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====Killer Wine Yeast====
Many wine yeast strains are known to be "killer" yeast strains that produce toxins that kill other nearby yeast cells in order to give the killer yeast strains a competitive edge over sensitive strains (this the term for these toxins has been changed to "zymocides" in science <ref name="Stewart_2018">[https://link.springer.com/chapter/10.1007/978-3-319-69126-8_10 "Killer (Zymocidal) Yeasts." Brewing and Distilling Yeasts. Graham G. Stewart. 2018.]</ref>, but is also sometimes called "mycocins" or "zymocins" <ref name="Boynton_2019">[https://onlinelibrary.wiley.com/doi/pdf/10.1002/yea.3398 The ecology of killer yeasts: interference competition in natural habitats. Primrose J. Boynton. 2019. DOI: https://doi.org/10.1002/yea.3398.]</ref>). Unwanted killer strains of ''Saccharomyces'' have been known to cause stuck wine fermentations by killing off the sensitive wine strain that was pitched into the wine by the winemaker <ref name="Bajaj_2017">[https://link.springer.com/chapter/10.1007/978-981-10-2621-8_7 Biology of Killer Yeast and Technological Implications. Bijender Kumar Bajaj, Satbir Singh. 2017.]</ref>. In ''Saccharomyces'', killer strains are genetically determined to secrete toxins (in the form of extracellular proteins or glycoproteins) called 'mycocins' that kill sensitive strains (there is no evidence that these toxins affect humans). These killer strains are immune to their own toxin. The mycocin toxins can act on sensitive strains in a number of ways: by blocking DNA synthesis and preventing cell division, inhibiting the synthesis of beta-glucans (β-1,6-glucan) that a part of their cell wall formation, and by causing ions to leak through the cell wall. In low dosages, which is typical in the natural environment, toxin triggers active cell death ([http://www.biology-pages.info/A/Apoptosis.html apoptosis]), while large dosages cause necrotic cell killing ([https://en.wikipedia.org/wiki/Necrosis necrosis]). One study in wine found that the use of killer strains to outcompete sensitive strains resulted in off-flavors from yeast autolysis <ref>[http://www.nature.com/nrmicro/journal/v4/n3/full/nrmicro1347.html Yeast viral killer toxins: lethality and self-protection. Manfred J. Schmitt & Frank Breinig. 2006.]</ref><ref name="Hatoum2012">[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3525881/ Rima Hatoum, Steve Labrie, and Ismail Fliss. 2012.]</ref>. Another study found that a lager strain that was genetically modified to secrete killer toxins was able to eliminate all cells of a sensitive ale strain within 24 hours of beer fermentation at a pitching rate of 99% sensitive ale strain to 1% killer lager strain, indicating that even a small amount of killer cells is enough to kill a larger population of sensitive cells <ref name="Stewart_2018" />. Ratios of killer cells to sensitive cells that have shown to completely eliminate or nearly eliminate the sensitive population on lab media includes 1:1, 1;10, and 1:100. In a starter of a sensitive strain that had 10% cells of a killer strain introduced, the viability of the sensitive strain was greatly reduced <ref name="Bajaj_2017" />. Neutral strains do not produce toxins, nor are they killed by them <ref>[https://books.google.com/books?hl=en&lr=&id=mvORN6OXHh4C&oi=fnd&pg=PA93&dq=Bussey,+H.+1981.+Physiology+of+killer+factor+in+yeast.+Adv.+Microb.+Physiol.+22:93-121&ots=jUY4T9NpgB&sig=aw-Y1um0KsDnGe6rRe5PTWIDYdI#v=onepage&q&f=false Advances in Microbial Physiology, Volume 22. Academic Press, Sep 15, 1981. Pg 94-95.]</ref>. Almost all domesticated ale and lager strains are sensitive to the toxins produced by killer strains <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1179271825434363/?comment_id=1179424538752425&offset=0&total_comments=5&comment_tracking=%7B%22tn%22%3A%22R%22%7D Conversation with Bryan of Sui Generis Blog on MTF on Killer Factor for Saccharomyces. 11/16/2015.]</ref><ref>[http://onlinelibrary.wiley.com/doi/10.1002/j.2050-0416.1973.tb03515.x/pdf Strains of Yeast Lethal to Brewery Yeasts. A.P. Maule and P.D. Thomas. 1972.]</ref>.
