Saccharomyces cerevisiae Cells Lacking the Zinc Vacuolar Transporter Zrt3 Display Improved Ethanol Productivity in Lignocellulosic Hydrolysates

Yeast-based bioethanol production from lignocellulosic hydrolysates (LH) is an attractive and sustainable alternative for biofuel production. However, the presence of acetic acid (AA) in LH is still a major problem. Indeed, above certain concentrations, AA inhibits yeast fermentation and triggers a regulated cell death (RCD) process mediated by the mitochondria and vacuole. Understanding the mechanisms involved in AA-induced RCD (AA-RCD) may thus help select robust fermentative yeast strains, providing novel insights to improve lignocellulosic ethanol (LE) production. Herein, we hypothesized that zinc vacuolar transporters are involved in vacuole-mediated AA-RCD, since zinc enhances ethanol production and zinc-dependent catalase and superoxide dismutase protect from AA-RCD. In this work, zinc limitation sensitized wild-type cells to AA-RCD, while zinc supplementation resulted in a small protective effect. Cells lacking the vacuolar zinc transporter Zrt3 were highly resistant to AA-RCD, exhibiting reduced vacuolar dysfunction. Moreover, zrt3Δ cells displayed higher ethanol productivity than their wild-type counterparts, both when cultivated in rich medium with AA (0.29 g L−1 h−1 versus 0.11 g L−1 h−1) and in an LH (0.73 g L−1 h−1 versus 0.55 g L−1 h−1). Overall, the deletion of ZRT3 emerges as a promising strategy to increase strain robustness in LE industrial production.

High Potential of Pichia kluyveri and Other Pichia Species in Wine Technology

The surfaces of grapes are covered by different yeast species that are important in the first stages of the fermentation process. In recent years, non-Saccharomyces yeasts such as Torulaspora delbrueckii, Lachancea thermotolerans, Metschnikowia pulcherrima, and Pichia kluyveri have become popular with regard to winemaking and improved wine quality. For that reason, several manufacturers started to offer commercially available strains of these non-Saccharomyces species. P. kluyveri stands out, mainly due to its contribution to wine aroma, glycerol, ethanol yield, and killer factor. The metabolism of the yeast allows it to increase volatile molecules such as esters and varietal thiols (aroma-active compounds), which increase the quality of specific varietal wines or neutral ones. It is considered a low- or non-fermentative yeast, so subsequent inoculation of a more fermentative yeast such as Saccharomyces cerevisiae is indispensable to achieve a proper fermented alcohol. The impact of P. kluyveri is not limited to the grape wine industry; it has also been successfully employed in beer, cider, durian, and tequila fermentation, among others, acting as a promising tool in those fermentation processes. Although no Pichia species other than P. kluyveri is available in the regular market, several recent scientific studies show interesting improvements in some wine quality parameters such as aroma, polysaccharides, acid management, and color stability. This could motivate yeast manufacturers to develop products based on those species in the near future.

The Important Contribution of Non-Saccharomyces Yeasts to the Aroma Complexity of Wine: A Review

Non-Saccharomyces yeast plays an important role in the initial stages of a wild ferment, as they are found in higher abundance in the vineyard than Saccharomyces cerevisiae. As such, there has been a focus in recent years to isolate these yeast species and characterize their effect on wine fermentation and subsequent aroma. This effect on wine aroma is often species and strain dependent, as the enzymatic profile of each yeast will determine which aroma compounds are formed as secondary metabolites. Semi-fermentative yeast, such as Hanseniaspora spp., Candida spp. and Metschnikowia pulcherrima, are commonly in high abundance in fresh grape must and have diverse enzymatic profiles, however they show a weak tolerance to ethanol, limiting their impact to the initial stages of fermentation. Fully fermentative non-Saccharomyces yeast, characterized by high ethanol tolerance, are often found at low abundance in fresh grape must, similar to Saccharomyces cerevisiae. Their ability to influence the aroma profile of wine remains high, however, due to their presence into the final stages of fermentation. Some fermentative yeasts also have unique oenological properties, such as Lanchancea thermotolerans and Schizosaccharomyces pombe, highlighting the potential of these yeast as inoculants for specific wine styles.

Looking at the Origin: Some Insights into the General and Fermentative Microbiota of Vineyard Soils

In winemaking processes, there is a current tendency to develop spontaneous fermentations taking advantage of the metabolic diversity of derived from the great microbial diversity present in grape musts. This enological practice enhances wine complexity, but undesirable consequences or deviations could appear on wine quality. Soil is a reservoir of important microorganisms for different beneficial processes, especially for plant nutrition, but it is also the origin of many of the phytopathogenic microorganisms that affect vines. In this study, a meta-taxonomic analysis of the microbial communities inhabiting vineyard soils was realized. A significant impact of the soil type and climate aspects (seasonal patterns) was observed in terms of alpha and beta bacterial diversity, but fungal populations appeared as more stable communities in vineyard soils, especially in terms of alpha diversity. Focusing on the presence and abundance of wine-related microorganisms present in the studied soils, some seasonal and soil-dependent patterns were observed. The Lactobacillaceae family, containing species responsible for the malolactic fermentation, was only present in non-calcareous soils samples and during the summer season. The study of wine-related fungi indicated that the Debaryomycetaceae family dominates the winter yeast population, whereas the Saccharomycetaceae family, containing the most important fermentative yeast species for winemaking, was detected as dominant in summer.

Whole-Genome Sequence of Starmerella bacillaris PAS13, a Nonconventional Enological Yeast with Antifungal Activity

ABSTRACT Starmerella bacillaris is a fermentative yeast commonly found in vineyards. Here, we present the draft genome sequence of S. bacillaris PAS13, a nonconventional enological yeast with a potential role as a biocontrol agent. This gene sequence will provide insights into the genetic basis of yeast activity against gray mold disease (Botrytis cinerea).

Evolution of ecological dominance of yeast species in high-sugar environments

In budding yeasts, fermentation in the presence of oxygen evolved around the time of a whole genome duplication (WGD) and is thought to confer dominance in high-sugar environments because ethanol is toxic to many species. While there are many fermentative yeast species, only Saccharomyces cerevisiae consistently dominates wine fermentations. In this study, we use co-culture experiments and intrinsic growth rate assays to examine the relative fitness of non-WGD and WGD yeast species across environments to assess when S. cerevisiae's ability to dominate high-sugar environments arose. We show that S. cerevisiae dominates nearly all other non-WGD and WGD species except for its sibling species S. paradoxus in both grape juice and a high-sugar rich medium. Of the species we tested, S. cerevisiae and S. paradoxus have evolved the highest ethanol tolerance and intrinsic growth rate in grape juice. However, the ability of S. cerevisiae and S. paradoxus to dominate certain species depends on the temperature and the type of high-sugar environment. Our results indicate that dominance of high-sugar environments evolved much more recently than the WGD, most likely just prior to or during the differentiation of Saccharomyces species, and that evolution of multiple traits contributes to S. cerevisiae's ability to dominate wine fermentations.