Yeast Killer Factors: Biology and Relevance in Food Biotechnology

Yeast killer factors: Biology and relevance in food biotechnology

Maja Starovič

Academic internship report

Msc Food technology: Food biotechnology and biorefining

October 2020

Mentors: Ramón González García, Instituto de Ciencias de la Vid y del Vino prof. dr. Eddy J. Smid, Wageningen University & Research

Table of contents

1 Yeast killer toxins ...... 1 1.1 Saccharomyces cerevisiae killer system ...... 1 1.1.1 Saccharomyces cerevisiae killer toxins ...... 1 2 Non- Saccharomyces cerevisiae killer factors...... 6 2.1 Non- Saccharomyces cerevisiae killer yeast ...... 6 2.2 Toxin entry into the target cell and the killing mechanism ...... 7 2.2.1 Killer toxins from Pichia ...... 7 1.2.1 Killer toxins from Kluyveromyces lactis ...... 8 1.2.2 Killer toxins from Candida ...... 9 1.2.3 Killer toxins from other non-Saccharomyces yeast ...... 9 3 Toxin immunity ...... 9 4 Role of killer yeast against common wine spoilage yeast ...... 11 4.1 Spoilage yeast in wine industry ...... 11 4.1.1 Brettanomyces bruxellensis ...... 11 4.2 Killer yeast used in wine industry ...... 12 4.2.1 Torulaspora delbrueckii ...... 13 4.2.2 Candida ...... 14 4.2.3 Pichia ...... 15 3.2.5 Kluyveromyces spp...... 16 5 Killer yeast in other biotechnological applications ...... 17 6 Influence of environmental conditions on killer toxin production and activity ...... 18 6.1 Temperature and pH ...... 18 6.2 Other conditions ...... 20 7 Molecular biology of killer yeast ...... 20 7.1 Molecular biology techniques applied to killer yeast...... 21 8 Conclusion ...... 23 9 Reference list ...... 25

Abstract

In the winemaking, yeast are the microorganisms that lead the fermentation process of grape juice. The starter culture that is inoculated into the grape juice is usually Saccharomyces cerevisiae, however other yeasts are also being applied. Moreover, yeast are not only considered as microorganisms to start a fermentation process, but also as producers of killer toxins. These yeasts are named killer yeast. They have lately received a great deal of attention as natural preservatives, since they proved to kill certain pathogens that cause unwanted changes in the characteristics of wine, other fermented food products, agricultural products and they were even investigated for medical purposes. In the wine industry, SO2 is currently mostly used preservative that efficiently kills nearly all of the pathogens. Since this agent is potentially toxic, researchers are trying to find proper killer yeasts that can be included in a certain winemaking step in order to prevent or kill unwanted microorganisms and thus avoid major economic losses. The killer and immunity mechanism has been mostly investigated for S. cerevisiae killer toxins (K1,K2 and K28). The killer phenotype of these toxins is determined by a medium sized double stranded satellite RNA virus (dsRNA). dsRNAs are identified as M- viruses (M1, M2 and M28) that encode K1, K2 and K28 toxins, respectively. They are encapsidated in virus-like particles (VLPs). M virus encodes the toxin, while L-A virus is responsible for replication, encapsidation and maintenance of both mycoviruses.

1 Yeast killer toxins Yeast that produce killer toxins (known as killer factors or killer proteins) are recognized as killer yeast (El-Banna, El Sahn, & Shehata, 2011; Garcia,’ Esteve-Zarzoso, & Arroyo, 2016). In agriculture and food industries the interest is to apply yeast killer toxins as bioprotective agents which prevent the growth of competing microorganisms. To use them as antimicrobials, killer yeast can be included as a fermentation starter or as purified killer toxins (Chessa et al., 2017). They are mostly being used in the winemaking process, as a partial substitute to SO2. This compound is a commonly used chemical antimicrobial agent which has shown to have carcinogenic and cytotoxic effects to humans and animals. For this reason, winemakers started to select their S. cerevisiae or non-S. cerevisiae yeast starter cultures based on their killing phenotype (Novotna, Flegelova, & Janderova, 2004; Carboni et al. 2020).

First killer character was discovered in S. cerevisiae, in 1960s (Garcia, Esteve-Zarzoso, & Arroyo, 2016). Shortly after, they found out that also other yeast species secrete killer toxins. The toxins were found in Zygosaccharomyces bailli, Hanseniaspora uvarum, Ustilago maydis, Candida, Cryptococcus, Debaryomyces, Hansenula, Kluyveromyces, Metschnikowia, Pichia, Torulopsis, Williopsis, Zygosaccharomyces, Aureobasidium, Zygowilliopsis and Mrakia (Liu et al. 2013; Schmitt & Breinig, 2006).

In case where there are different microbes present in the same niche, they start to compete. Some strains or species, known as killer yeast, have therefore developed a competitive advantage of possessing dsRNA viruses which encode killer toxins. These cells than gain advantage by killing sensitive cells. Killer yeast producing the toxins are immune to their own toxin and the reason for it is understood only in some cases (Belda et al., 2017).

1.1 Saccharomyces cerevisiae killer system Yeast killer toxins are either small basic proteins or larger multimeric protein complexes (glycoproteins) that kill sensitive cells (yeast, fungi or bacteria) via two-step mode of action and without cell-cell contact (Schmitt & Breinig, 2002; El-Banna, El Sahn, & Shehata, 2011; Liu et al., 2013; Garcia, Esteve-Zarzoso, & Arroyo, 2016; Schaffrath, Meinhardt, & Klassen, 2018). They are mediated by specific cell wall receptors of a target cell and immune to their own toxin, while being susceptible to the toxins excreted by other yeast species (Magliani et al., 1997; Schmitt & Breinig, 2006).

1.1.1 Saccharomyces cerevisiae killer toxins In S. cerevisiae there are three major killer viruses (ScV-M1, ScV-M2 and ScV-M28) and each of them encodes a specific killer toxin (K1, K2 and K28, respectively) (Schmitt & Breinig, 2006). These toxins are classed in K1, K2, K28 and Klus groups based on their lack of cross

immunity, their molecular mode of action and their killing profiles (Schmitt & Breinig, 2002; Ramirez et al. 2015).

K1 and K28 toxin belong to A/B family of toxins due to their entry into the cell by endocytosis and retrograde trafficking for toxicity. Shiga, Cholera and Ricin toxins also belong to this family. It is common for A/B toxins to contain one or more β subunits responsible for entry into the cell and intracellular targeting (Carrol et al. 2009).

1.1.1.1 L-A mycoviruses and satellite M dsRNAs

The killer phenotype of toxins secreted by S. cerevisiae is determined by a medium sized double stranded RNA virus (dsRNA) belonging to Totiviridae family (Schmitt et al. 2006; Liu et al. 2013). These satellite dsRNAs are identified as M-viruses (M1, M2 and M28) that encode K1, K2 and K28 toxins, respectively. They are encapsidated in virus-like particles (VLPs) (Magliani et al. 1997). In certain yeast, linear dsDNA plasmids carry the toxin genes (Kluyveromyces lactis, P. acaciae, P. inositovora, D. robertsiae, Trichosporon pullulans, etc.) and in some others they stay in the chromosomal DNA (Williopsis saturnus, Pichia anomala, Pichia kluyveri, Pichia membranifaciens, etc..). (Liu et al. 2013; Belda et al. 2017)

Depending on the killer type, the killer toxin-encoding genes are located in different parts of a cell. They are mostly encoded in the nucleus; however, some are encoded in the cytoplasm by genetic elements containing virus-like double stranded DNA or dsRNA (S. cerevisiae, Hansenula uvarum, Z. bailii and U. maydis )(Liu et al. 2013; Schaffrath et al. 2018).

