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.

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.

Fermentation assays reveal differences in sugar and (off-) flavor metabolism across different Brettanomyces bruxellensis strains.

Brettanomyces (Dekkera) bruxellensis is an ascomycetous yeast of major importance in the food, beverage and biofuel industry. It has been isolated from various man-made ecological niches that are typically characterized by harsh environmental conditions such as wine, beer, soft drink, etc. Recent comparative genomics studies revealed an immense intraspecific diversity, but it is still unclear whether this genetic diversity also leads to systematic differences in fermentation performance and (off-)flavor production, and to what extent strains have evolved to match their ecological niche. Here, we present an evaluation of the fermentation properties of eight genetically diverse B. bruxellensis strains originating from beer, wine and soft drinks. We show that sugar consumption and aroma production during fermentation are determined by both the yeast strain and composition of the medium. Furthermore, our results indicate a strong niche adaptation of B. bruxellensis, most clearly for wine strains. For example, only strains originally isolated from wine were able to thrive well and produce the typical Brettanomyces-related phenolic off-flavors 4-ethylguaiacol and 4-ethylphenol when inoculated in red wine. Sulfite tolerance was found as a key factor explaining the observed differences in fermentation performance and off-flavor production. Sequence analysis of genes related to phenolic off-flavor production, however, revealed only marginal differences between the isolates tested, especially at the amino acid level. Altogether, our study provides novel insights in the Brettanomyces metabolism of flavor production, and is highly relevant for both the wine and beer industry.

Tuning Chocolate Flavor through Development of Thermotolerant Saccharomyces cerevisiae Starter Cultures with Increased Acetate Ester Production.

Microbial starter cultures have extensively been used to enhance the consistency and efficiency of industrial fermentations. Despite the advantages of such controlled fermentations, the fermentation involved in the production of chocolate is still a spontaneous process that relies on the natural microbiota at cocoa farms. However, recent studies indicate that certain thermotolerant Saccharomyces cerevisiae cultures can be used as starter cultures for cocoa pulp fermentation. In this study, we investigate the potential of specifically developed starter cultures to modulate chocolate aroma. Specifically, we developed several new S. cerevisiae hybrids that combine thermotolerance and efficient cocoa pulp fermentation with a high production of volatile flavor-active esters. In addition, we investigated the potential of two strains of two non-Saccharomyces species that produce very large amounts of fruity esters (Pichia kluyveri and Cyberlindnera fabianii) to modulate chocolate aroma. Gas chromatography-mass spectrometry (GC-MS) analysis of the cocoa liquor revealed an increased concentration of various flavor-active esters and a decrease in spoilage-related off-flavors in batches inoculated with S. cerevisiae starter cultures and, to a lesser extent, in batches inoculated with P. kluyveri and Cyb. fabianii. Additionally, GC-MS analysis of chocolate samples revealed that while most short-chain esters evaporated during conching, longer and more-fat-soluble ethyl and acetate esters, such as ethyl octanoate, phenylethyl acetate, ethyl phenylacetate, ethyl decanoate, and ethyl dodecanoate, remained almost unaffected. Sensory analysis by an expert panel confirmed significant differences in the aromas of chocolates produced with different starter cultures. Together, these results show that the selection of different yeast cultures opens novel avenues for modulating chocolate flavor.

A large set of newly created interspecific Saccharomyces hybrids increases aromatic diversity in lager beers.

Lager beer is the most consumed alcoholic beverage in the world. Its production process is marked by a fermentation conducted at low (8 to 15°C) temperatures and by the use of Saccharomyces pastorianus, an interspecific hybrid between Saccharomyces cerevisiae and the cold-tolerant Saccharomyces eubayanus. Recent whole-genome-sequencing efforts revealed that the currently available lager yeasts belong to one of only two archetypes, "Saaz" and "Frohberg." This limited genetic variation likely reflects that all lager yeasts descend from only two separate interspecific hybridization events, which may also explain the relatively limited aromatic diversity between the available lager beer yeasts compared to, for example, wine and ale beer yeasts. In this study, 31 novel interspecific yeast hybrids were developed, resulting from large-scale robot-assisted selection and breeding between carefully selected strains of S. cerevisiae (six strains) and S. eubayanus (two strains). Interestingly, many of the resulting hybrids showed a broader temperature tolerance than their parental strains and reference S. pastorianus yeasts. Moreover, they combined a high fermentation capacity with a desirable aroma profile in laboratory-scale lager beer fermentations, thereby successfully enriching the currently available lager yeast biodiversity. Pilot-scale trials further confirmed the industrial potential of these hybrids and identified one strain, hybrid H29, which combines a fast fermentation, high attenuation, and the production of a complex, desirable fruity aroma.

