Domestication and Divergence of Saccharomyces cerevisiae Beer Yeasts.

Whereas domestication of livestock, pets, and crops is well documented, it is still unclear to what extent microbes associated with the production of food have also undergone human selection and where the plethora of industrial strains originates from. Here, we present the genomes and phenomes of 157 industrial Saccharomyces cerevisiae yeasts. Our analyses reveal that today's industrial yeasts can be divided into five sublineages that are genetically and phenotypically separated from wild strains and originate from only a few ancestors through complex patterns of domestication and local divergence. Large-scale phenotyping and genome analysis further show strong industry-specific selection for stress tolerance, sugar utilization, and flavor production, while the sexual cycle and other phenotypes related to survival in nature show decay, particularly in beer yeasts. Together, these results shed light on the origins, evolutionary history, and phenotypic diversity of industrial yeasts and provide a resource for further selection of superior strains. PAPERCLIP.

Locus-specific proteome decoding reveals Fpt1 as a chromatin-associated negative regulator of RNA polymerase III assembly.

Transcription of tRNA genes by RNA polymerase III (RNAPIII) is tuned by signaling cascades. The emerging notion of differential tRNA gene regulation implies the existence of additional regulatory mechanisms. However, tRNA gene-specific regulators have not been described. Decoding the local chromatin proteome of a native tRNA gene in yeast revealed reprogramming of the RNAPIII transcription machinery upon nutrient perturbation. Among the dynamic proteins, we identified Fpt1, a protein of unknown function that uniquely occupied RNAPIII-regulated genes. Fpt1 binding at tRNA genes correlated with the efficiency of RNAPIII eviction upon nutrient perturbation and required the transcription factors TFIIIB and TFIIIC but not RNAPIII. In the absence of Fpt1, eviction of RNAPIII was reduced, and the shutdown of ribosome biogenesis genes was impaired upon nutrient perturbation. Our findings provide support for a chromatin-associated mechanism required for RNAPIII eviction from tRNA genes and tuning the physiological response to changing metabolic demands.

Mutational robustness and the role of buffer genes in evolvability.

Organisms rely on mutations to fuel adaptive evolution. However, many mutations impose a negative effect on fitness. Cells may have therefore evolved mechanisms that affect the phenotypic effects of mutations, thus conferring mutational robustness. Specifically, so-called buffer genes are hypothesized to interact directly or indirectly with genetic variation and reduce its effect on fitness. Environmental or genetic perturbations can change the interaction between buffer genes and genetic variation, thereby unmasking the genetic variation's phenotypic effects and thus providing a source of variation for natural selection to act on. This review provides an overview of our understanding of mutational robustness and buffer genes, with the chaperone gene HSP90 as a key example. It discusses whether buffer genes merely affect standing variation or also interact with de novo mutations, how mutational robustness could influence evolution, and whether mutational robustness might be an evolved trait or rather a mere side-effect of complex genetic interactions.

The androgen receptor depends on ligand-binding domain dimerization for transcriptional activation.

Whereas dimerization of the DNA-binding domain of the androgen receptor (AR) plays an evident role in recognizing bipartite response elements, the contribution of the dimerization of the ligand-binding domain (LBD) to the correct functioning of the AR remains unclear. Here, we describe a mouse model with disrupted dimerization of the AR LBD (ARLmon/Y ). The disruptive effect of the mutation is demonstrated by the feminized phenotype, absence of male accessory sex glands, and strongly affected spermatogenesis, despite high circulating levels of testosterone. Testosterone replacement studies in orchidectomized mice demonstrate that androgen-regulated transcriptomes in ARLmon/Y mice are completely lost. The mutated AR still translocates to the nucleus and binds chromatin, but does not bind to specific AR binding sites. In vitro studies reveal that the mutation in the LBD dimer interface also affects other AR functions such as DNA binding, ligand binding, and co-regulator binding. In conclusion, LBD dimerization is crucial for the development of AR-dependent tissues through its role in transcriptional regulation in vivo. Our findings identify AR LBD dimerization as a possible target for AR inhibition.

FLO1 is a variable green beard gene that drives biofilm-like cooperation in budding yeast.

