Chromosomal location of Lg-FLO1 in bottom-fermenting yeast and the FLO5 locus of industrial yeast

Aims: To determine the chromosomal location and entire sequence of Lg-FLO1, the expression of which causes the flocculation of bottom-fermenting yeast. Methods and Results: Two cosmid clones carrying DNA from a bottom-fermenting yeast chromosome VIII right-arm end were selected by colony hybridization. Sequencing revealed that the clones contained DNA derived from a Saccharomyces cerevisiae type chromosome VIII and a Saccharomyces bayanus type chromosome VIII, both from bottom-fermenting yeast. Conclusions: Lg-FLO1 is located on the S. cerevisiae type chromosome VIII at the same position as the FLO5 gene of the laboratory yeast S. cerevisiae S288c. The unique chromosome VIII structure of bottom-fermenting yeast is conserved among other related strains. FLO5 and Lg-FLO1 promoter sequences are identical except for the presence of three 42 bp repeats in the latter, which are associated with gene activity. Flocculin genes might have been generated by chromosomal recombination at these repeats. Significance and Impact of the Study: This is the first report of the exact chromosomal location and entire sequence of Lg-FLO1. This information will be useful in the brewing industry for the identification of normal bottom-fermenting yeast. Moreover, variations in the FLO5 locus among strains are thought to reflect yeast evolution.     

Combination of 10% EDTA, Photosan, and a blue light hand-held photopolymerizer to inactivate leading oral bacteria in dentistry in vitro

Aims:  To study the yeast diversity of Nigerian palm wines by comparison with other African strains.
Methods and Results:  Twenty-three Saccharomyces cerevisiae strains were obtained from palm wine samples collected at four locations in eastern Nigeria, and characterized using different molecular techniques: internal transcribed spacer restriction fragment length polymorphism and sequence analysis, pulsed field gel electrophoresis, inter delta typing and microsatellite multilocus analysis. These techniques revealed that palm wine yeasts represent a group of closely related strains that includes other West African isolates (CBS400, NCYC110, DVPG6044). Population analysis revealed an excess of homozygote strains and an allelic richness similar to wine suggestive of local domestication. Several other African yeast strains were not connected to this group. Ghana sorghum beer strains and other African strains (DBVPG1853 and MUCL28071) displayed strikingly high relatedness with European bread, beer or wine strains, and the genome of strain MUCL30909 contained African and wine-type alleles, indicating its hybrid origin.
Conclusions:  Nigerian palm wine yeast represents a local specific yeast flora, whereas a European origin or hybrid was suspected for several other Africa isolates.
Significance and Impact of the Study:  This study presents the first genetic characterization of an autochthonous African palm wine yeast population and confirms the idea that human intervention has favoured yeast migration.     

The Chromosome End in Yeast: Its Mosaic Nature and Influence on Recombinational Dynamics

Yeast chromosome ends are composed of several different repeated elements. Among six clones of chromosome ends from two strains of Saccharomyces cerevisiae, at least seven different repeated sequence families were found. These included the previously identified Y'' and X elements. Some families are highly variable in copy number and location between strains of S. cerevisiae, while other elements appear constant in copy number and location. Three repeated sequence elements are specific to S. cerevisiae and are not found in its evolutionarily close relative, Saccharomyces paradoxus. Two other repeated sequences are found in both S. cerevisiae and S. paradoxus. None of those described here is found (by low stringency DNA hybridization) in the next closest species, Saccharomyces bayanus. The loosely characterized X element is now more precisely defined. X is a composite of at least four small (ca. 45-140 bp) sequences found at some, but not all, ends. There is also a potential ``core'''' X element of approximately 560 bp which may be found at all ends. Distal to X, only one of six clones had (TG(1-3))(n) telomere sequence at the junction between X and Y''. The presence of these internal (TG(1-3))(n) sequences correlates with the ability of a single Y'' to expand into a tandem array of Y''s by unequal sister chromatid exchange. The presence of shared repeated elements proximal to the X region can override the strong preference of Y''s to recombine ectopically with other Y''s of the same size class. The chromosome ends in yeast are evolutionarily dynamic in terms of subtelomeric repeat structure and variability.     

