Prevention of Yeast Spoilage in Feed and Food by the Yeast Mycocin HMK

The yeast Williopsis mrakii produces a mycocin or yeast killer toxin designated HMK; this toxin exhibits high thermal stability, high pH stability, and a broad spectrum of activity against other yeasts. We describe construction of a synthetic gene for mycocin HMK and heterologous expression of this toxin in Aspergillus niger. Mycocin HMK was fused to a glucoamylase protein carrier, which resulted in secretion of biologically active mycocin into the culture media. A partial purification protocol was developed, and a comparison with native W. mrakii mycocin showed that the heterologously expressed mycocin had similar physiological properties and an almost identical spectrum of biological activity against a number of yeasts isolated from silage and yoghurt. Two food and feed production systems prone to yeast spoilage were used as models to assess the ability of mycocin HMK to act as a biocontrol agent. The onset of aerobic spoilage in mature maize silage was delayed by application of A. niger mycocin HMK on opening because the toxin inhibited growth of the indigenous spoilage yeasts. This helped maintain both higher lactic acid levels and a lower pH. In yoghurt spiked with dairy spoilage yeasts, A. niger mycocin HMK was active at all of the storage temperatures tested at which yeast growth occurred, and there was no resurgence of resistant yeasts. The higher the yeast growth rate, the more effective the killing action of the mycocin. Thus, mycocin HMK has potential applications in controlling both silage spoilage and yoghurt spoilage caused by yeasts.     

Application of monoclonal antibodies to the isolation and characterization of a killer toxin secreted by Hansenula mrakii

A strain of yeast, Hansenula mrakii, secretes a toxin that kills sensitive yeasts, such as Saccharomyces cerevisiae Monoclonal antibodies raised against the toxin had both binding and neutralizing activities. The toxin in culture media was isolated by an affinity column of monoclonal antibody. The toxin is a basic polypeptide with an isoelectric point at pH 9.1, and devoid of mannosides. It is composed of 88 amino acid residues with a molecular size of 10 721 Da. The monoclonal antibodies could be applicable to the analysis of biologically active sites on the toxin, in an attempt to synthesize chemically a small peptide with killer activity and little immunogenicity.     

Unusual properties of the halotolerant yeast Candida nodaensis Killer toxin, CnKT

CnKT, the Killer toxin from the extreme halotolerant yeast Candida nodaensis, presents a strong salt-stimulated phenotype and is a resilient toxin, able to cope with very diverse and aggressive environmental conditions. This zymocin is active in a broad range of pH and temperature and tolerates freezing and conservation for long periods of time. CnKT stability is increased under very high ionic strength and its activity is stimulated by sodium ions, which might interfere in the zymocin structure/stability. All these characteristics make CnKT a promising candidate for several biotechnological applications, e.g. in the high-salt food products preservation from spoilage by other yeasts.     

Prevention of aerobic spoilage of maize silage by a genetically modified killer yeast, Kluyveromyces lactis, defective in the ability to grow on lactic acid.

In this study, we propose a new process of adding a genetically modified killer yeast to improve the aerobic stability of silage. Previously constructed Kluyveromyces lactis killer strain PCK27, defective in growth on lactic acid due to disruption of the gene coding for phosphoenolpyruvate carboxykinase, a key enzyme for gluconeogenesis, inhibited the growth of Pichia anomala inoculated as an aerobic spoilage yeast and prevented a rise in pH in a model of silage fermentation. This suppressive effect of PCK27 was not only due to growth competition but also due to the killer protein produced. From these results, we concluded that strain PCK27 can be used as an additive to prolong the aerobic stability of maize silage. In the laboratory-scale experiment of maize silage, the addition of a killer yeast changed the yeast flora and significantly reduced aerobic spoilage.     

