Discovery of a rapidly evolving yeast defense factor, KTD1, against the secreted killer toxin K28.

Secreted protein toxins are widely used weapons in conflicts between organisms. Elucidating how organisms genetically adapt to defend themselves against these toxins is fundamental to understanding the coevolutionary dynamics of competing organisms. Within yeast communities, "killer" toxins are secreted to kill nearby sensitive yeast, providing a fitness advantage in competitive growth environments. Natural yeast isolates vary in their sensitivity to these toxins, but to date, no polymorphic genetic factors contributing to defense have been identified. We investigated the variation in resistance to the killer toxin K28 across diverse natural isolates of the Saccharomyces cerevisiae population. Using large-scale linkage mapping, we discovered a novel defense factor, which we named KTD1. We identified many KTD1 alleles, which provided different levels of K28 resistance. KTD1 is a member of the DUP240 gene family of unknown function, which is rapidly evolving in a region spanning its two encoded transmembrane helices. We found that this domain is critical to KTD1's protective ability. Our findings implicate KTD1 as a key polymorphic factor in the defense against K28 toxin.

The prevalence of killer yeasts and double-stranded RNAs in the budding yeast Saccharomyces cerevisiae.

Killer toxins are antifungal proteins produced by many species of "killer" yeasts, including the brewer's and baker's yeast Saccharomyces cerevisiae. Screening 1270 strains of S. cerevisiae for killer toxin production found that 50% are killer yeasts, with a higher prevalence of yeasts isolated from human clinical samples and winemaking processes. Since many killer toxins are encoded by satellite double-stranded RNAs (dsRNAs) associated with mycoviruses, S. cerevisiae strains were also assayed for the presence of dsRNAs. This screen identified that 51% of strains contained dsRNAs from the mycovirus families Totiviridae and Partitiviridae, as well as satellite dsRNAs. Killer toxin production was correlated with the presence of satellite dsRNAs but not mycoviruses. However, in most killer yeasts, whole genome analysis identified the killer toxin gene KHS1 as significantly associated with killer toxin production. Most killer yeasts had unique spectrums of antifungal activities compared to canonical killer toxins, and sequence analysis identified mutations that altered their antifungal activities. The prevalence of mycoviruses and killer toxins in S. cerevisiae is important because of their known impact on yeast fitness, with implications for academic research and industrial application of this yeast species.

The ecology of killer yeasts: Interference competition in natural habitats.

Killer yeasts are ubiquitous in the environment: They have been found in diverse habitats ranging from ocean sediment to decaying cacti to insect bodies and on all continents including Antarctica. However, environmental killer yeasts are poorly studied compared with laboratory and domesticated killer yeasts. Killer yeasts secrete so-called killer toxins that inhibit nearby sensitive yeasts, and the toxins are frequently assumed to be tools for interference competition in diverse yeast communities. The diversity and ubiquity of killer yeasts imply that interference competition is crucial for shaping yeast communities. Additionally, these toxins may have ecological functions beyond use in interference competition. This review introduces readers to killer yeasts in environmental systems, with a focus on what is and is not known about their ecology and evolution. It also explores how results from experimental killer systems in laboratories can be extended to understand how competitive strategies shape yeast communities in nature. Overall, killer yeasts are likely to occur everywhere yeasts are found, and the killer phenotype has the potential to radically shape yeast diversity in nature.

Production of a novel killer toxin from Saccharomyces eubayanus using agro-industrial waste and its application against wine spoilage yeasts.

