Use of the Saccharomycopsis schoenii MET17 promoter for regulated heterologous gene expression.

The ability to regulate the expression of genes is a central tool for the characterization of fungal genes. This is of particular interest to study genes required for specific processes or the effect of genes expressed only under specific conditions. Saccharomycopsis species show a unique property of necrotrophic mycoparasitism that is activated upon starvation. Here we describe the use of the MET17 promoter of S. schoenii as a tool to regulate gene expression based on the availability of methionine. Conditional expression was tested using lacZ and GFP reporter genes. Gene expression could be strongly down-regulated by the addition of methionine or cysteine to the growth medium and upregulated by starvation for methionine. We used X-gal (5-bromo-4-chloro-3-indolyl-ß-d-galactopyranoside) to detect lacZ-expression in plate assays and ONPG (ortho-nitrophenyl-ß-galactopyranoside) as a substrate for ß-galactosidase in liquid-phase assays. For in vivo expression analyses we used fluorescence microscopy for the detection and localization of a MET17-driven histone H4-GFP reporter gene. With these assays we demonstrated the usefulness of the MET17 promoter to regulate expression of genes based on methionine availability. In silico analyses revealed similar promoter motifs as found in MET3 genes of Saccharomyces cerevisiae and Ashbya gossypii. This suggests a regulation of the MET17 promoter by CBF1 and MET31/MET32 in conjunction with the transcriptional activator MET4, which were also identified in the S. schoenii genome.

This article describes the characterization of the S. schoenii MET17 promoter for regulated gene expression.

Visualizing Metabolism in Biotechnologically Important Yeasts with dDNP NMR Reveals Evolutionary Strategies and Glycolytic Logic.

Saccharomyces cerevisiae has long been a pillar of biotechnological production and basic research. More recently, strides to exploit the functional repertoire of nonconventional yeasts for biotechnological production have been made. Genomes and genetic tools for these yeasts are not always available, and yeast genomics alone may be insufficient to determine the functional features in yeast metabolism. Hence, functional assays of metabolism, ideally in the living cell, are best suited to characterize the cellular biochemistry of such yeasts. Advanced in cell NMR methods can allow the direct observation of carbohydrate influx into central metabolism on a seconds time scale: dDNP NMR spectroscopy temporarily enhances the nuclear spin polarization of substrates by more than 4 orders of magnitude prior to functional assays probing central metabolism. We use various dDNP enhanced carbohydrates for in-cell NMR to compare the metabolism of S. cerevisiae and nonconventional yeasts, with an emphasis on the wine yeast Hanseniaspora uvarum. In-cell observations indicated more rapid exhaustion of free cytosolic NAD+ in H. uvarum and alternative routes for pyruvate conversion, in particular, rapid amination to alanine. In-cell observations indicated that S. cerevisiae outcompetes other biotechnologically relevant yeasts by rapid ethanol formation due to the efficient adaptation of cofactor pools and the removal of competing reactions from the cytosol. By contrast, other yeasts were better poised to use redox neutral processes that avoided CO2-emission. Beyond visualizing the different cellular strategies for arriving at redox neutral end points, in-cell dDNP NMR probing showed that glycolytic logic is more conserved: nontoxic precursors of cellular building blocks formed high-population intermediates in the influx of glucose into the central metabolism of eight different biotechnologically important yeasts. Unsupervised clustering validated that the observation of rapid intracellular chemistry is a viable means to functionally classify biotechnologically important organisms.

Isolation of Saccharomycopsis species from plant material.

