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Sulfiting agents are among the most widely used preservatives in the food and beverages industries, including winemaking, and one of their main functions is inhibition of spoilage microorganisms. We have used a whole genome quantitative fitness analysis in order to improve our knowledge on yeast tolerance to sulfites. Apart from the contribution of sulfite efflux to tolerance, results point to vesicle-mediated transport, autophagy and vacuolar activity as the main cellular functions required to survive sulfite challenges. The involvement of autophagic and vacuolar functions in sulfite tolerance was further confirmed by pairwise competition using a newly constructed atg2-defective strain, as well as by showing induction of ATG8 expression by sulfite. Autophagy is required for the turnover of proteins and subcellular structures damaged by sulfite. In addition, the requirement for vacuolar functions might be related to its role in intracellular pH homeostasis. Finally, the involvement of the sulfite pump Ssu1 and the transcription factor Fzf1 in sulfite tolerance by Saccharomyces cerevisiae was confirmed; a result that validates the experimental approach used in this work. These findings have relevance for understanding sulfite toxicity and tolerance, as well as for the eventual design of strategies aiming to control yeast spoilage. ; This work was funded by the Spanish Ministry of Science, Innovation and Universities and European Union through grant AGL2015‐63629‐R (MINECO/FEDER UE); and Junta de Andalucía through grant AGR6544. JT is funded by FGCSIC by the COMFUTURO program

Industrial wine yeast strains show genome particularities, with strains showing polyploid genomes or chromosome copy number variations, being easier to identify. Although these genomic structures have classically been considered transitory steps in the genomic adaptation to new environmental conditions, they may be more stable than thought. These yeasts are highly specialized strains able to cope with the different stresses associated with the fermentation process, from the high osmolarity to the final ethanol content. In this work, we use adaptive laboratory evolution, focusing on the initial steps of the fermentation process, where growth rate is maximum, to provide new insights into the role of the different genomic and chromosomic rearrangements that occur during adaptation to wine conditions, and providing an understanding of the chronology of the different evolutionary steps. ; This work was funded by the Spanish Ministry of Science, Innovation and Universities by the CoolWine Project (ERA CoBioTech) and grants AGL2012-32064 and AGL2015-63629-R (co-financed by FEDER funds). AM was the recipient to a FPI fellowship from the Spanish Government. ; Peer reviewed

The growing interest of the winemaking industry on the use of non-Saccharomyces starters has prompted several studies about the physiological features of this diverse group of microorganisms. The fact that the proposed use of these new starters will almost invariably involve either simultaneous or sequential inoculation with Saccharomyces cerevisiae has also driven the attention to the potential biological interactions between different starters during wine fermentation. Our current understanding is that alternative yeast starters will affect wine features by both direct and indirect mechanisms (through metabolic or other types of interactions with S. cerevisiae). There are still few studies addressing the question of yeast–yeast interactions in winemaking by a transcriptomic approach. In a previous report, we revealed early responses of S. cerevisiae and Torulaspora delbrueckii to the presence of each other under anaerobic conditions, mainly the overexpression of genes related with sugar consumption and cell proliferation. We have now studied the response, under aerobic conditions, of S. cerevisiae to other two non-Saccharomyces species, Hanseniaspora uvarum and Candida sake, keeping T. delbrueckii as a reference; and always focusing on the early stages of the interaction. Results point to some common features of the way S. cerevisiae modifies its transcriptome in front of other yeast species, namely activation of glucose and nitrogen metabolism, being the later specific for aerobic conditions. ; This work was supported by grant AGL2015-63629-R (MINECO/FEDER, UE), YeSVitE consortium (EU project, 7FP-IRSES-GA no. 612441), JC is the recipient of a MINECO Formación Postdoctoral contract from the Spanish Government. ; Peer reviewed

