S. pastorianus genomes is an advantageous trait supporting opportunities for genetic alteration in otherwise sterile strains.

Packing a punch: understanding how flavours are produced in lager fermentations.

Beer is one of the most popular beverages in the world and it has an irreplaceable place in culture. Although invented later than ale, lager beers dominate the current market. Many factors relating to the appearance (colour, clarity and foam stability) and sensory characters (flavour, taste and aroma) of beer, and other psychological determinants affect consumers' perception of the product and defines its drinkability. This review takes a wholistic approach to scrutinise flavour generation in the brewing process, focusing particularly on the contribution of the raw ingredients and the yeasts to the final flavour profiles of lager beers. In addition, we examine current developments to improve lager beer flavour profiles for the modern consumers.

Evolutionary journey and characterisation of a novel pan-gene associated with beer strains of Saccharomyces cerevisiae.

The sequencing of over a thousand Saccharomyces cerevisiae genomes revealed a complex pangenome. Over one third of the discovered genes are not present in the S. cerevisiae core genome but instead are often restricted to a subset of yeast isolates and thus may be important for adaptation to specific environmental niches. We refer to these genes as "pan-genes," being part of the pangenome but not the core genome. Here, we describe the evolutionary journey and characterisation of a novel pan-gene, originally named hypothetical (HYPO) open-reading frame. Phylogenetic analysis reveals that HYPO has been predominantly retained in S. cerevisiae strains associated with brewing but has been repeatedly lost in most other fungal species during evolution. There is also evidence that HYPO was horizontally transferred at least once, from S. cerevisiae to Saccharomyces paradoxus. The phylogenetic analysis of HYPO exemplifies the complexity and intricacy of evolutionary trajectories of genes within the S. cerevisiae pangenome. To examine possible functions for Hypo, we overexpressed a HYPO-GFP fusion protein in both S. cerevisiae and Saccharomyces pastorianus. The protein localised to the plasma membrane where it accumulated initially in distinct foci. Time-lapse fluorescent imaging revealed that when cells are grown in wort, Hypo-gfp fluorescence spreads throughout the membrane during cell growth. The overexpression of Hypo-gfp in S. cerevisiae or S. pastorianus strains did not significantly alter cell growth in medium-containing glucose, maltose, maltotriose, or wort at different concentrations.

The hybrid genomes of Saccharomyces pastorianus: A current perspective.

Saccharomyces pastorianus is a recently evolved interspecies hybrid of Saccharomyces cerevisiae and Saccharomyces eubayanus used in the production of lager-type beers and has a long-standing history with the brewing industry. At least two distinct types of lager yeasts (Groups I and II) have been identified based on chromosome content and structure. One important feature of the genomes of lager yeasts is the presence of a set of hybrid chromosomes that emerged as a result of homeologous recombination events between the parental chromosomes. The unique genetic composition of the hybrid genomes of S. pastorianus affords interesting opportunities for evolution, adaptation and survival of the hybrids. The co-expression of S. eubayanus, S. cerevisiae and hybrid gene alleles, together with gene dosage effects resulting from the presence of multiple copies of individual genes, creates a complex algorithm for gene expression, cellular biochemistry and physiology. The recent availability of genome sequences for three Group I and ten Group II lager yeast strains provides an opportunity to decipher this complex algorithm and understand how it impacts on the final fermentation product: flavoursome beer. Copyright © 2017 John Wiley & Sons, Ltd.

Recombination sites on hybrid chromosomes in Saccharomyces pastorianus share common sequence motifs and define a complex evolutionary relationship between group I and II lager yeasts.

Saccharomyces pastorianus, referred to as lager yeasts, are hybrids of S. cerevisiae and S. eubayanus. Isolates within the species are divided into two groups (I and II) based on chromosome structure and composition. Following the hybridisation, the parental chromosomes underwent homeologous recombination, generating a set of hybrid chromosomes unique to the species. Here, we assessed the recombination events in seven lager yeast genomes to more clearly define the evolutionary route of lager yeasts. Meta-analysis of the recombination epicentres, as well as a detailed analysis of recombination events at the MAT locus, reveals a more complex evolutionary relationship between the group I and II lager yeasts than previously considered and identifies several divergent routes of evolution leading to the current S. pastorianus strains. We show that recombination epicentres contain sequential runs of pyrimidines, often flanked by purines, on one strand of the DNA, and identify two common sequence motifs present in >80% of the recombination epicentres, indicating that a common mechanism might account for the recombination events. Taken together, the data support a sequential hybridisation model of evolution for the two types of lager yeasts and suggest that the genomes of this newly emerged species are highly dynamic and continually evolving.

