Killer toxin of Kluyveromyces phaffii DBVPG 6076 as a biopreservative agent to control apiculate wine Yeasts.

The use of Kluyveromyces phaffii DBVPG 6076 killer toxin against apiculate wine yeasts has been investigated. The killer toxin of K. phaffii DBVPG 6076 showed extensive anti-Hanseniaspora activity against strains isolated from grape samples. The proteinaceous killer toxin was found to be active in the pH range of 3 to 5 and at temperatures lower than 40 degrees C. These biochemical properties would allow the use of K. phaffii killer toxin in wine making. Fungicidal or fungistatic effects depend on the toxin concentration. Toxin concentrations present in the supernatant during optimal conditions of production (14.3 arbitrary units) exerted a fungicidal effect on a sensitive strain of Hanseniaspora uvarum. At subcritical concentrations (fungistatic effect) the saturation kinetics observed with the increased ratio of killer toxin to H. uvarum cells suggest the presence of a toxin receptor. The inhibitory activity exerted by the killer toxin present in grape juice was comparable to that of sulfur dioxide. The findings presented suggest that the K. phaffii DBVPG 6076 killer toxin has potential as a biopreservative agent in wine making.     

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 β-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 β-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.

     

Pichia anomala DBVPG 3003 Secretes a Ubiquitin-Like Protein That Has Antimicrobial Activity

The yeast strain Pichia anomala DBVPG 3003 secretes a killer toxin (Pikt) that has antifungal activity against Brettanomyces/Dekkera sp. yeasts. Pikt interacts with β-1,6-glucan, consistent with binding to the cell wall of sensitive targets. In contrast to that of toxin K1, secreted by Saccharomyces cerevisiae, Pikt killer activity is not mediated by an increase in membrane permeability. Purification of the toxin yielded a homogeneous protein of about 8 kDa, which showed a marked similarity to ubiquitin in terms of molecular mass and N-terminal sequences. Pikt is also specifically recognized by anti-bovine ubiquitin antibodies and, similar to ubiquitin-like peptides, is not absorbed by DEAE-cellulose. However, Pikt differs from ubiquitin in its sensitivity to proteolytic enzymes. Therefore, Pikt appears to be a novel ubiquitin-like peptide that has killer activity.     

Killer character in Kluyveromyces yeasts: Activity on Kloeckera apiculata

The screening for ‘killer’ character among 23 strains of the genus Kluyveromyces showed the presence of three ‘killer’ strains (K. phaffii; K. vanudenii and K. wikenii). Their ‘killer’ activity against yeasts of other genera is in agreement with the results of others studies. Particularly K. phaffii inhibited many strains of Kloeckera apiculata.     

Pichia anomala and Kluyveromyces wickerhamii killer toxins as new tools against Dekkera/Brettanomyces spoilage yeasts

Two yeast killer toxins active on spoilage yeasts belonging to the genus Dekkera/Brettanomyces are here described for the first time. The two toxins produced by Pichia anomala (DBVPG 3003) and Kluyveromyces wickerhamii (DBVPG 6077), and named Pikt and Kwkt, respectively, differ for molecular weight and biochemical properties. Interestingly, the fungicidal effect exerted by Pikt and Kwkt against Dekkera bruxellensis is stable for at least 10 days in wine. Thus, a potential application for the two toxins as antimicrobial agents active on Dekkera/Brettanomyces during wine ageing and storage can be hypothesised.     

Killer toxin producing strains of the yeasts Hanseniaspora uvarum and Pichia kluyveri

By heat treatment killer strains of the type K1 of Saccharomyces cerevisiae that are known to harbour dsRNA plasmids were completely cured, whereas only a small fraction of the clones of the killer type K2 had lost the dsRNA dependent killer character. The K2 killers but not the strains of killer type K1 were easily cured by cycloheximide. Killer strains of Hanseniaspora uvarum were not curable by heat treatment. Curing was successfull with cycloheximide or 5-fluorouracil. Two double-stranded RNA plasmids were detected in the killer strains of H. uvarum. The smaller dsRNA plasmid was absent in the strains that were cured of their killer character by 5-fluorouracil. The killer character of H. uvarum was transferred to S. cerevisiae by spheroplast fusion. The fusion products showing the killer character contained both dsRNA plasmids, obviously the smaller plasmid (M-dsRNA) carries the genes for killer toxin formation. Killer strains of Pichia kluyveri were not curable of their killer character, in these strains no dsRNA plasmids were detected.This paper was kindly supported by a grant from the Deutsche Forschungsgemeinschaft     