The budding yeast, Saccharomyces cerevisiae, has emerged as an archetype of eukaryotic cell biology. Here we show that S. cerevisiae is also a model for the evolution of cooperative behavior by revisiting flocculation, a self-adherence phenotype lacking in most laboratory strains. Expression of the gene FLO1 in the laboratory strain S288C restores flocculation, an altered physiological state, reminiscent of bacterial biofilms. Flocculation protects the FLO1 expressing cells from multiple stresses, including antimicrobials and ethanol. Furthermore, FLO1(+) cells avoid exploitation by nonexpressing flo1 cells by self/non-self recognition: FLO1(+) cells preferentially stick to one another, regardless of genetic relatedness across the rest of the genome. Flocculation, therefore, is driven by one of a few known "green beard genes," which direct cooperation toward other carriers of the same gene. Moreover, FLO1 is highly variable among strains both in expression and in sequence, suggesting that flocculation in S. cerevisiae is a dynamic, rapidly evolving social trait.

Variable Glutamine-Rich Repeats Modulate Transcription Factor Activity.

Excessive expansions of glutamine (Q)-rich repeats in various human proteins are known to result in severe neurodegenerative disorders such as Huntington's disease and several ataxias. However, the physiological role of these repeats and the consequences of more moderate repeat variation remain unknown. Here, we demonstrate that Q-rich domains are highly enriched in eukaryotic transcription factors where they act as functional modulators. Incremental changes in the number of repeats in the yeast transcriptional regulator Ssn6 (Cyc8) result in systematic, repeat-length-dependent variation in expression of target genes that result in direct phenotypic changes. The function of Ssn6 increases with its repeat number until a certain threshold where further expansion leads to aggregation. Quantitative proteomic analysis reveals that the Ssn6 repeats affect its solubility and interactions with Tup1 and other regulators. Thus, Q-rich repeats are dynamic functional domains that modulate a regulator's innate function, with the inherent risk of pathogenic repeat expansions.

Metabolic engineering of Yarrowia lipolytica for terpenoids production: advances and perspectives.

Terpenoids are a large family of natural products with diversified structures and functions that are widely used in the food, pharmaceutical, cosmetic, and agricultural fields. However, the traditional methods of terpenoids production such as plant extraction and chemical synthesis are inefficient due to the complex processes, high energy consumption, and low yields. With progress in metabolic engineering and synthetic biology, microbial cell factories provide an interesting alternative for the sustainable production of terpenoids. The non-conventional yeast, Yarrowia lipolytica, is a promising host for terpenoid biosynthesis due to its inherent mevalonate pathway, high fluxes of acetyl-CoA and NADPH, and the naturally hydrophobic microenvironment. In this review, we highlight progress in the engineering of Y. lipolytica as terpenoid biomanufacturing factories, describing the different terpenoid biosynthetic pathways and summarizing various metabolic engineering strategies, including progress in genetic manipulation, dynamic regulation, organelle engineering, and terpene synthase variants.

Network hubs affect evolvability.

The regulatory processes in cells are typically organized into complex genetic networks. However, it is still unclear how this network structure modulates the evolution of cellular regulation. One would expect that mutations in central and highly connected modules of a network (so-called hubs) would often result in a breakdown and therefore be an evolutionary dead end. However, a new study by Koubkova-Yu and colleagues finds that in some circumstances, altering a hub can offer a quick evolutionary advantage. Specifically, changes in a hub can induce significant phenotypic changes that allow organisms to move away from a local fitness peak, whereas the fitness defects caused by the perturbed hub can be mitigated by mutations in its interaction partners. Together, the results demonstrate how network architecture shapes and facilitates evolutionary adaptation.

An Integrated Approach Reveals DNA Damage and Proteotoxic Stress as Main Effects of Proton Radiation in S. cerevisiae.

Proton radiotherapy (PRT) has the potential to reduce the normal tissue toxicity associated with conventional photon-based radiotherapy (X-ray therapy, XRT) because the active dose can be more directly targeted to a tumor. Although this dosimetric advantage of PRT is well known, the molecular mechanisms affected by PRT remain largely elusive. Here, we combined the molecular toolbox of the eukaryotic model Saccharomyces cerevisiae with a systems biology approach to investigate the physiological effects of PRT compared to XRT. Our data show that the DNA damage response and protein stress response are the major molecular mechanisms activated after both PRT and XRT. However, RNA-Seq revealed that PRT treatment evoked a stronger activation of genes involved in the response to proteotoxic stress, highlighting the molecular differences between PRT and XRT. Moreover, inhibition of the proteasome resulted in decreased survival in combination with PRT compared to XRT, not only further confirming that protons induced a stronger proteotoxic stress response, but also hinting at the potential of using proteasome inhibitors in combination with proton radiotherapy in clinical settings.