Breeding Strategy To Generate Robust Yeast Starter Cultures for Cocoa Pulp Fermentations.

Cocoa pulp fermentation is a spontaneous process during which the natural microbiota present at cocoa farms is allowed to ferment the pulp surrounding cocoa beans. Because such spontaneous fermentations are inconsistent and contribute to product variability, there is growing interest in a microbial starter culture that could be used to inoculate cocoa pulp fermentations. Previous studies have revealed that many different fungi are recovered from different batches of spontaneous cocoa pulp fermentations, whereas the variation in the prokaryotic microbiome is much more limited. In this study, therefore, we aimed to develop a suitable yeast starter culture that is able to outcompete wild contaminants and consistently produce high-quality chocolate. Starting from specifically selected Saccharomyces cerevisiae strains, we developed robust hybrids with characteristics that allow them to efficiently ferment cocoa pulp, including improved temperature tolerance and fermentation capacity. We conducted several laboratory and field trials to show that these new hybrids often outperform their parental strains and are able to dominate spontaneous pilot scale fermentations, which results in much more consistent microbial profiles. Moreover, analysis of the resulting chocolate showed that some of the cocoa batches that were fermented with specific starter cultures yielded superior chocolate. Taken together, these results describe the development of robust yeast starter cultures for cocoa pulp fermentations that can contribute to improving the consistency and quality of commercial chocolate production.

Large-scale selection and breeding to generate industrial yeasts with superior aroma production.

The concentrations and relative ratios of various aroma compounds produced by fermenting yeast cells are essential for the sensory quality of many fermented foods, including beer, bread, wine, and sake. Since the production of these aroma-active compounds varies highly among different yeast strains, careful selection of variants with optimal aromatic profiles is of crucial importance for a high-quality end product. This study evaluates the production of different aroma-active compounds in 301 different Saccharomyces cerevisiae, Saccharomyces paradoxus, and Saccharomyces pastorianus yeast strains. Our results show that the production of key aroma compounds like isoamyl acetate and ethyl acetate varies by an order of magnitude between natural yeasts, with the concentrations of some compounds showing significant positive correlation, whereas others vary independently. Targeted hybridization of some of the best aroma-producing strains yielded 46 intraspecific hybrids, of which some show a distinct heterosis (hybrid vigor) effect and produce up to 45% more isoamyl acetate than the best parental strains while retaining their overall fermentation performance. Together, our results demonstrate the potential of large-scale outbreeding to obtain superior industrial yeasts that are directly applicable for commercial use.

Assessing genetic diversity among Brettanomyces yeasts by DNA fingerprinting and whole-genome sequencing.

Brettanomyces yeasts, with the species Brettanomyces (Dekkera) bruxellensis being the most important one, are generally reported to be spoilage yeasts in the beer and wine industry due to the production of phenolic off flavors. However, B. bruxellensis is also known to be a beneficial contributor in certain fermentation processes, such as the production of certain specialty beers. Nevertheless, despite its economic importance, Brettanomyces yeasts remain poorly understood at the genetic and genomic levels. In this study, the genetic relationship between more than 50 Brettanomyces strains from all presently known species and from several sources was studied using a combination of DNA fingerprinting techniques. This revealed an intriguing correlation between the B. bruxellensis fingerprints and the respective isolation source. To further explore this relationship, we sequenced a (beneficial) beer isolate of B. bruxellensis (VIB X9085; ST05.12/22) and compared its genome sequence with the genome sequences of two wine spoilage strains (AWRI 1499 and CBS 2499). ST05.12/22 was found to be substantially different from both wine strains, especially at the level of single nucleotide polymorphisms (SNPs). In addition, there were major differences in the genome structures between the strains investigated, including the presence of large duplications and deletions. Gene content analysis revealed the presence of 20 genes which were present in both wine strains but absent in the beer strain, including many genes involved in carbon and nitrogen metabolism, and vice versa, no genes that were missing in both AWRI 1499 and CBS 2499 were found in ST05.12/22. Together, this study provides tools to discriminate Brettanomyces strains and provides a first glimpse at the genetic diversity and genome plasticity of B. bruxellensis.

Phenotypic evaluation of natural and industrial Saccharomyces yeasts for different traits desirable in industrial bioethanol production.

