Saccharomyces Cerevisiae K2 toxin fusion with GFP

Abstract

Killer yeasts are considered as potential biocontrol agents to avoid or reduce wine spoilage by undesirable species. One of the biological mechanisms for the regulation of population dynamics in complex microbial ecosystems is the production of toxins capable to kill or inhibit other microorganisms. The toxins synthesized by yeasts, known as killer factor, are proteins, whose action is mediated by specific receptors in the cell wall of the sensitive microorganism. K2 killer protein of Saccharomyces cerevisiae is the most frequent toxin among yeast dominating in the vineyard-winery ecosystem. This protein is produced at negligible levels in yeast, making difficult to detect and isolate it in amounts sufficient for investigation. In the course of our research, the S. cerevisae K2 toxin encoding gene was tagged at its carboxyl terminus by GFP, and the chimeric construct was subcloned into the yeast episomal vector. The hybrid protein-possesing plasmid was introduced into E. coli and S. cerevisiae cells. Based on the fluorescence, expression of the GFP was observed in both bacteria and yeast cells. However, following the killer activity and immunity tests we found that the chimeric protein K2-GFP did not show killer activity and demonstrated only partial immunity to K2. This can be explained by the fact, that C-terminus of killer protein is highly sensitive for interruption and affect the functionality of the toxin.