Saccharomycopsis species are natural organic sulphur auxotrophs. Their genomes do not encode genes for the uptake and assimilation of sulphate and thus these species cannot grow on media lacking e.g. methionine. Due to the similarity between sulphate and selenate, uptake and assimilation of selenate occurs through the same pathway starting from sulphate transporters encoded by the homologs of the SUL1 and SUL2 genes in S. cerevisiae. Lack of these transporters renders Saccharomycopsis species resistant to selenate levels that are toxic to other microorganisms. We used this feature to enrich environmental samples for Saccharomycopsis species. This led to the isolation of S. schoenii, S. lassenensis and a hitherto undescribed Saccharomycopsis species with limited by-catch of other yeasts, mainly belonging to Metschnikowia and Hanseniaspora. We performed growth and predation assays to characterize the potential of these new isolates as predacious yeasts. Most Saccharomycopsis species are temperature sensitive and cannot grow at 37°C; with the exception of S. lassenensis strains. Predation assays with S. schoenii and S. cerevisiae as prey indicated that predation was enhanced at 20°C compared to 30°C. We crossed an American isolate of S. schoenii with our German isolate using marker directed breeding. Viable progeny indicated that both strains are interfertile and belong to the same biological species. S. lassenensis is heterothallic, while S. schoenii and the new Saccharomycopsis isolate, for which we suggest the name S. geisenheimensis sp. nov., are homothallic.

Mosaic Genome of a British Cider Yeast.

Hybrid formation and introgressions had a profound impact on fermentative yeasts domesticated for beer, wine and cider fermentations. Here we provide a comparative genomic analysis of a British cider yeast isolate (E1) and characterize its fermentation properties. E1 has a Saccharomyces uvarum genome into which ~102 kb of S. eubayanus DNA were introgressed that replaced the endogenous homologous 55 genes of chromosome XIV between YNL182C and YNL239W. Sequence analyses indicated that the DNA donor was either a lager yeast or a yet unidentified S. eubayanus ancestor. Interestingly, a second introgression event added ~66 kb of DNA from Torulaspora microellipsoides to the left telomere of SuCHRX. This region bears high similarity with the previously described region C introgression in the wine yeast EC1118. Within this region FOT1 and FOT2 encode two oligopeptide transporters that promote improved nitrogen uptake from grape must in E1, as was reported for EC1118. Comparative laboratory scale grape must fermentations between the E1 and EC1118 indicated beneficial traits of faster consumption of total sugars and higher glycerol production but low acetic acid and reduced ethanol content. Importantly, the cider yeast strain produced high levels of fruity ester, including phenylethyl and isoamyl acetate.

The genome sequence of the Champagne Epernay Geisenheim wine yeast reveals its hybrid nature.

Lager yeasts are hybrids between Saccharomyces cerevisiae and S. eubayanus. Wine yeast biodiversity, however, has only recently been discovered to include besides pure S. cerevisiae strains also hybrids between different Saccharomyces yeasts as well as introgressions from non-Saccharomyces species. Here, we analysed the genome of the Champagne Epernay Geisenheim (CEG) wine yeast. This yeast is an allotetraploid (4n - 1) hybrid of S. cerevisiae harbouring a substantially reduced S. kudriavzevii genome contributing only 1/3 of a full genome equivalent. We identified a novel oligopeptide transporter gene, FOT4, in CEG located on chromosome XVI. FOT genes were originally derived from Torulaspora microellipsoides and FOT4 arose by non-allelic recombination between adjacent FOT1 and FOT2 genes. Fermentations of CEG in Riesling and Müller-Thurgau musts were compared with the S. cerevisiae Geisenheim wine yeast GHM, which does not carry FOT genes. At low temperature (10°C), CEG completed fermentations faster and produced increased levels of higher alcohols (e.g. isoamyl alcohol). At higher temperature (18°C), CEG produced higher amounts of the pineapple-like alkyl esters i-butyric and propionic acid ethyl esters compared to GHM. The hybrid nature of CEG thus provides advantages in grape must fermentations over S. cerevisiae wine yeasts, especially with regard to aroma production.

PCR-based gene targeting in Hanseniaspora uvarum.

Lack of gene-function analyses tools limits studying the biology of Hanseniaspora uvarum, one of the most abundant yeasts on grapes and in must. We investigated a rapid PCR-based gene targeting approach for one-step gene replacement in this diploid yeast. To this end, we generated and validated two synthetic antibiotic resistance genes, pFA-hygXL and pFA-clnXL, providing resistance against hygromycin and nourseothricin, respectively, for use with H. uvarum. Addition of short flanking-homology regions of 56-80 bp to these selection markers via PCR was sufficient to promote gene targeting. We report here the deletion of the H. uvarum LEU2 and LYS2 genes with these marker genes via two rounds of consecutive transformations, each resulting in the generation of auxotrophic strains (leu2/leu2; lys2/lys2). The hereby constructed leucine auxotrophic leu2/leu2 strain was subsequently complemented in a targeted manner, thereby further validating this approach. PCR-based gene targeting in H. uvarum was less efficient than in Saccharomyces cerevisiae. However, this approach, combined with the availability of two marker genes, provides essential tools for directed gene manipulations in H. uvarum.