Functional expression of human HMG-CoA reductase in Saccharomyces cerevisiae: a system to analyse normal and mutated versions of the enzyme in the context of statin treatment

Aims:  Statins – inhibitors of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase – are known to reduce blood cholesterol levels. In this paper, we present a Saccharomyces cerevisiae expression system, which enables quick evaluation of the sensitivity of the wild-type and/or mutant forms of human HMG-CoA reductase towards statins or other drugs.
Methods and results:  We analysed the sequence of the HMG-CoA reductase gene in DNA extracted from blood samples of 16 patients with cardiovascular disorders. We applied the yeast system to examine the sensitivity of the wild-type and mutated versions of the hHMG-CoA reductase to different types of statins.
Conclusion:  The yeast and mammalian HMG-CoA reductases demonstrate structural and functional conservation, and expression of human HMG-CoA reductase in yeast complements the lethal phenotype of strains lacking the HMG1 and HMG2 genes.
Significance and Impact of the Study:  These data indicate that a yeast expression system can serve to study the influence of selected mutations in human HMG-CoA reductase on the sensitivity of the enzyme to commonly prescribed statins. Our results suggest that this model system is suitable for the development and selection of lipid-lowering drugs as well as for the examination of DNA sequence variations in the context of statin therapy.     

Effect of cultivation conditions on trehalose content and viability of brewing yeast following preservation via filter paper or lyophilization methods

The effects of variations in cultivation conditions on trehalose concentration and the viability of brewing yeasts following preservation by filter paper or lyophilization methods were evaluated. In case of filter paper preservation, the cultivation period had no affect on yeast viability, while agitation and aeration during cultivation had a positive effect regarding viability of the bottom-fermenting strains, Rh and Frank. For effective preservation, it was necessary to harvest yeast cells from the stationary phase during cultivation. For lyophilization preservation, the yeast strains tested showed a negative effect on viability, independent of strain or cultivation method. No significant correlation was found between trehalose concentration and yeast viability following either filter paper or lyophilization preservation. However, the filter paper preservation method was suitable for both bottom and top brewing yeast strains with regard to feasibility, viability, and maintenance of the yeast’s specific character.     

Isolation and characterization of ethanol-producing yeasts from fruits and tree barks

Aims:  Isolation and identification of yeasts converting xylose to ethanol.
Methods and Results:  A total of 374 yeasts were isolated from a variety of rotten fruits and barks of trees. Out of these, 27 yeast strains were able to assimilate xylose and produce 0·12–0·38 g of ethanol per gram of xylose. Based on phylogenetic analysis of D1/D2 domain sequence of LSU (Large Subunit) rRNA gene and phenotypic characteristics the ethanol-producing strains were identified as member(s) of the genera Pichia, Candida , Kluyveromyces, Issatchenkia, Zygosacchraomyces , Clavispora, Debaryomyces , Metschnikowia , Rhodotorula and Cryptococcus.
Conclusion:  Yeast strains producing ethanol from xylose have been isolated from a variety of rotten fruits and barks of trees and identified.
Significance and Impact of the Study:  Environmental isolates of yeasts which could convert xylose to ethanol could form the basis for bio-fuel production and proper utilization of xylan rich agricultural and forest wastes.     

芝麻香型白酒大曲中酵母菌的分离与鉴定

为了开发利用白酒大曲中的酵母菌资源,采用稀释涂布平板法,从芝麻香型白酒大曲中分离得到15株酵母菌株。利用26S rRNA基因序列分析技术对其进行分类鉴定。结果表明,其中6株酵母菌为酿酒酵母（Saccharomyces cerevisiae）,6株为库德里阿兹威氏毕赤酵母（Pichia kudriavzevii）,3株为热带假丝酵母（Candida tropicalis）;并对代表菌株Y1、Y2及Y4进行了形态观察;最后通过随机扩增多态性DNA标记（random amplified polymorphic DNA, RAPD）对分离得到的酵母菌进行分型,表明6株酿酒酵母分属于5个株型,6株库德里阿兹威氏毕赤酵母分属于5个株型,3株热带假丝酵母分属于3个株型。     

Role of bottom-fermenting brewer's yeast KEX2 in high temperature resistance and poor proliferation at low temperatures

Variants of bottom-fermenting brewer's yeast that grew at high temperatures and showed poor proliferation and fermentation at low temperatures were isolated. Similar variants of laboratory yeast were also isolated and found to be incapable of mating. The KEX2 gene was cloned by complementation. It was shown to be responsible for these traits, because a KEX2 disruptant of Saccharomyces cerevisiae (S. cerevisiae) laboratory yeast grew poorly at low temperatures and was resistant to high temperatures. In addition, a Saccharomyces bayanus (S. bayanus)-type KEX2 (Sb-KEX2) disruptant of bottom-fermenting brewer's yeast grew poorly at low temperatures and was resistant to high temperatures. The KEX2 gene product plays an important role in proliferation of yeast at low temperatures, which is an important trait of bottom-fermenting brewer's yeast. These findings advance our understanding of the proliferation of yeast at low temperatures, especially that of bottom-fermenting brewer's yeast.     

