Long-Term Adaption to High Osmotic Stress as a Tool for Improving Enological Characteristics in Industrial Wine Yeast.

Industrial wine yeasts owe their adaptability in constantly changing environments to a long evolutionary history that combines naturally occurring evolutionary events with human-enforced domestication. Among the many stressors associated with winemaking processes that have potentially detrimental impacts on yeast viability, growth, and fermentation performance are hyperosmolarity, high glucose concentrations at the beginning of fermentation, followed by the depletion of nutrients at the end of this process. Therefore, in this study, we subjected three widely used industrial wine yeasts to adaptive laboratory evolution under potassium chloride (KCl)-induced osmotic stress. At the end of the evolutionary experiment, we evaluated the tolerance to high osmotic stress of the evolved strains. All of the analyzed strains improved their fitness under high osmotic stress without worsening their economic characteristics, such as growth rate and viability. The evolved derivatives of two strains also gained the ability to accumulate glycogen, a readily mobilized storage form of glucose conferring enhanced viability and vitality of cells during prolonged nutrient deprivation. Moreover, laboratory-scale fermentation in grape juice showed that some of the KCl-evolved strains significantly enhanced glycerol synthesis and production of resveratrol-enriched wines, which in turn greatly improved the wine sensory profile. Altogether, these findings showed that long-term adaptations to osmotic stress can be an attractive approach to develop industrial yeasts.

The utility of iPBS retrotransposons markers to analyze genetic variation in yeast.

Yeasts are one of the main organisms in the food industry and effective components of many ecosystems. The method for identifying and detecting certain yeast species or strains is a crucial step for the food industry and should be simple, reliable, fast, and inexpensive. In our study, inter-priming binding sites (iPBS) retrotransposon marker system was employed to elucidate the genetic variability at intraspecies and interspecies levels among 112 yeast strains belonging to eight species previously obtained from fermented foods. The molecular identification of yeast strains was firstly confirmed by sequencing the D1/D2 domain of the 26S rRNA. The eight selected retrotransposon-based primers produced 278 bands, all of which were polymorphic with an average of 34.75 polymorphic fragments per primer. The averages of polymorphism information contents and the resolving power values for the iPBS marker system were 0.23 and 10.11, respectively. The genetic parameters within each yeast species obtained from iPBS markers were observed as; the percentage of polymorphic loci for each species ranging from 19.23% to 71.21%, Nei's gene diversity from 0.085 to 0.228, while Shannon's information index values ranging from 0.125 to 0.349. The value of gene flow (0.09) and genetic variation among the populations (0.85) showed higher genetic variation among the species. UPGMA analyses demonstrated considerable genetic variability in the yeast strains, clustered them according to their species, and revealed the intraspecific variation. Each of the selected iPBS primer provided enough species-discrimination. Present evaluations suggest the utility of iPBS marker system to estimate the genetic variation of yeast strains. This study is a preliminary point for further studies on the identification methodology, and population genetics of yeast species having importance in the food industry with iPBS markers.

Enhanced Production of High-Value Cyclopropane Fatty Acid in Yeast Engineered for Increased Lipid Synthesis and Accumulation.

The unique strained ring structure in cyclopropane fatty acids (CFA) conveys oxidative stability and lubricity to lipids. These attributes are highly valuable for industrial applications such as cosmetics and specialist lubrication but there is currently no commercial source of the lipid. Here, built on recently engineered strains of Saccharomyces cerevisiae, the authors have developed an efficient strategy for CFA production. Expression of the Escherichia coli cyclopropane fatty acid synthetase (Ec.CFAS) in the engineered yeast resulted in formation of cis-9,10-methylene-hexadecanoic and octadecanoic acids in both the phospholipid (PL) and triacylglycerol (TAG) fractions. CFA concentration in TAG of engineered yeast is 12 mg CFA g-1 DCW (fourfold above the strain expressing CFAS only). The yield of CFA increases from 13.2 to 68.3 mg L-1 , the highest reported in yeast, using a two-stage bioprocess strategy that separated cell growth from the lipid modification stage. Strategies for further improvement of this valuable lipid are proposed.

Synthetic biology stretching the realms of possibility in wine yeast research.