Involvement of [beta]-glucans in the wide-spectrum antimicrobial activity of Williopsis saturnus var. mrakii MUCL 41968 killer toxin

BACKGROUND: Williopsis saturnus var. mrakii MUCL 41968 secretes a 85-kDa glycoprotein killer toxin (WmKT) that displays a cytocidal activity against a wide range of microorganisms, making WmKT a promising candidate for the development of new antimicrobial molecules. Although the killing mechanism of WmKT is still unknown, the toxin was recently proposed to bind to the surface of sensitive microorganisms through the recognition of beta-glucans. Indeed, Saccharomyces cerevisiae strains sensitive to the toxin become resistant when mutated in their beta-glucan synthesis pathway. MATERIALS AND METHODS: To investigate the interaction of WmKT with beta-glucans, we examined in agar diffusion assays the WmKT activity in the presence of enzymes displaying beta-glucanase activity. The toxin activity was also investigated using spheroplasts derived from sensitive yeast cells. The hydrolytic activity of WmKT was studied using specific glucosidase inhibitors as well as various sugar molecules covalently linked to p-nitrophenyl as potential substrates. Finally, the ultrastructural modifications induced by WmKT activity on sensitive yeasts were assessed by scanning electron microscopy. RESULTS: The data reported here support the hypothesis that WmKT binds to sensitive cells using surface-exposed beta-glucans. Indeed beta-glucanase exerts an antagonistic effect on WmKT activity and spheroplasts derived from WmKT-sensitive yeast cells are shown to be resistant to WmKT, suggesting that cell wall beta-glucans are required for WmKT lethal effect. Because WmKT exhibits amino acid sequence similarities with proteins suspected to be glucanase, we also investigated the effect of castanospermine, a potent glucosidase inhibitor, on WmKT activity. Castanospermine completely abolished WmKT killer activity as well as its hydrolytic enzymatic activity against p-nitrophenyl beta-D-glucopyranoside. The scanning electron microscopy analysis of sensitive yeast cells treated with the toxin reveals that WmKT causes cell wall modifications similar to those observed with zymolyase. CONCLUSION: The results reported in this study show that WmKT activity requires an interaction between the mycocin and the cell wall beta-glucans. Moreover, they indicate that WmKT acts on sensitive yeast cells through a hydrolytic activity directed against cell wall beta-glucans that disrupts the yeast cell wall integrity leading to death.     

The incidence of killer activity of non-Saccharomyces yeasts towards indigenous yeast species of grape must: potential application in wine fermentation

Fourteen killer yeasts were assayed for their ability to kill species of yeast that are commonly associated with fermenting grape must and wine. A total of 147 of a possible 364 killer-sensitive interactions were observed at pH 4.5. Of the killer yeasts studied, Pichia anomala NCYC 434 displayed the broadest killing range. At a pH value comparable with those of wine ferments, pH 3.5, the incidence of killer-sensitive interactions was reduced by 700% across all the yeasts. Williopsis saturnus var. mrakii CBS 1707 exhibited the broadest killing range at the lower pH, killing more than half of the tester strains. Intraspecific variation in sensitivity to killer yeasts was observed in all species where more than one strain was tested. Also, in strains of Pichia anomala, Kluyveromyces lactis and Pichia membranifaciens, the three species in which more than one killer yeast was analysed, intraspecific variation in killer activity was observed.     

Fungicidal activity of yeast isolated from chal

A Kluyveromyces strain secreting a fungicidal proteinaceous toxin has been isolated. Its maximal activity is observed at pH 5.0 and an increased osmotic pressure. This agent has been identified as a mycocin; it is active towards species belonging to the genus Kluyveromyces and some representatives of taxonomically related taxa.     

Interactions between killer yeasts and pathogenic fungi

Abstract A total of 17 presumptive killer yeast strains were tested in vitro for growth inhibitory and killing activity against a range of fungal pathogens of agronomic, environmental and clinical significance. Several yeasts were identified which displayed significant activity against important pathogenic fungi. For example, isolates of the opportunistic human pathogen, Candida albicans , were generally very sensitive to Williopsis mrakii killer yeast activity, whilst killer strains of Saccharomyces cerevisiae and Pichia anomala markedly inhibited the growth of certain wood decay basidiomycetes and plant pathogenic fungi. Results indicate that such yeasts, together with their killer toxins, may have potential as novel antimycotic biocontrol agents.     