The effect of pyrimethanil on the growth of wine yeasts

Aims:  The toxicity of the fungicide pyrimethanil on the growth of wine yeasts was evaluated using in vivo and in vitro experimentation.
Methods and Results:  The effect of pyrimethanil in the must was studied during the spontaneous wine fermentation of three consecutive vintages and by the cultivation of Hanseniaspora uvarum and Saccharomyces cerevisiae yeasts in a liquid medium. The residues of the fungicide were measured using gas chromatography-mass spectrometry system and the sugar concentration in the must using HPLC-RI. Molecular and standard methods were used for identifying the yeast species. Although the pyrimethanil residues in grapes were below the maximum residue limits, they significantly affected the reduced utilization of sugars in the first days of fermentation. Its residues controlled the growth of H. uvarum during the fermentation and during in vitro cultivation as well.
Conclusions:  The fungicide pyrimethanil had an effect on the course and successful conclusion of spontaneous wine fermentation that was correlated with the initial concentration of yeasts in the must.
Significance and Impact of the Study:  The impact of pyrimethanil on the indigenous mixed yeast flora in fermenting must was investigated for the first time. The results showed that its residues might play an important role in the growth and succession of yeast during spontaneous wine fermentation.     

Microsatellite PCR profiling of Saccharomyces cerevisiae strains during wine fermentation

AIMS: Use of microsatellite PCR to monitor populations of Saccharomyces cerevisiae strains during fermentation of grape juice. METHOD AND RESULTS: Six commercial wine strains of S. cerevisiae were screened for polymorphism at the SC8132X locus using a modified rapid PCR identification technique. The strains formed four distinct polymorphic groups that could be readily distinguished from one another. Fermentations inoculated with mixtures of three strains polymorphic at the SC8132X locus were monitored until sugar utilization was complete, and all exhibited a changing population structure throughout the fermentation. CONCLUSIONS: Rapid population quantification demonstrated that wine fermentations are dynamic and do not necessarily reflect the initial yeast population structure. One or more yeast strains were found to dominate at different stages of the fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: The population structure of S. cerevisiae during mixed culture wine fermentation is dynamic and could modify the chemical composition and flavour profile of wine.     

Petite mutation in aged and oxidatively stressed ale and lager brewing yeast

Aims:  To determine the role of oxidative stress and chronological ageing on the propensity of brewing yeast strains to form respiratory deficient 'petites'.
Methods and Results:  Four industrial yeast strains (two ale and two lager strains) were exposed to oxidative stress in the form of H₂O₂ (5 mmol l⁻¹) for two hours. Cell viability and occurrence of petites were determined by the slide culture and TTC-overlay techniques, respectively. Increases in petite frequency were observed but only in those strains sensitive to oxidative stress. Chronological ageing under aerobic conditions led to an increase in petites in strains sensitive to oxidative stress. No such increase was observed under anaerobic conditions.
Conclusion:  Ageing may contribute to mitochondrial DNA damage and increase the propensity of brewing yeast cells to become respiratory deficient. Tolerant strains may be less likely to generate petites as a result of serial re-pitching.
Significance and Impact of the Study:  Continuous re-use of brewing yeast is associated with an increase in the frequency of petites within brewery yeast slurries, a phenomenon resulting in reduced fermentative capacity. The cause of petite generation during brewery handling is unknown. We show that endogenous oxidative stress has the potential to generate petites within brewing yeast populations.     

Isolation, characterization and identification of lactic acid bacteria and yeasts from sour Mifen, a traditional fermented rice noodle from China

Aims:  Considering the effect of natural fermentation on the textural improvement of fermented rice noodles in China and South Asia, and given the lack of reports concerning microbial populations and structure in the fermentation process, this study aims to determine the number of viable micro-organisms and identify the species isolated from the local factories, and to assess their potential use as a starter culture from their enzymatic profiles.
Methods and Results:  Fourteen samples from three local factories were analysed for the presence of micro-organisms. A total of 170 lactic acid bacteria (LAB) and 96 yeasts were isolated from the factories. The isolates were phenotypically characterized by using API 50 CHL kits, API 20 Strep kits, API ID 32 C kits and by performing additional biochemical tests. The enzymatic profiles of isolates were assessed by using API ZYM kits. Lactobacillus plantarum and Saccharomyces cerevisiae were identified as predominant species in the fermented supernatants. A majority of the isolates of LAB and yeasts displayed activities of α-glucosidase, β-glucosidase, lipase and trypsin.
Conclusions:  The microbial composition and strain characteristics present in the fermentation supernatant demonstrate that a majority of micro-organisms have the ability to digest starch, sugar, protein or lipid. It supports our previous work in which the rice starch was modified and purified by fermentation and thus improves the texture of rice noodles.
Significance and Impact of the Study:  The dominant strains would be important in developing a starter culture. The results can form the basis for the improvement of product quality and consistency.     

