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Adaptive Evolution of Non-Saccharomyces Yeasts to Produce Wines with Low Ethanol Content Catarina Rocha

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Adaptive Evolution of Non-Saccharomyces Yeasts to Produce Wines with Low Ethanol Content Catarina Rocha Adaptive Evolution of Non-Saccharomyces Yeasts to Produce Wines with Low Ethanol Content Catarina Rocha Abstract This work describes the implementation of adaptive evolution, a non-genetic engineering approach, applied to non- Saccharomyces yeasts to originate variants that produce reduced levels of ethanol, during alcoholic fermentation of grape must. Sub-lethal concentrations of potassium chloride and furfural were used as evolutive pressures. Both KCl and furfural impose stresses that affect the cell stability compromising the redox balance. The natural response of cell to regenerate NAD+ is increasing the production of glycerol. The original populations of two non-Saccharomyces strains, Metschnikowia pulcherrima 134|MET and Lachancea thermotolerans 483|LCH were subjected to adaptive evolution. In parallel, Saccharomyces cerevisiae 771|SAC strain was also put under adaptive evolution as a comparative evolutive line. At each 50 generations, the evolved populations were analyzed for ethanol and glycerol production and glucose and fructose consumption by enzymatic assays. The evolved populations under adaptive evolution did not show relevant differences regarding to the production of ethanol or glycerol, when compared with their originals. Additionally, ethyl methanesulfonate (EMS) was used as a mutagen to create a population of mutagenized cells. To assess the metabolome of yeasts, by identifying the metabolites present inside the cells and the ones exported to the environment, Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FTICR-MS) was applied along growth. In future, new adaptive evolution lines will be started with the EMS mutagenized cells and FTICR-MS Spectrometry will be used to compare the original yeast populations with the prospective evolved ones. Keywords: Non-Saccharomyces yeasts; Wine; Adaptive Evolution; Mutagenesis; Mass Spectrometry. 1. Introduction maturity and aromatic profile of grapes leading to wines not well balanced (Ozturk & Anli, 2014; Kutyna et al., 2010). Amongst all industrial applications of yeasts, the more From the point of view of consumers, high alcohol significant is the production of alcoholic beverages such as consumption is associated with health issues, and in wine and beer. Though some wineries still produce wine in countries were taxes are levied according to ethanol a traditional way, relying on natural microbiota of grapes, at content, the costs of wines are higher (Coulter, 2013; industrial scale the unpredictability of this approach led Kutyna et al., 2010). producers to choose selected pure yeast cultures with The concern with the increasing of ethanol content in known properties. This approach makes possible to wines has led to the development of technologies for produce consistent wine flavour and predictable wine producing wines with reduced ethanol concentrations. quality (Pretorius, 2000; Kutyna et al., 2010; Pérez-Torrado (Varela et al., 2008). et al., 2015). Viticultural techniques aim to reduce the amount of Presently, winemakers require cost-competitive wine sugar content in ripening grapes, which lead to lower production which led to the development of yeast strains production of ethanol during fermentation. The earlier fruit with improved fermentative performance. Saccharomyces harvest, the shading of vines and the manipulation of soil cerevisiae is the most used starter culture in winery compositions are some practices to obtain grapes with less industry. Some of these improvements rely on the increase sugar content (Ozturk & Anli, 2014; Novello & Palma, 2013; of ethanol and stress tolerance, fermentation efficiency and Kliewer & Dokoozlian, 2005; Salamon, 2006). more recently, glycerol overproduction and lower ethanol Physical practices include reduction of fermentable yields (Pérez-Torrado et al., 2015; McBryde et al., 2006). sugars from grapes prior to fermentation and removal of In recent years, alcohol level in wines produced around ethanol from wine after fermentation. Among the current the world has increased, mainly because of the increase of pre-fermentation techniques, nanofiltration and glucose weather temperatures as a consequence of global oxidase treatment of grape must are the most used (Varela warming. Warm climates led to the development of grapes et al., 2015)). Filtration, distillation, freeze concentration, with high sugar concentrations and affect the phenolic evaporation and extraction of ethanol are some of the pos- 2 fermentation techniques used to lower the ethanol content changed in a stable manner, resulting in a mutation of wines (Pickering et al., 2001)). (Chambers et al., 2007; Ennis, 