Comparative proteomic analysis of a Candida albicans DSE1 mutant under filamentous and non‐filamentous conditions

Candida albicans is a common opportunistic pathogen that causes a variety of diseases in immunocompromised hosts. In a pathogen, cell wall proteins are important virulence factors. We previously characterized Dse1 as a cell wall protein necessary for virulence and resistance to cell surface‐disrupting agents, such as Calcofluor white, chitin deposition, proper adhesion and biofilm formation. In the absence of decomplexation, our objectives were to investigate differential proteomic expression of a DSE1 mutant strain compared to the wild‐type strain. The strains were grown under filamentous and non‐filamentous conditions. The extracted cell proteome was subjected to tryptic digest, followed by generation of peptide profiles using MALDI–TOF MS. Generated peptide profiles were analysed and unique peaks for each strain and growth condition mined against a Candida database, allowing protein identification. The DSE1 mutant was shown to lack the chitin biosynthesis protein Chs5, explaining the previously observed decrease in chitin biosynthesis. The wild‐type strain expressed Pra1, involved in pH response and zinc acquisition, Atg15, a lipase involved in virulence, and Sod1, required for oxidative stress tolerance, in addition to proteins involved in protein biosynthesis, explaining the increase in total protein content observed compared to the mutants strain. The mutant, on the other hand, expressed glucoamylase 1, a cell wall glycoprotein involved in carbohydrate metabolism cell wall degradation and biofilm formation. As such, MALDI–TOF MS is a reliable technique in identifying mutant‐specific protein expression in C. albicans. Copyright © 2014 John Wiley & Sons, Ltd.     

Enhancement of β-glucosidase activity on the cell-surface of sake yeast by disruption of SED1

We determined the genetic background that would result in a more optimal display of heterologously expressed β-glucosidase (BGL) on the cell surface of yeast Saccharomyces cerevisiae. Amongst a collection of 28 strains carrying deletions in genes for glycosylphosphatidyl inositol (GPI)-anchored proteins, the Δsed1 and Δtos6 strains had significantly higher BGL-activity whilst maintaining wild type growth. Absence of Sed1p, which might facilitate incorporation of anchored BGL on the cell-surface, could also influence the activity of BGL on the cell surface with the heterologous gene being placed under the control of the SED1 promoter. For the evaluation of its industrial applicability we tested this system in heterologous and homogenous SED1-disruptants of sake yeast, a diploid S. cerevisiae strain, in which either the SED1 ORF or the complete gene including the promoter was deleted by use of the high-efficiency loss of heterozygosity method. Evaluation of disruptants displaying BGL showed that deletion of the SED1 ORF enhanced BGL activity on the cell surface, while additional deletion of the SED1 promoter increased further BGL activity on the cell surface. Compared to heterozygous disruption, homozygous disruption resulted generally in a higher BGL activity. Thus, homozygous deletion of both SED1 gene and promoter resulted in the most efficient display of BGL reaching a 1.6-fold increase of BGL-activity compared to wild type.     

Impact of lignin composition on cell‐wall degradability in an Arabidopsis mutant

An Arabidopsis mutant that does not deposit syringyl‐type lignin was used to test the hypothesis that lignin composition impacts cell‐wall degradability. Two lines of the ferulate‐5‐hydroxylase‐deficient fah1 mutant and the wild‐type control line were grown in the greenhouse. In Experiment 1, the plants were harvested at the mature seed stage. For Experiment 2, plants were harvested 5, 6, 7 and 8 weeks after sowing. In both experiments stems were collected and analysed for cell‐wall concentration and composition, and in vitro degradability of cell‐wall polysaccharide components by rumen micro‐organisms. The absence of syringyl‐type lignin was confirmed for the mutant lines by nitrobenzene oxidation and pyrolysis‐GC‐MS. Lignin concentration was the same for all three Arabidopsis lines, at all stages of maturity. The Arabidopsis stems were similar to forage legumes in that the potentially degradable cell‐wall fraction was very quickly degraded. Cell‐wall polysaccharide degradability did not differ among the Arabidopsis lines in the first experiment after 24‐h fermentations, but the cell‐wall polysaccharides of the fah1‐2 mutant line were less degradable after 96‐h than either the wild‐type or the fah1‐5 mutant. In contrast, in Experiment 2 no differences among lines were found for cell‐wall polysaccharide degradability after either 24‐ or 96‐h fermentations; however, signficantly higher levels of ester‐bound ferulic acid were found in the walls of the fah1 mutant lines. As expected, increasing stem maturity was correlated with reduced degradation of cell‐wall polysaccharides. These experiments indicate that either lignin composition, as measured by syringyl‐to‐guaiacyl ratio, does not alter cell‐wall degradability in Arabidopsis, or that the fah1 mutation has other effects on the cell walls of these mutants such that the impact of the change in syringyl‐to‐guaiacyl ratio is masked. © 1999 Society of Chemical Industry     

