title: Biotechnological exploitation of Tetrapisispora phaffii killer toxin: heterologous production in Komagataella phaffii (Pichia pastoris).
abstract: The use of natural antimicrobials from plants, animals and microorganisms to inhibit the growth of pathogenic and spoilage microorganisms is becoming more frequent. This parallels the increased consumer interest towards consumption of minimally processed food and 'greener' food and beverage additives. Among the natural antimicrobials of microbial origin, the killer toxin produced by the yeast Tetrapisispora phaffii, known as Kpkt, appears to be a promising natural antimicrobial agent. Kpkt is a glycoprotein with β-1,3-glucanase and killer activity, which induces ultrastructural modifications to the cell wall of yeast of the genera Kloeckera/Hanseniaspora and Zygosaccharomyces. Moreover, Kpkt maintains its killer activity in grape must for at least 14 days under winemaking conditions, thus suggesting its use against spoilage yeast in wine making and the sweet beverage industry. Here, the aim was to explore the possibility of high production of Kpkt for biotechnological exploitation. Molecular tools for heterologous production of Kpkt in Komagataella phaffii GS115 were developed, and two recombinant clones that produce up to 23 mg/L recombinant Kpkt (rKpkt) were obtained. Similar to native Kpkt, rKpkt has β-glucanase and killer activities. Moreover, it shows a wider spectrum of action with respect to native Kpkt. This includes effects on Dekkera bruxellensis, a spoilage yeast of interest not only in wine making, but also for the biofuel industry, thus widening the potential applications of this rKpkt.
authors.affiliation: Department of Agriculture, University of Sassari, Sassari, Italy.
authors.forename: Rossella
authors.initials: R
authors.lastname: Chessa
authors.affiliation: Department of Agriculture, University of Sassari, Sassari, Italy.
authors.forename: Sara
authors.initials: S
authors.lastname: Landolfo
authors.affiliation: Department of Life and Environmental Sciences, Polytechnic University of Marche, Ancona, Italy.
authors.forename: Maurizio
authors.initials: M
authors.lastname: Ciani
authors.affiliation: Department of Agriculture, University of Sassari, Sassari, Italy.|Enology and Viticulture Research Group, University of Sassari, Sassari, Italy.
authors.forename: Marilena
authors.initials: M
authors.lastname: Budroni
authors.affiliation: Department of Agriculture, University of Sassari, Sassari, Italy.|Enology and Viticulture Research Group, University of Sassari, Sassari, Italy.
authors.forename: Severino
authors.initials: S
authors.lastname: Zara
authors.affiliation: Department of Agriculture, University of Sassari, Sassari, Italy.|Department of Molecular Biology and Genetics, Faculty of Science & Letters, Istanbul Technical University, Istanbul, Turkey.|Dr. Orhan Ocalgiray Molecular Biology, Biotechnology and Genetics Research Center (ITU-MOBGAM), Istanbul Technical University, Istanbul, Turkey.
authors.forename: Murat
authors.initials: M
authors.lastname: Ustun
authors.affiliation: Department of Molecular Biology and Genetics, Faculty of Science & Letters, Istanbul Technical University, Istanbul, Turkey.|Dr. Orhan Ocalgiray Molecular Biology, Biotechnology and Genetics Research Center (ITU-MOBGAM), Istanbul Technical University, Istanbul, Turkey.
authors.forename: Zeynep Petek
authors.initials: ZP
authors.lastname: Cakar
authors.affiliation: Department of Agriculture, University of Sassari, Sassari, Italy. imannazzu@uniss.it.|Enology and Viticulture Research Group, University of Sassari, Sassari, Italy. imannazzu@uniss.it.
authors.forename: Ilaria
authors.identifier: http://orcid.org/0000-0003-3361-2057
authors.initials: I
authors.lastname: Mannazzu
mesh_terms: D001709:Biotechnology / Q000379:methods*; D002473:Cell Wall / Q000187:drug effects; D003603:Cytotoxins / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D055646:Killer Factors, Yeast / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D007716:Kluyveromyces / Q000378:metabolism*; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects; D004718:Saccharomycetales / Q000235:genetics / Q000378:metabolism; D014920:Wine / Q000382:microbiology; D015003:Yeasts / Q000187:drug effects; D020068:Zygosaccharomyces / Q000187:drug effects
chemical_list: D003603:Cytotoxins; D055646:Killer Factors, Yeast; D011994:Recombinant Proteins
keywords: Dekkera bruxellensis; Kpkt; Natural antimicrobial; Pichia pastoris; Spoilage yeast; β-Glucanase