d-Xylose consumption by nonrecombinant Saccharomyces cerevisiae: A review

Xylose is the second most abundant sugar in nature. Its efficient fermentation has been considered as a critical factor for a feasible conversion of renewable biomass resources into biofuels and other chemicals. The yeast Saccharomyces cerevisiae is of exceptional industrial importance due to its excellent capability to ferment sugars. However, although S. cerevisiae is able to ferment xylulose, it is considered unable to metabolize xylose, and thus, a lot of research has been directed to engineer this yeast with heterologous genes to allow xylose consumption and fermentation. The analysis of the natural genetic diversity of this yeast has also revealed some nonrecombinant S. cerevisiae strains that consume or even grow (modestly) on xylose. The genome of this yeast has all the genes required for xylose transport and metabolism through the xylose reductase, xylitol dehydrogenase, and xylulokinase pathway, but there seems to be problems in their kinetic properties and/or required expression. Self-cloning industrial S. cerevisiae strains overexpressing some of the endogenous genes have shown interesting results, and new strategies and approaches designed to improve these S. cerevisiae strains for ethanol production from xylose will also be presented in this review.     

Screening of native S. cerevisiae strains in the production of Pajarete wine: a tradition of Atacama Region,Chile

Pajarete is a Chilean wine with an appellation of origin. Although it has organoleptic properties, intensive utilization of Saccharomyces cerevisiae commercial yeast through the years has presumably produced the loss of native strains that may be associated with Pajarete oenologic uniqueness. In order to evaluate the effect of re-incorporation of indigenous strains into Pajarete winemaking, native S. cerevisiae strains were isolated and selected based on their properties shown during small and large laboratory scale fermentation, and then evaluated in industrial bioreactors. From an initial set of 312 isolates, a single native strain was selected based on taxonomy, fermentation performance, aroma, residual sugars, and production of alcohol for incorporation into market scale.     

Use of PCR‐DHPLC (Polymerase Chain Reaction—Denaturing High Performance Liquid Chromatography) for the Rapid Differentiation of Industrial Saccharomyces pastorianus and Saccharomyces cerevisiae Strains

Strain specific detection and control of Saccharomyces pastorianus and Saccharomyces cerevisiae starter cultures is of great importance for the fermentation industry. The preconditions of strain specific fermentation characteristics can be ensured by periodic analysis and confirmation of the strain identity. With regard to industrial S. pastorianus and S. cerevisiae strains and a focus on brewing strains, the differentiation methods most available are time‐consuming and not very discriminative. In this work PCR‐DHPLC analysis was investigated as a novel approach for the differentiation of industrially used S. pastorianus and S. cerevisiae strains. The PCR‐DHPLC‐system was specific for S. cerevisiae strains and S. pastorianus hybrid strains that contain IGS2 rDNA, which originates from the S. cerevisiae ancestor. For the DNA of 177 strains of 41 non‐target species, which are typical for beverage and fermentation surroundings, the absence of PCR‐amplificates could be confirmed by DHPLC analysis. It was shown that single strains of S. cerevisiae and S. pastorianus could be differentiated. A strain specific differentiation within the group of top‐fermenting Saccharomyces cerevisiae strains could also be performed. For the group of bottom fermenting S. pastorianus brewing strains, strain‐to‐strain specific differences in the DHPLC chromatograms could be observed which can be used to differentiate and to compare two single strains with each other, although the comparison of chromatograms of an unknown S. pastorianus strain with a set of known S. pastorianus chromatograms could only reveal tendencies towards grouping into types. The differential DHPLC chromatogram characteristics (fluorescence intensities, number of peaks/side‐peaks/peak‐shoulders) within S. pastorianus are present, but not as distinctive as for S. cerevisiae. Additionally PCR‐DHPLC has advantages compared to other differentiation methods, such as species specificity, speed (2.5 h for one sample) and precision with the described limits.     