Killer toxin of Hanseniaspora uvarum

The yeast Hanseniaspora uvarum liberates a killer toxin lethal to sensitive strains of the species Saccharomyces cerevisiae. Secretion of this killer toxin was inhibited by tunicamycin, an inhibitor of N-glycosylation, although the mature killer protein did not show any detectable carbohydrate structures. Culture supernatants of the killer strain were concentrated by ultrafiltration and the extracellular killer toxin was precipitated with ethanol and purified by ion exchange chromatography. SDS-PAGE of the electrophoretically homogenous killer protein indicated an apparent molecular mass of 18,000.Additional investigations of the primary toxin binding sites within the cell wall of sensitive yeast strains showed that the killer toxin of Hanseniaspora uvarum is bound by -1, 6-d-glucans.     

Utilization of KHR killer as genetic marker for purity test of starter yeast during fermentation of grape musts

An excellent wine yeast, Saccharomyces cerevisiae W3, which had KHR killer, was added as a starter yeast into grape must and behavior of the starter strain and wild yeasts was investigated during fermentation by using KHR killer as a genetic marker. The KHR killer was detected only in the strain W3 and not in other wine and wild yeast strains. Accordingly, the frequency of starter yeast W3 was monitored throughout the fermentation of grape musts by using KHR killer, W3 was discriminated efficiently from wild yeasts during fermentation by KHR killer activity and proved to lead the fermentation as a dominant yeast until their termination.     

Construction of Killer Wine Yeast Strain

A double-stranded RNA plasmid which confers the superkiller phenotype was transferred into a wine yeast (Montrachet strain 522) and its leucine-requiring derivative (strain 694) by cytoduction, using the protoplast fusion technique. The killer wine yeast constructed completely suppressed the growth of killer-sensitive strains of Saccharomyces cerevisiae in yeast extract-peptone-glucose medium at pH 4.5, whereas the killer effect was somewhat decreased at pH 3.5. The wine yeast harboring the killer factor also inhibited the growth of killer-sensitive cells satisfactorily when it was grown in grape juice.     

Identification of indigenous yeast flora isolated from the five winegrape varieties harvested in Xiangning,China

Inoculated fermentation by selected indigenous yeast strains from a specific location could provide the wine with unique regional sensory characteristics. The identification and differentiation of local yeasts are the first step to understand the function of yeasts and develop a better strain-selection program for winemaking. The indigenous yeasts in five grape varieties, Chardonnay, Cabernet Franc, Cabernet Sauvignon, Marselan, and Merlot cultivated in Xiangning, Shanxi, China were investigated. Eight species of seven genera including Aureobasidium pullulans, Candida zemplinina, Hanseniaspora uvarum, Hanseniaspora occidentalis, Issatchenkia terricola, Metschnikowia pulcherrima, Pichia kluyveri, and Saccharomyces cerevisiae were identified using Wallerstein Laboratory Nutrient medium with sequencing of the 26S rDNA D1/D2 domain. H. uvarum and S. cerevisiae were the predominant species, while most non-Saccharomyces species were present in the whole fermentation process at different levels among the grape varieties. The genotypes of S. cerevisiae from each microvinification were determined by using interdelta sequence analysis. The 102 isolates showed eight different genotypes, and genotype III was the predominant genotype found. The distribution of S. cerevisiae strains during the fermentation of Marselan was also studied. Six genotypes were observed among the 92 strains with different genotypes of competitiveness at different sampling stages. Genotype V demonstrated the potential for organizing starter strains and avoiding inefficient fermentation. In general, this study explored the yeast species in the grapes grown in Xiangning County and provided important information of relationship of local yeast diversity and its regional wine sensory characteristics.     

Construction and properties of K1 type killer wine yeasts

Summary With the use of a protoplast fusion technique the killer character of K1 type was transferred into four industrial Saccharomyces wine yeasts. The prototrophic yeast strains active against standard sensitive and K2 killer Saccharomyces strains, resistant to K1 killer toxin were constructed with no changes in technological properties.     