The juicing industry generates large amounts of waste that mostly lack commercial value and, in the absence of waste treatment policies, produces environmental pollution. Also, microbiological spoilage is a major concern in the wine industry and control tools are limited. Taking these challenges into account, agro-industrial waste coming from ultrafiltrated apple and pear juice were used to grow Saccharomyces eubayanus and to produce its killer toxin (SeKT). A Plackett-Burman screening was performed in order to optimize SeKT production in ultrafiltrated apple and pear juice. The optimized medium was characterized: 75% v/v WUJ, 0.5% m/v KH2PO4, 0.5% m/v MgSO4, 0.5% m/v (NH4)SO4, 0.5% g/L urea, 10% v/v glycerol and 0.1% v/v Triton X-100. SeKT produced in WUJ optimised medium was used to perform killer assays against wine spoilage yeasts and showed antagonistic activity against Brettanomyces bruxellensis, Pichia guilliermondii, Pichia manshurica and Pichia membranifaciens. Different inhibition percentages against spoilage species in a wine environment (49-69%) were detected and preserved for at least 48 h. For the first time, this work reports the ability of S. eubayanus to produce a killer toxin with potential use as a biocontrol tool in winemaking. Producing SeKT using agro-industrial waste as an alternative medium to cultivate S. eubayanus would have industrial, economic and ecological benefits.

Autoselection of cytoplasmic yeast virus like elements encoding toxin/antitoxin systems involves a nuclear barrier for immunity gene expression.

Cytoplasmic virus like elements (VLEs) from Kluyveromyces lactis (Kl), Pichia acaciae (Pa) and Debaryomyces robertsiae (Dr) are extremely A/T-rich (>75%) and encode toxic anticodon nucleases (ACNases) along with specific immunity proteins. Here we show that nuclear, not cytoplasmic expression of either immunity gene (PaORF4, KlORF3 or DrORF5) results in transcript fragmentation and is insufficient to establish immunity to the cognate ACNase. Since rapid amplification of 3' ends (RACE) as well as linker ligation of immunity transcripts expressed in the nucleus revealed polyadenylation to occur along with fragmentation, ORF-internal poly(A) site cleavage due to the high A/T content is likely to prevent functional expression of the immunity genes. Consistently, lowering the A/T content of PaORF4 to 55% and KlORF3 to 46% by gene synthesis entirely prevented transcript cleavage and permitted functional nuclear expression leading to full immunity against the respective ACNase toxin. Consistent with a specific adaptation of the immunity proteins to the cognate ACNases, cross-immunity to non-cognate ACNases is neither conferred by PaOrf4 nor KlOrf3. Thus, the high A/T content of cytoplasmic VLEs minimizes the potential of functional nuclear recruitment of VLE encoded genes, in particular those involved in autoselection of the VLEs via a toxin/antitoxin principle.

DNA damage induced by the anticodon nuclease from a Pichia acaciae killer strain is linked to ribonucleotide reductase depletion.

Virus like element (VLE) encoded killer toxins of Pichia acaciae and Kluyveromyces lactis kill target cells through anticodon nuclease (ACNase) activity directed against tRNA(Gln) and tRNA(Glu) respectively. Not only does tRNA cleavage disable translation, it also affects DNA integrity as well. Consistent with DNA damage, which is involved in toxicity, target cells' mutation frequencies are elevated upon ACNase exposure, suggesting a link between translational integrity and genome surveillance. Here, we analysed whether ACNase action impedes the periodically and highly expressed S-phase specific ribonucleotide reductase (RNR) and proved that RNR expression is severely affected by PaT. Because RNR catalyses the rate-limiting step in dNTP synthesis, mutants affected in dNTP synthesis were scrutinized with respect to ACNase action. Mutations elevating cellular dNTPs antagonized the action of both the above ACNases, whereas mutations lowering dNTPs aggravated toxicity. Consistently, prevention of tRNA cleavage in elp3 or trm9 mutants, which both affect the wobble uridine modification of the target tRNA, suppressed the toxin hypersensitivity of a dNTP synthesis mutant. Moreover, dNTP synthesis defects exacerbated the PaT ACNase sensitivity of cells defective in homologous recombination, proving that dNTP depletion is responsible for subsequent DNA damage.

Production of fluorescent and cytotoxic K28 killer toxin variants through high cell density fermentation of recombinant Pichia pastoris.

