Origins, evolution, domestication and diversity of Saccharomyces beer yeasts

Yeasts have been used for food and beverage fermentations for thousands of years. Today, numerous different strains are available for each specific fermentation process. However, the nature and extent of the phenotypic and genetic diversity and specific adaptations to industrial niches have only begun to be elucidated recently. In Saccharomyces, domestication is most pronounced in beer strains, likely because they continuously live in their industrial niche, allowing only limited genetic admixture with wild stocks and minimal contact with natural environments. As a result, beer yeast genomes show complex patterns of domestication and divergence, making both ale (S. cerevisiae) and lager (S. pastorianus) producing strains ideal models to study domestication and, more generally, genetic mechanisms underlying swift adaptation to new niches

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

Contribution of Eat1 and Other Alcohol Acyltransferases to Ester Production in Saccharomyces cerevisiae

Esters are essential for the flavor and aroma of fermented products, and are mainly produced by alcohol acyl transferases (AATs). A recently discovered AAT family named Eat (Ethanol acetyltransferase) contributes to ethyl acetate synthesis in yeast. However, its effect on the synthesis of other esters is unknown. In this study, the role of the Eat family in ester synthesis was compared to that of other Saccharomyces cerevisiae AATs (Atf1p, Atf2p, Eht1p, and Eeb1p) in silico and in vivo. A genomic study in a collection of industrial S. cerevisiae strains showed that variation of the primary sequence of the AATs did not correlate with ester production. Fifteen members of the EAT family from nine yeast species were overexpressed in S. cerevisiae CEN.PK2-1D and were able to increase the production of acetate and propanoate esters. The role of Eat1p was then studied in more detail in S. cerevisiae CEN.PK2-1D by deleting EAT1 in various combinations with other known S. cerevisiae AATs. Between 6 and 11 esters were produced under three cultivation conditions. Contrary to our expectations, a strain where all known AATs were disrupted could still produce, e.g., ethyl acetate and isoamyl acetate. This study has expanded our understanding of ester synthesis in yeast but also showed that some unknown ester-producing mechanisms still exist

A Cas3-base editing tool for targetable in vivo mutagenesis

DATA AVAILABILITY : Data supporting the findings of thiswork are availablewithin the paper and its Supplementary Information files. The whole genome sequencing data and the Oxford Nanopore sequencing data generated in this study, as well as the Sanger sequencing data of the SEC14 locus, have been deposited in the NCBI Sequence Read Archive under accession code PRJNA974923. All yeast strains and plasmids described in this work are available upon request. Source data are provided with this paper.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.https://www.nature.com/ncomms/am2024BiochemistryGeneticsMicrobiology and Plant PathologyNon

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

Exploring natural and artificial diversity of Saccharomyces cerevisiae for industrial fermentation processes