Prevention of Aerobic Spoilage of Maize Silage by a Genetically Modified Killer Yeast, Kluyveromyces lactis, Defective in the Ability To Grow on Lactic Acid

In this study, we propose a new process of adding a genetically modified killer yeast to improve the aerobic stability of silage. Previously constructed Kluyveromyces lactis killer strain PCK27, defective in growth on lactic acid due to disruption of the gene coding for phosphoenolpyruvate carboxykinase, a key enzyme for gluconeogenesis, inhibited the growth of Pichia anomala inoculated as an aerobic spoilage yeast and prevented a rise in pH in a model of silage fermentation. This suppressive effect of PCK27 was not only due to growth competition but also due to the killer protein produced. From these results, we concluded that strain PCK27 can be used as an additive to prolong the aerobic stability of maize silage. In the laboratory-scale experiment of maize silage, the addition of a killer yeast changed the yeast flora and significantly reduced aerobic spoilage.     

A Kluyveromyces lactis mycocin active at neutral pH

A strain of Kluyveromyces lactis was found to secrete a fungicidal mycocin active in the pH range from 6 to 9 and exhibiting the highest activity at pH of approximately 7. A few yeast species of the families Saccharomycetaceae and Wickerhamomycetaceae were sensitive to the mycocin. Some genera and species were heterogeneous in this respect. UV treatment of the mycocinogenic strain resulted in loss of its antifungal activity. Although prokaryotes were not sensitive to the mycocin, the strain under study inhibited growth of some bacteria.     

Williopsis saturnus yeast killer toxin does not kill Streptococcus pneumoniae

Streptococcus pneumoniae is an important human bacterial pathogen, and the increase in antibiotic resistance demands the development of new antimicrobial compounds. Several reports have suggested that yeast killer toxins show activity against bacteria and we therefore investigated the activity of K9 killer toxin from the yeast Williopsis saturnus var. mrakii NCYC 500 against S. pneumoniae. However, no inhibition of bacterial growth was observed with concentrated K9 preparations in agar diffusion assays and in liquid culture. Although cell morphology was slightly affected by K9 treatment, no effect on cellular viability was detectable, and K9 had no stimulatory effect on cell lysis induced by β-lactams or Triton X-100. This indicated that K9 did not contribute to cell wall damage. Moreover, flow cytometry was used as a sensitive assessment of integrity of cells exposed to killer toxin. No significant damage of S. pneumoniae cells was evident, although minor changes in fluorescence suggested that K9 killer toxin may interact with bacterial surface components.     

Specificity of sensitivity to mycocin from Tilletiopsis flava BKM Y-2838

The mycocinogenous strain Tilletiopsis flava VKM Y-2823 was found to possess fungicidal activity at pH 3.5-4.5, which was retained after curing the strain by eliminating the extrachromosomal genetic elements. The mycocin produced by the strain had a molecular mass of more than 10 kDa and was readily inactivated by heating and treatment with protease K. This mycocin was found to be active against species of the anamorphic genus Tilletiopsis. The overwhelming majority of other representatives of the order Tilletiales, as well as ascomycetous and basidiomycetous yeasts, which either form or did not from ballistospores of the orders Sporidiales and Tremellales, were resistant to it.     

Linear mitochondrial deoxyribonucleic acid from the yeast Hansenula mrakii.