We have recently shown that ethanol yields in winemaking can be reduced by taking advantage of the respiratory metabolism of some non-Saccharomyces yeast species. Using an orthogonal design we have now addressed the impact of three environmental factors (temperature, nitrogen source, and oxygen supply level) on the aerobic metabolism in synthetic must of Saccharomyces cerevisiae, Metschnikowia pulcherrima, Kluyveromyces lactis, and Candida sake. An integrative parameter, Efficacy (efficacy for alcohol level reduction) was designed to simplify comparisons between strains or growth conditions. It integrates sugar consumption, ethanol yield, and acetic acid production data. We found a high relative impact of nitrogen source availability and temperature, as compared to aeration conditions, for several fermentation parameters, including ethanol yield. However, increasing oxygen supply showed a positive impact in terms of alcohol reduction and Efficacy for all the strains tested. The best results across assays were obtained for C. sake CBS 5093, with high sugar consumption rates, associated to low ethanol yields, and very low acetic acid production. Processes involving this yeast strain would benefit from high aeration levels and low nitrogen source availability; while fermentation temperatures would have little impact on its Efficacy for alcohol level reduction. ; This work was supported by grants MINECOAGL2012-32064, INIARM2012-00007-00-00, and MINECO RTC-2014-2186-2 from the Spanish Government. A.J.R. is the recipient of a MINECO training contract, FPI; T.R. was the recipient of an Erasmus Program grant funded by the EU. ; Peer Reviewed

Contiene además supplementary material.-- This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. ; We have developed a wine fermentation procedure that takes advantage of the metabolic features of a previously characterized Metschnikowia pulcherrima strain in order to reduce ethanol production. It involves the use of M. pulcherrima/Saccharomyces cerevisiae mixed cultures, controlled oxygenation conditions during the first 48 h of fermentation, and anaerobic conditions thereafter. The influence of different oxygenation regimes and initial inoculum composition on yeast physiology and final ethanol content was studied. The impact of oxygenation on yeast physiology goes beyond the first aerated step and influences yields and survival rates during the anaerobic stage. The activity of M. pulcherrima in mixed oxygenated cultures resulted in a clear reduction in ethanol yield, as compared to S. cerevisiae. Despite relatively low initial cell numbers, S. cerevisiae always predominated in mixed cultures by the end of the fermentation process. Strain replacement was faster under low oxygenation levels. M. pulcherrima confers an additional advantage in terms of dissolved oxygen, which drops to zero after a few hours of culture, even under highly aerated conditions, and this holds true for mixed cultures. Alcohol reduction values about 3.7 % (v/v) were obtained for mixed cultures under high aeration, but they were associated to unacceptable volatile acidity levels. In contrast, under optimized conditions, only 0.35 g/L acetic acid was produced, for an alcohol reduction of 2.2 % (v/v), and almost null dissolved oxygen during the process. ; Authors would like to thank Cristina Juez Ojeda andMiguel Ángel Fernández Recio for excellent technical assistance, the YeSVitE consortium (EU project, 7FP-IRSES-GA no. 612441) for helpful discussions, and Laura López Ocaña (CECT) for help with yeast species confirmation. This work was supported by grants MINECO AGL2012-32064 and INIA RM2012-00007-00-00 from the Spanish Government. M.Q. was the recipient of a CSIC training contract, JAEDoc, co-funded by the European Social Fund of the EU. ; Peer reviewed

This is an open access article under the CC BY-NC-ND license. ; Respiration of sugars by non-Saccharomyces yeasts has been recently proposed for lowering alcohol levels in wine. Development of industrial fermentation processes based on such an approach requires, amongst other steps, the identification of yeast strains which are able to grow and respire under the relatively harsh conditions found in grape must. This work describes the characterization of a collection of non-Saccharomyces yeast strains in order to identify candidate yeast strains for this specific application. It involved the estimation of respiratory quotient (RQ) values under aerated conditions, at low pH and high sugar concentrations, calculation of yields of ethanol and other relevant metabolites, and characterization of growth responses to the main stress factors found during the first stages of alcoholic fermentation. Physiological features of some strains of Metschnikowia pulcherrima or two species of Kluyveromyces, suggest they are suitable for lowering ethanol yields by respiration. The unsuitability of Saccharomyces cerevisiae strains for this purpose was not due to ethanol yields (under aerated conditions they are low enough for a significant reduction in final ethanol content), but to the high acetic acid yields under these growth conditions. According to results from controlled aeration fermentations with one strain of M. pulcherrima, design of an aeration regime allowing for lowering ethanol yields though preserving grape must components from excessive oxidation, would be conceivable. ; This work was funded by the AGL2012-32064 and RM2012-00007-00-00 projects from the Spanish Government. M.Q. was the recipient of a CSIC training JAE-Doc contract, co-funded by the European Social Fund of the EU. ; Peer reviewed