Saccharomyces cerevisiae is the organism of choice for many food and beverage fermentations because it thrives in high-sugar and high-ethanol conditions. However, the conditions encountered in bioethanol fermentation pose specific challenges, including extremely high sugar and ethanol concentrations, high temperature, and the presence of specific toxic compounds. It is generally considered that exploring the natural biodiversity of Saccharomyces strains may be an interesting route to find superior bioethanol strains and may also improve our understanding of the challenges faced by yeast cells during bioethanol fermentation. In this study, we phenotypically evaluated a large collection of diverse Saccharomyces strains on six selective traits relevant for bioethanol production with increasing stress intensity. Our results demonstrate a remarkably large phenotypic diversity among different Saccharomyces species and among S. cerevisiae strains from different origins. Currently applied bioethanol strains showed a high tolerance to many of these relevant traits, but several other natural and industrial S. cerevisiae strains outcompeted the bioethanol strains for specific traits. These multitolerant strains performed well in fermentation experiments mimicking industrial bioethanol production. Together, our results illustrate the potential of phenotyping the natural biodiversity of yeasts to find superior industrial strains that may be used in bioethanol production or can be used as a basis for further strain improvement through genetic engineering, experimental evolution, or breeding. Additionally, our study provides a basis for new insights into the relationships between tolerance to different stressors.

Combinatorial optimization of gene expression through recombinase-mediated promoter and terminator shuffling in yeast.

Microbes are increasingly employed as cell factories to produce biomolecules. This often involves the expression of complex heterologous biosynthesis pathways in host strains. Achieving maximal product yields and avoiding build-up of (toxic) intermediates requires balanced expression of every pathway gene. However, despite progress in metabolic modeling, the optimization of gene expression still heavily relies on trial-and-error. Here, we report an approach for in vivo, multiplexed Gene Expression Modification by LoxPsym-Cre Recombination (GEMbLeR). GEMbLeR exploits orthogonal LoxPsym sites to independently shuffle promoter and terminator modules at distinct genomic loci. This approach facilitates creation of large strain libraries, in which expression of every pathway gene ranges over 120-fold and each strain harbors a unique expression profile. When applied to the biosynthetic pathway of astaxanthin, an industrially relevant antioxidant, a single round of GEMbLeR improved pathway flux and doubled production titers. Together, this shows that GEMbLeR allows rapid and efficient gene expression optimization in heterologous biosynthetic pathways, offering possibilities for enhancing the performance of microbial cell factories.

Orthogonal LoxPsym sites allow multiplexed site-specific recombination in prokaryotic and eukaryotic hosts.

Site-specific recombinases such as the Cre-LoxP system are routinely used for genome engineering in both prokaryotes and eukaryotes. Importantly, recombinases complement the CRISPR-Cas toolbox and provide the additional benefit of high-efficiency DNA editing without generating toxic DNA double-strand breaks, allowing multiple recombination events at the same time. However, only a handful of independent, orthogonal recombination systems are available, limiting their use in more complex applications that require multiple specific recombination events, such as metabolic engineering and genetic circuits. To address this shortcoming, we develop 63 symmetrical LoxP variants and test 1192 pairwise combinations to determine their cross-reactivity and specificity upon Cre activation. Ultimately, we establish a set of 16 orthogonal LoxPsym variants and demonstrate their use for multiplexed genome engineering in both prokaryotes (E. coli) and eukaryotes (S. cerevisiae and Z. mays). Together, this work yields a significant expansion of the Cre-LoxP toolbox for genome editing, metabolic engineering and other controlled recombination events, and provides insights into the Cre-LoxP recombination process.

Novel Saccharomyces cerevisiae variants slow down the accumulation of staling aldehydes and improve beer shelf-life.

Beer quality generally diminishes over time as staling compounds accumulate through various oxidation reactions. Here, we show that refermentation, a traditional practice where Saccharomyces cerevisiae cells are added to beer prior to bottling, diminishes the accumulation of staling aldehydes. However, commonly used beer yeasts only show a limited lifespan in beer. Using high-throughput screening and breeding, we were able to generate novel S. cerevisiae hybrids that survive for over a year in beer. Extensive chemical and sensory analyses of the two most promising hybrids showed that they slow down the accumulation of staling aldehydes, such as furfural and trans-2-nonenal and significantly increased beer flavor stability for up to 12 months. Moreover, the strains did not change the original flavor of the beer, highlighting their potential to be integrated in existing products. Together, these results demonstrate the ability to breed novel microbes that function as natural and sustainable anti-oxidative food preservatives.

A Cas3-base editing tool for targetable in vivo mutagenesis.

