# The Biology of _Pichia membranifaciens_ Killer Toxins
## 6 Applications, Conclusions and Future Perspectives of _P. membranarificiens_ Killer Toxins
(2015) and [PERSON] et al. (2015) conclude that a synergistic effect was operating using PMKT [185] and Pkkp (from _P. kluyveri_) [8] killer toxins, respectively, in combination with potassium metabisulfite, against wine spoilage yeasts such as _Zygosaccharomyces_ or _Dekkera/Brettanomyces_ species. Recently, [PERSON] and coworkers described the utility of mixed-inoculation with killer and sensitive yeast strains to speed cell death during second fermentation of sparkling-wines to release of certain compounds from autolyzed yeast cells [181]. In summary, the killer activity of _P. membranitaciens_ shows a broad spectrum of potential applications, sustained by a considerable diversity of mechanisms of action. However, a marked ability of these killer toxins restricts their application to specific environments and matrixes providing adequate chemical physical conditions. These requirements are basically found in food, post-harvest, and fermentative environments, even though the industrial application of killer toxins is far from a reality, since their large-scale production is still expensive. For this reason, the current tendency is only based in applying killer yeasts instead of their toxins. Furthermore, killer resistant strains have been described since the initial studies on killer toxins and, in many surveys, it is possible to found, over a panel of potential sensitive yeast isolates, that some strains are resistant to that potential biocontrol agents [20]. In accordance, these toxins could be less valuable for biocontrol in real applications. For those purposes, regarding toxin stability in both production and application stages, adaptive and directed evolution approaches are of great interest in the development and isolation of phenotypically improved variants. It is possible to evolve populations in laboratory conditions, where it is important that the selection conditions match the industrial parameters as closely as possible [191]. Since temperature and pH are the main limiting factors, especially for clinical applications, adaptive processes should be carried out with _P. membranitaciens_ killer strains to obtain toxins with increased stability at human physiological conditions. Additionally, killer toxins identified as leads for therapeutic uses should also be modulated to reduce their antigenicity [192]. For that purpose, the molecular structure and mechanism of action of _P. membranitaciens_ killer toxins should be studied in depth, in order to identify key residues causing toxicity and antigenicity, and determine their stability. Finally, the cellular response of the target pathogenic/spoilage microorganism to killer toxins needs to be studied species by species to understand the basis of their susceptibility. Additional studies are currently under development to increase the background of knowledge accumulated during the last two decades on the features of _P. membranitaciens_ killer toxins, in the hope of understanding how to work with these killer toxins that have potential biotechnological applications. Elucidation of the molecular mechanisms of their action will be helpful to develop the strategies to fight more and more harmful spoilage yeasts. The studies referenced in this review were supported by Grants from the European Community (AIR CT93-0830), Universidad Complutense-Community of Madrid (PR1/03-11632; CCG07-UCM/AGR-2612, GR45/06; GR69/06; GR74/07; GR58/08, GR35/10-A), and CDTI-MINECO-Centre for the Development of Industrial Technology (IDI-20130192; IDI-20140448; IDI-20160102). The authors declare no conflict of interest.