Pilar Morales

Selection of non-Saccharomyces yeast strains for reducing alcohol levels in wine by sugar respiration.

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.

Non-conventional Yeast Species for Lowering Ethanol Content of Wines.

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 non-conventional 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.

Purification and characterization of a xylanase and an arabinofuranosidase from Bacillus polymyxa

Abstract Two hemicellulases from Bacillus polymyxa were purified and characterized: a xylanase with a molecular mass of 61 kD and pl of 4.7 and an arabinofuranosidase with a molecular mass of 166 kD and pl of 4.7. The xylanase, which showed increased thermostability in the presence of MgCl2, showed a typical endo-action mode on xylans from several sources. The arabinofuranosidase was only active on (1→5)-α- l -arabinooligosaccharides but not on linear (1→5)-α- l -arabinan, arabinogalactan, and arabinoxylan. However, it was able to release arabinose from arabinoxylan when an active endoxylanase was also present in hydrolysis assays.

Metabolic Flux Analysis during the Exponential Growth Phase of Saccharomyces cerevisiae in Wine Fermentations

As a consequence of the increase in global average temperature, grapes with the adequate phenolic and aromatic maturity tend to be overripe by the time of harvest, resulting in increased sugar concentrations and imbalanced C/N ratios in fermenting musts. This fact sets obvious additional hurdles in the challenge of obtaining wines with reduced alcohols levels, a new trend in consumer demands. It would therefore be interesting to understand Saccharomyces cerevisiae physiology during the fermentation of must with these altered characteristics. The present study aims to determine the distribution of metabolic fluxes during the yeast exponential growth phase, when both carbon and nitrogen sources are in excess, using continuous cultures. Two different sugar concentrations were studied under two different winemaking temperature conditions. Although consumption and production rates for key metabolites were severely affected by the different experimental conditions studied, the general distribution of fluxes in central carbon metabolism was basically conserved in all cases. It was also observed that temperature and sugar concentration exerted a higher effect on the pentose phosphate pathway and glycerol formation than on glycolysis and ethanol production. Additionally, nitrogen uptake, both quantitatively and qualitatively, was strongly influenced by environmental conditions. This work provides the most complete stoichiometric model used for Metabolic Flux Analysis of S. cerevisiae in wine fermentations employed so far, including the synthesis and release of relevant aroma compounds and could be used in the design of optimal nitrogen supplementation of wine fermentations.

Purification and characterization of an arabinofuranosidase from Bacillus polymyxa expressed in Bacillus subtilis.

Two polypeptides showing α-l-arabinofuranosidase activity have been purified to homogeneity from culture supernatants of a Bacillus subtilis clone harbouring the xynD gene [Gosalbes et al. (1991) J Bacteriol 173: 7705–7710] from Bacillus polymyxa. Both polypeptides, with determined molecular masses of 64 kDa and 53 kDa, share the same sequence at their N termini, which also coincides with the sequence deduced for the mature protein from the previously determined sequence of nucleotides (Gosalbes et al. 1991). The two polypeptides have been biochemically characterized. Arabinose is the unique product released from arabinose-containing xylans which are substrates for both enzyme forms. Other natural arabinose-containing polysaccharides, such as arabinogalactans, are not attacked by them but some artificial arabinose derivatives are good substrates for both polypeptides. Their arabinose-releasing activity on arabinoxylans facilitates the hydrolysis of the xylan backbone by some endoxylanases from Bacillus polymyxa.

Genome-wide study of the adaptation of Saccharomyces cerevisiae to the early stages of wine fermentation.