Saccharomyces cerevisiae as a Model System for Eukaryotic Cell Biology, from Cell Cycle Control to DNA Damage Response.

The yeast Saccharomyces cerevisiae has been used for bread making and beer brewing for thousands of years. In addition, its ease of manipulation, well-annotated genome, expansive molecular toolbox, and its strong conservation of basic eukaryotic biology also make it a prime model for eukaryotic cell biology and genetics. In this review, we discuss the characteristics that made yeast such an extensively used model organism and specifically focus on the DNA damage response pathway as a prime example of how research in S. cerevisiae helped elucidate a highly conserved biological process. In addition, we also highlight differences in the DNA damage response of S. cerevisiae and humans and discuss the challenges of using S. cerevisiae as a model system.

Integrated Multi-Omics Analysis of Mechanisms Underlying Yeast Ethanol Tolerance.

For yeast cells, tolerance to high levels of ethanol is vital both in their natural environment and in industrially relevant conditions. We recently genotyped experimentally evolved yeast strains adapted to high levels of ethanol and identified mutations linked to ethanol tolerance. In this study, by integrating genomic sequencing data with quantitative proteomics profiles from six evolved strains (data set identifier PXD006631) and construction of protein interaction networks, we elucidate exactly how the genotype and phenotype are related at the molecular level. Our multi-omics approach points to the rewiring of numerous metabolic pathways affected by genomic and proteomic level changes, from energy-producing and lipid pathways to differential regulation of transposons and proteins involved in cell cycle progression. One of the key differences is found in the energy-producing metabolism, where the ancestral yeast strain responds to ethanol by switching to respiration and employing the mitochondrial electron transport chain. In contrast, the ethanol-adapted strains appear to have returned back to energy production mainly via glycolysis and ethanol fermentation, as supported by genomic and proteomic level changes. This work is relevant for synthetic biology where systems need to function under stressful conditions, as well as for industry and in cancer biology, where it is important to understand how the genotype relates to the phenotype.

Gene Loss Predictably Drives Evolutionary Adaptation.

Loss of gene function is common throughout evolution, even though it often leads to reduced fitness. In this study, we systematically evaluated how an organism adapts after deleting genes that are important for growth under oxidative stress. By evolving, sequencing, and phenotyping over 200 yeast lineages, we found that gene loss can enhance an organism's capacity to evolve and adapt. Although gene loss often led to an immediate decrease in fitness, many mutants rapidly acquired suppressor mutations that restored fitness. Depending on the strain's genotype, some ultimately even attained higher fitness levels than similarly adapted wild-type cells. Further, cells with deletions in different modules of the genetic network followed distinct and predictable mutational trajectories. Finally, losing highly connected genes increased evolvability by facilitating the emergence of a more diverse array of phenotypes after adaptation. Together, our findings show that loss of specific parts of a genetic network can facilitate adaptation by opening alternative evolutionary paths.

Old yeasts, young beer-The industrial relevance of yeast chronological life span.

Much like other living organisms, yeast cells have a limited life span, in terms of both the maximal length of time a cell can stay alive (chronological life span) and the maximal number of cell divisions it can undergo (replicative life span). Over the past years, intensive research revealed that the life span of yeast depends on both the genetic background of the cells and environmental factors. Specifically, the presence of stress factors, reactive oxygen species, and the availability of nutrients profoundly impact life span, and signaling cascades involved in the response to these factors, including the target of rapamycin (TOR) and cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathways, play a central role. Interestingly, yeast life span also has direct implications for its use in industrial processes. In beer brewing, for example, the inoculation of finished beer with live yeast cells, a process called "bottle conditioning" helps improve the product's shelf life by clearing undesirable carbonyl compounds such as furfural and 2-methylpropanal that cause staling. However, this effect depends on the reductive metabolism of living cells and is thus inherently limited by the cells' chronological life span. Here, we review the mechanisms underlying chronological life span in yeast. We also discuss how this insight connects to industrial observations and ultimately opens new routes towards superior industrial yeasts that can help improve a product's shelf life and thus contribute to a more sustainable industry.

The Pupal Parasitoid Trichopria drosophilae Is Attracted to the Same Yeast Volatiles as Its Adult Host.

