Saccharomyces cerevisiae and grape juice are 'natural companions' and make a happy wine marriage. However, this relationship can be enriched by allowing 'wild' non-Saccharomyces yeast to participate in a sequential manner in the early phases of grape must fermentation. However, such a triangular relationship is complex and can only be taken to 'the next level' if there are no spoilage yeast present and if the 'wine yeast' - S. cerevisiae - is able to exert its dominance in time to successfully complete the alcoholic fermentation. Winemakers apply various 'matchmaking' strategies (e.g. cellar hygiene, pH, SO2 , temperature and nutrient management) to keep 'spoilers' (e.g. Dekkera bruxellensis) at bay, and allow 'compatible' wild yeast (e.g. Torulaspora delbrueckii, Pichia kluyveri, Lachancea thermotolerans and Candida/Metschnikowia pulcherrima) to harmonize with potent S. cerevisiae wine yeast and bring the best out in wine. Mismatching can lead to a 'two is company, three is a crowd' scenario. More than 40 of the 1500 known yeast species have been isolated from grape must. In this article, we review the specific flavour-active characteristics of those non-Saccharomyces species that might play a positive role in both spontaneous and inoculated wine ferments. We seek to present 'single-species' and 'multi-species' ferments in a new light and a new context, and we raise important questions about the direction of mixed-fermentation research to address market trends regarding so-called 'natural' wines. This review also highlights that, despite the fact that most frontier research and technological developments are often focussed primarily on S. cerevisiae, non-Saccharomyces research can benefit from the techniques and knowledge developed by research on the former.

bOver recent decades, the average ethanol concentration of wine has increased, largely due to consumer preference for wine styles associated with increased grape maturity; sugar content increases with grape maturity, and this translates into increased alcohol content in wine. However, high ethanol content impacts wine sensory properties, reducing the perceived complexity of flavors and aromas. In addition, for health and economic reasons, the wine sector is actively seeking technologies to facilitate the production of wines with lower ethanol content. Nonconventional yeast species, in particular, non-Saccharomyces yeasts, have shown potential for producing wines with lower alcohol content. These yeast species, which are largely associated with grapes preharvest, are present in the early stages of fermentation but, in general, are not capable of completing alcoholic fermentation. We have evaluated 50 different non-Saccharomyces isolates belonging to 24 different genera for their capacity to produce wine with a lower ethanol concentration when used in sequential inoculation regimes with a Saccharomyces cerevisiae wine strain. A sequential inoculation of Metschnikowia pulcherrima AWRI1149 followed by an S. cerevisiae wine strain was best able to produce wine with an ethanol concentration lower than that achieved with the single-inoculum, wine yeast control. Sequential fermentations utilizing AWRI1149 produced wines with 0.9% (vol/vol) and 1.6% (vol/vol) (corresponding to 7.1 g/liter and 12.6 g/liter, respectively) lower ethanol concentrations in Chardonnay and Shiraz wines, respectively. In Chardonnay wine, the total concentration of esters and higher alcohols was higher for wines generated from sequential inoculations, whereas the total concentration of volatile acids was significantly lower. In sequentially inoculated Shiraz wines, the total concentration of higher alcohols was higher and the total concentration of volatile acids was reduced compared with those in control S. cerevisiae wines, whereas the total concentrations of esters were not significantly different.