Cytoplasm of a host cell that secretes K1, K2, K28 and Klus toxins is infected with dsRNA, M-toxin encoding killer virus and L-A helper virus. M virus encodes the toxin, while L-A virus is responsible for replication, encapsidation and maintenance of both mycoviruses (Magliani et al. 1997; Gier et al. 2020). M-viruses consist of satellite double stranded RNA (dsRNA) and are responsible for immunity and killer activity (Magliani et al. 1997; Schmitt & Breinig, 2002). Therefore, the production of K1, K2 and K28 toxins depends on the presence of these viruses and also on the L-A helper virus in order to be replicated and maintained within the cytoplasm of a target sensitive cell. A yeast that contains both, M-dsRNA, and L-A viruses, is an immune killer yeast (Schmitt & Breinig, 2002). Satellite dsRNAs and L-A viruses can be transmitted into the cell during cell division, sporogenesis or cell fusion. Maintenance and expression of the killer type are achieved in the host cell (Magliani et al. 1997).

1.1.1.1.2 Replication of L-A and M drRNA viruses L-A helper virus replicates autonomously, and its maintenance does not depend on the coexistence of M viruses (Schmitt & Breinig, 2002). Its positive strands are synthetized in the viral particles by end-to-end transcription of dsRNA. The transcription is done by the

transcriptase activity of Gag-Pol fusion protein. The two (+) L-A strands formed are afterwards extruded from the particles into the cytoplasm. After the extrusion, L-A virus encodes Gag, the capsid protein and Pol, the RNA- dependent RNA polymerase which is later expressed as Gag-Pol fusion protein. However, some of the positive strands get encapsidated in the new viral particles where they are used as a template for negative strands synthesis (Figure 1). Most (+) L-A strands are translated into capsid protein (Gag) and few (1-2%) are translated into Gag-Pol fusion protein. Subsequently, the dsRNA is encapsidated in the new VLPs due to the interaction with Gag-Pol protein. The new VLPs persist in the cytoplasm of an infected yeast cell (Magliani et al. 1997; Becker & Schmitt, 2017; Schaffrath et al. 2018; Gier et al. 2020). ScV-L-A virus like particles contain only one L-A (+) strands and their composition is the same as of the mature virions. After the assembly, the L-A (-) strand is synthetized by the replicase activity of Pol. Finally, dsRNA genome is complete, and each virion contains a single copy of ScV-L-A. The replication of M-virion is similar, however due to the smaller genome size than that of L-A, the final VLP contains two copies of M-dsRNA. The replication of M dsRNA occurs inside the virions. It starts in vivo by synthesis of M (+) strand which is then extruded into the cytoplasm. The (+) strand is either translated into the unprocessed toxin precursor or bound by Gag-Pol for encapsidation into VLPs. The replication is finished by the synthesis of M (-) strand and thus construction of dsRNA (Magliani et al. 1997; Becker and Schmitt, 2017). M and L-A viruses compete between each other for Gag and Gag-Pol viral proteins, because they are required for single stranded RNA (ssRNA) encapsidation, virion assembly, extrusion from the particles into the cytosol, negative strand RNA synthesis (replication), positive strand RNA synthesis (transcription), ssRNA translation and ssRNA binding (Schmitt et al. 2006).

Figure 1. Replication system of the killer and the helper virus in S. cerevisiae (taken from Becker & Schmitt, 2017).

1.1.1.2 Maturation of toxins and excretion from the host cell

As it was shown also for other killer toxins of S. cerevisiae, M-dsRNA (ScV-M28 satellite/killer virus) of K28 encodes the unprocessed K28 toxin precursor. The (+) strand of ScV-M28 carries the genetic information for the unprocessed K28 pptox in a single open reading frame (ORF). In K1 and K2 secreting S. cerevisiae yeast a single open reading frame (ORF) also encodes each toxin (K1, K2) which is then synthesized as a single polypeptide preprotoxin. In K28 secreting S. cerevisiae, the M (+) strand is then translated into K28 pptox which is subsequently imported into the secretory pathway of its host yeast cell, where the maturation and final secretion occur (Magliani et al. 1997; Becker and Schmitt, 2017; Giesselmann, Becker, & Schmitt, 2017).

The toxins are primarily translated as pptox (precursor proteins) and subsequently each of them enters the maturation process using the secretory pathway of a host cell. Prior to the maturation process, pptox of K28 is still unprocessed. It is build-up of an N-terminal signal peptide which serves for efficient transport into the ER (Endoplasmic reticulum) lumen, a proregion (δ-subunit) and the two subunits of a mature toxin (α and β). The α subunit serves for the killing toxicity, while the β subunit is responsible for efficient toxin-cell surface bind and toxin internalization. The proregion does not affect the toxicity of α subunit nor the maturation process of K1 toxin. The N-glycosylated γ-sequence is located between β and α subunit and it mediates proper precursor folding and the formation of a disulfide bond. In

the process of maturation, a signal peptidase at the ER membrane cleaves this bond and subsequently, the signal N- terminal peptide is removed. Hence, the single disulphide bond is then covalently linked between α and β subunit which creates a biologically active α/β heterodimer. In the next step, the pptox enters the Golgi where it is catalysed by the activities of Kex2p and Kex1p. This leads to the formation of and α/β heterodimer protein toxin, that is covalently linked by a disulphide bond and its final secretion. β-subunit’s carboxyterminal ER retention motif (HDEL) is necessary for host cell intoxication and intracellular toxin transport. The mature toxin is finally released from the cell and secreted into the medium (Breinig et al., 2006; Becker and Schmitt, 2017; Giesselmann, Becker, & Schmitt, 2017; Gier et al., 2019; Gier, Schmitt, & Breinig, 2020).

1.1.1.3 Entry of toxins into the target cell and mode of killing action

The cell wall receptors are essential for most killer toxin activity. They are separated into two classes: primary (on the cell wall) and secondary (on the plasma membrane). The identified primary receptors are 1,3-β-D-glucan, 1,6-β-D-glucan, mannoprotein and chitin. The secondary receptors are less known. Different yeast or their toxins have different primary or secondary receptors (Liu et al., 2013; Belda et al., 2017). In S. cerevisiae, β-1,6-D-glucan on the cell wall of a target cell acts as a primary toxin receptor for K1 and K2 toxins. Likewise, for some other yeasts (K. phaffi, W. saturnus var. mrakii, Hanseniaspora uvarum and P. membranifaciens) β-glucans act as binding components (Liu et al., 2013).

K1, K2 and K28 toxin act in two-step receptor mediated way that finally leads to lethal effect. Hydrophobic β subunit and α subunit are both necessary for the receptor binding. The former one is responsible for the primary binding and the latter one is multifunctional, responsible for killer activity, immunity, and binding (Gier et al., 2020).

1.1.1.3.1 K1 and K2 toxin entry into the target cell K1 and K2 toxins have a very similar mechanism of action, however the fact that they are able to kill each other suggest that there are still differences between them. The major differences are related to the target cell interaction and immunity mechanism (Novotna, Flegelova & Janderova, 2004; Schaffrath, Meinhardt, & Klassen, 2018).

The first binding step of K1 and K2 toxin is pH dependent, with an optimum pH of around 4.6. The absorption occurs with high velocity and low affinity and it does not require energy. On the contrary, the absorption in the second step occurs with high affinity and low velocity and the interaction between the toxin and the plasma membrane receptor is energy dependent. The primary membrane receptor, β-1,6-D-glucan includes killer resistance (KRE) genes. KRE1 product is necessary for K1 and K2 toxin action, whereas the KRE2 product only for K1 toxin. After binding to the cell wall, K1 or K2 binds to the cytoplasmic membrane where it

interacts with Kre1p, the second membrane receptor, an O-glycolsylated and GPI-anchored protein. Afterwards, voltage-independent cation transmembrane channels are formed which causes an uncontrolled influx of protons together with an efflux of potassium ions forms, causing ion leakage and consequently, cell death. By doing this it disrupts the membrane function, releasing either K1 or K2 toxin, ATP and other metabolites. The killer toxin causes loss in the membrane fluidity and right after the rise of it, making the membrane more susceptible to the toxin. The essential step of the killing action is the interaction between the killer toxin and membrane proteins. The channel formation is caused by the two strongly hydrophobic regions near the C terminus of the α subunit which act as a membrane-spanning domain. The disruption of the membrane defines the lethal effect of K1 and K2 toxins (Magliani et al., 1997; Novotna, Flegelova & Janderova, 2004; Breinig et al., 2006; Liu et al., 2013; Gier, Schmitt, & Breinig, 2020).