Production of Beer with a Genetically Engineered Strain of S. cerevisiae with Modified Beta Glucanase Expression

This study used a recombinant Saccharomyces cerevisiae strain, which expressed both β‐glucanase enzyme and reduced Pro‐teinase A expression during wort fermentations. The genetic stability and fermentation features of the strain were examined. The recombinant strain's proteinase A activity was reduced compared to the parent strain; β‐glucanase was produced throughout the fermentation. The fermentation with the recombinant S. cerevisiae strain exhibited a larger reduction in β‐glucan content than what was observed with the control strain, with β‐glucan degradation above 80%. The foam stability period was reduced when the beer produced by the recombinant S. cerevisiae was stored for 3 months. SDS‐PAGE analysis of the beer proteins indicated that lipid transfer protein 1 had disappeared. Fermentation studies indicated that based on the parameters examined, this recombinant strain was suitable for industrial beer production.     

Saccharomyces pastorianus: genomic insights inspiring innovation for industry

A combination of biological and non‐biological factors has led to the interspecific hybrid yeast species Saccharomyces pastorianus becoming one of the world's most important industrial organisms. This yeast is used in the production of lager‐style beers, the fermentation of which requires very low temperatures compared to other industrial fermentation processes. This group of organisms has benefited from both the whole‐genome duplication in its ancestral lineage and the subsequent hybridization event between S. cerevisiae and S. eubayanus, resulting in strong fermentative ability. The hybrid has key traits, such as cold tolerance and good maltose‐ and maltotriose‐utilizing ability, inherited either from the parental species or originating from genetic interactions between the parent genomes. Instability in the nascent allopolyploid hybrid genome may have contributed to rapid evolution of the yeast to tolerate conditions prevalent in the brewing environment. The recent discovery of S. eubayanus has provided new insights into the evolutionary history of S. pastorianus and may offer new opportunities for generating novel industrially‐beneficial lager yeast strains. Copyright © 2014 John Wiley & Sons, Ltd.     

Breeding of lager yeast with Saccharomyces cerevisiae improves stress resistance and fermentation performance

Lager beer brewing relies on strains collectively known as Saccharomyces carlsbergensis, which are hybrids between S. cerevisiae and S. eubayanus‐like strains. Lager yeasts are particularly adapted to low‐temperature fermentations. Selection of new yeast strains for improved traits or fermentation performance is laborious, due to the allotetraploid nature of lager yeasts. Initially, we have generated new F1 hybrids by classical genetics, using spore clones of lager yeast and S. cerevisiae and complementation of auxotrophies of the single strains upon mating. These hybrids were improved on several parameters, including growth at elevated temperature and resistance against high osmolarity or high ethanol concentrations. Due to the uncertainty of chromosomal make‐up of lager yeast spore clones, we introduced molecular markers to analyse mating‐type composition by PCR. Based on these results, new hybrids between a lager and an ale yeast strain were isolated by micromanipulation. These hybrids were not subject to genetic modification. We generated and verified 13 hybrid strains. All of these hybrid strains showed improved stress resistance as seen in the ale parent, including improved survival at the end of fermentation. Importantly, some of the strains showed improved fermentation rates using 18°Plato at 18–25°C. Uniparental mitochondrial DNA inheritance was observed mostly from the S. cerevisiae parent. Copyright © 2012 John Wiley & Sons, Ltd.     

Emergence of [GAR+] cells in yeast from sake brewing affects the fermentation properties

In the traditional (kimoto) method of sake (Japanese rice wine) brewing, Saccharomyces cerevisiae yeast cells are exposed to lactate, which is produced by lactic acid bacteria in the seed mash. Lactate promotes the appearance of glucose-repression-resistant [GAR⁺] cells. Herein, we compared the resistance to glucose repression among kimoto, industrial, and laboratory yeast strains. We observed that the frequencies of the spontaneous emergence of [GAR⁺] cells among the kimoto strains were higher than those among the industrial and laboratory strains. The fermentation ability of a kimoto yeast (strain U44) was lower than that of an industrial strain (K701), as [GAR⁺] cells generally showed slower ethanol production. The addition of lactate decreased the fermentation abilities of the K701 strain by increasing the number of [GAR⁺] cells, but it did not affect those of the U44 strain. These results suggest that lactate controlled fermentation by promoting the appearance of [GAR⁺] cells in the industrial sake strains but not in the kimoto strains.     