Aerobic fermentation was previously proposed to reduce the ethanol content of wine. The main constraint found for Saccharomyces cerevisiae to be used under these conditions was the high levels of acetic acid produced by all S. cerevisiae strains previously tested. This work addressed the identification of S. cerevisiae wine yeast strains suitable for aerobic fermentation and the optimization of fermentation conditions to obtain a reduced ethanol yield with acceptable volatile acidity. This approach unveiled a great diversity in acetic acid yield for different S. cerevisiae strains under aerobic conditions, with some strains showing very low volatile acidity. Three strains were selected for further characterization in bioreactors, with natural grape must, under aerobic and anaerobic conditions. Ethanol yields were lower under aerobic than under anaerobic conditions for all strains, and acetic acid levels were low for two of them. Strain-dependent changes in volatile compounds were also observed between aerobic and anaerobic conditions. Finally, the process was optimized at laboratory scale for one strain. This is the first report of S. cerevisiae wine strains showing low acetic acid production under aerobic conditions and paves the way for simplified aerobic fermentation protocols aimed to reducing the alcohol content of wines. ; This work was funded by the Spanish Government through grants AGL 2015-63629-R (co-financed by FEDER funds), PCI 2018-092949 (co-funded by ERA-CoBioTech). JT is funded by FGCSIC by the COMFUTURO program. AMG predoctoral contract is funded by Consejería de Desarrollo Económico e Innovación de la Comunidad Autónoma de La Rioja, EC was recipient of JAE-Intro fellowship from the Spanish National Research Council (CSIC).

Resumen del trabajo presentado en el EMBL Conference Molecular Mechanisms in Evolution and Ecology, celebrado de forma virtual del 30 de septiembre al 2 de octubre de 2020 ; Respiro-fermentative metabolism has been proposed as a tool to decrease the alcoholic content in wines. Saccharomyces cerevisiae strains -despite the Crabtree effect- can reduce significantly their ethanol yield under aerobic conditions, as compared to standard fermentation settings. However, an exacerbated acetic acid production associated with this condition limits the usefulness of this species for this application. In previous works, recombinant wine yeast strains defective for some carbon catabolite repression related genes showed a reduced acetic acid yield in the presence of oxygen (Curiel et al., 2016). But the GMO status also precludes its general applicability in winemaking. In this work, we used Adaptative Laboratory Evolution (ALE) to obtain partially derepressed S. cerevisiae wine strains from four different backgrounds -three of them commercially available. This was expected to act as an indirect selection for strains showing reduced acetic acid yield under aerobic conditions. The selective pressure was established by 2-deoxy-D-glucose (2-DG), using galactose as the only carbon source. The experiment was run in three replicates for each genetic background by repeated batch subculture for ~100 generations with growing concentrations of 2-DG. All the evolved populations showed improved growth on different carbon sources under carbon catabolite repression control -both fermentable and non-fermentable-, in the presence of either 2-DG or glucosamine (another non-metabolizable glucose analogue). Almost all the evolved populations were able to perform a complete fermentation of natural must, being slightly slower compared with the parental strain. In addition, all the evolved populations showed reduced acetic acid yield and a slight but significant increase in glycerol production. Singlecolony isolated from grape juice fermentations were tested for tolerance to the most common wine-industry factor stresses. Selected candidates were confirmed for acetic acid production under aerobic conditions significantly lower than the parental strain and for some of them lower than the original evolved population, below commercial wine standard thresholds. We show here a methodology to obtain in a quick manner industrial strains suitable to ferment under aerobic conditions by using a combined ALE and fermentation screening techniques. This work was funded by the Spanish Government through grant PCI2018¿092949 (cofunded by ERA¿CoBioTech). JT is funded by FGCSIC by the COMFUTURO program. AMG holds a training contract funded by La Rioja Government. Curiel JA, Salvadó Z, Tronchoni J, Morales P, Rodrigues AJ, Quirós M. Microb Cell Fact.2016;15:156