BACKGROUND: Virus infected killer strains of the baker's yeast Saccharomyces cerevisiae secrete protein toxins such as K28, K1, K2 and Klus which are lethal to sensitive yeast strains of the same or related species. K28 is somewhat unique as it represents an α/ß heterodimeric protein of the A/B toxin family which, after having bound to the surface of sensitive target cells, is taken up by receptor-mediated endocytosis and transported through the secretory pathway in a retrograde manner. While the current knowledge on yeast killer toxins is largely based on genetic screens for yeast mutants with altered toxin sensitivity, in vivo imaging of cell surface binding and intracellular toxin transport is still largely hampered by a lack of fluorescently labelled and biologically active killer toxin variants. RESULTS: In this study, we succeeded for the first time in the heterologous K28 preprotoxin expression and production of fluorescent K28 variants in Pichia pastoris. Recombinant P. pastoris GS115 cells were shown to successfully process and secrete K28 variants fused to mCherry or mTFP by high cell density fermentation. The fluorescent K28 derivatives were obtained in high yield and possessed in vivo toxicity and specificity against sensitive yeast cells. In cell binding studies the resulting K28 variants caused strong fluorescence signals at the cell periphery due to toxin binding to primary K28 receptors within the yeast cell wall. Thereby, the ß-subunit of K28 was confirmed to be the sole component required and sufficient for K28 cell wall binding. CONCLUSION: Successful production of fluorescent killer toxin variants of S. cerevisiae by high cell density fermentation of recombinant, K28 expressing strains of P. pastoris now opens the possibility to study and monitor killer toxin cell surface binding, in particular in toxin resistant yeast mutants in which toxin resistance is caused by defects in toxin binding due to alterations in cell wall structure and composition. This novel approach might be easily transferable to other killer toxins from different yeast species and genera. Furthermore, the fluorescent toxin variants described here might likewise represent a powerful tool in future studies to visualize intracellular A/B toxin trafficking with the help of high resolution single molecule imaging techniques.

Biotechnological exploitation of Tetrapisispora phaffii killer toxin: heterologous production in Komagataella phaffii (Pichia pastoris).

The use of natural antimicrobials from plants, animals and microorganisms to inhibit the growth of pathogenic and spoilage microorganisms is becoming more frequent. This parallels the increased consumer interest towards consumption of minimally processed food and 'greener' food and beverage additives. Among the natural antimicrobials of microbial origin, the killer toxin produced by the yeast Tetrapisispora phaffii, known as Kpkt, appears to be a promising natural antimicrobial agent. Kpkt is a glycoprotein with ß-1,3-glucanase and killer activity, which induces ultrastructural modifications to the cell wall of yeast of the genera Kloeckera/Hanseniaspora and Zygosaccharomyces. Moreover, Kpkt maintains its killer activity in grape must for at least 14 days under winemaking conditions, thus suggesting its use against spoilage yeast in wine making and the sweet beverage industry. Here, the aim was to explore the possibility of high production of Kpkt for biotechnological exploitation. Molecular tools for heterologous production of Kpkt in Komagataella phaffii GS115 were developed, and two recombinant clones that produce up to 23 mg/L recombinant Kpkt (rKpkt) were obtained. Similar to native Kpkt, rKpkt has ß-glucanase and killer activities. Moreover, it shows a wider spectrum of action with respect to native Kpkt. This includes effects on Dekkera bruxellensis, a spoilage yeast of interest not only in wine making, but also for the biofuel industry, thus widening the potential applications of this rKpkt.

Postharvest biocontrol ability of killer yeasts against Monilinia fructigena and Monilinia fructicola on stone fruit.