There is increasing evidence that microorganisms, particularly fungi and bacteria, emit volatile compounds that mediate the foraging behaviour of insects and therefore have the potential to affect key ecological relationships. However, to what extent microbial volatiles affect the olfactory response of insects across different trophic levels remains unclear. Adult parasitoids use a variety of chemical stimuli to locate potential hosts, including those emitted by the host's habitat, the host itself, and microorganisms associated with the host. Given the great capacity of parasitoids to utilize and learn odours to increase foraging success, parasitoids of eggs, larvae, or pupae may respond to the same volatiles the adult stage of their hosts use when locating their resources, but compelling evidence is still scarce. In this study, using Saccharomyces cerevisiae we show that Trichopria drosophilae, a pupal parasitoid of Drosophila species, is attracted to the same yeast volatiles as their hosts in the adult stage, i.e. acetate esters. Parasitoids significantly preferred the odour of S. cerevisiae over the blank medium in a Y-tube olfactometer. Deletion of the yeast ATF1 gene, encoding a key acetate ester synthase, decreased attraction of T. drosophilae, while the addition of synthetic acetate esters to the fermentation medium restored parasitoid attraction. Bioassays with individual compounds revealed that the esters alone were not as attractive as the volatile blend of S. cerevisiae, suggesting that other volatile compounds also contribute to the attraction of T. drosophilae. Altogether, our results indicate that pupal parasitoids respond to the same volatiles as the adult stage of their hosts, which may aid them in locating oviposition sites.

Moulded by humans: The domestication of blue-veined cheese fungi.

It is hard to imagine a world without food-associated microbes. The production of bread, wine, beer, salami, coffee, chocolate, cheese and many other foods and beverages all rely on specific microbes. In cheese, myriad microbial species collaborate to yield the complex organoleptic properties that are appreciated by millions of people worldwide. In the early days of cheese making, these complex communities emerged spontaneously from the natural flora associated with the raw materials, the equipment, the production environment or craftsmen involved in the production process. However, in some cases, the microbes shifted their natural habitat to the new cheese-associated environment. The most obvious cause of this is backslopping, where part of a fermented product is used to inoculate the next batch. In addition, some microbes may simply adhere to the tools used in the production process. These microbial communities gradually adapted to the novel man-made niches, a process referred to as "domestication." Domestication is associated with specific genomic and phenotypic changes and ultimately leads to lineages that are genetically and phenotypically distinct from their wild ancestors. In this issue of Molecular Ecology, Dumas et al. have investigated a prime example of cheese-associated microbes, the fungus Penicillium roqueforti. The authors identified several hallmarks of domestication in the genome and phenome of this species, allowing them to hypothesize about the origin of blue-veined cheese fungi domestication, and the specific evolutionary processes involved in adaptation to the cheese matrix.

On the duration of the microbial lag phase.

When faced with environmental changes, microbes enter a lag phase during which cell growth is arrested, allowing cells to adapt to the new situation. The discovery of the lag phase started the field of gene regulation and led to the unraveling of underlying mechanisms. However, the factors determining the exact duration and dynamics of the lag phase remain largely elusive. Naively, one would expect that cells adapt as quickly as possible, so they can resume growth and compete with other organisms. However, recent studies show that the lag phase can last from several hours up to several days. Moreover, some cells within the same population take much longer than others, despite being genetically identical. In addition, the lag phase duration is also influenced by the past, with recent exposure to a given environment leading to a quicker adaptation when that environment returns. Genome-wide screens in Saccharomyces cerevisiae on carbon source shifts now suggest that the length of the lag phase, the heterogeneity in lag times of individual cells, and the history-dependent behavior are not determined by the time it takes to induce a few specific genes related to uptake and metabolism of a new carbon source. Instead, a major shift in general metabolism, and in particular a switch between fermentation and respiration, is the major bottleneck that determines lag duration. This suggests that there may be a fitness trade-off between complete adaptation of a cell's metabolism to a given environment, and a short lag phase when the environment changes.

Taming wild yeast: potential of conventional and nonconventional yeasts in industrial fermentations.