1.1.1.3.2 K28 toxin entry into the target cell On the other hand, the K28 killer toxin binds to the cell surface and it enters the cell by endocytosis (Schmitt et al. 2006). After the bind to the membrane receptor Erd2p the toxin enters the cell where it targets an early endosomal compartment. Firstly, it travels the secretion pathway of a target cell in the opposite direction and in this way reaches its cytoplasm. The retrograde transport goes through Golgi and Endoplasmic reticulum. The export of the toxin from the ER into the cytosol is mediated by a transport channel, known as Sec61p complex or translocon (Schmitt et al. 2006). This channel also serves as a quality control by exporting and removing the misfolded proteins, which are later degraded in the cytosol. The cells that have a mutated Sec61p translocon are resistant to the toxin passage through this channel. The passage through the early secretory pathway is possible because the ER- targeting signal is initially masked by a terminal arginine residue. After it reaches the final part of the Golgi this residue is not necessary anymore and is therefore cleaved. When the toxin leaves the ER it reaches the nucleus, where the α subunit blocks the DNA synthesis which subsequently leads to arrest of the cell cycle in G1 and S-phase. Caspase mediated apoptosis follows and hence, cell death (Breinig et al., 2006; Schmitt et al. 2006; Liu et al., 2013; Gier et al. 2019; Gier, Schmitt, & Breinig, 2020). Caspases are cysteine proteases that mediate the process of programmed cell death, known as apoptosis. This process is regulated and controlled by cell’s genetics (Fink & Cookson, 2005).

2 Non- Saccharomyces cerevisiae killer factors 2.1 Non- Saccharomyces cerevisiae killer yeast

Killer factors of non-Saccharomyces yeast are important to consider since these yeasts are necessary in the early stages of wine fermentation. Only after the alcohol in grape juice rises,

S. cerevisiae start to outcompete the other yeast. At first all the non-Saccharomyces yeast were considered as spoilage yeast due to some species that produce undesirable compounds, affecting the final wine quality. However, later they found out that some of them release wanted compounds that increase the complexity and quality of wine. In many wine productions they are co-inoculated together with S. cerevisiae for optimum results (Garcia et al., 2016).

A diversity of cell wall receptors for killer toxins exist, evidencing that all the main cell wall components of yeasts could function as primary receptors for killer toxins. For example, (1,6)- β-D-glucans act as cell wall receptors for K1 and K2 killer toxins of S. cerevisiae, for Hanseniaspora uvarum killer toxin and for Pichia anomala DBVPG3003. (1,3)-β-D-glucans are primary receptors for Pichia anomala NCYC434, mannoproteins are receptors for K28 of S. cerevisiae and also Zygosaccharomyces bailii killer toxin, and, finally, chitin is the cell receptor for Kluyveromyces lactis killer toxin (Belda et al., 2017).

2.2 Toxin entry into the target cell and the killing mechanism

2.2.1 Killer toxins from Pichia PMKT and PMKT2 are low molecular mass proteins produced by Pichia membranifaciens, that bind to the primary receptors on the cell wall of a sensitive target yeast cell. PMKT2 binds to mannoproteins (Belda et al., 2017). Santos and Marquina (2004) found out that when this toxin bounded to a target yeast cell, the cell entered lag phase. They proposed this could be due to the formation of ion channels in the plasma membrane, the metabolic effects (dissipation of electrochemical gradients through the membrane) and lastly, large pores could be formed which lead to the leakage of large molecules. However, they noticed that many infected target yeast cells were still alive even when exposed to the toxin for a longer period. The reason for this could be that the cells were still able to repair some of the initial damage that the toxin had caused (Santos & Marquina, 2004; Wickner & Edskes, 2015).

In the research of Santos and Marquina (2004) killer toxin from P. membranifaciens CYC 1106 did not influence DNA replication in sensitive cells. They discovered that the killing factor of this toxin is the disruption of electrochemical gradients of the plasma membrane, which leads to a change in membrane permeability. The PMKT1 toxin from P. membranifaciens CYC 1106 binds to (1,6)-β-D-glucans on the cell wall. When it gets translocated to the plasma membrane it forms non-selective channels across the membrane which leads to the leakage of low-molecular weight metabolites and physiological ions (K+, Na+ and H+). This causes changes in cell homeostasis and hence, cell death. The lethal effect of PMKT secreted by P. membranifaciens CYC 1086 is also caused by disruption of the plasma membrane’s electrochemical gradients and subsequent cell death (Liu et al., 2013). Accordingly, P.

membranifaciens CYC 1086 killer toxin PMKT firstly binds to (1,6)-β-D-glucans located on the cell wall of a target yeast cell. The second bind is to the Cwp2p, a GPI-anchored protein on the cell membrane which acts as a secondary receptor (Liu et al., 2013; Belda et al., 2017).

The plasma membrane receptor for P. membranifaciens toxins, Cwp2p, plays a key role in the toxin killing action of PMKT by acting as a bridge between the primary receptors and the plasma membrane. This was proven when the mutant cells that did not possess this receptor appeared to be immune to the P. membranifaciens toxins (Belda et al., 2017).

It was revealed that PMKT2 toxin does not have an effect on the G1 or G2 phase, but it rather arrests the sensitive cells in their early S-phase (the stage of a nascent bud and pre- replicated 1n DNA). Shortly after the initiation of the S-phase, cells start to die off. The hypothesis is that during the first PMKT2 action the cell membrane does not get permeabilized. It starts to be permeable only after the cells become unviable due to the cell cycle arrest (Belda et al., 2017).

When incubated with S. cerevisiae, low dosages of PMKT and PMKT2 induce apoptosis and necrosis. The cells start their death process with PCD response and consequently die due to apoptosis. Their membrane becomes permeable, which confirms cell death or necrosis. However, when high dosages of these toxins are applied, the cells die due to the cell cycle arrest or loss of membrane permeability. This shows the dual mechanism of these two toxins. The exact mechanism of how the PCD is induced is still unknown. (Belda, Ruiz, Alonso, Marquina, & Santos, 2017).

P. membranifaciens was also able to kill Botrytis cinerea on apples. Out of many tested strains of P. membranifaciens, P. membranifaciens CYC 1106 had the widest spectrum of killing activity. In the research they tested both, the whole cells of P. membranifaciens and the purified toxin secreted by these cells. Both reported to efficiently limit the growth of B. cinerea, proving that the inhibiting effect was caused by the toxin. However, the effect was smaller when purified toxin was used, indicating an additional killing mechanism present in the whole cell. A synergy may exist, between the competition for nutrients and secretion of cell wall-degrading enzymes by P. membranifaciens. The fungal cell wall is an important component since it serves as a main protection against cell lysis (Santos, Sanchez, & Marquina, 2004).

1.2.1 Killer toxins from Kluyveromyces lactis

Zymocin, the toxin secreted by K. lactis consists of α, β and γ subunits. The α-subunit is responsible for the chitin binding, β-subunit for the uptake of the y-subunit into the target cell and the γ-subunit affects RNA-polymerase II-dependent transcription. The RNA-

polymerase II elongator is required to block the target cell in G1 phase. K. lactis toxin showed to be able to arrest cell proliferation by interfering with the cell cycle and DNA synthesis. The lethal effect of Zymocin is stopping the multiplication of yeast cells as unbudded cells by blocking completion of the G1 phase of the cell cycle (Santos & Marquina, 2004; Liu et al. 2013).

