Heterologous expression, bioreactor production and characterization of rKPKT: a ready-to-use antimicrobial compound of interest for the wine industry

The research activities here presented were carried out within the frame of “Programma Operativo Nazionale FSE-FESR Ricerca e Innovazione-Azione I.1-Dottorati innovativi con caratterizzazione Industriale” granted by the Italian Ministero dell‘Istruzione, dell’Università e della Ricerca (MIUR). This program aims at bridging the gap between University and industry by strengthening the collaboration among academic and industrial partners for the development of innovative solutions that contribute to the growth of specific productive sectors. The academic partners involved in the realization of this theses were the University of Sassari (Sassari, Italy) and the Brno Technical University (Brno, Czech Republic). The industrial partner was the Tenute Sella & Mosca S.p.A. This is the biggest winery in Sardinia (7,6 milioni bottles/year), strongly interested in searching for innovation of processes and products and in the technological transfer of innovation. In particular, this winery has been producing organic grapes for 4 years and is interested in the development of natural antimicrobials to be utilized in the wine industry.Based on that, and on the expertise on killer toxin characterization and exploitation acquired by researchers in the Laboratory of Microbiology of the Department of Agraria (University of Sassari), the general objective of this research was to explore the biotechnological potential of yeast killer toxins to be used as natural antimicrobials for the control of wine spoilage yeasts. In fact, although the antimicrobial activity of yeast killer toxins has been known for more than 50 years and many different authors have hypothesized their exploitation in the food and wine industry, their biotechnological potential has never been really explored. Previous works carried out in the Laboratory of Microbiology of the Department of Agriculture of the University of Sassari have contributed to shed light on the properties, mode of action and genetic determinant of Kpkt, a killer toxin produced by the yeast Tetrapisispora phaffii that has an extensive antimicrobial activity on wine spoilage yeasts. Based on these works, it was hypothesized a future for this toxin as natural antimicrobial in the wine industry where it can partially substitute sulphur dioxide thus reducing the final amount of this antimicrobial in wine. For that, considering that T. phaffii produces rather low amount of Kpkt, previous works showed that Komagataella phaffii, formerly Pichia pastoris, can be a suitable host for the heterologous production of the toxin.In this context, specific objectives of this research were: i) the development of further molecular tools for the production of recombinant (rKpkt) in K. phaffii; ii) the bioreactor production of rKpkt and its purification and characterization; iii) the production of a ready-to-use rKpkt containing preparation, and the evaluation of its effect on a variety of biological targets.After a review of the literature regarding natural antimicrobials and killer toxins, and K. phaffii as a host for heterologous expression in the introduction (chapter 1), and the description of the aims of the PhD thesis (chapter 2), the research activities carried out in the three years program are reported as follows.Chapter 3 regards the construction of new vectors for the heterologous production of rKpkt. For that, Kpkt coding sequence, optimized for the expression in K. phaffii, was cloned under the control of regulated (AOX1) or constitutive (PGK1) promoters and downstream of two different secretion signals (S. cerevisiae α-Factor and the T. phaffii native secretion signals). The resulting plasmids were transformed into K. phaffii strains GS115 and M12. Screening of transformants indicated that the integration of the expression cassette results in a low percentage of 2 recombinant clones showing killer activity. In fact, contrary to native Kpkt, recombinant version Kpkt (rKpkt) is toxic for K. phaffii GS115 and M12, while showing lower toxicity on recombinant killer clones.Chapter 4 reports on the bioreactor production and characterization of rKpkt. For that cultivation of recombinant clone#17 (rc#17) and the production of rKpkt were scaled up from baffled flask to bioreactor and rKpkt purification was attempted by immobilized metal affinity chromatography. Moreover, after having confirmed that rKpkt maintains β-glucanase activity and shows a wider spectrum of action in respect to its native counterpart, its cytotoxic effect on human cells was evaluated. Results obtained showed that rKpkt has limited toxicity to HaCaT cell line for concentrations up to 18 AU/ml thus corroborating the great potential of this toxin as a natural antimicrobial for use in the food and beverages industries.Chapter 5 regards the production of a lyophlized ready-to-use rKpkt preparation (LrKpkt) and the evaluation its effects in grape must and on a variety of biological targets. Results showed that LrKpkt maintains its killer activity for up to six months at 4 °C and it is easily soluble in sterile distilled water. When added to Cannonau and Vermentino grape musts LrKpkt inhibited the wild microflora, while showing limited or no effect on inoculated wine yeast starters. Moreover, besides being active on wine-related yeasts, LrKpkt exerted a strong microbicidal effects on a variety of bacterial species (lactic acid bacteria and food-borne pathogens). On the contrary, in accordance with its limited toxicity on HaCaT cell line, LrKpkt showed no lethal effect on Ceratitis capitata and Musca domestica, thus suggesting its low or null toxicity on multicellular eukaryotic model organisms. Indeed, additional studies are needed to further characterize LrKpkt. Nonetheless, the results here presented suggest that this ready-to-use antimicrobial compound may represent an interesting option for the management of microbial contaminations both in the wine and food industries. In chapter 6 challenges and perspectives on the topic treated are reported.In conclusion, the results here presented report on Kpkt heterologous production in K. phaffii, the possibility of producing a ready-to-use antimicrobial compound containing rKpkt and its low or null toxicity on higher Eukaryotic cells. Considering that the scarcity of studies regarding the effects of killer toxins on human consumers represents a constraint to their utilization in the food and beverages industries, these results, although preliminary, represent a further step towards the biotechnological exploitation of yeast killer toxins.