Improved gene-targeting efficiency upon starvation in Saccharomycopsis.

Commonly used fungal transformation protocols rely on the use of either electroporation or the lithium acetate/single strand carrier DNA/Polyethylene glycol/heat shock method. We have used the latter method previously in establishing DNA-mediated transformation in Saccharomycopsis schoenii, a CTG-clade yeast that exhibits necrotrophic mycoparasitism. To elucidate the molecular mechanisms of predation by Saccharomycopsis we aim at gene-function analyses to identify virulence-related pathways and genes. However, in spite of a satisfactory transformation efficiency our efforts were crippled by high frequency of ectopic integration of disruption cassettes. Here, we show that overnight starvation of S. schoenii cells, while reducing the number of transformants, resulted in a substantial increase in gene-targeting via homologous recombination. To demonstrate this, we have deleted the S. schoenii CHS1, HIS3 and LEU2 genes and determined the required size of the flanking homology regions. Additionally, we complemented the S. schoenii leu2 mutant with heterologous LEU2 gene from Saccharomycopsis fermentans. To demonstrate the usefulness of our approach we also generated a S. fermentans leu2 strain, suggesting that this approach may have broader applicability.

Co-Fermentations of Kveik with Non-Conventional Yeasts for Targeted Aroma Modulation.

Kveik are consortia of yeast used for farmhouse ale production in Western Norway. Yeast strains derived from these mixtures are known, for example, for their high fermentation rate, thermotolerance, lack of phenolic off flavor production (POF-) and strong flocculation phenotype. In this study, we used five single cell yeast isolates from different Kveik yeasts, analyzed their fermentation and flavor production, and compared it with a typical yeast used in distilleries using 20 °C and 28 °C as the fermentation temperatures. One of the isolates, Kveik No 3, showed an impairment of maltotriose utilization and thus a reduced ethanol yield. Kveik fermentations for spirit production often harbor bacteria for flavor enrichment. We sought to improve Kveik fermentations with non-conventional yeasts (NCY). To this end we co-fermented Kveik isolates with Hanseniaspora uvarum, Meyerozyma guilliermondii and Pichia kudriavzevii using 5:1 ratios (Kveik vs. NCY) at 20 °C. The combinations of Kveik No 1 with P. kudriavzevii and Kveik No 1 with Hanseniaspora uvarum showed substantially increased amounts of specific volatile aroma compounds that were previously identified in the NCYs. Our results indicate that Kveik isolates appear to be suitable for co-fermentations with certain NCY to enhance beer or spirit fermentations, increasing the potential of these yeasts for beverage productions.

Role of RIM101 for Sporulation at Alkaline pH in Ashbya gossypii.

Microorganisms need to sense and adapt to fluctuations in the environmental pH. In fungal species, this response is mediated by the conserved pacC/RIM101 pathway. In Aspergillus nidulans, PacC activates alkaline-expressed genes and represses acid-controlled genes in response to alkaline pH and has important functions in regulating growth and conidia formation. In Saccharomyces cerevisiae, the PacC homolog Rim101 is required for adaptation to extracellular pH and to regulate transcription of IME1, the Initiator of MEiosis. S. cerevisiae rim101 mutants are defective in sporulation. In Ashbya gossypii, a filamentous fungus belonging to the family of Saccharomycetaceae, little is known about the role of pH in regulating growth and sporulation. Here, we deleted the AgRIM101 homolog (AFR190C). Our analyses show that Rim101 is important for growth and essential for sporulation at alkaline pH in A. gossypii. Acidic liquid sporulation media were alkalinized by sporulating strains, while the high pH of alkaline media (starting pH = 8.6) was reduced to a pH ~ 7.5 by these strains. However, Agrim101 mutants were unable to sporulate in alkaline media and failed to reduce the initial high pH, while they were capable of sporulation in acidic liquid media in which they increased the pH like the wild type.

