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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

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

[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

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

[GAR+] prion-like elements partially relieve carbon catabolite repression in Saccharomyces cerevisiae. They have been hypothesized to contribute to wine yeast survival and alcohol level reduction, as well as communication with bacteria and stuck fermentation. In this work, we selected [GAR+] derivatives from several genetic backgrounds. They were characterized for phenotypic penetrance, heritability and confirmed as prion-like through curing by desiccation. In terms of fermentation kinetics, the impact of the prion on anaerobic wine fermentation (natural grape juice) was either neutral or negative, depending on the genetic background. Likewise, residual sugars were higher or similar for [GAR+] as compared to the cognate [gar-] strains. The prions had little or no impact on glycerol and ethanol yields; while acetic acid yields experienced the highest variations between [GAR+] and [gar-] strains. Strains analyzed under aerobic conditions followed the same pattern, with either little or no impact on fermentation kinetics, ethanol or glycerol yield; and a clearer influence on volatile acidity. Although no clear winemaking advantages were found for [GAR+] strains in this work, they might eventually show interest for some combinations of genetic background or winemaking conditions, e.g., for reducing acetic acid yield under aerated fermentation. ; This work was funded by the Spanish Ministry of Science, Innovation and Universities by grant AGL2015-63629-R (co-financed by FEDER funds). AM and AR were recipients to FPI contract, and JC of Formación Postdoctoral contract, all from the Spanish Government. JT was funded by FGCSIC by the COMFUTURO program. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI). ; Peer reviewed