Advances in microbial wine biotechnology have led to the recent commercialization of several non-Saccharomyces starter cultures. These are intended to be used in either simultaneous or sequential inoculation with Saccharomyces cerevisiae. The different types of microbial interactions that can be stablished during wine fermentation acquire an increased relevance in the context of these mixed-starter fermentations. We analysed the transcriptional response to co-cultivation of S. cerevisiae and Torulaspora delbrueckii. The study focused in the initial stages of wine fermentation, before S. cerevisiae completely dominates the mixed cultures. Both species showed a clear response to the presence of each other, even though the portion of the genome showing altered transcription levels was relatively small. Changes in the transcription pattern suggested a stimulation of metabolic activity and growth, as a consequence of the presence of competitors in the same medium. The response of S. cerevisiae seems to take place earlier, as compared to T. delbrueckii. Enhanced glycolytic activity of the mixed culture was confirmed by the CO production profile during these early stages of fermentation. Interestingly, HSP12 expression appeared induced by co-cultivation for both of S. cerevisiae and Torulaspora delbrueckii in the two time points studied. This might be related with a recently described role of Hsp12 in intercellular communication in yeast. Expression of S. cerevisiae PAU genes was also stimulated in mixed cultures. ; This work was supported by grant AGL2015-63629-R (MINECO/FEDER, UE), JAC is the recipient of a MINECO Formación Postdoctoral contract from the Spanish Government. ; Peer Reviewed

The respiratory metabolism of yeast species alternative to Saccharomyces cerevisiae has been explored in recent years as a tool to reduce ethanol content in grape wine. The efficacy of this strategy has been previously proven for mixed cultures of non-Saccharomyces and S. cerevisiae strains. In this work, we perform a transcriptomic analysis of the Crabtree-negative yeast Kluyveromyces lactis under tightly controlled growth conditions in order to better understand physiology of non-Saccharomyces yeasts during the fermentation of grape must under aerated conditions. Transcriptional changes in K. lactis are mainly driven by oxygen limitation, iron requirement, and oxidative stress. Oxidative stress appears as a consequence of the hypoxic conditions achieved by K. lactis once oxygen supply is no longer sufficient to sustain fully respiratory metabolism. This species copes with low oxygen and iron availability by repressing iron consuming pathways and activating iron transport mechanisms. Most of the physiological and transcriptomic features of K. lactis in aerobic wine fermentation are not shared with the Crabtree-positive yeast S. cerevisiae. ; This work was supported by the Spanish Government trough MINECO and FEDER (AGL2015-63629-R) funds: MINECO AGL2015-63629-R grants, MINECO training contract for A.J. Rodrigues, and MINECO Formación Postdoctoral contract for J.A. Curiel. ; Peer reviewed