The mitochondrial deoxyribonucleic acid (mtDNA) from a petite-negative yeast, Hansenula mrakii, was studied. A linear restriction map was constructed with 11 restriction enzymes. The linearity of the genome was confirmed by direct end labeling of the molecule, followed by restriction analysis. The molecular weight of the DNA was found to be 55,000 base pairs. This is the first linear mtDNA found in yeast species. Using specific gene probes obtained from Saccharomyces cerevisiae mtDNA, we have constructed a gene map of H. mrakii mtDNA. The arrangement of genes in this linear genome was very different from the circular mtDNA of other known yeasts.     

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.     

Occurrence of killer yeast strains in industrial and clinical yeast isolates

The secretion of proteinaceous toxins is a widespread characteristic in environmental and laboratory yeast isolates, a phenomenon called "killer system". The killer phenotype (K+) can be encoded by extrachromosomal genetic elements (EGEs) as double stranded DNA or RNA molecules (dsDNA, dsRNA) or in nuclear genes. The spectrum of action and the activity of killer toxins are influenced by temperature, salinity and pH of media. In the present work we determined the existence of K+ in a collection of S. cerevisiae and P. anomala yeasts isolated from environmental, industrial and clinical sources. The assays were performed in strains belonging to three yeast genera used as sensitive cells and under a wide range of pH and temperatures. Approximately 51 % of isolates tested showed toxicity against at least one sensitive yeast strain under the conditions tested. The K+ P. anomala isolates showed a wide spectrum of action and two of them had toxic activity against strains of the three yeast genera assayed, including C. albicans strains. In all S. cerevisiae K+ isolates an extrachromosomal dsRNA molecule (4.2 Kb) was observed, contrary to P. anomala K+ isolates, which do not possess any EGEs. The K+ phenotype is produced by an exported protein factor and the kinetics of killer activity production was similar in all isolates with high activity in the log phase of growth, decaying in the stationary phase.     

Oxygen requirements of the food spoilage yeast Zygosaccharomyces bailii in synthetic and complex media

Most yeast species can ferment sugars to ethanol, but only a few can grow in the complete absence of oxygen. Oxygen availability might, therefore, be a key parameter in spoilage of food caused by fermentative yeasts. In this study, the oxygen requirement and regulation of alcoholic fermentation were studied in batch cultures of the spoilage yeast Zygosaccharomyces bailii at a constant pH, pH 3.0. In aerobic, glucose-grown cultures, Z. bailii exhibited aerobic alcoholic fermentation similar to that of Saccharomyces cerevisiae and other Crabtree-positive yeasts. In anaerobic fermentor cultures grown on a synthetic medium supplemented with glucose, Tween 80, and ergosterol, S. cerevisiae exhibited rapid exponential growth. Growth of Z. bailii under these conditions was extremely slow and linear. These linear growth kinetics indicate that cell proliferation of Z. bailii in the anaerobic fermentors was limited by a constant, low rate of oxygen leakage into the system. Similar results were obtained with the facultatively fermentative yeast Candida utilis. When the same experimental setup was used for anaerobic cultivation, in complex YPD medium, Z. bailii exhibited exponential growth and vigorous fermentation, indicating that a nutritional requirement for anaerobic growth was met by complex-medium components. Our results demonstrate that restriction of oxygen entry into foods and beverages, which are rich in nutrients, is not a promising strategy for preventing growth and gas formation by Z. bailii. In contrast to the growth of Z. bailii, anaerobic growth of S. cerevisiae on complex YPD medium was much slower than growth in synthetic medium, which probably reflected the superior tolerance of the former yeast to organic acids at low pH.     