Altered Patterns of Maltose and Glucose Fermentation by Brewing and Wine Yeasts Influenced by the Complexity of Nitrogen Source

Maltose and glucose fermentations by industrial brewing and wine yeasts strains were strongly affected by the structural complexity of the nitrogen source. In this study, four Saccharomyces cerevisiae strains, two brewing and two wine yeasts, were grown in a medium containing maltose or glucose supplemented with a nitrogen source varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids) and peptides (peptone). Diauxie was observed at low sugar concentration for brewing and wine strains, independent of nitrogen supplementation, and the type of sugar. At high sugar concentrations altered patterns of sugar fermentation were observed, and biomass accumulation and ethanol production depended on the nature of the nitrogen source and were different for brewing and wine strains. In maltose, high biomass production was observed under peptone and casamino acids for the brewing and wine strains, however efficient maltose utilization and high ethanol production was only observed in the presence of casamino acids for one brewing and one wine strain studied. Conversely, peptone and casamino acids induced higher biomass and ethanol production for the two other brewing and wine strains studied. With glucose, in general, peptone induced higher fermentation performance for all strains, and one brewing and wine strain produced the same amount of ethanol with peptone and casamino acids supplementation. Ammonium salts always induced poor yeast performance. The results described in this paper suggest that the complex nitrogen composition of the cultivation medium may create conditions resembling those responsible for inducing sluggish/stuck fermentation, and indicate that the kind and concentration of sugar, the complexity of nitrogen source and the yeast genetic background influence optimal industrial yeast fermentation performance.     

Investigating flavour characteristics of British ale yeasts: techniques,resources and opportunities for innovation

Five British ale yeast strains were subjected to flavour profiling under brewery fermentation conditions in which all other brewing parameters were kept constant. Significant variation was observed in the timing and quantity of flavour‐related chemicals produced. Genetic tests showed no evidence of hybrid origins in any of the strains, including one strain previously reported as a possible hybrid of Saccharomyces cerevisiae and S. bayanus. Variation maintained in historical S. cerevisiae ale yeast collections is highlighted as a potential source of novelty in innovative strain improvement for bioflavour production. Copyright © 2014 John Wiley & Sons, Ltd.     

Effect of citrulline metabolism in Saccharomyces cerevisiae on the formation of ethyl carbamate during Chinese rice wine fermentation

Urea, as the main precursor of ethyl carbamate (EC), has received extensive attention. Here, we have metabolically engineered an industrial yeast strain – Saccharomyces cerevisiae N85 – to investigate the contribution of the EC precursor citrulline to the concentration of EC in Chinese rice wine. The results showed that the citrulline biosynthetic pathway of the modified strain N85‐arg3 was completely suppressed by deletion of ARG3, encoding ornithine carbamoyltransferase. However, there were no significant differences in the levels of citrulline or EC between N85‐arg3 and the parental strain N85 during fermentation. In addition, we over‐expressed ARG1 (encoding argininosuccinate synthase) and ARG4 (encoding argininosuccinate lyase) to construct the engineered strains N85^ARG1,4 and N85^ARG1,4‐arg3. The citrulline contents in Chinese rice wine fermented with N85^ARG1,4 and N85^ARG1,4‐arg3 were respectively 24.1 and 20.4% less than that of N85. However, the contents of EC were 23.8 and 28.5% more than that of N85. These results suggested that reducing the formation of EC during Chinese rice wine fermentation by genetically engineering citrulline metabolism in S. cerevisiae was not a viable proposition. Copyright © 2018 The Institute of Brewing & Distilling     

Breeding of high malate‐producing diploid sake yeast with a homozygous mutation in the VID24 gene