Evidence of different fermentation behaviours of two indigenous strains of Saccharomyces cerevisiae and Saccharomyces uvarum isolated from Amarone wine

Aims:  To explain the role of Saccharomyces cerevisiae and Saccharomyces uvarum strains (formerly Saccharomyces bayanus var. uvarum ) in wine fermentation.
Methods and Results:  Indigenous Saccharomyces spp. yeasts were isolated from Amarone wine (Italy) and analysed. Genotypes were correlated to phenotypes: Melibiose⁻ and Melibiose⁺ strains displayed a karyotype characterized by three and two bands between 225 and 365 kb, respectively. Two strains were identified by karyotype analysis (one as S. cerevisiae and the other as S. uvarum ). The technological characterization of these two strains was conducted by microvinifications of Amarone wine. Wines differed by the contents of ethanol, residual sugars, acetic acid, glycerol, total polysaccharides, ethyl acetate, 2-phenylethanol and anthocyanins. Esterase and β-glucosidase activities were assayed on whole cells during fermentation at 13° and 20°C. Saccharomyces uvarum displayed higher esterase activity at 13°C, while S. cerevisiae displayed higher β-glucosidase activity at both temperatures.
Conclusions:  The strains differed by important technological and qualitative traits affecting the fermentation kinetics and important aroma components of the wine.
Significance and Impact of the Study:  The contribution of indigenous strains of S. cerevisiae and S. uvarum to wine fermentation was ascertained under specific winemaking conditions. The use of these strains as starters in a winemaking process could differently modulate the wine sensory characteristics.     

HO gene polymorphism in Saccharomyces industrial yeasts and application of novel HO genes to convert homothallism to heterothallism in combination with the mating-type detection cassette

Southern blot analysis of industrial yeasts showed that all top-fermenting yeasts, distiller's yeasts and a proportion of wine yeasts tested in the present study produced a hybridization signal (approximately 7 kb), corresponding to a Saccharomyces cerevisiae-type HO gene (Sc-HO). It also showed that bottom-fermenting yeasts gave rise to 7-kb and 4-kb hybridization signals, corresponding to the Sc-HO gene and the lager yeast HO gene (Lg-HO), respectively. Two wine yeasts produced a 4-kb hybridization signal, corresponding to Lg-HO; and one wine yeast produced 2.5-kb and 1.5-kb hybridization bands, corresponding to a S. uvarum-type HO gene (Uv-HO). Partial nucleotide sequences of HO genes amplified from these wine yeasts perfectly matched those of Lg-HO and Uv-HO, respectively. HO disruption vectors were constructed by inserting a dominant selective marker PGK1p-neo and the mating-type detection cassette MFalpha1p-PHO5 within the Lg-HO or Uv-HO gene. From transformants carrying a single-disrupted ho gene, mating-competent progenies were easily obtained through meiosis. Moreover, mating-competent derivatives appearing at very low frequency could be obtained from a double-disrupted ho transformant without meiosis (even from a wine yeast lacking sporulation ability), because the sensitive phosphatase-staining method allowed detection of the Pho+ mating-competent derivatives from confluent colonies by the random spore method. Our study describes a rapid and convenient method for isolating mating-competent clones from industrial yeasts.     

Predominance of Saccharomyces uvarum during spontaneous alcoholic fermentation, for three consecutive years, in an Alsatian winery

AIMS: The purpose of this study was to determine the origin of the yeasts involved in the spontaneous alcoholic fermentation of an Alsatian wine. METHODS AND RESULTS: During three successive years, must was collected at different stages of the winemaking process and fermented in the laboratory or in the cellar. Saccharomyces yeasts were sampled at the beginning and at the end of the fermentations. Saccharomyces cerevisiae clones were genetically characterized by inter-delta PCR. Non-S. cerevisiae clones were identified as Saccharomyces uvarum by PCR-RFLP on MET2 gene and characterized at the strain level by karyotyping. The composition of the Saccharomyces population in the vineyard, after crushing and in the vat was analyzed. This led to three main results. First, the vineyard Saccharomyces population was rather homogeneous. Second, new non-resident strains had appeared in the must during the winemaking process. Finally, the yeast population in the vat only consisted in S. uvarum strains. CONCLUSION: This 3-year study has enabled us to show the involvement of indigenous S. uvarum in the alcoholic fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: This study gives a first insight into the polymorphism of S. uvarum strains involved in a spontaneous alcoholic fermentation.     

