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.

The zymocidial activity of Tetrapisispora phaffii in the control of Hanseniaspora uvarum during the early stages of winemaking.

AIMS: The yeast strain Tetrapisispora phaffii DBVPG 6706 (formerly Kluyveromyces phaffii) secretes a killer toxin (Kpkt) that has antimicrobial activity against apiculate yeasts. The aim of this study was to evaluate the killer activity of Kpkt towards Hanseniaspora uvarum under winemaking conditions. METHODS AND RESULTS: The zymocidial activity of Kpkt on H. uvarum was assayed in microfermentation trials inoculated with free and immobilized T. phaffii cells. The microbial evolution and fermentation profiles of the wines were evaluated to determine the effects of Kpkt on apiculate yeasts, in comparison with SO(2). The results indicate that the fungicidal activity of Kpkt against H. uvarum is stable for at least 14 days in wine, and the zymocin can control the proliferation of apiculate yeasts. The analytical composition of wines with the inoculum of T. phaffii immobilized cells did not differ from the wines with SO(2). In contrast to wines without this control of apiculate yeasts, an increase in ethyl acetate was seen. CONCLUSIONS: Tetrapisispora phaffii is an excellent candidate for the biological control of undesired proliferation of apiculate yeasts during the first steps of fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: Tetrapisispora phaffii cells in an immobilized form can be used as a biocontrol agent to reduce the need for SO(2) addition.