Development of Genetic Modification Tools for Hanseniasporauvarum.

Apiculate yeasts belonging to the genus Hanseniaspora are commonly isolated from viticultural settings and often dominate the initial stages of grape must fermentations. Although considered spoilage yeasts, they are now increasingly becoming the focus of research, with several whole-genome sequencing studies published in recent years. However, tools for their molecular genetic manipulation are still lacking. Here, we report the development of a tool for the genetic modification of Hanseniaspora uvarum. This was employed for the disruption of the HuATF1 gene, which encodes a putative alcohol acetyltransferase involved in acetate ester formation. We generated a synthetic marker gene consisting of the HuTEF1 promoter controlling a hygromycin resistance open reading frame (ORF). This new marker gene was used in disruption cassettes containing long-flanking (1000 bp) homology regions to the target locus. By increasing the antibiotic concentration, transformants were obtained in which both alleles of the putative HuATF1 gene were deleted in a diploid H. uvarum strain. Phenotypic characterisation including fermentation in Müller-Thurgau must showed that the null mutant produced significantly less acetate ester, particularly ethyl acetate. This study marks the first steps in the development of gene modification tools and paves the road for functional gene analyses of this yeast.

Sporulation in Ashbya gossypii.

Ashbya gossypii is a filamentous ascomycete belonging to the yeast family of Saccharomycetaceae. At the end of its growth phase Ashbya generates abundant amounts of riboflavin and spores that form within sporangia derived from fragmented cellular compartments of hyphae. The length of spores differs within species of the genus. Needle-shaped Ashbya spores aggregate via terminal filaments. A. gossypii is a homothallic fungus which may possess a and α mating types. However, the solo-MATa type strain is self-fertile and sporulates abundantly apparently without the need of prior mating. The central components required for the regulation of sporulation, encoded by IME1, IME2, IME4, KAR4, are conserved with Saccharomyces cerevisiae. Nutrient depletion generates a strong positive signal for sporulation via the cAMP-PKA pathway and SOK2, which is also essential for sporulation. Strong inhibitors of sporulation besides mutations in the central regulatory genes are the addition of exogenous cAMP or the overexpression of the mating type gene MATα2. Sporulation has been dissected using gene-function analyses and global RNA-seq transcriptomics. This revealed a role of Msn2/4, another potential PKA-target, for spore wall formation and a key dual role of the protein A kinase Tpk2 at the onset of sporulation as well as for breaking the dormancy of spores to initiate germination. Recent work has provided an overview of ascus development, regulation of sporulation and spore maturation. This will be summarized in the current review with a focus on the central regulatory genes. Current research and open questions will also be discussed.

Effect of Isomixing on Grape Must Fermentations of ATF1-Overexpressing Wine Yeast Strains.

Speeding up grape must fermentation would be of great economic benefit. We subjected Saccharomyces cerevisiae VIN13 and two recombinant VIN13-strains expressing ATF1 alleles under two different promoters (either PGK1 or HXT7) to four styles of grape must fermentations; we then assessed the effect of constantly stirring a must fermentation (isomixing). The four different fermentation setups were as follows: isomixed, closed in an ANKOM Rf Gas productions system; isomixed, open in a stirred tall tube cylinder; static, closed constituting a conventional fermentation in a wine bottle equipped with an airlock and static; and static, open in a tall tube cylinder (without stirring). We report on major fermentation parameters and the volatile aroma compositions generated in the finished wines. The primary fermentations of the strains subjected to constant stirring finished after 7 days, whereas the static fermentations reached dryness after 19 days. The wines derived from isomixed fermentations produced approximately 0.7% less ethanol compared to the unstirred fermentations. The speed that the isomixed fermentation took to reach completion may provide an alternative to static fermentations in the preparation of base wines for sparkling wine production. The observed increase of volatiles of isomixed fermentations merits further investigation.

Blending wine yeast phenotypes with the aid of CRISPR DNA editing technologies.