1.2.2 Killer toxins from Candida The killing mode of C. pyralidae killer toxins (CpKT1 and CpKT2) is not extensively researched. Based on the proven glucosidase activity of C. pyralidae, Mehlomakulu et al. (2016) suggest that CpKT1 binds to β- glucans on the cell wall and disrupts them. After the bind it causes membrane damage, although it still needs to be investigated if the consequence of this is permeabilization, such as leakage of nutrients, like it was found in some other toxins (PMKT, K1, K2). They were first to describe what happens to the surface of B. bruxellensis after being exposed to CpKT1. The cell surface exhibited wrinkling with indentations and wounds. However, after 56h of exposure to CpKT1, the cells started to recuperate their initial appearance. This finding is in accordance with their previous research (Mehlomakulu et al. 2014) where the activity of C. pyralidae killer toxins remained at 80% until 5 days and afterwards it started to decrease.

1.2.3 Killer toxins from other non-Saccharomyces yeast Toxins secreted by Williopsis sp. have a broad spectrum of killing action. The toxin produced by Williopsis saturnus DBVPG 4561 possess a wide antimycotic activity against Candida glabrata strains, Issatchenkia orientalis and Pichia guilliermondii KT4561. While the killer toxin produced by W. saturnus WC91-2 was lethal to the pathogenic yeast strain WCY, C. albicans, C. tropicalis, Cryptococcus aureus, Yarrowia lipolytica and Lodderomyces elongisporus. However, it did not kill Rhodotorula mucilaginosa, a common yeast found on grape skins, that is usually undesirable among wine makers (Belda et al. 2017; Viticulture and Enology, 2020).

Killer toxin secreted by H. mrakii kills the sensitive cells by inhibiting the synthesis of β-1,3- glucan in the cell wall. Toxins from other yeast may disrupt the cell wall integrity by specific β-glucanase activity or β-1,3-D-glucanase activity which both belong to hydrolases. (Liu et al. 2013)

3 Toxin immunity

The reasons why killer yeasts are resistant to their own toxins still needs to be demonstrated for most killer systems. Although in some cases, as described further on, it has been resolved (Meinhardt et al. 2015; Wickner et al. 2015; Belda et al. 2017; Gier et al. 2020).

For toxins secreted by bacteria it is known that they selectively kill their target eukaryotes and thus the immunity mechanism is not important for this purpose. However, the immunity of killer yeast against their own toxins is necessary for them to survive since they inhibit eukaryotic cell functions (Breinig et al. 2006).

The immunity of toxin-producing yeast to their own toxin is a part of a complex mechanism. On a molecular level it has so far been explained only for K28 toxin. The immunity against K28 is believed to be due to a mechanism connected to the post translational ER import of the toxin precursor molecule. This precursor then blocks the action of reinternalized mature toxin molecules in the cytosol (Gier et al. 2020).

Magliani et al. (1997) propose that for a susceptible yeast to become immune to the toxin, it needs a mutation in one of the KRE genes. One of the differences between the immune yeast and the susceptible one, is that the former’s spheroplasts are resistant to the toxin, while the spheroplasts of the latter one are sensitive to it. This indicates that the secondary receptors on the cytoplasmic membrane of the immune yeast are destroyed, masked, or modified in a way to prevent the damage by the toxin (Magliani et al. 1997).

K1 strains are immune due to their toxin precursor. The expression of the immunity requires α and γ components of the precursor. The latter one acts as an internal chaperon for protoxin and it is important for its folding, processing, and maturation. The γ subunit interacts with the α subunit and blocks its toxic activity, without restricting the receptor interaction until the final processing and secretion of the toxin. Hence, the γ subunit of the K1 toxin has an important role in the cell immunity. However, the exact mechanisms of the intrinsic immunity and toxic effect have not been demonstrated so far (Magliani et al. 1997; Gier et al. 2020).

Cytoplasmic VLEs (virus like elements) in Pichia acaciae, Kluyveromyces lactis and Debaryomyces robertsiae) encode specific immunity proteins that confer cells insensitive to their own toxin. These elements can be dsRNAs or DNA based (Wickner & Edskes, 2015; Belda et al., 2017). Meinhardt et al. (2015) explain their results by demonstrating the mechanism of VLE. They suggest that the toxin and immunity function participate in the evolutionary auto selection system. The mechanism of VLE actually works to prevent the host cells from incorporating the immunity gene into their nucleus. This is due to the higher A/T content in VLEs compared to the host cell. If the mechanism did not prevent the incorporation of the immunity into the chromosomal locus, the toxin action would escape from the cell in the form of VLEs. In other words, when the immunity gene is integrated, toxin encoding VLEs are lost, leaving behind an immune cell.

4 Role of killer yeast against common wine spoilage yeast

4.1 Spoilage yeast in wine industry Yeast killer toxins are mostly applied in wine fermentations against common spoilage yeast. It was shown that wine environment can be favourable for toxin stability, its activity and production. Most of the toxins prefer acidic pH and temperatures around 20°C (Ramirez et al., 2017; Schaffrath, Meinhardt & Klassen, 2018; Villalba et al., 2020).

The concept of spoilage yeast in fermented foods and beverages is quite complex. There is a thin line between the wanted flavours and aroma caused by the metabolites produced by certain microorganisms and the overaccumulation of these metabolites that is perceived as spoilage. Such an example is the production of 4-ethyl-phenol by Brettanomyces/Dekkera spp. It is desirable in quantities under a certain level when it still contributes to the needed complexity of wine aroma. Usually, the spoilage is defined during aging or storage because certain yeasts present in the wine must are essential for the fermentation. However, many yeasts can have detrimental effect also during and before the fermentation and thus the control of spoilage yeast should be examined throughout all the winemaking process. Yeast are therefore recognized as common contaminants in the winemaking that lead to wine spoilage. The consequences are film formation in stored wines, cloudiness or haziness, sediments and gas production in bottles, off-odours and off-tastes. Such species are P. membranifaciens, P. anomala, Candida spp. with aerobic or weakly fermentative metabolism that can grow well in the wine environment. If wine has insufficient amounts of sulphite to prevent their growth, they may cause film formation on the wine surface. P. anomala, M. pulcherrima and H. uvarum (Kloeckera apiculata) are problematic during the initial fermentation steps, because they produce high levels of ethyl acetate which leads to wine deterioration (Loureiro & Malfeito-Ferreira, 2003).

4.1.1 Brettanomyces bruxellensis B. bruxellensis are common spoilage yeast in winemaking that are recognized by off-odours and off-flavours which come from volatile phenols and tetrahydropyridines produced by this yeast (Mehlomakulu, Setati, & Divol, 2014; Mehlomakulu et al., 2016; Oro et al., 2016). They can develop at the end of alcoholic fermentation in wine or during the aging stage in wooden barrels. However, there are not naturally present on grapes. They produce unwanted off- flavours and smells which are revealed in the final wine and thus cause major economic losses (Santos, Sanchez, & Marquina, 2004). In the early stages of winemaking they grow slowly and are therefore present in low numbers. They can also enter into the viable but not culturable state (VBNC). For the listed reasons, the detection of this yeast may be challenging. Slow growth continues also during the fermentation period, however, during the aging stage it starts to thrive. Even the presence of SO2 and ethanol does not prevent it,

because this yeast enters the stage of slower metabolic activity which also makes it undetectable. Its normal growth is resumed at the end of fermentation and when aged in wooden barrels. Attempt of removing B. bruxellensis is done with filtration, decreasing the temperatures or barrel sanitization. However, these conditions are not that effective, and they do not prevent recontamination after the process itself. Furthermore, they impose unwanted effects on wine, such as loss of the body, viscosity and sensorial properties of red wine during aging. On the other hand, some preservatives were shown to be more efficient, but their use is limited due to a possible negative effect on the wine flavour (Comitini et al., 2004a; Mehlomakulu, Setati, & Divol, 2014; Mehlomakulu et al., 2016).