2001). Ethyl The biological practices involve the choice or methanesulfonate (EMS) is an alkylating agent that induces improvement of organisms that can be interesting. point mutations both in nuclear and mitochondrial genomes Nowadays the most economical, acceptable and of yeasts. EMS induces base-pair substitutions of both GC explored way to produce wines with lower concentration of to AT and AT to GC transition mutations and can cause ethanol is redirecting yeast carbon metabolism away from nonreversible base-pair insertions or deletions (Sega, ethanol production during alcoholic fermentation (Ozturk & 1984; Mobini-Dehkordi et al., 2008). Anli, 2014; Kutyna et al., 2010). The redox equilibrium Adaptive evolution (AE) defines a set of mutations that between NAD+ and NADH is the center of the metabolic occur in selective conditions. Populations cultured in an pathway that led to the production of ethanol. In yeasts environment with a sub-lethal concentration of a selection under normal fermentative conditions, NAD+ is mainly pressure will probably acquire certain mutations that lead to reduced during glycolysis and most of the NADH produced adaptation to these conditions (Pérez-Torrado et al., 2015; is oxidized during ethanol formation. The redox balance of McBryde et al., 2006). Favoring the production of glycerol yeast cells is linked with the production of metabolic sub- instead of ethanol creates new carbon sinks as metabolic products such as glycerol, acetaldehyde and acetic acid. end points for sugar-derived carbon and was shown as a Rerouting metabolic carbon flux towards desired end points possible way to reduce ethanol production (Pretorius, that maintain the redox balance, can lead to a decrease in 2000). ethanol production (Bakker et al., 2000; Kutyna et al., Hyperosmotic stress imposed to yeasts causes a 2010). decrease of growth rate and compromises cell integrity. Given that the glycolytic pathway is regulated by the Cells respond to the stress imposed by NaCl or KCl by expression of gene products, the application of genetic increasing the internal concentration of osmoprotective engineering to modify the expression of a target gene osmolytes such as glycerol. To allow the increased seems to be the easiest way to redirect the alcoholic production of glycerol, the cell needs to guarantee enough pathway (Schmidtke et al., 2012). However, there is a strict amount of cytoplasmatic NADH. The most direct way to regulation about the use of GMO in food and a general increase the amount of free NADH is redirecting alcoholic concern about the possible risks that genetically modified pathway to the oxidation of acetaldehyde to acetate instead food can bring to public health (Pérez-Torrado et al., 2015). of its reduction to ethanol (Ölz et al., 1993). The difficult acceptance of GMO-based food had open Another way to limit ethanol production is the utilization way to the development of non-GMO approaches. Species of metabolic blockers that inhibit the action of some of the genus Saccharomyces are the most important and enzymes. Furfural is an aromatic aldehyde that has an the most used yeasts, since they are able to grow in high inhibitory effect in the glycolytic pathway and it is toxic to sugar mediums at low pH and can survive to high ethanol yeast cells. The natural reaction of cells to its toxicity is concentrations (Pretorius, 2000). Although non- breaking down furfural to their corresponding and less toxic Saccharomyces species lack competitiveness as alcohol, furfuryl alcohol (Heer & Sauer, 2008; Ozturk & Anli, fermentative yeasts under oenological conditions, mainly 2014). Alcohol dehydrogenase promotes the reduction of because their lower resistance to increasing concentrations acetaldehyde into ethanol and, when present, convert of ethanol, they can be used with S. cerevisiae to directly furfural into furfuryl alcohol. Moreover, aldehyde ferment grape must. The non-Saccharomyces yeasts dehydrogenase can oxidized furfural to furoic acid produce considerable less amount of ethanol and may have especially under aerobic conditions. This process is also a beneficial influence on the volatile composition of wines one way of yeast cells to survive to the toxic effects of and S. cerevisiae prevents an incomplete fermentation of furfural, since furoic acid is less acid than acetic acid (Modig grapes controlling the fermentation after non- et al., 2002). Saccharomyces yeast start losing their viability (Quirós et The present work aims to set up adaptive evolution lines al., 2014; Pérez-Torrado et al., 2015; Ozturk & Anli, 2014). with KCl and furfural as evolutive pressures for each yeast Increasing the resistance of non-Saccharomyces strain and additionally to perform mutagenesis with EMS to yeasts to the stress conditions imposed by ethanol makes obtain mutagenized yeast cells for adaptive evolution. The them strong candidates to
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