The antagonistic effects of Debaryomyces hansenii KI2a, D. hansenii MI1a and Wickerhamomyces anomalus BS91 were tested against Monilinia fructigena and Monilinia fructicola in in vitro and in vivo trials. All yeast strains demonstrated antifungal activity at different levels depending on species, strain and pathogen. D hansenii KI2a and W. anomalus BS91 showed the highest biocontrol activity in vitro; the production of hydrolytic enzymes, killer toxins and volatile organic compounds (VOCs) were hypothesized as their main mechanisms of action against pathogens. D hansenii KI2a and W. anomalus BS91 significantly reduced brown rot incidence and severity on peach and plum fruits artificially inoculated with M. fructigena and M. fructicola, especially when applied 24 h before pathogen inoculation. On the opposite, D. hansenii MI1a exhibited weak antagonistic activity towards M. fructigena on peach and plum fruits and was ineffective against M. fructicola. The noticeable ability of W. anomalus BS91 to control brown rot could be also correlated with its high capacity to colonize the wound tissue and to increase its population density. Accordingly, the antagonistic strains of D. hansenii and W. anomalus could be proposed as active ingredients for the development of biofungicides against Monilinia species that are responsible for considerable economic losses in stone fruit crops.

Yeast ß-1,6-glucan is a primary target for the Saccharomyces cerevisiae K2 toxin.

Certain Saccharomyces cerevisiae strains secrete different killer proteins of double-stranded-RNA origin. These proteins confer a growth advantage to their host by increasing its survival. K2 toxin affects the target cell by binding to the cell surface, disrupting the plasma membrane integrity, and inducing ion leakage. In this study, we determined that K2 toxin saturates the yeast cell surface receptors in 10 min. The apparent amount of K2 toxin, bound to a single cell of wild type yeast under saturating conditions, was estimated to be 435 to 460 molecules. It was found that an increased level of ß-1,6-glucan directly correlates with the number of toxin molecules bound, thereby impacting the morphology and determining the fate of the yeast cell. We observed that the binding of K2 toxin to the yeast surface receptors proceeds in a similar manner as in case of the related K1 killer protein. It was demonstrated that the externally supplied pustulan, a poly-ß-1,6-glucan, but not the glucans bearing other linkage types (such as laminarin, chitin, and pullulan) efficiently inhibits the K2 toxin killing activity. In addition, the analysis of toxin binding to the intact cells and spheroplasts confirmed that majority of K2 protein molecules attach to the ß-1,6-glucan, rather than the plasma membrane-localized receptors. Taken together, our results reveal that ß-1,6-glucan is a primary target of K2 toxin and is important for the execution of its killing property.

rRNA fragmentation induced by a yeast killer toxin.

Virus like dsDNA elements (VLE) in yeast were previously shown to encode the killer toxins PaT and zymocin, which target distinct tRNA species via specific anticodon nuclease (ACNase) activities. Here, we characterize a third member of the VLE-encoded toxins, PiT from Pichia inositovora, and identify PiOrf4 as the cytotoxic subunit by conditional expression in Saccharomyces cerevisiae. In contrast to the tRNA targeting toxins, however, neither a change of the wobble uridine modification status by introduction of elp3 or trm9 mutations nor tRNA overexpression rescued from PiOrf4 toxicity. Consistent with a distinct RNA target, expression of PiOrf4 causes specific fragmentation of the 25S and 18S rRNA. A stable cleavage product comprising the first ∼ 130 nucleotides of the 18S rRNA was purified and characterized by linker ligation and subsequent reverse transcription; 3'-termini were mapped to nucleotide 131 and 132 of the 18S rRNA sequence, a region showing some similarity to the anticodon loop of tRNA(Glu)(UUC), the zymocin target. PiOrf4 residues Glu9 and His214, corresponding to catalytic sites Glu9 and His209 in the ACNase subunit of zymocin are essential for in vivo toxicity and rRNA fragmentation, raising the possibility of functionally conserved RNase modules in both proteins.

Hsp12p and PAU genes are involved in ecological interactions between natural yeast strains.

The coexistence of different yeasts in a single vineyard raises the question on how they communicate and why slow growers are not competed out. Genetically modified laboratory strains of Saccharomyces cerevisiae are extensively used to investigate ecological interactions, but little is known about the genes regulating cooperation and competition in ecologically relevant settings. Here, we present evidences of Hsp12p-dependent altruistic and contact-dependent competitive interactions between two natural yeast isolates. Hsp12p is released during cell death for public benefit by a fast-growing strain that also produces a killer toxin to inhibit growth of a slow grower that can enjoy the benefits of released Hsp12p. We also show that the protein Pau5p is essential in the defense against the killer effect. Our results demonstrate that the combined action of Hsp12p, Pau5p and a killer toxin is sufficient to steer a yeast community.