By applying different classical and fast protein liquid chromatographic techniques, three xylanases (β-1,4-d-xylan xylanhydrolase) were purified to homogeneity from the extracellular enzymatic complex of Bacillus polymyxa. The three enzymes (X34C, X34E, and X22) were small proteins of 34, 34, and 22 kDa and basic pIs 9.3, >9.3, and 9.0, respectively. X34C and X34E are closely related and seem to be isoforms of the same enzyme. However, they differ in some characteristics. The three enzymes had different pH and temperature optima. One of them, X34E, showed a high thermal stability. The Vmax values determined for X34C, X34E, and X22 enzymes on oat spelts xylan were 14.9, 85.5, and 64.0 U mg-1, respectively, and 16.1, 62.0, and 150.6 U mg-1 on birchwood xylan. When oat spelts xylan was the substrate used, Km values of 3.4, 2.4, and 1.9 mg ml-1 were obtained for X34C, X34E, and X22 enzymes, respectively, and 0.65, 6.3, and 0.32 mg ml-1 were the respective Km values determined with birchwood xylan as the substrate. The enzymes were nondebranching endo-β-xylanases. Xylose was one of the products of xylan hydrolysis by xylanases X34C and X34E, but this monosaccharide was not released by X22 enzyme. However, neither of the enzymes was able to degrade xylobiose ; This research was supported by grants Bio/88-0217 and ALI91- 0336 from Comisi6n Interministerial de Ciencia y Tecnologia (CICyT) of the Spanish Government ; Peer reviewed

In winemaking, the use of alternative yeast starters is becoming increasingly popular. They contribute to the diversity and complexity of wine sensory features and are typically used in combination with Saccharomyces cerevisiae , to ensure complete fermentation. This practice has drawn the interest on interactions between different oenological yeasts, which are also relevant in spontaneous and conventional fermentations, or in the vineyard. Although several interactions have been described and some mechanisms have been suggested, the possible involvement of extracellular vesicles (EVs) has not yet been considered. This work describes the production of EVs by six wine yeast species (S. cerevisiae , Torulaspora delbrueckii , Lachancea thermotolerans , Hanseniaspora uvarum , Candida sake and Metschnikowia pulcherrima ) in synthetic grape must. Proteomic analysis of EV‐enriched fractions from S. cerevisiae and T. delbrueckii showed enrichment in glycolytic enzymes and cell‐wall‐related proteins. The most abundant protein found in S. cerevisiae , T. delbrueckii and L. thermotolerans EV‐enriched fractions was the enzyme exo‐1,3‐β‐glucanase. However, this protein was not involved in the here‐observed negative impact of T. delbrueckii extracellular fractions on the growth of other yeast species. These findings suggest that EVs may play a role in fungal interactions during wine fermentation and other aspects of wine yeast biology. ; This work was funded by the Spanish Government through grants AGL2015-63629-R (co-financed by FEDER funds), PCI2018-092949 (co-funded by ERA-CoBioTech) and BES-2016-077557 (training contract for AM). JT is funded by FGCSIC by the COMFUTURO program. ; Peer reviewed

[Background] Aerobic fermentation of grape must, leading to respiro-fermentative metabolism of sugars, has been proposed as way of reducing alcohol content in wines. Two factors limit the usefulness of Saccharomyces cerevisiae for this application, the Crabtree effect, and excess volatile acidity under aerobic conditions. This work aimed to explore the impact on ethanol acetate production of different S. cerevisiae strains deleted for genes previously related with the Crabtree phenotype. [Results] Recombinant strains were constructed on a wine industrial genetic background, FX10. All yeast strains, including FX10, showed respiro-fermentative metabolism in natural grape must under aerobic conditions, as well as a concomitant reduction in ethanol yield. This indicates that the Crabtree effect is not a major constrain for reaching relevant respiration levels in grape must. Indeed, only minor differences in ethanol yield were observed between the original and some of the recombinant strains. In contrast, some yeast strains showed a relevant reduction of acetic acid production. This was identified as a positive feature for the feasibility of alcohol level reduction by respiration. Reduced acetic acid production was confirmed by a thorough analysis of these and some additional deletion strains (involving genes HXK2, PYK1, REG1, PDE2 and PDC1). Some recombinant yeasts showed altered production of glycerol and pyruvate derived metabolites. [Conclusions] REG1 and PDC1 deletion strains showed a strong reduction of acetic acid yield in aerobic fermentations. Since REG1 defective strains may be obtained by non-GMO approaches, these gene modifications show good promise to help reducing ethanol content in wines. ; This work was supported in part by the Spanish Government trough MINECO and FEDER funds: MINECO AGL2015-63629-R, MINECO training contract for AJR, and MINECO Formación Postdoctoral contract for JAC. The authors are also grateful Junta de Andalucía (P10_AGR6544) and to Laffort SA (Bordeaux-France) for financial support and for providing the S. cerevisie FX10 strain. ; Peer Reviewed