Effect of wine inhibitors on the proteolytic activity of papain from Carica papaya L. latex

The influence of potential inhibitors naturally present in wine on the proteolytic activity of papain from Carica papaya latex was investigated to evaluate its applicability in white wine protein haze stabilization. Enzymatic activity was tested against a synthetic tripeptide chromogenic substrate in wine‐like acidic medium that consisted of tartaric buffer (pH 3.2) supplemented with ethanol, free sulfur dioxide (SO₂), grape skin and seed tannins within the average ranges of concentrations that are typical in wine. The diagnosis of inhibition type, performed with the graphical method, demonstrated that all of tested wine constituents were reversible inhibitors of papain. The strongest inhibition was exerted by free SO₂, which acted as a mixed‐type inhibitor, similar to grape skin and seed tannins. Finally, when tested in table white wines, the catalytic activity of papain, even when if it was ascribable to the hyperbolic behavior of Michaelis‐Menten equation, was determined to be strongly affected by free SO₂ and total phenol level. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:48–54, 2015     

From grape berries to wine: population dynamics of cultivable yeasts associated to “Nero di Troia” autochthonous grape cultivar

The aim of this work was to study the biodiversity of yeasts isolated from the autochthonous grape variety called “Uva di Troia”, monitoring the natural diversity from the grape berries to wine during a vintage. Grapes were collected in vineyards from two different geographical areas and spontaneous alcoholic fermentations (AFs) were performed. Different restriction profiles of ITS–5.8S rDNA region, corresponding to Saccharomyces cerevisiae, Issatchenkia orientalis, Metschnikowia pulcherrima, Hanseniaspora uvarum, Candida zemplinina, Issatchenkia terricola, Kluyveromyces thermotolerans, Torulaspora delbrueckii, Metschnikowia chrysoperlae, Pichia fermentans, Hanseniaspora opuntiae and Hanseniaspora guilliermondii, were observed. The yeast occurrences varied significantly from both grape berries and grape juices, depending on the sampling location. Furthermore, samples collected at the end of AF revealed the great predominance of Saccharomyces cerevisiae, with a high intraspecific biodiversity. This is the first report on the population dynamics of ‘cultivable’ microbiota diversity of “Uva di Troia” cultivar from the grape to the corresponding wine (“Nero di Troia”), and more general for Southern Italian oenological productions, allowing us to provide the basis for an improved management of wine yeasts (with both non-Saccharomyces and Saccharomyces) for the production of typical wines with desired unique traits. A certain geographical-dependent variability has been reported, suggesting the need of local based formulation for autochthonous starter cultures, especially in the proportion of the different species/strains in the design of mixed microbial preparations.     

The K3 type killer strains of genusSaccharomyces for wine production

Using simultaneous fusion of protoplasts of three strains the auxotrophic K3 killer designated MH1 was prepared at a very low frequency and further employed for the transfer of the K3 killer character into a commercial wine yeast. A novelSaccharomyces Bratislava 1K3 strain with desirable technological and anti-yeast killer abilities was thus constructed. Attempts to prepare double K1/K3 killers were made. The obtained fusion products contained M₁ dsRNA and were able to produce only the K1 type killer toxin.     

Reducing diacetyl production of wine by overexpressing <Emphasis Type="Italic">BDH1</Emphasis> and <Emphasis Type="Italic">BDH2</Emphasis> in <Emphasis Type="Italic">Saccharomyces uvarum</Emphasis>

As a byproduct of yeast valine metabolism during fermentation, diacetyl can produce a buttery aroma in wine. However, high diacetyl concentrations generate an aromatic off-flavor and poor quality in wine. 2,3-Butanediol dehydrogenase encoded by BDH1 can catalyze the two reactions of acetoin from diacetyl and 2,3-butanediol from acetoin. BDH2 is a gene adjacent to BDH1, and these genes are regulated reciprocally. In this study, BDH1 and BDH2 were overexpressed in Saccharomyces uvarum to reduce the diacetyl production of wine either individually or in combination. Compared with those in the host strain WY1, the diacetyl concentrations in the recombinant strains WY1-1 with overexpressed BDH1, WY1-2 with overexpressed BDH2 alone, and WY1-12 with co-overexpressed BDH1 and BDH2 were decreased by 39.87, 33.42, and 46.71%, respectively. BDH2 was only responsible for converting diacetyl into acetoin, but not for the metabolic pathway of acetoin to 2,3-butanediol in S. uvarum. This study provided valuable insights into diacetyl reduction in wine.     

Tetrapisispora phaffii killer toxin is a highly specific β-glucanase that disrupts the integrity of the yeast cell wall

Background  

Killer yeasts have been used to combat contaminating wild yeasts in food, to control pathogenic fungi in plants, and in the medical field, to develop novel antimycotics for the treatment of human and animal fungal infections. Among these killer yeasts, Tetrapisispora phaffii (formerly known as Kluyveromyces phaffii) secretes a glycoprotein known as Kpkt that is lethal to spoilage yeasts under winemaking conditions. In the present study, the mode of action of Kpkt, and the specific damage produced by this toxin on sensitive yeasts is investigated.     