Yeast is the main driving force behind many food fermentation processes, including the production of beer, wine, sake and bread. Historically, these processes originate from uncontrolled, spontaneous fermentation reactions that rely on a complex mixture of microbes present in the environment. Because such spontaneous processes are inherently inconsistent, inefficient and the presence of undesired spoilage microbes regularly leads to the formation of off-flavors, most of today s industrial production utilizes defined starter cultures, often consisting of a specific domesticated strain of S. cerevisiae, S. bayanus, or S. pastorianus. Although this practice greatly improved process consistency, efficiency, and overall quality, the choice for a particular yeast strain for a specific industrial application is often based on historical, rather than scientific grounds, often resulting in the commercial application of a suboptimal strain. Moreover, new biotechnological yeast applications, such as the production of second-generation biofuels and other biochemicals, or the controlled fermentation of cocoa for the production of chocolate, confront yeast with completely new environments and challenges. Therefore, this study aims to identify or develop novel, superior yeast variants for both new and traditional fermentation processes.In the first chapter, we give a detailed, comprehensive literature overview of the natural biodiversity of Saccharomyces yeasts, describe the history of single-strain yeast starter cultures and discuss several methods to developed artificial yeast variants with altered characteristics. In Chapter 2, we describe the large-scale phenotypic investigation (mainly focusing on stress tolerance, aroma production and fermentation characteristics) of a broad collection of genetically diverse Saccharomyces yeasts originating from various niches. This way, we were able to identify some interesting patterns and correlations, and revealed some systematic differences between natural strains (so-called non-domesticated or wild strains) and strains from synthetic (man-made) fermentation environments (domesticated strains). For example, we were able to show that the production of fruity acetate esters is significantly higher in domesticated compared to natural strains, hinting towards positive selection for this trait during domestication. Moreover, the resulting dataset allowed selection of phenotypically interesting yeast strains directly employable in specific industrial settings, such as the selection of highly osmo- and ethanol-tolerant wine and wild strains for the production of second generation bioethanol. In Chapter 3 and 4, we further use this dataset as a platform to select parental strains for further phenotypic improvement through large-scale breeding programs. In these experiments, we mainly target the yeast s ability to produce high concentrations of isoamyl acetate (IA), the main responsible for the fruity flavors in fermented foods and beverages. This way, novel hybrid strains were developed for the production of highly aromatic ale beers (Chapter 3). Interestingly, many of these newly developed beer yeasts showed a strong heterosis effect for IA production, while retaining their overall fermentation performance. Additionally, novel hybrid yeasts for the production of specialty chocolate were produced (Chapter 4). These new variants combine beneficial traits from both parental strains: robustness in a cocoa environment and a high production of fruity aroma compounds. In a final chapter, we used Quantitative Trait Locus (QTL) mapping to identify genetic factors underlying the immense differences in IA production observed in S. cerevisiae strains. The genetic mechanisms underlying this complex (but industrially highly relevant) trait are currently insufficiently studied and identification of superior alleles might enable more targeted approaches of strain improvement, such as genetic engineering or marker-assisted breeding.In conclusion, this work provides a global overview of the Saccharomyces phenome (with the main focus on industrially relevant traits) and the exploitation of the resulting dataset for the development of superior yeast variants and the investigation of the genetic factors underlying natural diversity of aroma production.nrpages: 172status: publishe

"An exploration of some social mechanisms affecting domestic political actors’ Europeanisation: the Belgian case"

The principal idea of this paper is that the European socialisation of domestic political actors as a result of the growing involvement in European policy-making settings is not as self evident as early and current neofunctionalists often assume. Rather we suggest that the European socialisation of national actors is mediated by factors that relate to the actors’ domestic embeddedness. It seems that socialisation power of the European institutions is inferior to the mechanism or scope conditions that are situated at the domestic level. Therefore we believe that future studies on European socialisation should control more explicitly for preexisting dispositions as key determinants of potential attitude change. Related to this, we argue that socialisation studies should focus more extensively on (domestic) recruitment that may affect pre-socialisation and international socialisation

Stop that Noise and Turn Up the Antisense Transcription

Many genes are not only transcribed in the sense direction but also yield antisense transcripts. In this issue of Cell Reports, Huber et al. (2016) report that some of these transcripts may serve to suppress sense transcription and noise

The europeanisation of intergovernmental cooperation and conflict resolution in Belgium: the case of agriculture

This article analyses the genesis of formal procedures and institutional conditions within the agriculture policy domain in federal Belgium. Our process tracing starts in 1988 when a small number of agriculture competencies were handed over to the regions and ends in 2002 with the almost complete de‐federalisation of agriculture. In particular we argue that the fourth Belgian state reform in combination with the change of TEU‐article 146 (current article 203) (both in 1992) has set the institutional parameters for the almost complete de‐federalization of the agriculture policy‐domain in 2002. The case of agricultural policy demonstrates how sectoral changes are embedded within a more general process of institutional change and how, more in particular, domestic reform and European adaptation or Europeanisation evolves through a concatenated set of small incremental, at first hand seemingly unrelated, steps which may lock‐in actors into a specific set of institutional choices. With respect to the Belgian case, we demonstrate that Europe mitigates or softens the dual nature of Belgian federalism and that it stimulates cooperative forms of governance. © Koninklijke Brill NV, Leiden, The Netherlands, 2004.status: publishe