Comparative Analysis of the Antimicrobial Activities of Plant Defensin-Like and Ultrashort Peptides against Food-Spoiling Bacteria.

UNLABELLED: Antimicrobial peptides offer potential as novel therapeutics to combat food spoilage and poisoning caused by pathogenic and nonpathogenic bacteria. Our previous studies identified the peptide human beta-defensin 3 (HBD3) as a potent antimicrobial agent against a wide range of beer-spoiling bacteria. Thus, HBD3 is an excellent candidate for development as an additive to prevent food and beverage spoilage. To expand the repertoire of peptides with antimicrobial activity against bacteria associated with food spoilage and/or food poisoning, we carried out an in silico discovery pipeline to identify peptides with structure and activity similar to those of HBD3, focusing on peptides of plant origin. Using a standardized assay, we compared the antimicrobial activities of nine defensin-like plant peptides to the activity of HBD3. Only two of the peptides, fabatin-2 and Cp-thionin-2, displayed antimicrobial activity; however, the peptides differed from HBD3 in being sensitive to salt and were thermostable. We also compared the activities of several ultrashort peptides to that of HBD3. One of the peptides, the synthetic tetrapeptide O3TR, displayed biphasic antimicrobial activity but had a narrower host range than HBD3. Finally, to determine if the peptides might act in concert to improve antimicrobial activity, we compared the activities of the peptides in pairwise combinations. The plant defensin-like peptides fabatin-2 and Cp-thionin-2 displayed a synergistic effect with HBD3, while O3TR was antagonistic. Thus, some plant defensin-like peptides are effective antimicrobials and may act in concert with HBD3 to control bacteria associated with food spoilage and food poisoning. IMPORTANCE: Food spoilage and food poisoning caused by bacteria can have major health and economic implications for human society. With the rise in resistance to conventional antibiotics, there is a need to identify new antimicrobials to combat these outbreaks in our food supply. Here we screened plant peptide databases to identify peptides that share structural similarity with the human defensin peptide HBD3, which has known antimicrobial activity against food-spoiling bacteria. We show that two of the plant peptides display antimicrobial activity against bacteria associated with food spoilage. When combined with HBD3, the peptides are highly effective. We also analyzed the activity of an easily made ultrashort synthetic peptide, O3TR. We show that this small peptide also displays antimicrobial activity against food-spoiling bacteria but is not as effective as HBD3 or the plant peptides. The plant peptides identified are good candidates for development as natural additives to prevent food spoilage.

Loss of lager specific genes and subtelomeric regions define two different Saccharomyces cerevisiae lineages for Saccharomyces pastorianus Group I and II strains.

Lager yeasts, Saccharomyces pastorianus, are interspecies hybrids between S. cerevisiae and S. eubayanus and are classified into Group I and Group II clades. The genome of the Group II strain, Weihenstephan 34/70, contains eight so-called 'lager-specific' genes that are located in subtelomeric regions. We evaluated the origins of these genes through bioinformatic and PCR analyses of Saccharomyces genomes. We determined that four are of cerevisiae origin while four originate from S. eubayanus. The Group I yeasts contain all four S. eubayanus genes but individual strains contain only a subset of the cerevisiae genes. We identified S. cerevisiae strains that contain all four cerevisiae 'lager-specific' genes, and distinct patterns of loss of these genes in other strains. Analysis of the subtelomeric regions uncovered patterns of loss in different S. cerevisiae strains. We identify two classes of S. cerevisiae strains: ale yeasts (Foster O) and stout yeasts with patterns of 'lager-specific' genes and subtelomeric regions identical to Group I and II S. pastorianus yeasts, respectively. These findings lead us to propose that Group I and II S. pastorianus strains originate from separate hybridization events involving different S. cerevisiae lineages. Using the combined bioinformatic and PCR data, we describe a potential classification map for industrial yeasts.

Challenges for the production of bioethanol from biomass using recombinant yeasts.

