Saccharomyces cerevisiae biomass as a source of next-generation food preservatives: Evaluating potential proteins as a source of antimicrobial peptides

Saccharomyces cerevisiae is the main biotechnological tool for the production of Baker's or Brewer's biomasses, largely applied in beverage and fermented-food production. Through its gene expression reprogramming and production of compounds that inactivate the growth of other microorganisms, S. cerevisiae is able to grow in adverse environments and in complex microbial consortia, as in fruit pulps and root flour fermentations. The distinct set of up-regulated genes throughout yeast biomass propagation includes those involved in sugar fermentation, ethanol metabolization, and in protective responses against abiotic stresses. These high abundant proteins are precursors of several peptides with promising health-beneficial activities such as antihypertensive, antioxidant, antimicrobial, immunomodulatory, anti-obesity, antidiabetes, and mitogenic properties. An in silico investigation of these S. cerevisiae derived peptides produced during yeast biomass propagation or induced by physicochemical treatments were performed using four algorithms to predict antimicrobial candidates encrypted in abundantly expressed stress-related proteins encoded by different genes like AHP1, TSA1, HSP26, SOD1, HSP10, and UTR2, or metabolic enzymes involved in carbon source utilization, like ENO1/2, TDH1/2/3, ADH1/2, FBA1, and PDC1. Glyceraldehyde-3-phosphate dehydrogenase and enolase II are noteworthy precursor proteins, since they exhibited the highest scores concerning the release of antimicrobial peptide candidates. Considering the set of genes upregulated during biomass propagation, we conclude that S. cerevisiae biomass, a food-grade product consumed and marketed worldwide, should be considered a safe and nonseasonal source for designing next-generation bioactive agents, especially protein encrypting antimicrobial peptides that display broad spectra activity and could reduce the emergence of microbial resistance while also avoiding cytotoxicity.     

RRD1, a component of the TORC1 signalling pathway,affects anaesthetic response in Saccharomyces cerevisiae

The molecular mechanisms of action of volatile anaesthetics remain unknown despite clinical use for over 150 years. While many effects of these agents have been characterized, clear insight into how these effects relate to the physiological state of anaesthesia has not been established. Volatile anaesthetics arrest cell division in Saccharomyces cerevisiae in a manner that parallels the anaesthetic actions of these drugs in mammals. To gain additional insight into the cellular activities of these drugs, we isolated genes that, when present on multi‐copy plasmids, render S. cerevisiae resistant to the volatile anaesthetic isoflurane. One of these genes, RRD1, encodes a subunit of the Tap42p–Sit4p–Rrd1p phosphatase complex that functions in the target of rapamycin complex 1 (TORC1) signalling pathway. In addition, we show that mutations in two other genes encoding components of the TORC1 pathway, GLN3 and URE2, also affect yeast anaesthetic response. These findings suggest that TORC1‐mediated signalling is involved in cellular response to volatile anaesthetics in S. cerevisiae. Copyright © 2009 John Wiley & Sons, Ltd.     

Acetaldehyde tolerance in Saccharomyces cerevisiae involves the pentose phosphate pathway and oleic acid biosynthesis

To identify genes responsible for acetaldehyde tolerance, genome‐wide screening was performed using a collection of haploid Saccharomyces cerevisiae strains deleted in single genes. The screen identified 49 genes whose deletion conferred acetaldehyde sensitivity, and these were termed the genes required for acetaldehyde tolerance. We focused on six of these genes required for acetaldehyde tolerance, ZWF1, GND1, RPE1, TKL1 and TAL1, which encode enzymes in the pentose phosphate pathway (PPP), and OAR1, which encodes for NADPH‐dependent 3‐oxoacyl‐(acyl‐carrier‐protein) reductase. These genes were not only responsible for acetaldehyde tolerance but also turned out to be induced by acetaldehyde. Moreover, the content of oleic acid was remarkably increased in yeast cells under acetaldehyde stress, and supplementation of oleic acid into the media partially alleviated acetaldehyde stress‐induced growth inhibition of strains disrupted in the genes required for acetaldehyde tolerance and OLE1. Taken together, our data suggest that the supply of NADPH and the process of fatty acid biosynthesis are the key factors in acetaldehyde tolerance in the yeast, and that oleic acid plays an important role in acetaldehyde tolerance. Copyright © 2008 John Wiley & Sons, Ltd.     