Immunity factors for two related tRNAGln targeting killer toxins distinguish cognate and non-cognate toxic subunits.

The cytoplasmic virus-like element pWR1A from Debaryomyces robertsiae encodes a toxin (DrT) with similarities to the Pichia acaciae killer toxin PaT, which acts by importing a toxin subunit (PaOrf2) with tRNA anticodon nuclease activity into target cells. As for PaT, loss of the tRNA methyltransferase Trm9 or overexpression of tRNA(Gln) increases DrT resistance and the amount of tRNA(Gln) is reduced upon toxin exposure or upon induced intracellular expression of the toxic DrT subunit gene DrORF3, indicating DrT and PaT to share the same in vivo target. Consistent with a specific tRNase activity of DrOrf3, the protein cleaves tRNA(Gln) but not tRNA(Glu) in vitro. Heterologous cytoplasmic expression identified DrOrf5 as the DrT specific immunity factor; it confers resistance to exogenous DrT as well as to intracellular expression of DrOrf3 and prevents tRNA depletion by the latter. The PaT immunity factor PaOrf4, a homologue of DrOrf5 disables intracellular action of both toxins. However, the DrT protection level mediated by PaOrf4 is reduced compared to DrOrf5, implying a recognition mechanism for the cognate toxic subunit, leading to incomplete toxicity suppression of similar, but non-cognate toxic subunits.

Functional analysis of the C-II subgroup killer toxin-like chitinases in the filamentous ascomycete Aspergillus nidulans.

Chitinases are hydrolytic enzymes responsible for chitin polymer degradation. Fungal chitinases belong exclusively to glycoside hydrolases family 18 and they are categorized into three phylogenetic groups (A, B and C), which are further divided into subgroups (A-II to A-V, B-I to B-V and C-I to C-II). Subgroup C chitinases display similarity with the α/ß-subunit of the zymocin yeast killer toxin produced by Kluyveromyces lactis, suggesting a role of these enzymes in fungal-fungal interactions. In this study, we investigated the regulation and function of 4 Aspergillus nidulans subgroup C-II killer toxin-like chitinases by quantitative PCR and by constructing gene deletion strains. Our results showed that all 4 genes were highly induced during interactions with Botrytis cinerea and Rhizoctonia solani, compared to self-interactions. In addition, chiC2-2 and chiC2-3 were also induced during contact with Fusarium sporotrichoides, while none of these genes were induced during interactions with Phytophthora niederhauserii. In contrast, no difference in expression levels were observed between growth on glucose-rich media compared with media containing colloidal chitin, while all genes were repressed during growth on R. solani cell wall material. Phenotypic analysis of chitinase gene deletion strains revealed that B. cinerea biomass was significantly higher in culture filtrate derived from the ΔchiC2-2 strain compared to biomasses grown in media derived from A. nidulans wild type or the other chitinase gene deletion strains. The analysis also showed that all chitinase gene deletion strains displayed increased biomass production in liquid cultures, and altered response to abiotic stress. In summary, our gene expression data suggest the involvement of A. nidulans subgroup C-II chitinases in fungal-fungal interactions, which is further proven for ChiC2-2. In addition, lacking any of the 4 chitinases influenced the growth of A. nidulans.

A fungal anticodon nuclease ribotoxin exploits a secondary cleavage site to evade tRNA repair.

