Kpkt katil toksininin Saccharomyces cerevisiae ve Pichia pastoris hücrelerinde heterolog üretimi için ekspresyon vektörlerinin oluşturulması

In nature, microorganisms process several defense mechanisms to survive under limited nutritional conditions. Killer yeasts have one of the defense mechanisms. They produce toxins called killer toxins (KTs) against sensitive microorganisms. Killer toxins are produced by killer yeasts into extracellular environment and they show their activities by binding to specific compounds called killer toxin receptors (KTRs). Killer toxins can be produced as either proteins or glycoproteins. While killer yeasts have self-immune system to their own KTs, their cytocidal activities usually process in a two-step mechanism on sensitive strains. In the first step, they bind the receptors on cell-wall of the sensitive strains. These receptors are known as primary KTRs. Secondary KTRs found on cell membrane are the second targets of KTs. They bind to these receptors to kill the sensitive strains to enter the cell. Different action mechanisms have been presented for killer toxins and they depend on the genetic determinants as well as physical-chemical properties of KTs. Kpkt is one of the killer toxins produced by Tetrapisispora phaffii (formerly known as Kluyveromyces phaffii). Kpkt killer toxin is active against wine spoilage yeasts such as Kloeckera apiculata and Hanseniaspora uvarum. Therefore, Kpkt has a potential to be used in wine fermentation since it maintains its zymocidal activity for more than 14 days in wine. Kpkt is a glycoprotein and it has a β-glucanase activity. It shows its activity by hydrolyzing β-1,3- and β-1,6- glucans on cell-walls of the sensitive strains. Kpkt killer toxin is encoded by TpBGL2 gene in T. phaffii. The aim of the study was to construct expression vectors for heterologous production in Saccharomyces cerevisiae and Pichia pastoris. For this purpose, pYES2.1/V5-His-TOPO® vector was utilized for both extracellular and intracellular production in S. cerevisiae as well as pPIC9 vector was utilized for extracellular production in P. pastoris. In the first part of the study, suitable yeast strains resistant to Kpkt were selected for heterologous production of Kpkt. S. cerevisiae YPH501 and BY4741 along with P. pastoris GS115 strains were subjected to killer plate assay using YPD plates containing citrate-phosphate buffer (pH 4.6). P. pastoris GS115 appeared as a suitable strain after this plate assay. Then, S. cerevisiae YPH501 and BY4741 were grown in galactose containing media to observe their growth profiles in galactose containing media since pYES2.1/V5-His-TOPO® vector harbors GAL1 promoter which is active only in the presence of galactose. Finally, BY4741 was selected as a suitable strain for transformation. In this study, three different vectors were constructed: i) TpBGL2 gene in pYES2.1/V5-His-TOPO® under the control of GAL1 promoter; ii) TpBGL2 gene in pYES2.1/V5-His-TOPO® under the control of GAL1 promoter and downstream the  sequence; iii) TpBGL2 gene in pPIC9 under the control of AOX1 promoter and downstream the  sequence. To construct them, first DNA extraction was carried out from T. phaffii. Next, DNA quality was checked by PCR amplifying with ITS1 and ITS4 universal primers. First, TpBGL2 gene was amplified using BGL2F and BGL2R primers from T. phaffii total DNA in order to ligate it directly to pYES2.1/V5-His-TOPO® vector to construct the first vector (TpBGL2 gene in pYES2.1/V5-His-TOPO® under the control of GAL1 promoter) for intracellular expression in S. cerevisiae BY4741. After ligation, vectors were transformed into Escherichia coli cells and vectors were extracted from E. coli cells through minipreparation. The resulting vectors were further analyzed to investigate both the presence and the orientation of TpBGL2 gene in the vectors by constructing restriction patterns with MlyI and XbaI enzymes as well as by performing sequence analysis. In addition, PCR amplification was carried out using GAL1F and BGL2R primers. All results confirmed that the insert was in frame with GAL1 promoter. To construct the second and the third vectors, first TpBGL2 was amplified from T. phaffii total DNA with FW2 and RV2 primers having EcoRI and NotI restriction sites, respectively. TpBGL2 gene flanked by EcoRI and NotI restriction sites was ligated into pGEM-T Easy Vector by TA cloning. After transformation and cloning in E. coli cells, extracted primers were digested by EcoRI to confirm the presence of the insert. Next, both empty pPIC9 vectors and pGEM-T Easy vectors having TpBGL2 gene flanked by EcoRI and NotI restriction sites were digested by EcoRI and NotI enzymes. Then, they were ligated to construct the third vector (TpBGL2 gene in pPIC9 under the control of AOX1 promoter and downstream the  sequence). Both sequence analysis and PCR amplification with AOX1-BGL2R primer set confirmed that the insert was in frame with AOX1 promoter and  sequence. To construct the second vector (TpBGL2 gene in pYES2.1/V5-His-TOPO® under the control of GAL1 promoter and downstream the  sequence), TpBGL2 gene downstream the  sequence was amplified using ALFA and BGL2R primers and the amplicon was ligated into pYES2.1/V5-His-TOPO® vector. However, sequence analysis showed that the insert was in wrong orientation and no further studies were carried out with this vector. pYES2.1/V5-His-TOPO® + TpBGL2 vector was transformed into S. cerevisiae BY4741 strain for intracellular production of Kpkt killer toxin and the presence of the insert was confirmed by PCR analysis in S. cerevisiae transformants. The selection was carried out on YNB selective medium without uracil plates. One of the six transformants called T1 was cultivated in 2% (w/v) galactose and 2% (w/v) raffinose containing buffered yeast minimal medium. After 24 h and 48 h, sampling was carried out for crude extraction and for taking supernatant from the culture. Next, well plate assay was performed to investigate killer activity on buffered YPD plates spread by sensitive S. cerevisiae DBVPG 6500 strain to Kpkt killer toxin. T1 was also cultivated in 0.2% (w/v) galactose and 2% (w/v) raffinose containing buffered yeast minimal medium for 48 h and it was transferred into 2% (w/v) galactose containing minimal medium. After 24 h incubation, well plate assay was performed again for both crude extract and the supernatant of the culture. In addition, T1 was cultivated in 2% sucrose (w/v) containing buffered selective minimal medium and it was transferred into 2% (w/v) galactose containing buffered selective medium after 48 h of growth. Well plate assay was performed again after 24 h of growth in galactose containing medium. All studies showed that Kpkt killer toxin did not perform its killing activity when it was expressed intracellularly. Finally, the third vector (PIC9 + TpBGL2) was transformed into P. pastoris GS115 cells for extracellular expression of Kpkt in P. pastoris. To do that, P. pastoris cells were prepared to obtain competent cells for transformation which was carried out through electroporation. For transformation, linearized plasmids obtained by digesting with either SacI or BglII enzyme were utilized along with linearized empty pPIC9 vectors with the same enzymes. Selection was carried on minimal dextrose plates. Next, the transformants were grown on both minimal dextrose and minimal methanol plates to determine putative Mut+ (Methanol utilization plus) or MutS (Methanol utilization slow) phenotypes of the transformants since only Mut+ phenotypes can grow on methanol containing medium if the insertion results in functional AOX1 gene encoding alcohol oxidase, which is the main enzyme in methanol utilization pathway. Finally, transformants were subjected to PCR amplification using AOX1 and AOXR primers to investigate whether they have inserts. The evaluation of the killer activity of Kpkt killer toxin in P. pastoris is in progress.