It took several millennia to fully understand the scientific intricacies of the process through which grape juice is turned into wine. This yeast-driven fermentation process is still being perfected and advanced today. Motivated by ever-changing consumer preferences and the belief that the 'best' wine is yet to be made, numerous approaches are being pursued to improve the process of yeast fermentation and the quality of wine. Central to recent enhancements in winemaking processes and wine quality is the development of Saccharomyces cerevisiae yeast strains with improved robustness, fermentation efficiencies and sensory properties. The emerging science of Synthetic Biology - including genome engineering and DNA editing technologies - is taking yeast strain development into a totally new realm of possibility. The first example of how future wine strain development might be impacted by these new 'history-making' Synthetic Biology technologies, is the de novo production of the raspberry ketone aroma compound, 4-[4-hydroxyphenyl]butan-2-one, in a wine yeast containing a synthetic DNA cassette. This article explores how this breakthrough and the imminent outcome of the international Yeast 2.0 (or Sc2.0) project, aimed at the synthesis of the entire genome of a laboratory strain of S. cerevisiae, might accelerate the design of improved wine yeasts.

Technical note: use of internal transcribed spacer for ruminal yeast identification in dairy cows.

Molecular techniques are important tools for microbiological studies in different habitats, and the internal transcribed spacer (ITS) has been proved to be useful for analyzing fungal diversity. The aim of this study was to use the ITS region to generate ruminal yeast profile and to identify ruminal yeast. DNA from ruminal digesta was extracted to amplify the ribosomal ITS region. The profile from the PCR products was visualized and the excised bands from the profile were identified as the genera Millerozyma, Pichia, Rhizomucor and Hyphopichia. Overall, the ITS resulted to be a simple, fast and sensitive approach that allowed profiling and identification of ruminal yeast that have not been previously described (Millerozyma and Hyphopichia) in the rumen microbial community.

Oral application of freeze-dried yeast particles expressing the PCV2b Cap protein on their surface induce protection to subsequent PCV2b challenge in vivo.

Porcine circovirus type 2 (PCV2) is now endemic in every major pig producing country, causing PCV-associated disease (PCVAD), linked with large scale economic losses. Current vaccination strategies are based on the capsid protein of the virus and are reasonably successful in preventing PCVAD but fail to induce sterile immunity. Additionally, vaccinating whole herds is expensive and time consuming. In the present study a "proof of concept" vaccine trial was employed to test the effectiveness of powdered freeze-dried recombinant Saccharomyces cerevisiae yeast stably expressing the capsid protein of PCV2b on its surface as an orally applied vaccine. PCV2-free pigs were given 3 doses of vaccine or left un-vaccinated before challenge with a defined PCV2b strain. Rectal temperatures were measured and serum and faeces samples were collected weekly. At the end of the study, pigs were euthanized, tissue samples taken and tested for PCV2b load by qPCR and immunohistochemistry. The peak of viraemia in sera and faeces of unvaccinated pigs was higher than that of vaccinated pigs. Additionally more sIgA was found in faeces of vaccinated pigs than unvaccinated. Vaccination was associated with lower serum concentrations of TNFα and IL-1ß but higher concentrations of IFNα and IFNγ in comparison to the unvaccinated animals. At the end of the trial, a higher viral load was found in several lymphatic tissues and the ileum of unvaccinated pigs in comparison to vaccinated pigs. The difference between groups was especially apparent in the ileum. The results presented here demonstrate a possible use for recombinant S. cerevisiae expressing viral proteins as an oral vaccine against PCV2. A powdered freeze-dried recombinant S. cerevisiae used as an oral vaccine could be mixed with feed and may offer a cheap and less labour intensive alternative to inoculation with the additional advantage that no cooling chain would be required for vaccine transport and storage.

Evaluation of different genetic procedures for the generation of artificial hybrids in Saccharomyces genus for winemaking.