Analysis of the DNA Sequence of a 34 038 bp Region on the Left Arm of Yeast Chromosome XV

We report the DNA sequence of a 34 038 bp segment of Saccharomyces cerevisiae chromosome XV. Subsequent analysis revealed 20 open reading frames (ORFs) longer than 300 bp and two tRNA genes. Five ORFs correspond to genes previously identified in S. cerevisiae, including RPLA2, PRE6, MSE1, IFM1 and SCM2 (TAT2, TAP2, LTG3). Two putative proteins share considerable homology with other proteins in the current data libraries. ORF O2145 shows 41·2% identity with the glycophospholipid-anchored surface glycoprotein Gas1p of S. cerevisiae and ORF O2197 has 53·2% identity to chromosome segregation protein Dis3p of Schizosaccharomyces pombe. Accession Numbers for these sequences are provided in Table 1.©1997 John Wiley & Sons, Ltd.     

Genetic and enological analysis of selected Saccharomyces cerevisiae strains for wine production

The non‐wine Saccharomyces cerevisiae strain of 96581 was found to be a promising candidate for the production of white wine. It produced wines with fusel alcohols that were 57% higher than those produced by the wine yeasts studied and was also more efficient in the production of 2‐phenethyl acetate and 3‐methyl‐1‐butanol acetate. This study also shows that there is a difference in the ester‐formation efficiency for acetates relative to C6, C8 and C10 fatty acid esters for all the studied yeast strains. Therefore, it supports the view that other unidentified enzymes besides those regulated by ATF1 and ATF2 genes are involved in the production of ethyl esters of C6–C10 fatty acids. DNA analysis of the 25S, 18S, 5.8S and 5S ribosomal DNA genes in these strains showed high conservation. Despite the closely related nature of these yeast strains, the chemical profiles of the wines produced were significantly different.     

Candida albicans CaPTC6 is a functional homologue for Saccharomyces cerevisiae ScPTC6 and encodes a type 2C protein phosphatase

Type 2C protein phosphatases (PP2C) are monomeric enzymes and their activities require the presence of magnesium or manganese ions. There are seven PP2C genes, ScPTC1, ScPTC2, ScPTC3, ScPTC4, ScPTC5, ScPTC6 and ScPTC7, in Saccharomyces cerevisiae. PTC6 is highly conserved in pathogenic and nonpathogenic yeasts. In the current study we have demonstrated that the Candida albicans CaPTC6 gene could complement the functions of ScPTC6 in the rapamycin and caffeine sensitivities of S. cerevisiae cells, indicating that they are functional homologues. We have also demonstrated that the CaPTC6‐encoded protein is a typical PP2C enzyme and that CaPtc6p is localized in the mitochondrion of yeast‐form and hyphal cells. However, deletion of CaPTC6 neither affects cell and hyphal growth nor renders Candida cells sensitive to rapamycin and caffeine. Therefore, possibly with a functional redundancy to other mitochondrial phosphatases, CaPtc6p is likely to be involved in the regulation of a mitochondrial physiology. Copyright © 2009 John Wiley & Sons, Ltd.     

Two-dimensional electrophoretic separation of yeast proteins using a non-linear wide range (pH 3–10) immobilized pH gradient in the first dimension; reproducibility and evidence for isoelectric focusing of alkaline (pI >7) proteins

The proteome of the yeast Saccharomyces cerevisiae was analysed by two-dimensional (2D) polyacrylamide gel electrophoresis utilizing a non-linear immobilized pH gradient (3–10) in the first-dimensional separation. Cells were labelled by [³⁵S]methionine incorporation in the respiro-fermentative phase during exponential growth on glucose. Gels were run, visualized with phosphoimager technology and all resolved proteins automatically quantified. Proteins were well resolved over the whole pH interval, and evidence for isoelectric focusing on the basic side of the pattern was generated by sequencing of some spots, revealing the 2D positions of Tef1p, Pgk1p, Gpm1p, Tdh1p and Shm2p. Roughly 25% of the spots were resolved at the alkaline side of the pattern (pI>7). The position reproducibility was high and in the range 1–2 mm in the x-and y-dimension, respectively. No quantitative variation was linked to a certain size or charge class of resolved proteins, and the average quantitative standard deviation was 17±11%. The obtained immobilized pH gradient based pattern could easily be compared to the old ampholine-based 2D pattern, and the previously reported identifications could thus be transferred. Our yeast pattern currently contains 43 known proteins, all identified by protein sequencing. Utilizing these identified proteins, relevant pI and Mr scales in the pattern were constructed. Normalization of the expression of identified spots by compensating for the number of methionine residues a protein contains allowed stoichiometric comparisons. The most dominant proteins under these growth conditions were Tdh3p, Fba1p, Eno2p and Tef1p/Tef2p, all being expressed at more than 500 000 copies per cell. The differential carbon source response during exponential growth on either glucose, galactose or ethanol was examined for the alkaline proteins identified by micro-sequencing in this study. © 1997 John Wiley & Sons, Ltd.     