In ''Saccharomyces cerevisiae'', four toxins have been identified: K1, K2, K28, and Klus, the first three of which can only kill other strains/species of ''Saccharomyces''. The Klus toxin has been found to kill all strains of ''S. cerevisiae'' (including those that produce the previous three toxins), as well as yeast from other genera, such as ''Hanseniaspora spp.'', ''Kluyveromyces lactis'', ''Candida albicans'', ''Candida dubliniensis'', ''Candida kefir'' and ''Candida tropicalis''. Rodriguez et al. (2011) reported that out of 1,114 strains of ''S. cerevisiae'' isolated from spontaneous wine fermentations, 38% of them were killer positive with most producing K2. Only 7% of produced the Klus toxin (no commercial wine yeast strains have been reported to produce the Klus toxin that we know of) <ref name="Rodriguez">[http://aem.asm.org/content/77/5/1822.long A New Wine Saccharomyces cerevisiae Killer Toxin (Klus), Encoded by a Double-Stranded RNA Virus, with Broad Antifungal Activity Is Evolutionarily Related to a Chromosomal Host Gene. Nieves Rodríguez-Cousiño, Matilde Maqueda, Jesús Ambrona, Emiliano Zamora, Rosa Esteban and Manuel Ramírez. 2011]</ref>. The K1 toxin is most active between a pH of 4.6 and 4.8, while K2 and Klus are active around a pH of 4.0 to 4.3 <ref name="Rodriguez"></ref>. The activity of the toxin is greatest during the log phase of growth, and decays during the stationary phase of fermentation <ref name="Buyuksirit"></ref>. Generally, none of the toxins secreted by killer strains of ''Saccharomyces'' have been found to kill ''Brettanomyces'' <ref>[http://www.scielo.org.za/scielo.php?pid=S2224-79042015000100010&script=sci_arttext&tlng=pt Non-Saccharomyces killer toxins: Possible biocontrol agents against Brettanomyces in wine? S. Afr. J. Enol. Vitic. vol.36 n.1 Stellenbosch. 2015.]</ref>. One study from India reported that a wild ''S. cerevisiae'' strain caught from flowers killed another wild caught strain of ''Brettanoyces anomulus'', however, their methodology was not explicit and potentially not scientifically rigorous enough <ref>[http://nopr.niscair.res.in/handle/123456789/7735 Production and effect of killer toxin by Saccharomyces cerevisiae and Pichia kluyveri on sensitive yeasts and fungal pathogens. Dabhole, Madhusudan P, Joishy, K N. 2005.]</ref>. For example, this study did not use DNA fingerprinting to identify the wild yeast strains used in the study and instead relied on morphology and media selection, and they did not identify the type of toxin produced by the killer strain of wild ''S. cerevisiae''. They also reported that the ''B. anamulus'' strain did not ferment glucose, which is not typical for this species.
Several strains of ''Saccharomyces eubayanus'' isolated from seeds from monkey puzzle trees in Patagonia, Argentina, were found to secrete a killer toxin that kills ''Brettanomyces'' and ''Pichia''. One strain was found to produce a lot of the toxin, which is called "SeKT". ''S. cerervisiae'' strains, including strains that are sensitive to the above toxins, are not sensitive to this toxin. Mazzucco et al. (2019) found that SeKT toxin produced by this one strain of ''S. eubaynus'' in a special growth medium designed to maximize the SeKT toxin production (WUJ medium, which is "ultrafiltered" apple and pear juice) inhibited a strain of ''B. bruxellensis'' to around 50% growth after 48 hours in a wine growth medium. It also inhibited ''Pichia guilliermondii'', ''Pichia manshurica'', and ''Pichia membranifaciens'' by 50-70%. Note that the toxin was applied directly to the ''Brettanomyces'' and ''Pichia'' species, and not in a co-fermentation setting. Since ''S. cerevisiae'' strains are not effected by the SeKT toxin, it has been proposed as a way to limit ''Brettanomyces'' and ''Pichia'' in wine fermentations <ref>[https://www.ncbi.nlm.nih.gov/pubmed/30671692?dopt=Abstract Production of a novel killer toxin from Saccharomyces eubayanus using agro-industrial waste and its application against wine spoilage yeasts. Mazzucco MB, Ganga MA, Sangorrín MP. 2019. DOI: 10.1007/s10482-019-01231-5.]</ref>.