Malate is an important taste component of sake (a Japanese alcoholic beverage) that is produced by the yeast Saccharomyces cerevisiae during alcoholic fermentation. A variety of methods for generating high malate‐producing yeast strains have been developed to date. We recently reported that a high malate‐producing strain was isolated as a mutant sensitive to dimethyl succinate (DMS), and that a mutation in the vacuolar import and degradation protein (VID) 24 gene was responsible for high malate productivity and DMS sensitivity. In this work, the relationships between heterozygous and homozygous mutants of VID24 and malate productivity in diploid sake yeast were examined and a method was developed for breeding a higher malate‐producing strain. First a diploid yeast was generated with a homozygous VID24 mutation by genetic engineering. The homozygous integrants produced more malate during sake brewing and grew more slowly in DMS medium than wild‐type and heterozygous integrants. Thus, the genotype of the VID24 mutation influenced the level of malate production and sensitivity to DMS in diploid yeast. Then a homozygous mutant from a heterozygous mutant was obtained without genetic engineering by ultraviolet irradiation and culturing in DMS with nystatin enrichment. The non‐genetically modified sake yeast with a homozygous VID24 mutation exhibited a higher level of malate productivity than the parent heterozygous mutant strain. These findings provide a basis for controlling malate production in yeast, and thereby regulating malate levels in sake. Copyright © 2016 The Institute of Brewing & Distilling     

Saccharomyces cerevisiae associated with the spontaneous fermentation of tequila agave juice

Saccharomyces cerevisiae dominates the spontaneous fermentation of blue agave juice. Because of the batch heterogeneity, the aim of this work was to determine the strain diversity of S. cerevisiae among fermentations. During January and February 2015, agave juice was sampled in triplicate from four sampling points at a tequila distillery. The heterogeneity of yeast strains and the production of carbon dioxide were assessed during fermentation, whereas the amount of ethanol produced was measured at the end of the process. The fermentation cycle times varied widely (9 to 25 days), as did fermentation efficiency (2.5–45.5%). Yeast isolates were identified at the species level by ITS‐5.8S rRNA restriction fragment length polymorphism and differentiated at the strain level by random amplified polymorphic DNA. A total of 199 isolates were obtained and identified as S. cerevisiae, showing 69 different random amplified polymorphic DNA profiles. There was no clear dominance of any strain during fermentation. However, two strains (P1 and P2) were detected in all fermentation samples, suggesting their residency in the distillery, despite the deep‐cleaning applied to the tanks after each fermentation batch. According to the RAPD profiles, the number of strains isolated from fermentation samples increased from 17 in January to 25 in February. © 2018 The Institute of Brewing & Distilling     

Surfome analysis of a wild-type wine Saccharomyces cerevisiae strain

The yeast Saccharomyces cerevisiae, besides being an eukaryotic cell model, plays a fundamental role in the production of fermented foods. In the winemaking industry, yeast cell walls may be involved in numerous processes and contribute substantially to the final chemical and sensorial profiles of wines. Nonetheless, apart from mannoproteins, little is known on the protein components of the yeast cell wall and their changes during the fermentation of must into wine. In this work, we performed a dynamic analysis of the cell surface proteome (surfome) of an autochthonous wine yeast strain (previously selected as a wine fermentation starter) by shaving intact cells with trypsin and identifying tryptic peptides by means of nLC-ESI-LIT-MS/MS. Out of the 42 identified proteins, 16 and 14 were found to be specifically expressed in wine yeast surfome at the beginning and at the end of fermentation, respectively. The molecular functions of these specifically expressed proteins might help in explaining their roles in the cell wall as a response to the alcoholic fermentation-related stresses. Additionally, we provided the identification of 20 new potential cell wall related proteins. Globally, our results might provide new useful data for the selection and characterization of yeast strains to be used in the winemaking industry.     

