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 b-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 b-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.