To fight this unwanted yeast, the focus over the last decade has been on zymocins that can represent an alternative preservative to synthetic antimicrobial agents in the wine industry (Oro et al., 2016; Comitini & Ciani 2011; Santos et al., 2011; Mehlomakulu et al., 2014).

Besides SO2, dimethyl decarbonate and chitosan are also being used as preservatives. Although, dimethyl decarbonate was shown to be relatively toxic and it is difficult to handle. Whereas chitosan is effective only against certain strains of this yeast species (Mehlomakulu et al., 2016).

4.2 Killer yeast used in wine industry Industries that produce fermented drinks or food (wine, beer, or bread industry) are exceptionally interested in the killer strains, since certain steps, such as post-fermentative aging process, can get ruined by spoilage yeasts. Usually these yeast are of wild type that occur during the production and can cause off-odours, off-flavours, and other unwanted characteristics (Schmitt & Breinig, 2002; Mehlomakulu, Setati, & Divol, 2014; Schaffrath, Meinhardt, & Klassen, 2018). It was discovered that killer toxins secreted by S. eubayanus and Candida pyralidae can kill common spoilage yeasts in winemaking, such as B. bruxellensis, P. membranifaciens, Meyerozyma guilliermondii and Peronospora manshurica. Moreover, they discovered that wine-like environment is favourable for stability and activity of their toxins (Mehlomakulu, Setati, & Divol, 2014; Mazzucco, Ganga, & Sangorrin, 2019; Villalba et al. 2020). Another wine-spoilage yeast, Brettanomyces/Dekkera were efficiently killed by Kluyveromyces wickerhamii that produces Kwkt killer toxin (Liu et al. 2013).

In the past, strains of S. cerevisiae were the main focus of the research in oenology and the only ones used as starter cultures for wine fermentation. Recently, other non- Saccharomyces yeast, that were previously considered to be spoilage microorganisms, started to attract attention among researchers. Most of the research is focused on using them in co-culture with Saccharomyces species as this combination demonstrated to be beneficial for winemaking (Mostert et al., 2014).

The environmental conditions of wine fermentation and aging (low pH and low temperatures) are favourable for the majority of yeast killer toxins. Such an example is toxin KpKt from Tetrapisispora phaffii (former K. phaffii) which can inhibit Hanseniaspora uvarum present on grapes and grape juices. Its activity is even comparable to the antifungal activity of SO2. Moreover, KpKt was found to inhibit other yeast species, killing not only Kloeckera/Hanseniaspora and Zygosaccharomyces but also B. bruxellensis (Schaffrath et al., 2018).

Current processes in the wine industries that prevent the growth of spoilage microorganisms, involve microfiltration of wine and increased SO2 concentrations. As these methods are not appropriate to be used during wine aging the use of yeast killer toxins has gained major interest (Comitini, De, Pepe, Mannazzu, & Ciani, 2004a). To use them as antimicrobials, killer yeast can be included as a fermentation starter or as purified killer toxin (Chessa et al. 2017). Most killer toxins described in literature possess different modes of killer activity such as the membrane damage, glucanase activity, they may be inhibitors of β-1,3-glucansynthase, cell cycle arrest, inhibition of calcium uptake, or tRNase activity. The toxins from Pichia genus performs most of these actions. This implies the broad-spectrum of Pichia killer toxins and expands their application in various food industries. However, there are some restrictions upon using yeast killer toxins, such as high costs of purified large-scale toxin production and limitation to use them in specific environments with defined chemical and physical conditions (fermented and post-harvest food). Hence, the application is currently limited to the use of cells, rather than the purified toxins. The stability of P. membranifaciens toxins in production and application stages still represents a problem. Thus, the adaptive and directed evolution experiments could be helpful in the development of phenotypically appropriate varieties. The evolving populations would need to be prepared under winemaking conditions if they are intended to be used in the wine industry. For the therapeutic purposes, P. membranifaciens should be studied more for their toxicity, stability and antigenicity which should be done under physiological conditions (Belda et al., 2017).

4.2.1 Torulaspora delbrueckii T. delbrueckii is one of mostly used yeast in winemaking. It improves the complexity and enhances certain specific wine characteristics. Furthermore, it has a low production of acetaldehyde, acetoin, acetate, and ethyl acetate. Recently, this yeast has been used together with S. cerevisiae and L. thermotolerans for wine production. In the production of sparkling wines, T. delbrueckii showed to have positive effects on the foam properties (Ramirez et al., 2016; Garcia, Esteve-Zarzoso, & Arroyo, 2016). The problem of using this species is in its interaction and small proportion compared to other yeast species in wine must. This is also obvious from the fact that T. delbrueckii yeasts are rarely isolated from spontaneously

fermenting must. T. delbrueckii has a slower growth rate and less fermentation vigour compared to S. cerevisiae. Hence, in wine fermentation conditions it becomes quickly outcompeted by wild or inoculated S. cerevisiae. There are also interactions between these two species, that result in decline of T. delbrueckii population (Ramirez et al., 2015).

Ramirez et al. (2015) researched if the killer toxins secreted by T. delbrueckii (Kbarr-1) can kill S. cerevisiae and subsequently dominate in the fermentation process. The result of the joint fermentation in wine must resulted in improvement of wine quality. They recommend using a strain of T. delbrueckii that produces killer toxin Kbarr-1 in order to achieve supremacy of T. delbrueckii over S. cerevisiae during the fermentation of wine must. It was even shown that Kbarr-1 can kill S. cerevisiae killer strains (K1, K2, K28 and Klus). It also killed non-S. cerevisiae species (Hanseniaspora sp., Kluyveromyces lactis, Schizosaccharomyces pombe, Candida albicans, C. tropicalis, C. dubliniensis, C. kefir, C. glabrata, C. parasilopsis, C. krusei, Yarrowia lipolytica, and Hansenula mrakii). However, it was not lethal to Brettanomyces sp. The killer toxin Kbarr-1 showed a stronger killing activity than Klus or K2 toxins and a similar activity to K1 or K28 toxins. Interestingly, the killer dsRNA virus system of Kbarr-1 resembles the one found in S. cerevisiae (Ramirez et al., 2015).

The killing activity of T. delbrueckii was tested against some common wine spoilage yeast (P. membranifaciens, Candida glabrate, P. manshurica and P. guilliermondii) species, with a focus on B. bruxellensis. T. delbrueckii NPCC 1033 was chosen for the research due to its wide spectrum of killer action. The stability of this strain remained good in grape juice and wine for at least one day. They discovered that the binding sites on yeast cells for T. delbrueckii are probably β-1,6-glucans, and chitin. However, partial killer action could be also owed to the hydrolytic activity of this strain. Chitinase and β-glucanase caused degradation of cell wall polysaccharides, setting off the cell wall disruption. This led to initial apoptotic and after 24 hours, necrotic cell death in yeast target cells. In order to investigate the exact mechanism that caused yeast apoptosis, further research on purified toxin is needed. They also found that it was stable during all the stages of wine production. Even though the toxin was able to control the growth of wine spoilage yeast, it did not inhibit S. cerevisiae (Vilalba et al., 2016).

4.2.2 Candida CpKT1 and CpKT2 were isolated from two strains of Candida pyralidae and they showed an inhibitory activity against B. bruxellensis. Their activity was lost at higher temperatures and when incubated with proteinase K, which proves the proteinaceous nature of these toxins (Mehlomakulu, Setati, & Divol, 2014). CpKT1 was stable for 5 days, while CpKT2 for 20 days at 15 and 20°C. This is important to consider when applying it to a target yeast population. The researches stated that this amount of time should be enough to eliminate the targeted

yeast population, considering that in literature it was found that the yeast population can be diminished 24h after adding the toxin (Mehlomakulu, Setati, & Divol, 2014). The toxins examined in this research were not significantly influenced by the change of sugar and ethanol concentration. The stability and endurance of CpKT1, CpKT2, Pikt and Kwkt toxins showed that they can remain active throughout the whole fermentation. If the toxins are applied to the conditions resembling those in winemaking, CpKT1 and CpKT2 can potentially control the population of B. bruxellensis strains, while not inhibiting S. cerevisiae and lactic acid bacteria. The latter two are important for alcoholic and malolactic fermentation (Mehlomakulu, Setati, & Divol, 2014).