This work was designed to identify yeast cellular functions specifically affected by the stress factors predominating during the early stages of wine fermentation, and genes required for optimal growth under these conditions. The main experimental method was quantitative fitness analysis by means of competition experiments in continuous culture of whole genome barcoded yeast knockout collections. This methodology allowed the identification of haploinsufficient genes, and homozygous deletions resulting in growth impairment in synthetic must. However, genes identified as haploproficient, or homozygous deletions resulting in fitness advantage, were of little predictive power concerning optimal growth in this medium. The relevance of these functions for enological performance of yeast was assessed in batch cultures with single strains. Previous studies addressing yeast adaptation to winemaking conditions by quantitative fitness analysis were not specifically focused on the proliferative stages. In some instances our results highlight the importance of genes not previously linked to winemaking. In other cases they are complementary to those reported in previous studies concerning, for example, the relevance of some genes involved in vacuolar, peroxisomal, or ribosomal functions. Our results indicate that adaptation to the quickly changing growth conditions during grape must fermentation require the function of different gene sets in different moments of the process. Transport processes and glucose signaling seem to be negatively affected by the stress factors encountered by yeast in synthetic must. Vacuolar activity is important for continued growth during the transition to stationary phase. Finally, reduced biogenesis of peroxisomes also seems to be advantageous. However, in contrast to what was described for later stages, reduced protein synthesis is not advantageous for the early (proliferative) stages of the fermentation process. Finally, we found adenine and lysine to be in short supply for yeast growth in some natural grape musts.

Early transcriptional response to biotic stress in mixed starter fermentations involving Saccharomyces cerevisiae and Torulaspora delbrueckii

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 CO2 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.

Genome-wide identification of genes involved in growth and fermentation activity at low temperature in Saccharomyces cerevisiae

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.

An impaired ubiquitin ligase complex favors initial growth of auxotrophic yeast strains in synthetic grape must

We used experimental evolution in order to identify genes involved in the adaptation of Saccharomyces cerevisiae to the early stages of alcoholic fermentation. Evolution experiments were run for about 200 generations, in continuous culture conditions emulating the initial stages of wine fermentation. We performed whole-genome sequencing of four adapted strains from three independent evolution experiments. Mutations identified in these strains pointed to the Rsp5p-Bul1/2p ubiquitin ligase complex as the preferred evolutionary target under these experimental conditions. Rsp5p is a multifunctional enzyme able to ubiquitinate target proteins participating in different cellular processes, while Bul1p is an Rsp5p substrate adaptor specifically involved in the ubiquitin-dependent internalization of Gap1p and other plasma membrane permeases. While a loss-of-function mutation in BUL1 seems to be enough to confer a selective advantage under these assay conditions, this did not seem to be the case for RSP5 mutated strains, which required additional mutations, probably compensating for the detrimental effect of altered Rsp5p activity on essential cellular functions. The power of this experimental approach is illustrated by the identification of four independent mutants, each with a limited number of SNPs, affected within the same pathway. However, in order to obtain information relevant for a specific biotechnological process, caution must be taken in the choice of the background yeast genotype (as shown in this case for auxotrophies). In addition, the use of very stable continuous fermentation conditions might lead to the selection of a rather limited number of adaptive responses that would mask other possible targets for genetic improvement.

New insights into the advantages of ammonium as a winemaking nutrient.

Nitrogen limitation is the most common cause for stuck or sluggish fermentation in winemaking, and it is usually dealt with by supplementing grape juice with either ammonium salts or organic nutrients. These practices have a direct impact on both fermentation kinetics and the sensorial features of the final product. The aim of this work is to provide a detailed characterization of yeast physiology in response to ammonium supplementation during alcoholic fermentation. This is done by determining changes in metabolic rates on a high frequency basis, as a sensitive way to detect the impact of fermentation conditions on yeast physiology. Our results indicate that the choice of supplementation strategy has an impact on several enological parameters like fermentation length, volatile acidity, final glycerol content, and aroma profile. Interestingly, a higher proportion of ammonium relates with improved glycerol and volatile acidity, for the same global yeast assimilable nitrogen content. However, ammonium over-supplementation has a negative impact on quality related parameters, notably on volatile acidity and aroma complexity. Production kinetics and final content of several volatile compounds are also differentially influenced by standard or excess ammonium supplementation.