Comparison of RAPDs, AFLPs and SSR markers for the genetic analysis of yeast strains of Saccharomyces cerevisiae

We evaluated the usefulness of different molecular techniques for the genetic analysis of Saccharomyces cerevisiae strains. Three commonly used PCR-derived genetic methods, random amplified polymorphic DNA (RAPDs), amplified fragment length polymorphism (AFLPs) and simple sequence repeats (SSRs; microsatellites), were used to characterize 27 wine yeast strains of S. cerevisiae from the “Denominación de Origen Vinos de Madrid” (Spain). Using these methods, we were able to overcome certain limitations associated with classical taxonomic methods. Based on the presence or absence of amplified fragments for each genotype, AFLPs and SSRs showed a similar discriminatory power superior to that of the RAPDs. Genetic relationships between strains were also estimated using the three methods. In general, very poor correlations were found, reflecting the different genomic regions for which the methods are screened. Results are discussed in terms of which molecular technique is most appropriate for use with a particular aspect of genetic evaluation.     

PCR‐Based Differentiation and Homology of Brewing and Type Strains of the Genus Saccharomyces

Restriction fragment length polymorphism (RFLP) patterns of PCR‐amplified ribosomal RNA gene fragments (rDNA) and randomly amplified polymorphic DNA (RAPD) were applied for the analysis of 15 brewing and 6 related yeast strains of the genus Saccharomyces. One five‐base (ScrFI) and two four‐base cutting (HaeIII, MspI) restriction enzymes were used. The primers 21 and M13 core sequence were selected for RAPD analysis. PCR‐RFLP rDNA analysis with HaeIII, ScrFI and MspI differentiated the strains tested into four, five and four types of patterns, respectively and the analyses of the profiles showed 100% homology, between the yeast strains. One strain was an exception. Homological groups were observed for strains used in breweries globally, from a local production strain and from the isolates identified as S. cerevisiae. Using RAPD analysis, and according to discrete differences in the profiles, it was possible to divide twenty one strains into 15 and 20 groups with primer 21 and M13 respectively. RFLP‐PCR rDNA analysis was used to show similarities in closely related brewing strains, while RAPD analysis was used for differentiation of strains.     

Yeasts isolated from New Zealand vineyards and wineries

Background and Aims: The yeast flora from a range of New Zealand commercial wineries was surveyed to estimate the incidence of yeast species in grape juice. Methods and Results: Molecular analysis of the internal transcribed spacer region was performed for 1279 yeast colonies isolated from 17 different fresh grape juices sampled in eight New Zealand wineries between 2003 and 2009. The 17 juices contained at least 25 different species of yeast from nine genera. Microsatellite fingerprinting of Saccharomyces cerevisiae showed that some strains were identical to known commercial yeast varieties, but we also found evidence for local populations of S. cerevisiae common to individual wineries or regions. Five genotypes from Central Otago, New Zealand, were very closely related to a single sequenced strain derived from Chile, which in turn is related to European wine isolates. Conclusions: The yeast flora found in New Zealand grape juices is broadly similar to that found in wineries elsewhere around the world. Genotyping of S. cerevisiae suggests recent dispersal of both commercial and non-commercial yeast strains from Europe to New Zealand. Significance of the Study: These data are consistent with two human-mediated modes for the international dispersal of S. cerevisiae: one via the escape of strains traded commercially, and another via long distance dispersal of non-commercial strains.     

YEAST CLASSIFICATION*

Following a discussion of the need for systems of classification, which are inevitably to some extent subjective, a survey is made of primary and auxiliary diagnostic tests used in yeast classification. The possibilities and limitations of numerical taxonomy are discussed and an assessment is made of the value of current techniques used for identifying strains of brewing yeasts. Methods are described for detecting wild yeasts and for evaluating the suitability of different strains of brewing yeast. Reasons are given for retaining Saccharomyces carlsbergensis as a species in its own right rather than as a synonym of Sacch. uvarum.     

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.     