Mehlomakulu et al. (2016) performed a research on the population dynamics of S. cerevisiae (a chosen strain resistant to C. pyralidae toxins), B. bruxellensis and C. pyralidae co-culture in grape juice. They performed the experiment at pH 4.5 (optimum for C. pyralidae) and at the pH close to the one found in wine (3.5). Between 24h and 72h, in the co-culture of C. pyralidae and B. bruxellensis, the population of the latter largely decreased and after 72h it was undetectable. In the culture where also S. cerevisiae was added, the population of B. bruxellensis cells experienced a similar decline. In the absence of B. bruxellensis, S. cerevisiae and C. pyralidae affected each other’s growth only slightly. By proving the inhibitory effect of a purified toxin (CpKT1) on B. bruxellensis they confirmed that the antimicrobial effect of C. pyralidae was due to the toxin and not the competition for nutrients or some other mechanism. Although, the sensitive cells recovered after the toxin was not applied to them anymore. The outcome of the study proves that C. pyralidae can be efficiently used as a biocontrol tool against B. bruxellensis in grape juice.

4.2.3 Pichia The discovery of the killer toxins in P. membranifaciens opened the possibilities to use it in agro-food industry. Initially, they noticed that P. membranifaciens, when co-inoculated in grape wine together with B. cinerea, limited the growth of its mycelium. They suggested that the killing process happened due to the production of exo- and endo-β-1,3-glucanases by P. membranifaciens (Belda et al. 2017). Later, in a research by Santos, Sanchez, & Marquina (2004) they reported that the PMKT activity was responsible for the killer factor in grape wine and apple matrixes. This suggest that for the killer activity of P. membranifaciens, there are many factors involved, such as hydrolysing enzymes and killer toxins. P. membranifaciens appeared to be an efficient biological tool also against other fruit spoilage moulds, such as Penicillium expansum and in post-harvest fruit. Their toxins were proven to be efficient in winemaking conditions, specially PMKT against B. bruxellensis (Yap et al. 2000; Marquina, Santos, A., & Peinado, 2002; Santos, Sanchez, & Marquina, 2004). Killer toxin PMKT2 produced by P. membranifaciens CYC 1086 can be applied to wine fermentations, since it

does not affect the growth of S. cerevisiae, but it is inhibitory to certain fungi and spoilage yeast (Liu et al. 2013).

Also, killer toxins from other yeast species such as Ustilago maydis, Torulaspora delbrueckii (TdKT), Kluyveromyces wickerhamii (Kwkt), Wickerhamomyces anomalus (Pikt), Candida pyralidae (CpKT1) and the pulcherriminic acid produced by Metschnikowia pulcherrima were reported to be efficient against B. bruxellensis. The use of P. membranifaciens together with some other yeast species could therefore present a promising biocontrol tool against certain spoilage mould that causes problems in the wine industry (Belda et al. 2017).

3.2.5 Kluyveromyces spp. 3.2.5.1 Kluyveromyces wickerhamii Comitini et al. (2004a) first described two toxins active against Dekkera/Brettanomyces, a common spoilage yeast in the wine industry. DBVPG 3003 is produced by P. anomala and DBVPG 6077 by K. wickerhamii. The toxins are named Pikt and Kwkt, respectively. P. anomala (DBVPG 3003) and K. wickerhamii (DBVPG 6077) were therefore proposed be used during wine aging as a protective biocontrol agent.

K. wickerhamii’s (now T. phaffii) killer toxin KwKT showed an inhibitory activity against B. bruxellensis. However, some strains of Brettanomyces/Dekkera were resistant to it. They found out that the toxin from T. phaffii CS 4417 did not inhibit the growth of Hanseniaspora spp., even though in other literature (Ciani & Fatichenti, 2001; Comitini & Ciani, 2010) this effect was proven. The contrariness is probably due to a different media used for the screening in each research. Therefore, the killer activity is not only dependent on the killer and sensitive yeast but also on the type of media (Mehlomakulu, Setati, & Divol, 2014).

Comitini et al. (2004a) showed that P. anomala (DBVPG 3003) and K. wickerhamii (DBVPG 6077) revealed their killer activity against all the tested Dekkera/Brettanomyces strains. Besides, both of them were able to kill S. cerevisiae DBPVG 6500.

Oro et al. 2016 compared the antimicrobial effect of Kwkt toxin and SO2 against B. bruxellensis under physico-chemical conditions used in winemaking. After 60h of incubation both showed inhibition of B. bruxellensis growth. The treatment of B. bruxellensis with Kwkt resulted in 50% of the cells being death. However, out of other 50% only 3% were metabolically active.

After the treatment with SO2 and Kwkt they tried to recover B. bruxellensis cells in both media.

They found out that the cells previously treated with SO2 were able to recover after the removal of the stress agent (SO2) which indicates that they were in a VBNC state (viable but not culturable) when the stress was applied. On the contrary, the damage that was done on the cells by Kwkt could not be reversed even after the removal of the toxin. This proves the efficiency of Kwkt over SO2 against B. bruxellensis (Oro et al. 2016).

3.2.5.2 Kluyveromyces phaffii The killer toxin KpKt secreted by Kluyveromyces phaffii showed potential as a biocontrol agent in the wine industry due to its activity against some wine-spoilage yeast. (Comitini et al., 2004b). K. phaffii strain DBVPG 6076 produces a glycoprotein with β-glucanase activity that is known as Kpkt (Comitini et al. 2004b, 2009). This yeast strain is able to kill yeast from Zygosaccharomyces and Kloeckera/Hanseniaspora genera, that are known to cause wine spoilage. It is known to maintain the killer activity in wine for 14 days (Comitini and Ciani,

2009). Current chemical treatment of freshly pressed grape juice is done by applying SO2 in order to prevent the later spoilage by certain yeast. This agent is added also at the end of the fermentation process to prevent oxidation. However, the wine industry is considering about substituting a part of SO2 with natural antimicrobials, since it was shown that this agent can be toxic to humans and also the interest in natural antimicrobials is rising among consumers. One of the possible candidates, could be KpKt toxin which has a broad spectrum against spoilage yeast and can be active under winemaking conditions. It could be used instead of

SO2 in the pre-fermentative stage and hence the overall amount of SO2 would be considerably lower (Comitini et al., 2004b).

5 Killer yeast in other biotechnological applications

Besides the wine production, killer yeast showed potential to be applied in enviormental, medical and industrial biotechnology. They are already applied to the production of fermented vegetables, in biological control of post-harvest diseases, yeast bio-typing and as antimycotics in the medical field. Moreover, they can also be used as a model system to understand eukaryotic protein processing, secretion and expression of eukaryotic viruses. In agriculture and food industries the interest is to apply yeast killer toxins as bioprotective agents which prevent the growth of competing microorganisms (Marquina, Santos & Peinado, 2002; Belda et al., 2017). The important application is also in the field of genetics which includes fingerprinting of wine yeast and recombinant DNA technology (further explained in chapter 7) (Marquina, Santos & Peinado, 2002).

In biotechnological field of application, killer yeast have been applied to agriculture where they prevented wood-decay and plant pathogenic fungi, to beer and sake production for prevention of contaminants and they were also used as food preservatives against spoilage yeast found in yogurt (Lowes et al., 2000; Marquina, Santos & Peinado, 2002). To prevent the spoilage in silage, K. lactis zymocin and Cyb. Mrakii HM-1 toxin can be applied. Certain yeast, such as Wickerhamomyces anomalus produce glucanase toxins which are known for their antimicrobial activity against some pathogenic bacteria, yeasts, mycelial fungi and protozoas. They are used as a biocontrol agent against certain pathogenic fungi that appear on harvested

fruit during storage. Furthermore, an expression of some killer toxins in transgenic plants was done which led to crops resistant to a certain disease (Schaffrath et al., 2018).