Various other yeast species have the ability to produce toxins that effect a range of other yeasts (but generally not bacteria), including species from the genera ''Candida'', ''Cryptococcus'', ''Debaryomyces'', ''Hanseniaspora'', ''Hansenula'', ''Kluyveromyces'', ''Metschnikowia'', ''Pichia'', ''Ustilago'', ''Torulopsis'', ''Williopsis'', ''Zygosaccharomyces'', ''Aureobasidium'', ''Zygowilliopsis'', and ''Mrakia'' <ref name="Buyuksirit">[http://waset.org/publications/9999528/antimicrobial-agents-produced-by-yeasts Antimicrobial Agents Produced by Yeasts. T. Buyuksirit, H. Kuleasan. 2014.]</ref><ref name="Stewart_2018" />. For example, strains of the yeast species ''Candida pyralidae'' <ref name="Buyuksirit"></ref>, ''Wickerhamomyces anomalus'', ''Kluyveromyces wickeramii'', ''Torulaspora delbrueckii'' and ''Pichia membranifaciens'' have been found to produce toxin that inhibits ''Brettanomyces'' <ref name="Ciani_2016">[https://www.researchgate.net/publication/301581233_Yeast_Interactions_in_Inoculated_Wine_Fermentation Yeast Interactions in Inoculated Wine Fermentation. Maurizio Ciani, Angela Capece, Francesca Comitini, Laura Canonico, Gabriella Siesto and Patrizia Romano. 2016.]</ref>. In addition, the toxin produced by ''Wickerhamomyces anomalus'' and ''Williopsis markii'' have been found to inhibit a wide range of spoilage and pathogenic fungi <ref name="Hatoum2012"></ref>. Killer strains of ''S. cerevisiae'' and other yeast can occur naturally in the wild on fruit and can have a negative impact on other flora that are found in the same environment <ref name="Buyuksirit"></ref>. Strains of ''Torulaspora delbrueckii'' have been shown to kill killer strains of ''S. cerevisae'' (wine strains), as well as to kill ''Pichia'' species <ref name="Ciani_2016"></ref>. The occurrence of killer strains of yeast in the wild is also wide spread. For example, out of 210 yeasts from various genera isolated from molasses, 13 of them were killer strains. Out of 1,000 isolates of various ''Candida'' species isolated from human skin, 52 were killer strains. Out of 65 strains of various yeasts isolated from fermented foods, soil samples, and spoiled fruits/vegetables, 12 were killer strains <ref name="Bajaj_2017" />. It has been hypothesized that toxin production is ubiquitous throughout nearly all genera of yeast; the more studies that have been done on a particular genus of yeast, the more likely it is that toxin production has been found by species and strains within that genus. Yeasts that produce toxins have been found on every continent and in every natural habitat of yeast, including leaf surfaces, leaf litter, tree slime fluxes, fruits, cactus stems and cladodes, insect guts, mammal feces, leaf-cutting ant nests, lake water, ocean sediment, soil, wine, bakeries, and dairy products <ref name="Boynton_2019" />.
Scientists have used genetic modification to create ''S. cerevisiae'' strains that produce various killer toxins that can assist in completing fermentation in the baking, wine, distillation, and beer making processes. These yeasts are able to inhibit undesired yeast contaminants, preventing various off-flavors and other unwanted characteristics in the finished products. Ale and lager strains that have been modified to release these toxins have reportedly retained the positive fermentation and flavor characteristics of the original strains <ref name="Bajaj_2017" />. Branco et al. (2017 and 2019) discovered several strains of ''S. cerevisiae'' that excrete a biocin toxin that is active against several other genera of yeast, including ''Brettanomyces bruxellensis''. The toxin is composed of peptides derived from the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is a protein that serves many different roles in different species of microbes and animals. This toxin is produced by some strains of ''S. cerevisiae'' as they enter the stationary phase after primary fermentation. However, the amount of the toxin needed to inhibit ''B. bruxellensis'' was 10 times the amount that is produced naturally during fermentation. The researchers later genetically modified a strain of ''S. cerevisiae'' to over-produce the toxin, which they named "saccharomycin", at levels required to completely inhibit ''B. bruxellensis'' when co-pitched at a 1:1 ratio (10^5 cells/ml for both). This toxin was also reported to be highly active against ''Hanseniaspora guilliermondii'', ''Kluyveromyces marxianus'', ''Lactobacillus thermotolerans'' (inhibited at 250 μg/ml of toxin), while inhibition of ''Torulaspora delbrueckii'' and ''B. bruxellensis'' required very high amounts of the toxin (500 μg/ml and 1000-2000 μg/ml) <ref>[https://link.springer.com/article/10.1007%2Fs00253-016-7755-6 Antimicrobial properties and death-inducing mechanisms of saccharomycin, a biocide secreted by Saccharomyces cerevisiae. Patrícia Branco, Diana Francisco, Margarida Monteiro, Maria Gabriela Almeida, Jorge Caldeira, Nils Arneborg, Catarina Prista, Helena Albergaria. 2017. DOI: 10.1007/s00253-016-7755-6.]</ref><ref>[https://link.springer.com/article/10.1007/s00253-019-09657-7 Biocontrol of Brettanomyces/Dekkera bruxellensis in alcoholic fermentations using saccharomycin-overproducing Saccharomyces cerevisiae strains. Patrícia Branco, Farzana Sabir, Mário Diniz, Luísa Carvalho, Helena Albergaria, Catarina Prista. 2019.]</ref>.