Isolation, Purification, and Characterization of a Killer Protein fromSchwanniomyces occidentalis

Abstract

The yeast Schwanniomyces occidentalis produces a killer toxin lethal to sensitive strains of Saccharomyces cerevisiae. Killer activity is lost after pepsin and papain treatment, suggesting that the toxin is a protein. We purified the killer protein and found that it was composed of two subunits with molecular masses of approximately 7.4 and 4.9 kDa, respectively, but was not detectable with periodic acid-Schiff staining. A BLAST search revealed that residues 3 to 14 of the 4.9-kDa subunit had 75% identity and 83% similarity with killer toxin K2 from S. cerevisiae at positions 271 to 283. Maximum killer activity was between pH 4.2 and 4.8. Killer yeasts secrete toxins lethal to sensitive yeasts but are immune to their own toxins. Since first discovered in Saccharomyces cerevisiae (2), killer strains have been isolated from several yeast genera, including Candida (46), Cryptococcus (10), Hanseniaspora (33), Kluyveromyces (14), Pichia (27), Torulopsis (7), Ustilago (30), Williopsis (45), and Zygosaccharomyces (32). Based on killing and immunity interactions among killer yeasts, killer phenotypes are classified into at least 10 groups (48) and the responsible genes may be carried on a chromosome (S. cerevisiae KHS, KHR, Williopsis mrakii) (11,12,21), on a double-stranded RNA (dsRNA) (S. cerevisiae K1, K2, K28, Ustilage maydis, Hanseniaspora uvarum) (5,15,22,35,49), or on a linear double-stranded DNA (dsDNA) (Kluyveromyces lactis, Pichia inositovora, Pichia acaciae) (14,16,44).Schwanniomyces occidentalis produces amylolytic enzymes, including ␣-amylase and glucoamylase (8). It is one of the few yeasts capable of completely hydrolyzing soluble starch. Moreover, it can grow to high cell mass by utilizing cheap starch from plants such as cassava, corn, potato, sorghum, and wheat as the carbon source (40). S. occidentalis has been used to produce ethanol and single cell protein from starch fermentation (19,42). S. occidentalis has no detectable extracellular proteases and can secrete large proteins (40). It also has an established transformation system and available inducible promoters, which could make it a commercially important system for the production of heterologous proteins (40). For example, endoglucanase D recently has been successfully expressed and secreted in this system (31).Wild killer yeasts sometimes contaminate cultures of industrial yeasts, resulting in lagging or stopped fermentation and poor product quality (39). To avoid such complications, the use of industrial killer strains as starters has been suggested (17). Commercially interesting killer strains for sake brewing, wine making, alcohol fermentation, and lager, beer, and ale production have been constructed (29,36,39,47). Furthermore, the W. mrakii mycocin expressed by Aspergillus niger can reduce silage and yogurt spoilage caused by yeasts (25).The killer phenotype of S. occidentalis has not been described previously. Thus, our objectives in this study were as follows: (i) to screen killer strains from S. occidentalis for a killer phenotype; (ii...

Killer toxin from several food-derived Debaryomyces hansenii strains effective against pathogenic Candida yeasts

“…We found that mycocin from the strains Dh-274, Dh-237 andDh-65 exhibited temperature-dependent killer activity against C. albicans up to pH 5.5 and against C. tropicalis up to pH 6.0,with activity decreasing abruptly at nonpermissive temperatures and pH values. Most published studies on mycocin activity report that mycocins are generally stable only over narrow pH ranges and each mycocin has a defined optimal pH for killer activity against the sensitive yeast species (Chen et al, 2000;Soares and Sato, 2000). A previous report by Marquina et al (2001) stated that mycocin from D. hansenii isolated from olive brines had an optimal stability and activity against Candida boidinii (IGC3430) between pH 4.5 and pH 5.1, congruent with our observations.…”

Killer toxin of Pichia membranifaciens and its possible use as a biocontrol agent against grey mould disease of grapevine

“…These common properties are consistent with the proteinaceous nature of killer toxins, which is also evident from the susceptibility of most toxins to proteolytic enzymes. Most toxins are irreversibly inactivated above pH 5?0 and are stable only in a narrow pH range (Bevan et al, 1973;Chen et al, 2000;Marquina et al, 2001a;Middelbeek et al, 1979;Pfeiffer & Radler, 1984), although the killer toxin from Hansenula saturnus (Ohta et al, 1984) has a broad stability range. Moreover, differences in other properties indicate that the toxins of several yeasts are biochemically distinct (Pfeiffer & Radler, 1984).…”

Killer Toxin ofKluyveromyces phaffiiDBVPG 6076 as a Biopreservative Agent To Control Apiculate Wine Yeasts

“…Evidence from protease treatments suggests that Kpkt is a protein with sulfide bonds like several other killer factors (4,35), since papain has been shown to destroy the toxin and its activity. Like most toxins (28), Kpkt is unstable at both high temperatures and high pH values.…”