Multilocus sequence typing of oenological Saccharomyces cerevisiae strains

This study describes the application of a multilocus sequence typing (MLST) analysis for molecular discrimination at the strain level of Spanish wine yeast strains. The discrimination power of MLST is compared to mitochondrial RFLP analysis. Fragments of the ADP1, ACC1, RPN2, GLN4, and ALA1 genes were amplified by PCR from chromosomal DNA of 18 wine Saccharomyces cerevisiae strains. Ten polymorphic sites were found in the five loci analyzed showing 13 different genotypes, with 11 of them represented by only one strain. RFLP analysis of the same 18 wine yeast strains showed seventeen different mitochondrial patterns. Phylogenetic relationships among the strains analyzed, inferred by MLST data, showed wine isolates of S. cerevisiae as a rather homogeneous group. The discrimination potential of mitochondrial RFLP analysis was superior to the MLST scheme used in this work. However, MLST analysis allowed an easy construction of reliable phylogenetic trees. MLST analysis offers the possibility of typing wine S. cerevisiae strains simultaneously to the study of the genetic relationship among them.     

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.     

STRAINS OF YEAST LETHAL TO BREWERY YEASTS

Strains of yeast that are lethal to brewery ale and lager yeasts have been isolated from production-scale two-stage stirred continuous fermentors. These strains release a “killer” factor which is highly active in the pH range 3.8–4.2. When the level of infection reaches 2% the concentration of killer factor is sufficient to give a selective advantage in continuous fermentation, whereupon the proportion of killer yeasts rises and the brewery yeast is rapidly killed. The beer acquires a characteristic off-flavour which has been described as “herbal/phenolic”. Both flocculent and non-flocculent killer strains have been found and these show the characteristics of Saccharomyces cerevisiae but appear to ferment additional wort sugar(s), have an abormally small cell-size and are pleomorphic in mixed culture.     

The 2μm plasmid of laboratory yeast strains is a type-1/type-2 hybrid

Industrial yeast strains carry one of two homeologous 2μm plasmids designated as type-1 or type-2. The 2μm plasmid, Scp1, found in common laboratory strains of Saccharomyces cerevisiae is considered a type-2 plasmid, since the ori, STB, RAF and REP1 loci and intergenic sequences of the right-unique region of Scp1 are homologous to the corresponding loci in industrial strain type-2 plasmids. However, within both its 599 bp inverted repeats Scp1 has 142-bp sequences homologous to the bakers' yeast type-1 plasmid. DNA sequence analyses and oligonucleotide hybridizations indicate that the 142-bp insertion in Scp1 was probably due to homeologous recombination between type-1 and type-2 plasmids. These results suggest that some of the plasmid and chromosomal sequence polymorphisms seen in laboratory yeast strains result from homeologous recombination in their ancestral breeding stock.     

Applications of the Long and Accurate Polymerase Chain Reaction Method in Yeast Molecular Biology: Direct Sequencing of the Amplified DNA and Its Introduction into Yeast

A DNA fragment longer than 10 kb can be amplified by the long and accurate polymerase chain reaction (LA-PCR) method. We demonstrate here applications of this technique in molecular biological studies of Saccharomyces cerevisiae. We have shown that DNA fragments amplified by LA-PCR can be directly used as a template in the chain-termination sequencing protocol, making it possible to quickly identify the DNA insert of yeast genomic library clones. We have also shown that the amplified yeast DNA can easily be introduced into yeast by co-transformation with linearized vector DNA. Overlapping DNA between the amplified yeast fragment and the vector must be more than 20 bp long in order to obtain 90% or more correct recombinant plasmids. These results suggest that simple amplification of yeast clones by LA-PCR can replace the previous procedures of yeast clone recovery, consisting of transformation of Escherichia coli, propagation of plasmids in E. coli and preparation of plasmid DNA. © 1997 John Wiley & Sons, Ltd.     