In recent years, CRISPR/Cas9-based genetic editing has become a mainstay in many laboratories including manipulations done with yeast. We utilized this technique to generate a self-cloned wine yeast strain that overexpresses two genes of oenological relevance i.e. the glycerol-3-phosphate dehydrogenase 1 (GPD1) and the alcohol acetyltransferase 1 (ATF1) directly implicated in glycerol and acetate ester production respectively. Riesling wine made from the resulting strain showed increased glycerol and acetate ester levels compared to the parental strain. In addition, significantly less acetic acid levels were measured in wine made with yeast containing both genetic alterations compared to wine made with the strain that only overexpresses GPD1. Thus, this strain provides an alternative strategy for alleviating the accumulation of acetic acid once glycerol production is favoured during alcoholic fermentation with the addition of dramatically increasing acetate esters production.

Special Issue: Non-Conventional Yeasts: Genomics and Biotechnology.

Non-conventional yeasts, i.e., the vast biodiversity beyond already well-established model systems such as Saccharomyces cerevisiae, Candida albicans and Schizosaccharomyces pombe and a few others, are a huge and untapped resource of organisms. [...].

The Whiff of Wine Yeast Innovation: Strategies for Enhancing Aroma Production by Yeast during Wine Fermentation.

Despite being used chiefly for fermenting the sugars of grape must to alcohol, wine yeasts (most prominently Saccharomyces cerevisiae) play a pivotal role in the final aroma profiles of wines. Strain selection, intentionally incorporating non-Saccharomyces yeast in so-called mixed-culture fermentations, and genetic modifications of S. cerevisiae have all been shown to greatly enhance the chemical composition and sensory profile of wines. In this Review, we highlight how wine researchers employ fermenting yeasts to expand on the aroma profiles of the wines they study.

A script for initiating molecular biology studies with non-conventional yeasts based on Saccharomycopsis schoenii.

Non-conventional yeasts (NCYs), i.e. all yeasts other than Saccharomyces cerevisiae, are emerging as novel production strains and gain more and more attention to exploit their unique properties. Yet, these yeasts can hardly compete against the advanced methodology and genetic tool kit available for exploiting and engineering S. cerevisiae. Currently, for many NCYs one has to start from scratch to initiate molecular genetic manipulations, which is often time consuming and not straight-forward. More so because utilization of S. cerevisiae tools based on short-flank mediated homologous recombination or plasmid biology are not readily applicable in NCYs. Here we present a script with discrete steps that will lead to the development of a basic and expandable molecular toolkit for ascomycetous NCYs and will allow genetic engineering of novel platform strains. For toolkit development the highly efficient in vivo recombination efficiency of S. cerevisiae is utilized in the generation and initial testing of tools. The basic toolkit includes promoters, reporter genes, selectable markers based on dominant antibiotic resistance genes and the generation of long-flanking homology disruption cassettes. The advantage of having pretested molecular tools that function in a heterologous host facilitate NCY strain manipulations. We demonstrate the usefulness of this script on Saccharomycopsis schoenii, a predator yeast with useful properties in fermentation and fungal biocontrol.

Overexpression of RAD51 Enables PCR-Based Gene Targeting in Lager Yeast.

Lager beer fermentations rely on specific polyploid hybrids between Saccharomyces cerevisiae and Saccharomyces eubayanus falling into the two groups of S. carlsbergensis/Saaz-type and S. pastorianus/Frohberg-type. These strains provide a terroir to lager beer as they have long traditional associations and local selection histories with specific breweries. Lager yeasts share, based on their common origin, several phenotypes. One of them is low transformability, hampering the gene function analyses required for proof-of-concept strain improvements. PCR-based gene targeting is a standard tool for manipulating S. cerevisiae and other ascomycetes. However, low transformability paired with the low efficiency of homologous recombination practically disable targeted gene function analyses in lager yeast strains. For genetic manipulations in lager yeasts, we employed a yeast transformation protocol based on lithium-acetate/PEG incubation combined with electroporation. We first introduced freely replicating CEN/ARS plasmids carrying ScRAD51 driven by a strong heterologous promoter into lager yeast. RAD51 overexpression in the Weihenstephan 34/70 lager yeast was necessary and sufficient in our hands for gene targeting using short-flanking homology regions of 50 bp added to a selection marker by PCR. We successfully targeted two independent loci, ScADE2/YOR128C and ScHSP104/YLL026W, and confirmed correct integration by diagnostic PCR. With these modifications, genetic alterations of lager yeasts can be achieved efficiently and the RAD51-containing episomal plasmid can be removed after successful strain construction.