Yeasts are the main driving force behind several industrial food fermentation processes, including the production of beer, wine, sake, bread, and chocolate. Historically, these processes developed from uncontrolled, spontaneous fermentation reactions that rely on a complex mixture of microbes present in the environment. Because such spontaneous processes are generally inconsistent and inefficient and often lead to the formation of off-flavors, most of today's industrial production utilizes defined starter cultures, often consisting of a specific domesticated strain of Saccharomyces cerevisiae, S. bayanus, or S. pastorianus. Although this practice greatly improved process consistency, efficiency, and overall quality, it also limited the sensorial complexity of the end product. In this review, we discuss how Saccharomyces yeasts were domesticated to become the main workhorse of food fermentations, and we investigate the potential and selection of nonconventional yeasts that are often found in spontaneous fermentations, such as Brettanomyces, Hanseniaspora, and Pichia spp.

Apibacter muscae sp. nov., a novel bacterial species isolated from house flies.

We describe the isolation and characterization of three bacterial isolates from the common house fly, Musca domestica, caught in Londerzeel, Belgium and Huye District, Rwanda. Although isolated from distinct geographical locations, the strains show >99 % identical 16S rRNA gene sequences and are <95 % identical to type strains of Apibacter species. Whole-genome sequences were obtained for all three strains. The genomes are 2.4-2.5 Mb with a G+C content of ~30.3 mol%. Bacteriological and biochemical analysis of the strains demonstrate distinctly different characteristics compared to known Apibacter species. Particularly, the three strains investigated in this study can be distinguished from the known Apibacter species (Apibacter mensalisand Apibacter adventoris) through urease and ß-glucosidase activities. Whole-cell fatty acid methyl ester analysis shows that the fatty acid composition of the novel strains is also unique. On the basis of phylogenetic, genotypic and phenotypic data, we propose to classify these isolates as representatives of a novel species of the genus Apibacter, Apibacter muscae sp. nov., in reference to its prevalence in house flies, with strain G8T (=LMG 30898T=DSM 107922T) as the type strain.

Development and validation of a glass-silicon microdroplet-based system to measure sulfite concentrations in beverages.

Sulfite is often added to beverages as an antioxidant and antimicrobial agent. In fermented beverages, sulfite is also naturally produced by yeast cells. However, sulfite causes adverse health effects in asthmatic patients and accurate measurement of the sulfite concentration is therefore very important. Current sulfite analysis methods are time- and reagent-consuming and often require costly equipment. Here, we present a system allowing sensitive, ultralow-volume sulfite measurements based on a reusable glass-silicon microdroplet platform on which microdroplet generation, addition of enzymes through chemical-induced emulsion destabilization and pillar-induced droplet merging, emulsion restabilization, droplet incubation, and fluorescence measurements are integrated. In a first step, we developed and verified a fluorescence-based enzymatic assay for sulfite by measuring its analytical performance (LOD, LOQ, the dynamic working range, and the influence of salts, colorant, and sugars) and comparing fluorescent microplate readouts of fermentation samples with standard colorimetric measurements using the 5,5'-dithiobis-(2-nitrobenzoic acid) assay of the standard Gallery Plus Beermaster analysis platform. Next, samples were analyzed on the microdroplet platform, which also showed good correlation with the standard colorimetric analysis. Although the presented platform does not allow stable reinjection of droplets due to the presence of a tight array of micropillars at the fluidics entrances to prevent channel clogging by dust, removing the pillars, and integrating miniaturized pumps and optics in a future design would allow to use this platform for high-throughput, automated, and portable screening of microbes, plant, or mammalian cells. Graphical abstract ᅟ.

Variable tandem repeats accelerate evolution of coding and regulatory sequences.

Genotype-to-phenotype mapping commonly focuses on two major classes of mutations: single nucleotide polymorphisms (SNPs) and copy number variation (CNV). Here, we discuss an underestimated third class of genotypic variation: changes in microsatellite and minisatellite repeats. Such tandem repeats (TRs) are ubiquitous, unstable genomic elements that have historically been designated as nonfunctional "junk DNA" and are therefore mostly ignored in comparative genomics. However, as many as 10% to 20% of eukaryotic genes and promoters contain an unstable repeat tract. Mutations in these repeats often have fascinating phenotypic consequences. For example, changes in unstable repeats located in or near human genes can lead to neurodegenerative diseases such as Huntington disease. Apart from their role in disease, variable repeats also confer useful phenotypic variability, including cell surface variability, plasticity in skeletal morphology, and tuning of the circadian rhythm. As such, TRs combine characteristics of genetic and epigenetic changes that may facilitate organismal evolvability.