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

Abstract

The yeast strain Pichia anomala DBVPG 3003 secretes a killer toxin (Pikt) that has antifungal activity against Brettanomyces/Dekkera sp. yeasts. Pikt interacts with ␤-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.After its initial discovery in Saccharomyces cerevisiae (2), the killer phenotype has been found in yeasts belonging to the genera Candida, Cryptococcus, Debaryomyces, Hanseniaspora, Hansenula, Kloeckera, Kluyveromyces, Metschnikowia, Pichia, Rhodotorula, Schwanniomyces, Torulopsis, Trichosporon, Williopsis, and Zygosaccharomyces (9,16,19,25,28,53,56). The distribution of this character among natural yeast isolates and in laboratory strains is accompanied by killer toxins with large biodiversity, in terms of their biochemical characteristics, genetic determinants, and spectra of action (25). In spite of this diversity, all known killer toxins are proteins (1, 31) or glycoproteins (5,11,28,43,54) that kill sensitive cells via a two-step mode of action. During the first step, the killer toxin binds a receptor site on the cell wall of its sensitive target. In the second step, which has been far less characterized, the killer toxin is assumed to interact with receptors on the cell membrane and to kill the sensitive cells via different mechanisms. These include cell membrane permeabilization, cell cycle perturbation, inhibition of DNA synthesis, and inhibition of ␤-1,3-glucan synthetase activity (7,14,15,41,49,53).As the spectrum of action of some toxins has extended to microbial pathogens of clinical interest, killer toxins and/or killer toxin-like antibodies and mimotopes are of great relevance to medicine (8,25). Other toxins that exert a killing action on spoilage yeasts have interesting applications in the fermentative (46) and food and feed industries (10,11,12,23,32,44), where they can be used as "natural" food antimicrobials.In a previous study, we showed that Pichia anomala DBVPG 3003 produces a killer toxin (known as Pikt) that is active against Dekkera/Brettanomyces sp. yeasts (12). These yeasts can develop in white and red wines, resulting in unpleasant odors and tastes (18). Thus, they represent a major problem in the wine industry. Pikt is stable at acidic pH and in a range of temperatures between 20°C and 40°C (12), as are other toxins produced by different strains of P. anomala (25,26,37). Moreover, Pikt maintains its killing activity for ...

Non-Saccharomyces wine yeasts have a promising role in biotechnological approaches to winemaking

“…This preliminary characterisation showed that KwKt and Pikt are stable within a pH range from 3 to 5 and at temperatures below 35°C, and that they maintain their killer activities for at least 10 days in wine. Further characterisation of Pikt has indicated that this toxin is a ubiquitin-like protein with an apparent molecular mass of 8 kDa (De Ingeniis et al 2009). A more recent study (Comitini et al 2009a) carried out in grape must and wine showed that K. wickerhamii and P. anomala can indeed control Brettanomyces/Dekkera spoilage yeasts.…”

Challenges of the Non-Conventional Yeast Wickerhamomyces anomalus in Winemaking

“…The killer toxin Pikt produced by the P. anomala DBVPG 3003 strain was active on 15 isolates belonging to the genus Dekkera/Brettanomyces, and its fungicidal effect in wine was maintained during at least 10 days [69]. Further research revealed that this toxin presented a ubiquitin-like peptide structure with a molecular mass of approximately 8 kDa, and that it selectively interacts with β-1,6 glucans, which are the putative binding sites for Pikt on the cell wall of the sensitive targets [70]. Recently, the killer toxin KTCf20 secreted by the strain W. anomalus Cf20 was also suggested to bind to β-1,3 and β-1,6 glucans of the cell wall of sensitive strains.…”

Past, present and future research directions with Pichia anomala

“…There is an amazing diversity of characterised killer proteins, with molecular masses ranging from 8 kDa to more than 300 kDa (De Ingeniis et al 2009;Kagiyama et al 1988); however, this diversity in protein size may arise mainly from different levels of glycosylation. An extremely heat-stable killer protein has been observed in one strain (Vustin et al 1989).…”