Systematic analysis of yeast strains with possible defects in lipid metabolism

Lipids are essential components of all living cells because they are obligate components of biological membranes, and serve as energy reserves and second messengers. Many but not all genes encoding enzymes involved in fatty acid, phospholipid, sterol or sphingolipid biosynthesis of the yeast Saccharomyces cerevisiae have been cloned and gene products have been functionally characterized. Less information is available about genes and gene products governing the transport of lipids between organelles and within membranes or the turnover and degradation of complex lipids. To obtain more insight into lipid metabolism, regulation of lipid biosynthesis and the role of lipids in organellar membranes, a group of five European laboratories established methods suitable to screen for novel genes of the yeast Saccharomyces cerevisiae involved in these processes. These investigations were performed within EUROFAN (European Function Analysis Network), a European initiative to identify the functions of unassigned open reading frames that had been detected during the Yeast Genome Sequencing Project. First, the methods required for the complete lipid analysis of yeast cells based on chromatographic techniques were established and standardized. The reliability of these methods was demonstrated using tester strains with established defects in lipid metabolism. During these investigations it was demonstrated that different wild‐type strains, among them FY1679, CEN.PK2‐1C and W303, exhibit marked differences in lipid content and lipid composition. Second, several candidate genes which were assumed to encode proteins involved in lipid metabolism were selected, based on their homology to genes of known function. Finally, lipid composition of mutant strains deleted of the respective open reading frames was determined. For some genes we found evidence suggesting a possible role in lipid metabolism. Copyright © 1999 John Wiley & Sons, Ltd.     

Designer deletion and prototrophic strains derived from Saccharomyces cerevisiae strain W303‐1a

We report the construction of Saccharomyces cerevisiae strains isogenic to W303‐1a that are designed to allow efficient genetic analysis. To facilitate the generation of null alleles of target genes by PCR‐mediated gene disruption, we constructed designer deletion alleles of the ARG4, TRP1 and URA3 genes. In addition, a single pair of oligonucleotide primers were designed that can be used to amplify any of several marker genes for use in PCR‐mediated gene disruption. A new version of the ‘reusable’ hisG‐URA3‐hisG cassette was constructed for use in PCR‐mediated gene disruption. Finally, to facilitate the formation of isogenic diploids by selection, we constructed strains that contain combinations of wild‐type alleles of ADE2, HIS3, LEU2, TRP1 and URA3. Copyright © 1999 John Wiley & Sons, Ltd.     

Cloning and characterization of a novel NAD+‐dependent glyceraldehyde‐3‐phosphate dehydrogenase gene from Candida glycerinogenes and use of its promoter

A 3950 bp genomic fragment from Candida glycerinogenes, WL2002‐5, containing the CgGAP gene encoding a glyceraldehyde‐3‐phosphate dehydrogenase homologous to GAP genes in other yeasts using degenerate primers, was cloned and characterized with inverse PCR. Sequence analysis revealed a 1164 bp open reading frame encoding a putative peptide of 387 deduced amino acids, with a molecular mass of 36 kDa. The CgGAP protein consisted of an N‐terminal NAD⁺‐binding domain and a central catalytic domain. Six stress‐response elements were found in the upstream region of the CgGAP gene. The influence of CgGAP on glycolysis was investigated. Functional analysis revealed that Saccharomyces cerevisiae transformed with CgGAP was restored to the wild‐type phenotype when cultured in high‐osmolarity medium, suggesting that it is a functional GAP protein. Promoter studies in S. cerevisiae using the green fluorescent protein (gfp) gene as a reporter showed that the GAP promoter (P_CgGAP) is constitutively expressed in S. cerevisiae cells grown on glucose. Copyright © 2013 John Wiley & Sons, Ltd.     

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.