Lignocellulose biomass, one of the most abundant renewable resources on the planet, is an alternative sustainable energy source for the production of second-generation biofuels. Energy in the form of simple or complex carbohydrates can be extracted from lignocellulose biomass and fermented by microorganisms to produce bioethanol. Despite 40 years of active and cutting-edge research invested into the development of technologies to produce bioethanol from lignocellulosic biomass, the process remains commercially unviable. This review describes the achievements that have been made in generating microorganisms capable of utilizing both simple and complex sugars from lignocellulose biomass and the fermentation of these sugars into ethanol. We also provide a discussion on the current "roadblocks" standing in the way of making second-generation bioethanol a commercially viable alternative to fossil fuels.

Engineering Saccharomyces pastorianus for the co-utilisation of xylose and cellulose from biomass.

BACKGROUND: Lignocellulosic biomass is a viable source of renewable energy for bioethanol production. For the efficient conversion of biomass into bioethanol, it is essential that sugars from both the cellulose and hemicellulose fractions of lignocellulose be utilised. RESULTS: We describe the development of a recombinant yeast system for the fermentation of cellulose and xylose, the most abundant pentose sugar in the hemicellulose fraction of biomass. The brewer's yeast Saccharomyces pastorianus was chosen as a host as significantly higher recombinant enzyme activities are achieved, when compared to the more commonly used S. cerevisiae. When expressed in S. pastorianus, the Trichoderma reesei xylose oxidoreductase pathway was more efficient at alcohol production from xylose than the xylose isomerase pathway. The alcohol yield was influenced by the concentration of xylose in the medium and was significantly improved by the additional expression of a gene encoding for xylulose kinase. The xylose reductase, xylitol dehydrogenase and xylulose kinase genes were co-expressed with genes encoding for the three classes of T. reesei cellulases, namely endoglucanase (EG2), cellobiohydrolysase (CBH2) and ß-glucosidase (BGL1). The initial metabolism of xylose by the engineered strains facilitated production of cellulases at fermentation temperatures. The sequential metabolism of xylose and cellulose generated an alcohol yield of 82% from the available sugars. Several different types of biomass, such as the energy crop Miscanthus sinensis and the industrial waste, brewer's spent grains, were examined as biomass sources for fermentation using the developed yeast strains. Xylose metabolism and cell growth were inhibited in fermentations carried out with acid-treated spent grain liquor, resulting in a 30% reduction in alcohol yield compared to fermentations carried out with mixed sugar substrates. CONCLUSIONS: Reconstitution of complete enzymatic pathways for cellulose hydrolysis and xylose utilisation in S. pastorianus facilitates the co-fermentation of cellulose and xylose without the need for added exogenous cellulases and provides a basis for the development of a consolidated process for co-utilisation of hemicellulose and cellulose sugars.

The PolyA tail length of yeast histone mRNAs varies during the cell cycle and is influenced by Sen1p and Rrp6p.

Yeast histone mRNAs are polyadenylated, yet factors such as Rrp6p and Trf4p, required for the 3'-end processing of non-polyadenylated RNAs, contribute to the cell cycle regulation of these transcripts. Here, we investigated the role of other known 3'-end processing/transcription termination factors of non-polyadenylated RNA in the biogenesis of histone mRNAs, specifically the Nab3p/Nrd1p/Sen1p complex. We also re-evaluated the polyadenylation status of these mRNAs during the cell cycle. Our analysis reveals that yeast histone mRNAs have shorter than average PolyA tails and the length of the PolyA tail varies during the cell cycle; S-phase histone mRNAs possess very short PolyA tails while in G1, the tail length is relatively longer. Inactivation of either Sen1p or Rrp6p leads to a decrease in the PolyA tail length of histone mRNAs. Our data also show that Sen1p contributes to 3'-end processing of histone primary transcripts. Thus, histone mRNAs are distinct from the general pool of yeast mRNAs and 3'-end processing and polyadenylation contribute to the cell cycle regulation of these transcripts.

Recombination between homoeologous chromosomes of lager yeasts leads to loss of function of the hybrid GPH1 gene.