PaOrf2 and γ-toxin subunits of Pichia acaciae toxin (PaT) and Kluyveromyces lactis zymocin are tRNA anticodon nucleases. These secreted ribotoxins are assimilated by Saccharomyces cerevisiae, wherein they arrest growth by depleting specific tRNAs. Toxicity can be recapitulated by induced intracellular expression of PaOrf2 or γ-toxin in S. cerevisiae. Mutational analysis of γ-toxin has identified amino acids required for ribotoxicity in vivo and RNA transesterification in vitro. Here, we report that PaOrf2 residues Glu9 and His287 (putative counterparts of γ-toxin Glu9 and His209) are essential for toxicity. Our results suggest a similar basis for RNA transesterification by PaOrf2 and γ-toxin, despite their dissimilar primary structures and distinctive tRNA target specificities. PaOrf2 makes two sequential incisions in tRNA, the first of which occurs 3' from the mcm(5)s(2)U wobble nucleoside and depends on mcm(5). A second incision two nucleotides upstream results in the net excision of a di-nucleotide. Expression of phage and plant tRNA repair systems can relieve PaOrf2 toxicity when tRNA cleavage is restricted to the secondary site in elp3 cells that lack the mcm(5) wobble U modification. Whereas the endogenous yeast tRNA ligase Trl1 can heal tRNA halves produced by PaOrf2 cleavage in elp3 cells, its RNA sealing activity is inadequate to complete the repair. Compatible sealing activity can be provided in trans by plant tRNA ligase. The damage-rescuing ability of tRNA repair systems is lost when PaOrf2 can break tRNA at both sites. These results highlight the logic of a two-incision mechanism of tRNA anticodon damage that evades productive repair by tRNA ligases.

Site-directed mutagenesis of the heterotrimeric killer toxin zymocin identifies residues required for early steps in toxin action.

Zymocin is a Kluyveromyces lactis protein toxin composed of αßγ subunits encoded by the cytoplasmic virus-like element k1 and functions by αß-assisted delivery of the anticodon nuclease (ACNase) γ into target cells. The toxin binds to cells' chitin and exhibits chitinase activity in vitro that might be important during γ import. Saccharomyces cerevisiae strains carrying k1-derived hybrid elements deficient in either αß (k1ORF2) or γ (k1ORF4) were generated. Loss of either gene abrogates toxicity, and unexpectedly, Orf2 secretion depends on Orf4 cosecretion. Functional zymocin assembly can be restored by nuclear expression of k1ORF2 or k1ORF4, providing an opportunity to conduct site-directed mutagenesis of holozymocin. Complementation required active site residues of α's chitinase domain and the sole cysteine residue of ß (Cys250). Since ßγ are reportedly disulfide linked, the requirement for the conserved γ C231 was probed. Toxicity of intracellularly expressed γ C231A indicated no major defect in ACNase activity, while complementation of k1ΔORF4 by γ C231A was lost, consistent with a role of ß C250 and γ C231 in zymocin assembly. To test the capability of αß to carry alternative cargos, the heterologous ACNase from Pichia acaciae (P. acaciae Orf2 [PaOrf2]) was expressed, along with its immunity gene, in k1ΔORF4. While efficient secretion of PaOrf2 was detected, suppression of the k1ΔORF4-derived k1Orf2 secretion defect was not observed. Thus, the dependency of k1Orf2 on k1Orf4 cosecretion needs to be overcome prior to studying αß's capability to deliver other cargo proteins into target cells.

TpBGL2 codes for a Tetrapisispora phaffii killer toxin active against wine spoilage yeasts.

Tetrapisispora phaffii produces a killer toxin known as Kpkt that has extensive anti-Hanseniaspora/Kloeckera activity under winemaking conditions. Kpkt has a ß-glucanase activity and induces ultrastructural modifications in the cell wall of sensitive strains, with a higher specific cytocidal activity and a selective action towards target yeast cells. In this study, a two-step PCR-based approach was used to isolate the gene coding ß-glucanase of T. phaffii. Initially, a fragment of the open reading frame was isolated by degenerate PCR, with primers designed on the NH2 -terminal sequence of the protein and on conserved motifs of Bgl2p of Saccharomyces cerevisiae and Candida albicans. Subsequently, the entire sequence of the gene was obtained by inverse PCR. blast analyses of TpBGL2 highlight high identity with homologous genes in other yeast species, in which TpBGL2p shows no killer activity. However, gene disruption resulted in complete loss of the glucanase activity and the killer phenotype, thus confirming that TpBgl2p has a killer activity.