Several methods based on recombinant DNA techniques have been proposed for yeast strain improvement; however, the most relevant oenological traits depend on a multitude of loci, making these techniques difficult to apply. In this way, hybridization techniques involving two complete genomes became interesting. Natural hybrid strains between different Saccharomyces species have been detected in diverse fermented beverages including wine, cider and beer. These hybrids seem to be better adapted to fluctuating situations typically observed in fermentations due to the acquisition of particular physiological properties of both parental strains. In this work we evaluated the usefulness of three different hybridization methods: spore to spore mating, rare-mating and protoplast fusion for the generation of intra- and inter-specific stable hybrids, being the first report about the comparison of different methods to obtain artificial hybrids to be used in fermentations. Spore to spore mating is an easy but time-consuming method; hybrids generated with this technique could lack some of the industrially relevant traits present in the parental strains because of the segregation occurred during meiosis and spore generation prior to hybridization. Hybrids obtained by protoplast fusion get the complete information of both parents but they are currently considered as genetically modified organisms (GMOs). Finally, hybrids obtained by rare-mating are easily obtained by the optimized methodology described in this work, they originally contain a complete set of chromosomes of both parents and they are not considered as GMOs. Hybrids obtained by means of the three methodological approaches showed a high genetic variability; however, a loss of genetic material was detected in most of them. Based on these results, it became evident that a last crucial aspect to be considered in every hybridization program is the genetic stabilization of recently generated hybrids that guarantee its invariability during future industrial utilization. In this work, a wine yeast genetic stabilization process was developed and vegetatively stable hybrids were obtained.

Transcription profiling of sparkling wine second fermentation.

There is a specific set of stress factors that yeast cells must overcome under second fermentation conditions, during the production of sparkling wines by the traditional (Champenoise) method. Some of them are the same as those of the primary fermentation of still wines, although perhaps with a different intensity (high ethanol concentration, low pH, nitrogen starvation) while others are more specific to second fermentation (low temperature, CO(2) overpressure). The transcription profile of Saccharomyces cerevisiae during primary wine fermentation has been studied by several research groups, but this is the first report on yeast transcriptome under second fermentation conditions. Our results indicate that the main pathways affected by these particular conditions are related to aerobic respiration, but genes related to vacuolar and peroxisomal functions were also highlighted in this study. A parallelism between the transcription profile of wine yeast during primary and second fermentation is appreciated, with ethanol appearing as the main factor driving gene transcription during second fermentation. Low temperature seems to also influence yeast transcription profile under these particular winemaking conditions.

Utilización de Candida guilliermondii aislada del corozo chiquito (Bactris guineensis) en la producción de xilitol / Utilization of Candida guilliermondii isolated of corozo chiquito (Bactris guineensis) in the production of xylitol

La levadura Candida guilliermondii es objeto de estudio debido a su capacidad de producir xilitol aprovechando compuestos hemicelulósicos ricos en xilosa, dado esto, la cepa Candida guilliermondii aislada del fruto del corozo chiquito (Bactris guineensis) fue usada en este estudio con el fin de evaluar su capacidad para producir xilitol sobre un sustrato hidrolizado de cascarilla de arroz. El objetivo de este trabajo fue determinar los parámetros fermentativos como producción de xilitol, productividad volumétrica (Qp) y rendimiento de sustrato en producto (Yp/s) durante la fermentación con la cepa nativa Candida guilliermondii. Se emplearon 200 ml de medio de cultivo hidrolizado de cascarilla de arroz, el cual contenía una concentración de xilosa de 27,5 g/L. La fermentación se llevó a cabo bajo las siguientes condiciones: temperatura 30 ºC, pH del medio 5,8, agitación 120 rpm e inóculo adaptado de 3 g/L. Los resultados mostraron que después de 120 horas de fermentación se obtuvieron 2,6 g/L de xilitol con productividad volumétrica (Qp) de 0,02 g/L-h y rendimiento de sustrato en producto (Yp/s) de 0,13 g/g. De esta manera, la cepa nativa Candida guilliermondii, aislada del fruto de Corozo chiquito (Bactris guineensis), produjo xilitol bajo condiciones específicas de fermentación.

The yeast Candida guilliermondii has been studied due to its ability to produce xylitol in xylose-rich hemicellulosic compounds, Candida guilliermondii strain isolated from the fruit of Corozo chiquito (Bactris guineensis) was used in this study to assess their ability to xylitol production on these substrates. The aim of this study was to determine the fermentation parameters such as xylitol production, volumetric productivity (Qp) and yield of xylitol production (Yp/s) during fermentation with the native strain Candida guilliermondii. Was used 200 ml of culture medium rice husk hydrolysate, which contained a xylose concentration of 27.5 g/L. The fermentation was carried out under the following conditions: temperature 30 ºC, pH of 5.8, agitation 120 rpm and adapted inoculum of 3 g/L. The results showed that after 120 hours of fermentation 2.6 g / L of xylitol was achieved with volumetric productivity (Qp) 0.02 g/L-h and 0.13 g/g yield of xylitol production (Yp/s). The native strain Candida guilliermondii, isolated from the fruit of Corozo chiquito (Bactris guineensis) produced xylitol fermentation under specific conditions.