History,lineage and phenotypic differentiation of sake yeast

ABSTRACT

Sake yeast was first isolated as a single yeast strain, Saccharomyces sake, during the Meiji era. Yeast strains suitable for sake fermentation were subsequently isolated from sake brewers and distributed as Kyokai yeast strains. Sake yeast strains that produce characteristic flavors have been bred in response to various market demands and individual preferences. Interestingly, both genetic and morphological studies have indicated that sake yeast used during the Meiji era differs from new sake yeasts derived from Kyokai Strain No. 7 lineage. Here, we discuss the history of sake yeast breeding, from the discovery of sake yeast to the present day, to highlight the achievements of great Japanese scientists and engineers.     

Protein Sequences of Two Keto Ester Reductases: Possible Identity as Hypothetical Proteins

We determined the amino acid sequences of two keto ester reductases (YKER-V and -VI) purified from a cell-free extract of Saccharomyces cerevisiae. The N-terminal and internal amino acid sequences of YKER-VI (AcKR) were in agreement with the sequence of hypothetical 36.4-kDa protein (S. cerevisiae chromosome X reading frame ORF YJR105w) in yeast. The N-terminal amino acid sequence of YKER-V was also identical with that of the hypothetical protein coded by yeast chromosome XIV or II. These results suggested that two hypothetical proteins were expressed as keto ester reductases in yeast cells.     

Map-based cloning of the tomato genomic region that spans the Sw-5 tospovirus resistance gene in tomato

Two yeast artificial chromosomes (YACs) containing genomic DNA from tomato have been isolated using CT220, an RFLP marker which is tightly linked to the tomato spotted wilt virus resistance gene, Sw-5. High-resolution mapping of the YAC ends and internal YAC probes demonstrated that one of the YAC clones, TY257 (400 kb), spans Sw-5. By chromosome walking in a cosmid library, the position of Sw-5 has been delimited within the YAC to a maximal chromosomal segment of 100 kb, spanned by nine overlapping cosmid clones. Received: 13 March 1997 / Accepted: 11 may 1997     

Validation of the official control method based on Polymerase Chain Reaction (PCR) for identification of authorised probiotic yeast in animal feed

Council Directive 70/524/EEC regulates the application of probiotic (microorganisms) additives in feeding stuffs. In the present study a method for the differentiation and strain identification of authorised probiotic Saccharomyces cereviseae strains in feeding stuffs by Polymerase Chain Reaction (PCR) was validated. Four different samples of animal feeding stuffs containing yeast at levels between 10(5) to 10(7) CFU/g were examined. Samples were enumerated on chloramphenicol glucose yeast extract agar and colonies were selected from these plates for DNA extraction and subsequent analysis. The PCR method using delta sequence primers produced an 'amplified sequence polymorphism' characteristic for the test strain. Feeds supplemented with one of four probiotic yeast strains each were analysed by seven of nine invited laboratories. All laboratories returned valid results with the exception of one laboratory that had insufficiently separated bands on the gel. The method had a good reproducibility for probiotic yeast isolates from feed of all four authorised probiotic yeast strains (APYS) CBS 493.94, APYS CNCM 1-1079, APYS CNCM 1-1077, APYS NCYC SC47 and of a commercially available yeast reference strain, NCYC 81. The PCR method is to be considered by CEN and ISO as official control method for identification of authorised probiotic Saccharomyces cerevisiae strains from feeding stuffs.     

Mapping irradiation hybrids to cosmid and yeast artificial chromosome libraries by direct hybridization of Alu-PCR products.

A direct hybridization protocol is described for screening cosmid and yeast artificial chromosome libraries with pools of Alu-PCR products from somatic cell or irradiation hybrids. This method eliminates purification, cloning and analysis of each individual Alu-PCR product before library screening. A series of human X chromosome irradiation hybrids were mapped by this method, using a cosmid reference library for comparisons between overlapping hybrids to identify interesting clones for further analysis.