Killer activity of Saccharomyces cerevisiae strains: partial characterization and strategies to improve the biocontrol efficacy in winemaking.

Killer yeasts are considered potential biocontrol agents to avoid or reduce wine spoilage by undesirable species. In this study two Saccharomyces cerevisiae strains (Cf8 and M12) producing killer toxin were partially characterized and new strategies to improve their activity in winemaking were evaluated. Killer toxins were characterized by biochemical tests and growth inhibition of sensitive yeasts. Also genes encoding killer toxin were detected in the chromosomes of both strains by PCR. Both toxins showed optimal activity and production at conditions used during the wine-making process (pH 3.5 and temperatures of 15-25 °C). In addition, production of both toxins was higher when a nitrogen source was added. To improve killer activity different strategies of inoculation were studied, with the sequential inoculation of killer strains the best combination to control the growth of undesired yeasts. Sequential inoculation of Cf8-M12 showed a 45 % increase of killer activity on sensitive S. cerevisiae and spoilage yeasts. In the presence of ethanol (5-12 %) and SO2 (50 mg/L) the killer activity of both toxins was increased, especially for toxin Cf8. Characteristics of both killer strains support their future application as starter cultures and biocontrol agents to produce wines of controlled quality.

Strain prevalence and killer factor only partially influence the fermentation activity of pairwise Saccharomyces cerevisiae wine strains inoculation.

Commercial Saccharomyces cerevisiae starters are single-strain cultures widely used in winemaking to optimise the fermentation process and improve the organoleptic quality of wine. Unfortunately, the worldwide extensive use of a limited number of industrial strains led to the standardisation of the sensory properties, reducing the identity of wines. Therefore, the use of multi-strain S. cerevisiae starters can be an alternative tool to alter the sensory profile of wines, increasing the diversity of wine styles. However, this strategy may be interesting only if the overall fermentation kinetics is not affected. To date, there is a lack of information regarding the influence of multi-strain starters on the overall fermentation process in wine. In this context, killer toxins, affecting the viability of sensitive strains, can play a significant role. This study aimed to evaluate the effects of pairing eight wine strains of S. cerevisiae (two sensitive, three neutral and three killer) in co-fermentations compared to single-strain fermentations. Results evidenced that, among co-fermentations where the strain prevalence was significant, the killer strains constituted 79% to 100% of the total yeast population when co-inoculated with a sensitive one. However, in most of the cases, co-fermentations kinetics were similar to those of sensitive strains or worse than both strains. Thus, the presence of a killer strain alone is not sufficient to predict the overall fermentation progress, which is an essential information in winemaking. Interestingly, the neutral strain P304.4 was always prevalent, regardless of the second strain and, in most of the co-fermentations, the overall fermentation trend was similar to the P304.4 single-strain fermentation. Regardless of killer activity, our results suggest that the effect of strains on fermentative kinetics is still unpredictable, and further studies are needed to thoroughly explore strain to strain interactions in winemaking.

Killer yeasts: expanding frontiers in the age of synthetic biology.

Killer yeasts secrete protein toxins that are selectively lethal to other yeast and filamentous fungi. These exhibit exceptional genetic and functional diversity, and have several biotechnological applications. However, despite decades of research, several limitations hinder their widespread adoption. In this perspective we contend that technical advances in synthetic biology present an unprecedented opportunity to unlock the full potential of yeast killer systems across a spectrum of applications. By leveraging these new technologies, engineered killer toxins may emerge as a pivotal new tool to address antifungal resistance and food security. Finally, we speculate on the biotechnological potential of re-engineering host double-stranded (ds) RNA mycoviruses, from which many toxins derive, as a safe and noninfectious system to produce designer RNA.