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.