Fermentation at low temperatures is one of the most popular current winemaking practices because of its reported positive impact on the aromatic profile of wines. However, low temperature is an additional hurdle to develop Saccharomyces cerevisiae wine yeasts, which are already stressed by high osmotic pressure, low pH and poor availability of nitrogen sources in grape must. Understanding the mechanisms of adaptation of S. cerevisiae to fermentation at low temperature would help to design strategies for process management, and to select and improve wine yeast strains specifically adapted to this winemaking practice. The problem has been addressed by several approaches in recent years, including transcriptomic and other high-throughput strategies. In this work we used a genome-wide screening of S. cerevisiae diploid mutant strain collections to identify genes that potentially contribute to adaptation to low temperature fermentation conditions. Candidate genes, impaired for growth at low temperatures (12 °C and 18 °C), but not at a permissive temperature (28 °C), were deleted in an industrial homozygous genetic background, wine yeast strain FX10, in both heterozygosis and homozygosis. Some candidate genes were required for growth at low temperatures only in the laboratory yeast genetic background, but not in FX10 (namely the genes involved in aromatic amino acid biosynthesis). Other genes related to ribosome biosynthesis (SNU66 and PAP2) were required for low-temperature fermentation of synthetic must (SM) in the industrial genetic background. This result coincides with our previous findings about translation efficiency with the fitness of different wine yeast strains at low temperature. ; Funding from the Spanish Government trough MINECO and FEDER funds: MINECO AGL2012-32064 and AGL2015-63629-R grants, INIA RM2012-00007-00-00 grant, MINECO RTC-2014-2186-2 and MINECO PCIN-2015-143 grants is acknowledged. ; Peer reviewed

Rising sugar content in grape must, and the concomitant increase in alcohol levels in wine, are some of the main challenges affecting the winemaking industry nowadays. Among the several alternative solutions currently under study, the use of nonconventional yeasts during fermentation holds good promise for contributing to relieve this problem. Non-Saccharomyces wine yeast species comprise a high number or species, so encompassing a wider physiological diversity than Saccharomyces cerevisiae. Indeed, the current oenological interest of these microorganisms was initially triggered by their potential positive contribution to the sensorial complexity of quality wines, through the production of aroma and other sensory-active compounds. This diversity also involves ethanol yield on sugar, one of the most invariant metabolic traits of S. cerevisiae. This review gathers recent research on non-Saccharomyces yeasts, aiming to produce wines with lower alcohol content than those from pure Saccharomyces starters. Critical aspects discussed include the selection of suitable yeast strains (considering there is a noticeable intra-species diversity for ethanol yield, as shown for other fermentation traits), identification of key environmental parameters influencing ethanol yields (including the use of controlled oxygenation conditions), and managing mixed fermentations, by either the sequential or simultaneous inoculation of S. cerevisiae and non-Saccharomyces starter cultures. The feasibility, at the industrial level, of using non-Saccharomyces yeasts for reducing alcohol levels in wine will require an improved understanding of the metabolism of these alternative yeast species, as well as of the interactions between different yeast starters during the fermentation of grape must. ; Experimental work by researchers from ICVV is supported by the Spanish Government trough MINECO and FEDER funds: MINECO AGL2012-32064 and AGL2015-63629-R grants, INIA RM2012-00007-00-00 grant, MINECO RTC-2014-2186-2 grant, MINECO training contract for AR, MINECO Formación Postdoctoral contract for JC. Experimental work by the authors from Polytechnic University of Marche is supported by Ricerca Scientifica di Ateneo RSA2015. ; Peer Reviewed