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.

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.].

Interspecific hybridization facilitates niche adaptation in beer yeast.

Hybridization between species often leads to non-viable or infertile offspring, yet examples of evolutionarily successful interspecific hybrids have been reported in all kingdoms of life. However, many questions on the ecological circumstances and evolutionary aftermath of interspecific hybridization remain unanswered. In this study, we sequenced and phenotyped a large set of interspecific yeast hybrids isolated from brewing environments to uncover the influence of interspecific hybridization in yeast adaptation and domestication. Our analyses demonstrate that several hybrids between Saccharomyces species originated and diversified in industrial environments by combining key traits of each parental species. Furthermore, posthybridization evolution within each hybrid lineage reflects subspecialization and adaptation to specific beer styles, a process that was accompanied by extensive chimerization between subgenomes. Our results reveal how interspecific hybridization provides an important evolutionary route that allows swift adaptation to novel environments.

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

Today's beer market is challenged by a decreasing consumption of traditional beer styles and an increasing consumption of specialty beers. In particular, lager-type beers (pilsner), characterized by their refreshing and unique aroma and taste, yet very uniform, struggle with their sales. The development of novel variants of the common lager yeast, the interspecific hybrid Saccharomyces pastorianus, has been proposed as a possible solution to address the need of product diversification in lager beers. Previous efforts to generate new lager yeasts through hybridization of the ancestral parental species (S. cerevisiae and S. eubayanus) yielded strains with an aromatic profile distinct from the natural biodiversity. Unfortunately, next to the desired properties, these novel yeasts also inherited unwanted characteristics. Most notably is their phenolic off-flavor (POF) production, which hampers their direct application in the industrial production processes. Here, we describe a CRISPR-based gene editing strategy that allows the systematic and meticulous introduction of a natural occurring mutation in the FDC1 gene of genetically complex industrial S. cerevisiae strains, S. eubayanus yeasts and interspecific hybrids. The resulting cisgenic POF- variants show great potential for industrial application and diversifying the current lager beer portfolio.

Variable repeats in the eukaryotic polyubiquitin gene ubi4 modulate proteostasis and stress survival.

Ubiquitin conjugation signals for selective protein degradation by the proteasome. In eukaryotes, ubiquitin is encoded both as a monomeric ubiquitin unit fused to a ribosomal gene and as multiple ubiquitin units in tandem. The polyubiquitin gene is a unique, highly conserved open reading frame composed solely of tandem repeats, yet it is still unclear why cells utilize this unusual gene structure. Using the Saccharomyces cerevisiae UBI4 gene, we show that this multi-unit structure allows cells to rapidly produce large amounts of ubiquitin needed to respond to sudden stress. The number of ubiquitin units encoded by UBI4 influences cellular survival and the rate of ubiquitin-proteasome system (UPS)-mediated proteolysis following heat stress. Interestingly, the optimal number of repeats varies under different types of stress indicating that natural variation in repeat numbers may optimize the chance for survival. Our results demonstrate how a variable polycistronic transcript provides an evolutionary alternative for gene copy number variation.Eukaryotic cells rely on the ubiquitin-proteasome system for selective degradation of proteins, a process vital to organismal fitness. Here the authors show that the number of repeats in the polyubiquitin gene is evolutionarily unstable within and between yeast species, and that this variability may tune the cell's capacity to respond to sudden environmental perturbations.

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.

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 robot-assisted genome shuffling yields industrial Saccharomyces cerevisiae yeasts with increased ethanol tolerance.