Disruption of ubiquitin-related genes in laboratory yeast strains enhances ethanol production during sake brewing

Sake yeast can produce high levels of ethanol in concentrated rice mash. While both sake and laboratory yeast strains belong to the species Saccharomyces cerevisiae, the laboratory strains produce much less ethanol. This disparity in fermentation activity may be due to the strains' different responses to environmental stresses, including ethanol accumulation. To obtain more insight into the stress response of yeast cells under sake brewing conditions, we carried out small-scale sake brewing tests using laboratory yeast strains disrupted in specific stress-related genes. Surprisingly, yeast strains with disrupted ubiquitin-related genes produced more ethanol than the parental strain during sake brewing. The elevated fermentation ability conferred by disruption of the ubiquitin-coding gene UBI4 was confined to laboratory strains, and the ubi4 disruptant of a sake yeast strain did not demonstrate a comparable increase in ethanol production. These findings suggest different roles for ubiquitin in sake and laboratory yeast strains.     

Large-scale phenotypic analysis—the pilot project on yeast chromosome III

In 1993, a pilot project for the functional analysis of newly discovered open reading frames, presumably coding for proteins, from yeast chromosome III was launched by the European Community. In the frame of this programme, we have developed a large-scale screening for the identification of gene/protein functions via systematic phenotypic analysis. To this end, some 80 haploid mutant yeast strains were constructed, each carrying a targeted deletion of a single gene obtained by HIS3 or TRP1 transplacement in the W303 background and a panel of some 100 growth conditions was established, ranging from growth substrates, stress to, predominantly, specific inhibitors and drugs acting on various cellular processes. Furthermore, co-segregation of the targeted deletion and the observed phenotype(s) in meiotic products has been verified. The experimental procedure, using microtiter plates for phenotypic analysis of yeast mutants, can be applied on a large scale, either on solid or in liquid media. Since the minimal working unit of one 96-well microtiter plate allows the simultaneous analysis of at least 60 mutant strains, hundreds of strains can be handled in parallel. The high number of monotropic and pleiotropic phenotypes (62%) obtained, together with the acquired practical experience, have shown this approach to be simple, inexpensive and reproducible. It provides a useful tool for the yeast community for the systematic search of biochemical and physiological functions of unknown genes accounting for about a half of the 6000 genes of the complete yeast genome. © 1997 John Wiley & Sons, Ltd.     

LONG-CHAIN FATTY ACID COMPOSITION AS A CRITERION FOR YEAST DISTINCTION IN THE BREWING INDUSTRY

The diversity of yeasts isolated from brewing plants and its role on beer quality makes yeast distinction a major concern in industrial microbiological control. Several approaches have been tried to develop rapid and simple methods to perform such tasks. Among these, stands the utilization of long-chain fatty acid composition of total yeast biomass. In this paper results are reported showing the potential of this technique to characterize yeast flora isolated from industrial plants. Fatty acid profiles of brewing species are clearly differentiated from those of non-Saccharomyces strains using statistical data treatment by principal component analysis (PCA). Distinction between brewing and wild strains of Saccharomyces spp. was not apparent. In comparison, fatty acid profiling showed higher discriminating ability than growth on lysine medium for non-Saccharomyces strains. For distinction of S. cerevisiae var. diastaticus from other Saccharomyces strains, growth on starch medium showed to be necessary.     

Delimination of brewing yeast strains using different molecular techniques

In general, the genetic characteristics, the phenotype and the microbial purity of the production brewing yeast strains are among the most important factors in maintaining a consistently good quality of products. Analysis of restriction fragment length polymorphism (RFLP) patterns of 18S rRNA-coding DNA was investigated to group ale and lager strains. All production brewing yeast strains showed the same RFLP pattern as the type strain and synonym type strains of S. cerevisiae, and were quite different from the type and synonym type strains of S. pastorianus. Based on these data, all production brewing yeast strains investigated in this study appeared to belong to S. cerevisiae. Electrophoretic karyotyping and random amplified polymorphic DNA (RAPD) analysis appeared to be suitable methods for distinguishing not only the type and synonym type strain of S. cerevisiae and S. pastorianus, but also the ale and the lager strains.     

