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.

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.

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.

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.

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.

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.

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.

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.

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.].

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.

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.

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.

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.

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.

Characterization and Degradation of Pectic Polysaccharides in Cocoa Pulp.

Microbial fermentation of the viscous pulp surrounding cocoa beans is a crucial step in chocolate production. During this process, the pulp is degraded, after which the beans are dried and shipped to factories for further processing. Despite its central role in chocolate production, pulp degradation, which is assumed to be a result of pectin breakdown, has not been thoroughly investigated. Therefore, this study provides a comprehensive physicochemical analysis of cocoa pulp, focusing on pectic polysaccharides, and the factors influencing its degradation. Detailed analysis reveals that pectin in cocoa pulp largely consists of weakly bound substances, and that both temperature and enzyme activity play a role in its degradation. Furthermore, this study shows that pulp degradation by an indigenous yeast fully relies on the presence of a single gene (PGU1), encoding for an endopolygalacturonase. Apart from their basic scientific value, these new insights could propel the selection of microbial starter cultures for more efficient pulp degradation.

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.

Physiology, ecology and industrial applications of aroma formation in yeast.

Yeast cells are often employed in industrial fermentation processes for their ability to efficiently convert relatively high concentrations of sugars into ethanol and carbon dioxide. Additionally, fermenting yeast cells produce a wide range of other compounds, including various higher alcohols, carbonyl compounds, phenolic compounds, fatty acid derivatives and sulfur compounds. Interestingly, many of these secondary metabolites are volatile and have pungent aromas that are often vital for product quality. In this review, we summarize the different biochemical pathways underlying aroma production in yeast as well as the relevance of these compounds for industrial applications and the factors that influence their production during fermentation. Additionally, we discuss the different physiological and ecological roles of aroma-active metabolites, including recent findings that point at their role as signaling molecules and attractants for insect vectors.

High-throughput system-wide engineering and screening for microbial biotechnology.

Genetic engineering and screening of large number of cells or populations is a crucial bottleneck in today's systems biology and applied (micro)biology. Instead of using standard methods in bottles, flasks or 96-well plates, scientists are increasingly relying on high-throughput strategies that miniaturize their experiments to the nanoliter and picoliter scale and the single-cell level. In this review, we summarize different high-throughput system-wide genome engineering and screening strategies for microbes. More specifically, we will emphasize the use of multiplex automated genome evolution (MAGE) and CRISPR/Cas systems for high-throughput genome engineering and the application of (lab-on-chip) nanoreactors for high-throughput single-cell or population screening.