BACKGROUND: During the final phases of bioethanol fermentation, yeast cells face high ethanol concentrations. This stress results in slower or arrested fermentations and limits ethanol production. Novel Saccharomyces cerevisiae strains with superior ethanol tolerance may therefore allow increased yield and efficiency. Genome shuffling has emerged as a powerful approach to rapidly enhance complex traits including ethanol tolerance, yet previous efforts have mostly relied on a mutagenized pool of a single strain, which can potentially limit the effectiveness. Here, we explore novel robot-assisted strategies that allow to shuffle the genomes of multiple parental yeasts on an unprecedented scale. RESULTS: Screening of 318 different yeasts for ethanol accumulation, sporulation efficiency, and genetic relatedness yielded eight heterothallic strains that served as parents for genome shuffling. In a first approach, the parental strains were subjected to multiple consecutive rounds of random genome shuffling with different selection methods, yielding several hybrids that showed increased ethanol tolerance. Interestingly, on average, hybrids from the first generation (F1) showed higher ethanol production than hybrids from the third generation (F3). In a second approach, we applied several successive rounds of robot-assisted targeted genome shuffling, yielding more than 3,000 targeted crosses. Hybrids selected for ethanol tolerance showed increased ethanol tolerance and production as compared to unselected hybrids, and F1 hybrids were on average superior to F3 hybrids. In total, 135 individual F1 and F3 hybrids were tested in small-scale very high gravity fermentations. Eight hybrids demonstrated superior fermentation performance over the commercial biofuel strain Ethanol Red, showing a 2 to 7% increase in maximal ethanol accumulation. In an 8-l pilot-scale test, the best-performing hybrid fermented medium containing 32% (w/v) glucose to dryness, yielding 18.7% (v/v) ethanol with a productivity of 0.90 g ethanol/l/h and a yield of 0.45 g ethanol/g glucose. CONCLUSIONS: We report the use of several different large-scale genome shuffling strategies to obtain novel hybrids with increased ethanol tolerance and fermentation capacity. Several of the novel hybrids show best-parent heterosis and outperform the commonly used bioethanol strain Ethanol Red, making them interesting candidate strains for industrial 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.

Detailed analysis of the microbial population in Malaysian spontaneous cocoa pulp fermentations reveals a core and variable microbiota.

The fermentation of cocoa pulp is one of the few remaining large-scale spontaneous microbial processes in today's food industry. The microbiota involved in cocoa pulp fermentations is complex and variable, which leads to inconsistent production efficiency and cocoa quality. Despite intensive research in the field, a detailed and comprehensive analysis of the microbiota is still lacking, especially for the expanding Asian production region. Here, we report a large-scale, comprehensive analysis of four spontaneous Malaysian cocoa pulp fermentations across two time points in the harvest season and two fermentation methods. Our results show that the cocoa microbiota consists of a "core" and a "variable" part. The bacterial populations show a remarkable consistency, with only two dominant species, Lactobacillus fermentum and Acetobacter pasteurianus. The fungal diversity is much larger, with four dominant species occurring in all fermentations ("core" yeasts), and a large number of yeasts that only occur in lower numbers and specific fermentations ("variable" yeasts). Despite this diversity, a clear pattern emerges, with early dominance of apiculate yeasts and late dominance of Saccharomyces cerevisiae. Our results provide new insights into the microbial diversity in Malaysian cocoa pulp fermentations and pave the way for the selection of starter cultures to increase efficiency and consistency.

A Legionella pneumophila collagen-like protein encoded by a gene with a variable number of tandem repeats is involved in the adherence and invasion of host cells.

Legionella pneumophila is a Gram-negative, facultative intracellular pathogen and the causative agent of Legionnaires' disease, a severe pneumonia in humans. Analysis of the Legionella sequenced genomes revealed a gene with a variable number of tandem repeats (VNTRs), whose number varies between strains. We examined the strain distribution of this gene among a collection of 108 clinical, environmental and hot spring serotype I strains. Twelve variants were identified, but no correlation was observed between the number of repeat units and clinical and environmental strains. The encoded protein contains the C-terminal consensus motif of outer membrane proteins and has a large region of collagen-like repeats that is encoded by the VNTR region. We have therefore annotated this protein Lcl for Legionella collagen-like protein. Lcl was shown to contribute to the adherence and invasion of host cells and it was demonstrated that the number of repeat units present in lcl had an influence on these adhesion characteristics.