The generation of genetic diversity via mutagenesis is routinely used for protein engineering and pathway optimization. Current technologies for random mutagenesis often target either the whole genome or relatively narrow windows. To bridge this gap, we developed CoMuTER (Confined Mutagenesis using a Type I-E CRISPR-Cas system), a tool that allows inducible and targetable, in vivo mutagenesis of genomic loci of up to 55 kilobases. CoMuTER employs the targetable helicase Cas3, signature enzyme of the class 1 type I-E CRISPR-Cas system, fused to a cytidine deaminase to unwind and mutate large stretches of DNA at once, including complete metabolic pathways. The tool increases the number of mutations in the target region 350-fold compared to the rest of the genome, with an average of 0.3 mutations per kilobase. We demonstrate the suitability of CoMuTER for pathway optimization by doubling the production of lycopene in Saccharomyces cerevisiae after a single round of mutagenesis.

Interspecific hybridization as a driver of fungal evolution and adaptation.

Cross-species gene transfer is often associated with bacteria, which have evolved several mechanisms that facilitate horizontal DNA exchange. However, the increased availability of whole-genome sequences has revealed that fungal species also exchange DNA, leading to intertwined lineages, blurred species boundaries or even novel species. In contrast to prokaryotes, fungal DNA exchange originates from interspecific hybridization, where two genomes are merged into a single, often highly unstable, polyploid genome that evolves rapidly into stabler derivatives. The resulting hybrids can display novel combinations of genetic and phenotypic variation that enhance fitness and allow colonization of new niches. Interspecific hybridization led to the emergence of important pathogens of humans and plants (for example, various Candida and 'powdery mildew' species, respectively) and industrially important yeasts, such as Saccharomyces hybrids that are important in the production of cold-fermented lagers or cold-cellared Belgian ales. In this Review, we discuss the genetic processes and evolutionary implications of fungal interspecific hybridization and highlight some of the best-studied examples. In addition, we explain how hybrids can be used to study molecular mechanisms underlying evolution, adaptation and speciation, and serve as a route towards development of new variants for industrial applications.

Massive QTL analysis identifies pleiotropic genetic determinants for stress resistance, aroma formation, and ethanol, glycerol and isobutanol production in Saccharomyces cerevisiae.

BACKGROUND: The brewer's yeast Saccharomyces cerevisiae is exploited in several industrial processes, ranging from food and beverage fermentation to the production of biofuels, pharmaceuticals and complex chemicals. The large genetic and phenotypic diversity within this species offers a formidable natural resource to obtain superior strains, hybrids, and variants. However, most industrially relevant traits in S. cerevisiae strains are controlled by multiple genetic loci. Over the past years, several studies have identified some of these QTLs. However, because these studies only focus on a limited set of traits and often use different techniques and starting strains, a global view of industrially relevant QTLs is still missing. RESULTS: Here, we combined the power of 1125 fully sequenced inbred segregants with high-throughput phenotyping methods to identify as many as 678 QTLs across 18 different traits relevant to industrial fermentation processes, including production of ethanol, glycerol, isobutanol, acetic acid, sulfur dioxide, flavor-active esters, as well as resistance to ethanol, acetic acid, sulfite and high osmolarity. We identified and confirmed several variants that are associated with multiple different traits, indicating that many QTLs are pleiotropic. Moreover, we show that both rare and common variants, as well as variants located in coding and non-coding regions all contribute to the phenotypic variation. CONCLUSIONS: Our findings represent an important step in our understanding of the genetic underpinnings of industrially relevant yeast traits and open new routes to study complex genetics and genetic interactions as well as to engineer novel, superior industrial yeasts. Moreover, the major role of rare variants suggests that there is a plethora of different combinations of mutations that can be explored in genome editing.

Domestication of Industrial Microbes.

Domestication refers to artificial selection and breeding of wild species to obtain cultivated variants that thrive in man-made niches and meet human or industrial requirements. Several genotypic and phenotypic signatures of domestication have been described in crops, livestock and pets. However, domestication is not unique to plants and animals. Microbial diversity has also been shaped by the emergence of novel and highly specific man-made environments, like food and beverage fermentations. This allowed rapid adaptation and diversification of various microbes, such as certain Lactococcus, Lactobacillus, Oenococcus, Saccharomyces and Aspergillus species. During the domestication process, microbes gained the capacity to efficiently consume particular nutrients, cope with a multitude of industry-specific stress factors and produce desirable compounds, often at the cost of a reduction in fitness in their original, natural environments. Moreover, different lineages of the same species adapted to highly diverse niches, resulting in genetically and phenotypically distinct strains. In this Review, we discuss the basic principles of microbial domestication and describe how recent research is uncovering its genetic underpinnings.

Correction: Reducing phenolic off-flavors through CRISPR-based gene editing of the FDC1 gene in Saccharomyces cerevisiae x Saccharomyces eubayanus hybrid lager beer yeasts.

[This corrects the article DOI: 10.1371/journal.pone.0209124.].