A study was done on the relationship between pathogens that cause disease in marine animals and yeast killer toxins found in marine animals. The researchers discovered that certain killer yeast inhibited the growth of pathogenic bacteria or even killed pathogenic yeast in marine animals and thus opening up this topic for further research (Chi et al., 2010).

Industries that produce fermented drinks or food are especially interested in the killer strains, since some steps such as post-fermentative aging process can get spoiled by certain spoilage yeasts (Schaffrath et al., 2018). Fermented food presents a natural habitat for killer yeast which, if present in high numbers, outcompete the sensitive yeast. Commercial yeast are mostly sensitive to killer toxins which should be taken into account especially in continuous fermentation processes, where killer yeast have a competitive advantage. On the other hand, if commercial yeast are present in higher number than killer yeast in a batch process, they outcompete the killer strains due to the higher nutrient uptake (Marquina, Santos & Peinado, 2002).

As mentioned, yeast killer toxins are also being utilized for medical purposes. Some can be applied as antifungal agents for treatment of humans infected by pathogens such as Candida albicans or Cryptococcus neoformans that are found on skin and mucosal membranes. However, this application is limited due to the instability of these proteins when the temperatures are over 37°C or the pH is around neutral. Marquina, Santos & Peinado (2002) proposed to solve this problem by adding the toxins to buffered solutions and apply them to skin as such (Marquina, Santos & Peinado, 2002; Schaffrath et al., 2018).

6 Influence of environmental conditions on killer toxin production and activity

6.1 Temperature and pH Most toxins exhibit their highest killing activity at acidic pH values and low temperatures (Table 1), yet there are some exceptions that show higher activity at a wider pH range and higher temperatures. Such an example is killer toxin HM-1 produced by W. saturnus var. mraki IFO0895 that is stable at pH range 2-11 and at also at higher temperatures (100°C for 10 minutes), compared to the majority of the toxins. This stability is assumed to be due to its internal disulphide bridges (Belda et al., 2017).

The influence of pH on the toxic activity of killer yeast is an important factor when considering using them as a biocontrol agent in the winemaking process. For example, K2 produced by S. cerevisiae showed to be more effective than K1 at pH value found in wine (~3.5) and it thus

makes it more suitable to use it as an antimicrobial agent against wine spoilage yeast. Moreover, K2 is also active at a wider pH range (2.8 - 4 .8) than K1 (Schaffrath, Meinhardt, & Klassen, 2018). Furthermore, K28 as well as the other zymocins, have a low pH optimum and are therefore also suitable to be added to the grape juice which has a pH in the range 2.8 – 3.8. Besides that, K2 and K28 toxins did not exhibit any undesired effects on wine quality (Pretorius, 2000).

The pH value affects the activity of killer toxins also by the means of binding. When PMKT is exposed to low pH values that still allow for its activity, it is positively charged. As such, it can strongly bind to the negative charge present on the secondary plasma membrane receptor, Cwp2p. Moreover, at low pH values PMKT also causes conductance in liposomes which leads to channel formation and consequently leakage of common physiological ions (Santos & Marquina, 2004; Belda et al. 2017).

Pichia ’s toxins exhibit higher activity at low pH values and low temperatures (Belda et al., 2017). The highest toxin production and stability of PMKT and PMKT2 was observed at pH 4.5 and at 20°C. PMKT was stable at pH interval between 3.0 and 4.8 and at temperatures below 20°C. Above these values its stability and production was lost. PMKT2 was active at pH values between 2.5 and 4.8 and temperatures below 20°C as well. However, its stability when exposed to higher temperatures for a short time was not that affected as it was for PMKT. The toxin instability was measured as protein denaturation (Belda et al., 2017). Its killer activity against B. bruxellensis remained stable under pH and temperature ranges found in wine, showing its potential as a biocontrol agent in the wine production (Comitini & Ciani, 2011). P. anomala (DBVPG 3003) and K. wickerhamii (DBVPG 6077) maintained their killer activity at pH of wine, as well. The strongest killing activity was observed at pH 4.4 and Pikt was more resistant to higher T than Kpkt (Comitini et al., 2004a).

The strongest activity of Kbarr-1 produced by T. delbrueckii was measured at pH 4.7, and at 12°C against S. cerevisiae K2 producer, at pH 4 and 20°C against S. cerevisiae K28 producer and at pH 4.7 and 20°C against H. mrakii, C . kefir, C. glabrata, C. dubliniensis, and Y. lipolytica strains (Ramirez et al., 2015). SeKT secreted by S. eubayanus was the most active at pH range 3 - 4.5 and it started to decrease when the pH rose. The activity was lower at temperatures above 26°C (Vilalba et al., 2020). CpKT1 was the most stable at pH 4.5, while CpKT2 at pH 4.0. Both were active at acidic pH, between 3.5 and 4.5 and the strongest stability and activity were measured between 15 and 20°C (Mehlomakulu et al., 2014). KpKt produced by Tetrapisispora phaffii is active under wine making conditions (pH around 3.5 and low temperatures) against certain yeast species, including Hanseniaspora uvarum, which are most frequently found on grapes and grape juice (Ciani and Fatichenti, 2001).

Kwkt secreted by Kluyveromyces wickerhamii and Pikt secreted by Wickerhamomyces anomalus showed their optimal activity at pH 4.4 and remained active in the pH range 3-5 (Oro et al., 2016). Kwkt and Pikt were active against B. bruxellensis in the range of their pH optimum (4.4), but also at lower pH value (3.2). On the other hand, SO2 that is pH dependent, had a much lower toxic effect at the higher pH value (Oro et al., 2016).

Yeast P. K. W. saturnus S. membranifaci T. delbrueckii C. pyralidae wickerhamii var. mraki eubayanus ens

Killer PMKT and Kbarr-1 Kpkt HM-1 SeKT CpKT1 CpKT2 toxin PMKT2 pH 4.5 4 – 4.7 4.4 2-11 3-4.5 4.5 4.0 (stable) Temper- 20°C 12-20°C __ Up to 100°C Below 26°C 15-20°C ature (stable) Table 1. Optimal pH and temperature at which listed toxins have the highest activity.

6.2 Other conditions P. membranifaciens that was isolated from olive brines showed that it possesses killer activity. Moreover, many other yeast (39%) isolated from the same habitat showed to have a killer character. High amounts of salt in olive brines create anthropic environment, in which halotolerant yeast can develop. It was found that concentrations of NaCl comparable to the ones found in olive brines increase the killer activity and spectra of yeast isolated from this environment. This implies that under these circumstances the killer toxins can create a great advantage (Belda et al., 2017).

7 Molecular biology of killer yeast

Molecular techniques used on yeast are already well known and established. They make It possible to genetically engineer yeast in order to resemble the genetic mutations observed in disease or disorder of interest (Oliver and Castrillo, 2019). Synthetic biological systems are of great interest, since they can provide a better understanding of the natural biological systems. Moreover, they are useful to engineer pathways important for agriculture or medicine. In the field of genetic engineering, CRISPR-Cas tool has been proven to be efficient in many complex yeast systems. For instance, CRISPR-Cas has been applied to S. cerevisiae, a frequently used microorganism in this field, for the wholesale editing of the yeast genome, engineering of mutations and complex metabolic pathways (Oliver and Castrillo, 2019).

Bussey and Meaden (1985) were one of the first authors to describe the process of a selection system for plasmid preservation. They selected the plasmids by expressing a cDNA encoding the yeast killer toxin and immunity gene (M1 from S. cerevisiae) in sensitive yeast strains.

Under the killer toxin producing conditions, the selective advantage was at the transformants with killer toxin-immunity gene, while the ones that lost their plasmids, died.