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Problematic fermentations are common in the wine industry. Assimilable nitrogen deficiency is the most prevalent cause of sluggish fermentations and can reduce fermentation rates significantly. A lack of nitrogen diminishes a yeast's metabolic activity, as well as the biomass yield, although it has not been clear which of these two interdependent factors is more significant in sluggish fermentations. Under winemaking conditions with different initial nitrogen concentrations, metabolic flux analysis was used to isolate the effects. We quantified yeast physiology and identified key metabolic fluxes. We also performed cell concentration experiments to establish how biomass yield affects the fermentation rate. Intracellular analysis showed that trehalose accumulation, which is highly correlated with ethanol production, could be responsible for sustaining cell viability in nitrogen-poor musts independent of the initial assimilable nitrogen content. Other than the higher initial maintenance costs in sluggish fermentations, the main difference between normal and sluggish fermentations was that the metabolic flux distributions in nitrogen-deficient cultures revealed that the specific sugar uptake rate was substantially lower. The results of cell concentration experiments, however, showed that in spite of lower sugar uptake, adding biomass from sluggish cultures not only reduced the time to finish a problematic fermentation but also was less likely to affect the quality of the resulting wine as it did not alter the chemistry of the must.Sluggish and stuck fermentations are common in the wine industry. Factors that affect the yeast growth rate and that lead to problematic fermentations include limited nutrient contents, ethanol toxicity, fatty acid toxicity, temperature extremes (5), the ratio of nitrogen sources to carbon sources in the medium (21), and the initial quantity and quality of the amino acids (29). Sluggish cultures may prolong the process time from days to weeks. In stuck fermentations, high levels of residual sugar are left following the arrest of fermentation. Several difficulties occur at the cellular level; these include diminished sugar transport capacity, the inability of yeasts to produce compatible solutes in response to high osmolality (20,28), and cell membrane integrity issues in the presence of high concentrations of ethanol (8). As such mechanisms may act simultaneously in cells and to various extents, it is difficult to isolate and reproduce the mechanisms in stuck and sluggish fermentations.Quantitatively, nitrogen is the second most abundant nutrient in wine fermentations. It is essential for yeast metabolism and growth. Consequently, a lack of nitrogen triggers sluggish fermentations (1,8,13). In previous studies workers found differences in nitrogen-related curves (e.g., curves of biomass versus assimilable nitrogen) which indicated that the fermentation rate and biomass yield functions are distinct (9, 10; W. Agenbach, Proc. S. Afr. Soc. Enol. Vitic., p. 66-88, 1977). In other studies the ...

The yeast Saccharomyces cerevisiae synthesises a variety of volatile aroma compounds during wine fermentation. In this study, the influence of fermentation temperature on (1) the production of yeast-derived aroma compounds and (2) the expression of genes involved in aroma compounds' metabolism (ADH1, PDC1, BAT1, BAT2, LEU2, ILV2, ATF1, ATF2, EHT1 and IAH1) was assessed, during the fermentation of a defined must at 15 and 28 degrees C. Higher concentrations of compounds related to fresh and fruity aromas were found at 15 degrees C, while higher concentrations of flowery related aroma compounds were found at 28 degrees C. The formation rates of volatile aroma compounds varied according to growth stage. In addition, linear correlations between the increases in concentration of higher alcohol and their corresponding acetates were obtained. Genes presented different expression profiles at both temperatures, except ILV2, and those involved in common pathways were co-expressed (ADH1, PDC1 and BAT2; and ATF1, EHT1 and IAH1). These results demonstrate that the fermentation temperature plays an important role in the wine final aroma profile, and is therefore an important control parameter to fine-tune wine quality during winemaking.

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SummaryMycolic acids are vital components of the cell wall of the tubercle bacillus Mycobacterium tuberculosis and are required for viability and virulence. While mycolic acid biosynthesis is studied extensively, components involved in mycolate transport remain unidentified. We investigated the role of large membrane proteins encoded by mmpL genes in mycolic acid transport in mycobacteria and the related corynebacteria. MmpL3 was found to be essential in mycobacteria and conditional depletion of MmpL3 in Mycobacterium smegmatis resulted in loss of cell wall mycolylation, and of the cell wall-associated glycolipid, trehalose dimycolate. In parallel, an accumulation of trehalose monomycolate (TMM) was observed, suggesting that mycolic acids were transported as TMM. In contrast to mycobacteria, we found redundancy in the role of two mmpL genes, in Corynebacterium glutamicum; a complete loss of trehalose-associated and cell wall bound corynomycolates was observed in an NCgl0228-NCgl2769 double mutant, but not in individual single mutants. Our studies highlight the role of mmpL genes in mycolic acid metabolism and identify potential new targets for anti-TB drug development.

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