Kpkt is a yeast killer toxin, naturally produced by Tetrapisispora phaffii, with possible applications in winemaking due to its antimicrobial activity on wine-related yeasts including Kloeckera/Hanseniaspora, Saccharomycodes and Zygosaccharomyces. Here, Kpkt coding gene was expressed in Komagataella phaffii (formerly Pichia pastoris) and the bioreactor production of the recombinant toxin (rKpkt) was obtained. Moreover, to produce a ready-to-use preparation of rKpkt, the cell-free supernatant of the K. phaffii recombinant killer clone was 80-fold concentrated and lyophilized. The resulting preparation could be easily solubilized in sterile distilled water and maintained its killer activity for up to six months at 4 °C. When applied to grape must, it exerted an extensive killer activity on wild wine-related yeasts while proving compatible with the fermentative activity of actively growing Saccharomyces cerevisiae starter strains. Moreover, it displayed a strong microbicidal effect on a variety of bacterial species including lactic acid bacteria and food-borne pathogens. On the contrary it showed no lethal effect on filamentous fungi and on Ceratitis capitata and Musca domestica, two insect species that may serve as non-mammalian model for biomedical research. Based on these results, bioreactor production and lyophilization represent an interesting option for the exploitation of this killer toxin that, due to its spectrum of action, may find application in the control of microbial contaminations in the wine and food industries.

Kpkt是一种由酵母菌（Tetrapisispora phaffii）天然产生的酵母杀伤毒素，由于其对与酒相关的酵母菌（包括Kloeckera / Hanseniaspora，Saccharomyomys和Zygosaccharomyces）具有抗菌活性，因此有可能在酿酒中应用。在这里，Kpkt编码基因在根瘤菌（以前的毕赤酵母）中表达，并获得了重组毒素（rKpkt）的生物反应器生产。此外，为了生产现成的rKpkt制剂，即K. phaffii的无细胞上清液将重组杀手克隆浓缩80倍并冻干。所得制剂可轻松溶于无菌蒸馏水中，并在4°C下保持长达六个月的杀灭活性。当应用于葡萄汁时，它对野生葡萄酒相关的酵母具有广泛的杀伤活性，同时证明与活跃生长的酿酒酵母起始菌株的发酵活性兼容。此外，它对包括乳酸菌和食源性病原体在内的多种细菌都表现出强大的杀菌作用。相反，它对丝状真菌，人参角藻和家蝇无致死作用。，这两种昆虫可以作为生物医学研究的非哺乳动物模型。基于这些结果，生物反应器的生产和冻干是这种杀手毒素的开发的一个有趣的选择，由于其作用范围广，可用于控制葡萄酒和食品工业中的微生物污染。