Kluyveromyces wickerhamii killer toxin: purification and activity towards Brettanomyces/Dekkera yeasts in grape must

Brettanomyces/Dekkera yeasts have been identified as part of the grape yeast flora. They are well known for colonizing the cellar environmental and spoiling wines, causing haze, turbidity and strong off-flavours in wines and enhancing the volatile acidity. As the general practices applied to combat Brettanomyces/Dekkera yeasts are not particularly appropriate during wine ageing and storage, a biological alternative to curtailing their growth would be welcomed in winemaking. In this study, we investigated the Kluyveromyces wickerhamii killer toxin (Kwkt) that is active against Brettanomyces/Dekkera spoilage yeasts. Purification procedures allowed the identification of Kwkt as a protein with an apparent molecular mass of 72 kDa and without any glycosyl residue. Interestingly, purified Kwkt has fungicidal effects at low concentrations under the physicochemical conditions of winemaking. The addition of 40 and 80 mg L(-1) purified Kwkt showed efficient antispoilage effects, controlling both growth and metabolic activity of sensitive spoilage yeasts. At these two killer toxin concentrations, compounds known to contribute to the 'Brett' character of wines, such as ethyl phenols, were not produced. Thus, purified Kwkt appears to be a suitable biological strategy to control Brettanomyces/Dekkera yeasts during fermentation, wine ageing and storage.     

Production of fluorescent and cytotoxic K28 killer toxin variants through high cell density fermentation of recombinant <Emphasis Type="Italic">Pichia pastoris</Emphasis>

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/β 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 β-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.

     

The Species-Specific Acquisition and Diversification of a K1-like Family of Killer Toxins in Budding Yeasts of the Saccharomycotina

Killer toxins are extracellular antifungal proteins that are produced by a wide variety of fungi, including Saccharomyces yeasts. Although many Saccharomyces killer toxins have been previously identified, their evolutionary origins remain uncertain given that many of these genes have been mobilized by double-stranded RNA (dsRNA) viruses. A survey of yeasts from the Saccharomyces genus has identified a novel killer toxin with a unique spectrum of activity produced by Saccharomyces paradoxus. The expression of this killer toxin is associated with the presence of a dsRNA totivirus and a satellite dsRNA. Genetic sequencing of the satellite dsRNA confirmed that it encodes a killer toxin with homology to the canonical ionophoric K1 toxin from Saccharomyces cerevisiae and has been named K1-like (K1L). Genomic homologs of K1L were identified in six non-Saccharomyces yeast species of the Saccharomycotina subphylum, predominantly in subtelomeric regions of the genome. When ectopically expressed in S. cerevisiae from cloned cDNAs, both K1L and its homologs can inhibit the growth of competing yeast species, confirming the discovery of a family of biologically active K1-like killer toxins. The sporadic distribution of these genes supports their acquisition by horizontal gene transfer followed by diversification. The phylogenetic relationship between K1L and its genomic homologs suggests a common ancestry and gene flow via dsRNAs and DNAs across taxonomic divisions. This appears to enable the acquisition of a diverse arsenal of killer toxins by different yeast species for potential use in niche competition.     

<Emphasis Type="Italic">PiggyBac</Emphasis> transposon-mediated mutagenesis and application in yeast <Emphasis Type="Italic">Komagataella phaffii</Emphasis>

Objective

Around one-fourth of the Komagataella phaffii genes encode hypothetical proteins with unknown functions. However, lack of powerful tools for genetic screening in K. phaffii significantly limits the functional analysis of these unknown genes. Transposon mutagenesis has been utilized as an insertional mutagenesis tool in many other organisms and would be extremely valuable if it could be applied in K. phaffii.

Results

In this study, we investigated in K. phaffii the transposition activity and efficiency of piggyBac (PB) transposon, a DNA transposon from the cabbage looper moth Trichoplusia ni through the integrated-plasmid system. We also designed a binary-plasmid system which could generate stable mutants. Finally we evaluated the quality of this mutagenesis system by a simple screening for functional genes involved in K. phaffii carbon catabolite repression.

Conclusions

Our results demonstrate that PB-mediated mutagenesis could be a feasible and useful tool for functional gene screening in K. phaffii.