Anaerobic lactic acid degradation during ensilage of whole crop maize inoculated with lactobacillus buchneri inhibits yeast growth and improves aerobic stability

Aerobic deterioration of silages is initiated by (facultative) aerobic micro-organisms, usually yeasts, that oxidize the preserving organic acids. In this study, a Lactobacillus buchneri strain isolated from maize silage was evaluated for its potential as a bacterial inoculant that enhances aerobic stability of silages. In four experiments, chopped whole crop maize (30-43% dry matter (DM)) was inoculated with Lact. buchneri and ensiled in laboratory silos. Uninoculated silages served as controls. Analysis of silages treated with Lact. buchneri at levels of 103-106 cfu g-1 after about 3 months of anaerobic storage showedthat acetic acid and 1-propanol contents increased with inoculum levels above 104 cfu g-1,whereas lactic acid decreased. Propionic acid, silage pH and DM loss increased withinoculum levels above 105 cfu g-1. Time course experiments with maize inoculated with Lact. buchneri at 4 x 104-2 x 105 cfu g-1 showed that up to 7-14 d after ensiling, Lact. buchneri had no effect on silage characteristics. Thereafter, the lactic acid content of the inoculated silages declined and, simultaneously, acetic acid and, to a lesser extent, propionic acid and 1-propanol, accumulated. Inoculation reduced survival of yeasts during the anaerobic storage phase and inhibited yeast growth when the silage was exposed to O2, resulting in a substantial improvement in aerobic stability. The results indicate that the use of Lact. buchneri as a silage inoculant can enhance aerobic stability by inhibition of yeasts. The ability of the organism to ferment lactic acid to acetic acid appears to be an important underlying principle of this effect.     

The pdr12 ABC transporter is required for the development of weak organic acid resistance in yeast.

Exposure of Saccharomyces cerevisiae to sorbic acid strongly induces two plasma membrane proteins, one of which is identified in this study as the ATP-binding cassette (ABC) transporter Pdr12. In the absence of weak acid stress, yeast cells grown at pH 7.0 express extremely low Pdr12 levels. However, sorbate treatment causes a dramatic induction of Pdr12 in the plasma membrane. Pdr12 is essential for the adaptation of yeast to growth under weak acid stress, since Deltapdr12 mutants are hypersensitive at low pH to the food preservatives sorbic, benzoic and propionic acids, as well as high acetate levels. Moreover, active benzoate efflux is severely impaired in Deltapdr12 cells. Hence, Pdr12 confers weak acid resistance by mediating energy-dependent extrusion of water-soluble carboxylate anions. The normal physiological function of Pdr12 is perhaps to protect against the potential toxicity of weak organic acids secreted by competitor organisms, acids that will accumulate to inhibitory levels in cells at low pH. This is the first demonstration that regulated expression of a eukaryotic ABC transporter mediates weak organic acid resistance development, the cause of widespread food spoilage by yeasts. The data also have important biotechnological implications, as they suggest that the inhibition of this transporter could be a strategy for preventing food spoilage.     

Specificity of Yeast Sensitivity to the Mycocin of Tilletiopsis flava VKM Y-2823

The mycocinogenous strain Tilletiopsis flava VKM Y-2823 was found to possess fungicidal activity at pH 3.5–4.5, which was retained after curing the strain by eliminating the extrachromosomal genetic elements. The mycocin produced by the strain had a molecular mass of more than 10 kDa and was readily inactivated by heating and treatment with protease K. This mycocin was found to be active against species of the anamorphic genus Tilletiopsis. The overwhelming majority of other representatives of the order Tilletiales, as well as ascomycetous and basidiomycetous (including ballistosporous) yeasts of the orders Sporidiales and Tremellales, were resistant to it.     

Killer toxin from Hansenula mrakii selectively inhibits cell wall synthesis in a sensitive yeast

Hansenula mrakii secretes extracellularly a killer toxin which kills sensitive Saccharomyces cerevisiae. In protoplasts of this yeast, the killer toxin selectively inhibited the synthesis of alkali-insoluble acid-insoluble polysaccharides consisting mainly of beta-glucan, but did not inhibit either the synthesis of other cell wall polysaccharides, such as mannan, chitin and alkali-insoluble acid-soluble polysaccharides, or the synthesis of protein. Consistent with these results, the toxin was inhibitory to the beta-(1,3)-glucan synthetase activity of a cell-free extract from sensitive S. cerevisiae.