Yeasts used in the production of lagers contain complex allopolyploid genomes, resulting from the fusion of two different yeast species closely related to Saccharomyces cerevisiae and Saccharomyces bayanus. Recombination between the homoeologous chromosomes has generated a number of hybrid chromosomes. These recombination events provide potential for adaptive evolution through the loss or gain of gene function. We have examined the genotypic and phenotypic effects of one of the conserved recombination events that occurred on chromosome XVI in the region of YPR159W and YPR160W. Our analysis shows that the recombination event occurred within the YPR160W gene, which encodes the enzyme glycogen phosphorylase and generates a hybrid gene that does not produce mature mRNA and is nonfunctional due to frameshifts in the coding region. The loss of function of the hybrid gene leads to glycogen levels similar to those found in haploid yeast strains. The implications for the control of glycogen levels in fermentative yeasts are discussed.

Chapter 6: The genomes of lager yeasts.

Yeasts used in the production of lagers belong to the genus Saccharomyces pastorianus. Species within this genus arose from a natural hybridization event between two yeast species that appear to be closely related to Saccharomyces cerevisiae and Saccharomyces bayanus. The resultant hybrids contain complex allopolyploid genomes and retain genetic characteristics of both parental species. Recent genome analysis using both whole genome sequencing and competitive genomic hybridization techniques has revealed the underlying composition of lager yeasts genomes. There appear to be at least 36 unique chromosomes, many of which are lager specific, resulting from recombination events between the homeologous parental chromosomes. The recombination events are limited to a defined set of genetic loci, which are highly conserved within strains of lager yeasts. In addition to the hybrid chromosomes, several non-reciprocal chromosomal translocations and inversions are also observed. Remarkably, in response to exposure to environmental stresses such as high temperatures and high osmotic pressure, the genomes appear to be highly dynamic and undergo recombination events at defined loci and alterations in the telomeric regions. The ability of environmental stress to alter the structure and composition of the genomes of lager yeasts may point to mechanisms of adaptive evolution in these species.

Deletion of the nuclear exosome component RRP6 leads to continued accumulation of the histone mRNA HTB1 in S-phase of the cell cycle in Saccharomyces cerevisiae.

The nuclear exosome, a macromolecular complex of 3' to 5' exonucleases, is required for the post-transcriptional processing of a variety of RNAs including rRNAs and snoRNAs. Additionally, this complex forms part of a nuclear surveillance network where it acts to degrade any aberrantly processed mRNAs in the nucleus. The exosome complex has been implicated in the biogenesis pathway of general messenger RNAs through its interaction with the 3'-end processing machinery. During the cell cycle, yeast histone mRNAs accumulate in the S-phase and are rapidly degraded as cells enter the G2-phase. To determine if the exosome contributes to the cyclic turnover of yeast histone mRNAs, we examined the pattern of accumulation of 'HTB1' mRNA during the cell cycle in a deletion strain of 'RRP6', a component of the nuclear exosome. Our results show that cells lacking Rrp6p continue to accumulate HTB1 mRNA as the cell cycle proceeds. This continued accumulation appears to result from a delay in exit from S-phase in rrp6 cells. The accumulation of HTB1 mRNA in rrp6 cells is influenced by the interaction of the nuclear exosome with the 3'-end processing machinery although there is no evidence for differential regulation of histone mRNA 3'-end processing during the yeast cell cycle.

Fermentation innovation through complex hybridization of wild and domesticated yeasts.

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.

Sinusitis: a possible link with adalimumab.

The experience with anti-TNF agents is relatively short; and with time, we are learning more about the frequency of occurrence of different adverse events as the original trials were either too small or too brief. We report a case series of four patients who suffered from chronic inflammatory arthritis [rheumatoid arthritis (n = 3) and psoriatic arthritis (n = 1)]. Their inflammatory arthritis remained refractory to increasing doses of methotrexate up to 20 mg weekly and required an advance in treatment to TNF antagonists. However, within a few weeks of commencing these patients on adalimumab, they developed newly diagnosed recurring sinusitis. All these patients were assessed by otorhinolaryngologists, and had clinically confirmed diagnosis. The sinusitis remained refractory to standard medications; however, it resolved after the discontinuation of adalimumab. Although FDA and Irish Pharmaceutical Health Association describe that adalimumab use increases the risk of non-serious infections marginally and most of the patients continued on Humira (adalimumab) after the infection was resolved, however, our recent observation raises the concern of probable higher incidence.