K. lactis strains carry k1 and k2 plasmids, which have been investigated the most thoroughly. They are both located in the cytoplasm and present in multi-copy. The replication and transcription processes of these plasmids function on their own, independently of the host cell. They encode the immunity factor and the genes that determine the subunits of the K. lactis killer toxins (Schaffrath, Sasnauskas, & Meacock, 2000). It has been observed that the transcription of killer plasmids genes is not compatible with host-encoded RNA polymerases (RNPs) and is thus independent of it. Therefore, when they tried to clone k1 and k2 genes with their equivalent promoters into nuclear yeast vectors they were generally not expressed. k1 and k2 were then successfully genetically manipulated by allelic replacement, gene shuffling and site-specific mutagenesis in vivo (Schaffrath, Sasnauskas, & Meacock, 2000).

The toxin secreted by K. lactis and its linear plasmids related with the killer character are quite different from the other known yeast systems. K1 and K2 toxins secreted by this yeast have a high amount of A/T content, proteins are covalently linked at 5’ terminus of both DNA strands and they have terminal inverted repeat sequences. Indeed, their structure is quite particular and the fact that they utilize plasmid-encoded DNA polymerases (DNPs) implies that they multiply in a virus-like mode via protein-priming of DNA replication (Stark et al., 1989; Schaffrath, Sasnauskas, & Meacock, 2000).

7.1 Molecular biology techniques applied to killer yeast

As stated in chapter 4.2.1, S. cerevisiae produces K2 toxin which is a promising antimicrobial agent for biotechnological applications. In spite of the negligible amounts of this extracellular protein produced by S. cerevisiae, K2 still possesses a high biological activity. Nonetheless, the small concentrations make it difficult to isolate the sufficient amounts of K2 needed for the research (Podoliankaite et al., 2014; Carboni et al., 2020). Thus, Podoliankaite et al. (2014) did an experiment on cloning, expression, and purification of S. cerevisiae killer toxin K2 using a bacterial system of E. coli. These bacteria are often used to produce desired proteins for the requirements of industrial upscaling, due to their simplicity and low production costs (Podoliankaite et al., 2014). The major problem they encountered was the introduction of plasmid with full pptox gene into E. coli and low expression level of the target protein. When they tried to remove C-terminal of K2 protein, the killing activity was abrogated and had a negative impact on the bacterial cell proliferation. Hence, they solved this problem by directing the killer pptox into IBs (Inclusion bodies) to keep the protein as non-toxic and thus enable it to be accumulated in the cytoplasm of E. coli. Moreover, IBs allow for efficient and simple separation from the host proteins and are essential for high-level expression. The

recovery (return to their active biological state) of the protein starts with isolation and solubilization of IBs and ends with refolding into protein’s native state (Podoliankaite et al., 2014).

Giesselmann et al. (2017) successfully fluorescently labelled the killer toxin K28 by fusing it with fluorescent proteins (mCherry or mTFP) and performing a heterologous expression in P. pastoris yeast. In vivo expression was needed, so the toxin is properly posttranslationally processed and folded using the secretory pathway of the host cell, resembling the natural toxin processing and secretion in S. cerevisiae. They added the fluorescent tag between the β-subunit and its C-terminal HDELR motif, because the α-subunit is known to be sensitive to the tag addition. mCherry was used for fluorescent labelling due to its insensitivity towards C- and N-terminal fusions and stability at low pH (pH optimum of K28 is around 4.7). P. pastoris processed the K28 tagged variant and was not negatively affected by the mCherry fusion. This resulted in high-level production and secretion of fluorescent and bioactive toxin recombinant yeast that had a killing effect against some sensitive strains. Due to the fluorescent signals on the periphery of the cell they successfully demonstrated that the β- subunit is necessary for the cell binding of K28. The authors propose that this method could be implemented onto the other killer yeast as well, as a tool for live cell imaging and high- resolution microscopy for purposes of cell-surface binding analyses. Furthermore, with more research in this field, the intracellular toxin trafficking could be visualized using a high- resolution single molecule fluorescence microscopy.

The production of Kpkt produced by T. phaffii appeared to be small, but nevertheless effective against Kloeckera/Hanseniaspora and Zygosaccharomyces. Furthermore, it was shown to possess its killer activity even after 14 days in grape must under winemaking conditions (Chessa et al., 2017). However, the drawback of using T. phaffii is that it is not suitable for direct application in wine production (Carboni et al., 2020). In order to overcome the problem of small load of Kpkt, Chessa et al. (2017) delve into the research of the possibility to increase the production of Kpkt toxin for biotechnological applications. They explored the option of producing a recombinant (r)Kpkt in Komagatella phaffii GS115 (formerly Pichia pastoris) by developing specific molecular tools and two recombinant clones that produce rKpkt. The recombinant rKpkt showed a larger killer and β-glucanase activity compared to the native Kpkt and it even killed some strains of S. cerevisiae and D. bruxellensis that were insensitive to Kpkt. Therefore, the heterologous production of Kpkt together with optimization described by the authors, has potential to be applied into wine and sweet beverage industry in order to combat possible pathogens (Chessa et al., 2017).

Carboni et al. (2020) used the method for the production of the recombinant rKpkt as described by Chessa et al. (2017). The production was carried out in bioreactors for the

interest of upscaling and the toxin coding gene was expressed in Komagatella phaffii. In order to prepare a ready-to use antimicrobial compound they concentrated and lyophilized the cell- free supernatant containing rKpkt. The obtained lyophilized rKpkt inhibited the growth of certain wine-related yeast, filamentous fungi and even some gram-positive and gram- negative bacteria. As desired, it did not influence the growth of S. cerevisiae starter yeast. The production in bioreactors compared to the media successfully increased the production of rKpkt toxin. An advantage that lyophilized toxin offers to the industry is that it can be stored at 4°C for up to six months and its killer activity can be revived upon the solubilization in water (Carboni et al., 2020).

8 Conclusion

The described studies showed a great potential of killer yeast to be applied in the winemaking process as natural preservatives. They killed the most common pathogens (B. bruxellensis, Zygosaccharomyces, Kloeckera/Hanseniaspora, P. expansum, Candida spp., H. mrakii, Y. lipolytica) found in wine production and even showed better action than SO2 against B. bruxellensis. The environmental conditions of wine production (pH and temperature) appeared to be suitable for the activity of killer yeast and even their optimum pH range is wider than that of SO2. B. bruxellensis, a common wine spoilage yeast was inhibited by various killer toxins (PMKT, CPKT1 and CPKT2 and Kwkt). However, some of the killer yeasts were also able to kill S. cerevisiae which should be considered when applying them to a fermented food product of which fermentation is initiated by this yeast. The killing mechanism of killer yeast is best understood for S. cerevisiae killer toxins (K1,K2 and K28). For the other yeast toxins, such as CpKT1 and CPKT2, Kwkt and Kpkt, the mechanism is poorly known and thus should be investigated more in order to apply these toxins in biotechnological processes. The immunity system of killer yeast is however, the least known, even for S. cerevisiae killer toxins. Despite all the listed positive aspects of yeast killer toxins there are still some limitations to the biotechnological application of killer yeast. It is difficult to upscale their production, they are not easy to handle and there is limited data on their spectrum of action and safety (Manazzu et al. 2019). In the researches mostly live cells are being used, instead of the purified toxins, because they are less expensive and do not need defined physico-chemical conditions. In order to overcome these obstacles, researchers try to develop different molecular techniques on killer yeast. The fluorescent labelling with fluorescent microscopy showed a promising tool for toxin trafficking inside a yeast cell. Furthermore, the use of E. coli bacterial system for the production of K2 resulted in an increased and less expensive production of this toxin, compared to the production in native S. cerevisiae. Likewise, Kpkt toxin, secreted by T. phaffii was expressed in K. phaffii which also showed a higher production compared to the native one. Nevertheless, the use of killer toxins for medical purposes is not

established yet, since the toxins should be tested more for their stability, antifungicity and toxicity.

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