Multi-omics characterization of the necrotrophic mycoparasite Saccharomycopsis schoenii.

Pathogenic yeasts and fungi are an increasing global healthcare burden, but discovery of novel antifungal agents is slow. The mycoparasitic yeast Saccharomycopsis schoenii was recently demonstrated to be able to kill the emerging multi-drug resistant yeast pathogen Candida auris. However, the molecular mechanisms involved in the predatory activity of S. schoenii have not been explored. To this end, we de novo sequenced, assembled and annotated a draft genome of S. schoenii. Using proteomics, we confirmed that Saccharomycopsis yeasts have reassigned the CTG codon and translate CTG into serine instead of leucine. Further, we confirmed an absence of all genes from the sulfate assimilation pathway in the genome of S. schoenii, and detected the expansion of several gene families, including aspartic proteases. Using Saccharomyces cerevisiae as a model prey cell, we honed in on the timing and nutritional conditions under which S. schoenii kills prey cells. We found that a general nutrition limitation, not a specific methionine deficiency, triggered predatory activity. Nevertheless, by means of genome-wide transcriptome analysis we observed dramatic responses to methionine deprivation, which were alleviated when S. cerevisiae was available as prey, and therefore postulate that S. schoenii acquired methionine from its prey cells. During predation, both proteomic and transcriptomic analyses revealed that S. schoenii highly upregulated and translated aspartic protease genes, probably used to break down prey cell walls. With these fundamental insights into the predatory behavior of S. schoenii, we open up for further exploitation of this yeast as a biocontrol yeast and/or source for novel antifungal agents.

Expansion of a Telomeric FLO/ALS-Like Sequence Gene Family in Saccharomycopsis fermentans.

Non-Saccharomyces species have been recognized for their beneficial contribution to fermented food and beverages based on their volatile compound formation and their ability to ferment glucose into ethanol. At the end of fermentation brewer's yeast flocculate which provides an easy means of separation of yeasts from green beer. Flocculation in Saccharomyces cerevisiae requires a set of flocculation genes. These FLO-genes, FLO1, FLO5, FLO9, FLO10, and FLO11, are located at telomeres and transcription of these adhesins is regulated by Flo8 and Mss11. Here, we show that Saccharomycopsis fermentans, an ascomycete yeast distantly related to S. cerevisiae, possesses a very large FLO/ALS-like Sequence (FAS) family encompassing 34 genes. Fas proteins are variable in size and divergent in sequence and show similarity to the Flo1/5/9 family. Fas proteins show the general build with a signal peptide, an N-terminal carbohydrate binding PA14 domain, a central region differing by the number of repeats and a C-terminus with a consensus sequence for GPI-anchor attachment. Like FLO genes in S. cerevisiae, FAS genes are mostly telomeric with several paralogs at each telomere. We term such genes that share evolutionary conserved telomere localization "telologs" and provide several other examples. Adhesin expression in S. cerevisiae and filamentation in Candida albicans is regulated by Flo8 and Mss11. In Saccharomycopsis we identified only a single protein with similarity to Flo8 based on sequence similarity and the presence of a LisH domain.

The mycoparasitic yeast Saccharomycopsis schoenii predates and kills multi-drug resistant Candida auris.

Candida auris has recently emerged as a multi-drug resistant fungal pathogen that poses a serious global health threat, especially for patients in hospital intensive care units (ICUs). C. auris can colonize human skin and can spread by physical contact or contaminated surfaces and equipment. Here, we show that the mycoparasitic yeast Saccharomycopsis schoenii efficiently kills both sensitive and multi-drug resistant isolates of C. auris belonging to the same clade, as well as clinical isolates of other pathogenic species of the Candida genus suggesting novel approaches for biocontrol.