Concentration of amino acids in wine after the end of fermentation by Saccharomyces cerevisiae strains

Both quantitative and qualitative differences in the utilization and release of assimilable nitrogen by two wine strains of Saccharomyces cerevisiae (cerevisiae and capensis) under different conditions of oxygen were observed. These differences were influenced by the presence of oxygen at the beginning of the fermentation, and by the strain of S cerevisiae. The release of some amino acids post‐fermentation may be the result of reoxidation of NAD(P)H in order to maintain a normal redox balance. Copyright © 2003 Society of Chemical Industry     

Possible involvement of GDI1 protein, a GDP dissociation inhibitor related to vesicle transport, in an amelioration of zinc toxicity in Saccharomyces cerevisiae

The GDI1 protein related vesicle transport system was studied to investigate the possibility that an exclusion of toxic zinc (Zn) from the cytoplasm ameliorates Zn toxicity in Saccharomyces cerevisiae (yeast). A temperature‐sensitive gdi1 mutant (originally called sec19), in which the GDP dissociation inhibitor becomes inactive at the non‐permissive temperature (37 °C), was more sensitive to Zn than its parental GDI1 strain at 32 °C (a moderately non‐permissive temperature). The relative efflux of cytoplasmic Zn in the gdi1 mutant was lower than that in the control strain. Treatment with a vesicle transport‐specific inhibitor, Brefeldin A, caused an increase of Zn sensitivity and a decrease of Zn efflux in these strains. It is therefore suggested that the GDI1‐related vesicle transport system contributes to Zn tolerance in yeast. Furthermore, changes in the number of Zn‐specific fluorescent granules (zincosomes) were observed by zinquin staining in the mutant cells under Zn treatment at 32 °C and 37 °C. We concluded that the GDI1 protein is implicated in control of vesicle numbers. Collectively, the results suggest that the GDI1protein is involved in Zn efflux via small vesicle trafficking and contributes to the control of cytoplasmic Zn content, allowing yeast to survive in the presence of toxic Zn. Copyright © 2011 John Wiley & Sons, Ltd.     

Enhancement of urea uptake in Chinese rice wine yeast strain N85 by the constitutive expression of DUR3 for ethyl carbamate elimination

Most of the fermented alcoholic beverages, particularly Chinese rice wine, contain the potentially human carcinogenic compound ethyl carbamate (EC). As a major EC precursor in Chinese rice wine, urea in fermentations can be transported into the yeast cell by urea permease and finally metabolized by urea carboxylase and allophanate hydrolase in vivo. To eliminate EC in Chinese rice wines, the present study constructed high urea uptake yeast strains N1‐D, N2‐D and N‐D, by introducing a strong promoter (PGK1p) into the urea permease gene (DUR3) of the industrial Chinese rice wine yeast N85, and by the restoration of the URA3 gene at the same time. With these self‐cloned, high urea uptake strains, the urea and EC in the terminal Chinese rice wine samples were reduced to different extents. With two copies of overexpressed DUR3, the N‐D strain could reduce the urea and the EC by 53.4 and 26.1%, respectively. No difference in fermentation characteristics was found between the engineered strains and the parental industrial yeast strain N85. These results could help to optimize the genetic manipulation strategy for EC elimination in Chinese rice wine production. Copyright © 2015 The Institute of Brewing & Distilling     

Isolation of glutathione biosynthesis-deficient mutants of Saccharomyces cerevisiae and their use in baker's yeast production

 Glutathione biosynthesis-deficient mutants of Saccharomyces cerevisiae 0511 were obtained by mutation under specific conditions. A total of 3388 strains were isolated and among them were found 46 mutants sensitive to methylglyoxal. The intracellular glutathione concentration of mutant strain S. cerevisiae 3033 was 0.0172 g/g dry weight, which was a decrease of >76% compared to that of the parent. The growth of mutant strains S. cerevisiae 3033 and S. cerevisiae 1116 in the medium with glutathione present and absent was compared to that of the parent strain. The sensibility of the baker's yeast strains studied to antifoaming agents, butanol and acetic acid was also investigated. The relationship between glutathione presence in the cell and the sensitivity of strain S. cerevisiae 3033 to antifoaming agents and butanol was ascertained, while such a connection with the presence of acetic acid in the molasses medium used for baker's yeast cultivation was not observed. The higher sensitivity of strain S. cerevisiae 3033 to some chemical compounds in the molasses nutrition medium was shown. Received: 2 November 1999 / Revised version: 15 February 2000