Biodiversity of Yeast Mycobiota in “Sucuk,” a Traditional Turkish Fermented Dry Sausage: Phenotypic and Genotypic Identification,Functional and Technological Properties

In this study, yeasts from Turkish fermented sucuks were identified and their functional and technological properties were evaluated. Two hundred fifty‐five yeast isolates were obtained from 35 different sucuk samples from different regions of Turkey. The yeast isolates were determined as genotypic using 2 different polymerase chain reaction (PCR) methods (rep‐PCR and RAPD‐PCR). Functional and technological properties of including proteolytic, lipolytic, and catalase activities, tolerance to NaCl and bile, as well as growing rates at different temperature and pH conditions selected yeast strains were also evaluated. Candida zeylanoides and Debaryomyces hansenii were dominant strains in sucuk samples. All C. zeylanoides and D. hansenii tested could grow at the condition of 15% NaCl and 0.3% bile salt. However, none of the strains were able to grow at 37 °C, even though catalase activity, weak proteolytic and lipolytic activities was still observed. D. hansenii were able to grow only at pH 3, while some of C. zeylanoides could grow at lower pH levels (pH 2). Three and 4 strains of C. zeylanoides showed β‐hemolysis activity and nitrate reduction ability to nitrite, respectively. D. hansenii did not have properties, which are β‐hemolysis, nitrate reduction, or hydrogen sulfide production. Overall, diverse yeast mycobiota present in Turkish fermented sucuk and their functional and technological properties were revealed with this study.     

RESTRICTION ANALYSIS OF 2 μm DNA FROM DIVERSE STRAINS OF YEAST

2 μm plasmids from an ale yeast, a lager yeast and a strain of Saccharomyces diastaticus have been characterised by restriction analysis. Plasmids were similar to each other and conformed to a pattern which has been reported for well-characterised genetic stocks. The structure of 2 μm DNA, therefore, has been conserved in diverse strains of Saccharomyces. Preparative procedures used here are likely to be applicable to the detection and characterisation of plasmid DNA from a range of yeast genera. The apparent yield of 2 μm was increased when strains were grown at elevated temperature.     

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     

ASSESSMENT OF POTENTIAL PROBIOTIC PROPERTIES OF LACTIC ACID BACTERIA AND YEAST STRAINS

ABSTRACT

Four lactic acid bacteria (LAB) and three yeast strains isolated from a traditional Bulgarian cereal-based fermented beverage were assessed for potential probiotic properties. Acid and bile resistance, antipathogenic activity and antibiotic resistance of the strains were evaluated. Tolerance to low pH values (2.0–3.0) and high bile concentrations (0.2–2.0%) of the LAB and yeast strains varied, but all strains kept viable throughout the experiments. Antagonistic activity towards most of the eight test-pathogens was observed for one LAB (Lactobacillus plantarum B28) and two yeast strains (Candida rugosa Y28 and Candida lambica Y30). Antibiotic resistance (39 antibiotics) of the LAB strains was variable, but showed their potential for therapeutic application.     

Brewing characteristics of haploid strains isolated from sake yeast Kyokai No. 7

Sake yeast exhibit various characteristics that make them more suitable for sake brewing compared to other yeast strains. Since sake yeast strains are Saccharomyces cerevisiae heterothallic diploid strains, it is likely that they have heterozygous alleles on homologous chromosomes (heterozygosity) due to spontaneous mutations. If this is the case, segregation of phenotypic traits in haploid strains after sporulation and concomitant meiosis of sake yeast strains would be expected to occur. To examine this hypothesis, we isolated 100 haploid strains from Kyokai No. 7 (K7), a typical sake yeast strain in Japan, and compared their brewing characteristics in small‐scale sake‐brewing tests. Analyses of the resultant sake samples showed a smooth and continuous distribution of analytical values for brewing characteristics, suggesting that K7 has multiple heterozygosities that affect brewing characteristics and that these heterozygous alleles do segregate after sporulation. Correlation and principal component analyses suggested that the analytical parameters could be classified into two groups, indicating fermentation ability and sake flavour. Copyright © 2008 John Wiley & Sons, Ltd.