Evaluación del efecto de diferentes cepas de levadura (Montrachet, K1-V1116, EC-1118, 71B-1122 y IVC-GRE ®) y clarificantes sobre los atributos sensoriales del vino de naranja criolla (Citrus sinensis) / Evaluation of effect of various strains of yeast (Montrachet, K1-V1116, EC-1118, 71B-1122 y IVC-GRE ®) and clearer on the sensory attributes orange wine (Citrus sinensis)

En este trabajo se evaluó el efecto de diferentes cepas de levadura (Montrachet, K1-V1116, EC-1118, 71B-1122 y IVC-GRE ®) sobre los atributos sensoriales del vino de naranja. Estos atributos fueron medidos utilizando la escala modificada de UC Davis. En una prueba de ordenamiento para determinar el mejor tratamiento de clarificación se determinó que la gelatina por sí sola no causa efecto sobre el atributo apariencia general, la combinación de la gelatina y la microfiltración tienen un efecto positivo sobre la apariencia del vino de naranja. Los cinco vinos tratados con diferentes levaduras presentaron diferencias significativas sobre la puntuación total, acidez total, sabor y calidad en general. En términos del efecto de las levaduras, la evaluación sensorial realizada a los vinos mostró que el de naranja con la levadura K1-V1116 fue el que sobresalió en términos de puntuación en los promedios de casi todos los atributos analizados por el panel sensorial.

In this Wort was evaluated the effect of different types of strains of yeast (Montrachet, K1-V1116, EC-1118, 71B-1122 y IVC-GRE) over the sensorial attributes of orange wines were also studied. These attributes were measured in a modified scale of UC Davis. By using an order test in order to know the best cleared treatment, it was determined that gelatin by itself does not cause any effect over the general quality attribute, but the combination of gelatin and microfiltration, cause a positive effect over the orange wine appearance. The five wines treated with different yeasts presented significant differences on individual scores, total acidy, flavor and general quality of the UC Davis scale. The sensorial evaluation of wines showed that the yeast K1-V1116 produced the best rated orange wine. This wine was significantly different over many attributes when compared with the other wines evaluated by the sensorial panel.

Analysis of the genomic response of a wine yeast to rehydration and inoculation.

We used DNA microarrays to study the transcriptome of a wine yeast before and after rehydration and during the first hours following inoculation of a synthetic must. There was a substantial transcriptional remodeling during this period, including 1,874 genes regulated more than threefold. Dried yeasts displayed an expression profile typical of respiratory-grown cells starved for nitrogen and carbon and which had been highly stressed. During rehydration, many genes involved in biosynthetic pathways, in transcription or in protein synthesis were coordinately induced while genes subject to glucose repression were down-regulated. The transcriptional response was very rapid indicating that yeast quickly recovered the capacity to sense environmental signals and to respond appropriately. Our data show that genes involved in the general stress response were repressed during rehydration while acid stress specific genes were induced probably in response to organic acid accumulation. The glycolytic genes and acid stress-responsive genes were simultaneously and transiently repressed after inoculation into the fermentation medium suggesting that regulation of glycolytic genes may correspond to an adjustment to the energetic needs of the cells. Surprisingly, inoculation into the must did not trigger a stress response despite the high concentrations of sugars.

Biological function of modified nucleotides in tRNA molecules--synthesis and biological activity of the analogues of yeast alanyl tRNA with I34 replaced by A34 or G34.

Analogues of yeast alanyl tRNA with I34 replaced by A34 or G34 were synthesized. Synthetic analogues of yeast alanyl tRNT occupy the same position as the natural yeast alanyl tRNA on polyacrylamide gel electrophoresis, and their purity is about 95% after electrophoresis on a 10% or 20% polyacrylamide gel. The two terminal and nearest neighbour nucleotides of the analogues are all correct. The accepting activity of the synthetic analogues is similar to that of the reconstituted natural yeast alanyl tRNA. The incorporation activity of alanine into proteins of the synthetic analogues is about 30% of that of the natural of reconstituted natural yeast alanyl tRNA when I34 is replaced by A, and is 90% when I34 is replaced by G. The reason of the variation in biological function of the analogues of yeast alanyl tRNA after I34 replaced by A or G was discussed.