Genome‐wide screen of Saccharomyces cerevisiae for killer toxin HM‐1 resistance

We screened a set of Saccharomyces cerevisiae deletion mutants for resistance to killer toxin HM‐1, which kills susceptible yeasts through inhibiting 1,3‐beta‐glucan synthase. By using HM‐1 plate assay, we found that eight gene‐deletion mutants had higher HM‐1‐resistance compared with the wild‐type. Among these eight genes, five—ALG3, CAX4, MNS1, OST6 and YBL083C—were associated with N‐glycan formation and maturation. The ALG3 gene has been shown before to be highly resistant to HM‐1. The YBL083C gene may be a dubious open reading frame that overlaps partially the ALG3 gene. The deletion mutant of the MNS1 gene that encodes 1,2‐alpha‐mannosidase showed with a 13‐fold higher HM‐1 resistance compared with the wild‐type. By HM‐1 binding assay, the yeast plasma membrane fraction of alg3 and mns1 cells had less binding ability compared with wild‐type cells. These results indicate that the presence of the terminal 1,3‐alpha‐linked mannose residue of the B‐chain of the N‐glycan structure is essential for interaction with HM‐1. A deletion mutant of aquaglyceroporin Fps1p also showed increased HM‐1 resistance. A deletion mutant of osmoregulatory mitogen‐activated protein kinase Hog1p was more sensitive to HM‐1, suggesting that high‐osmolarity glycerol pathways plays an important role in the compensatory response to HM‐1 action. Copyright © 2010 John Wiley & Sons, Ltd.     

Human NKCC2 cation–Cl– co‐transporter complements lack of Vhc1 transporter in yeast vacuolar membranes

Cation–chloride co‐transporters serve to transport Cl^– and alkali metal cations. Whereas a large family of these exists in higher eukaryotes, yeasts only possess one cation–chloride co‐transporter, Vhc1, localized to the vacuolar membrane. In this study, the human cation–chloride co‐transporter NKCC2 complemented the phenotype of VHC1 deletion in Saccharomyces cerevisiae and its activity controlled the growth of salt‐sensitive yeast cells in the presence of high KCl, NaCl and LiCl. A S. cerevisiae mutant lacking plasma‐membrane alkali–metal cation exporters Nha1 and Ena1‐5 and the vacuolar cation–chloride co‐transporter Vhc1 is highly sensitive to increased concentrations of alkali–metal cations, and it proved to be a suitable model for characterizing the substrate specificity and transport activity of human wild‐type and mutated cation–chloride co‐transporters. Copyright © 2013 John Wiley & Sons, Ltd.     

Evaluating potential applications of indigenous yeasts and their β‐glucosidases

The potential applications of wild yeast strains with β‐glucosidase activity were investigated by assaying their enzymatic production under simulated oenological conditions, coupled with the exploration of the potential applications of the β‐glucosidases by studying the enzymatic activity and stability under similar oenological conditions. The assay of enzymatic locations revealed that the β‐glucosidase activities from these wild strains occurred in the extracellular fraction, and in whole and permeabilized cells. The effects of different oenological factors on β‐glucosidase production indicated that the F6 Trichosporon asahii strain had higher β‐glucosidase production than the other strains under low pH conditions. However, the F35 Hanseniaspora uvarum strain and the F30 Saccharomyces cerevisiae strain showed higher β‐glucosidase production under high‐sugar conditions. Furthermore, the influence of oenological factors on the activity and stability of the β‐glucosidases revealed that the enzyme from the F6 T. asahii strain had a stronger low‐pH‐value resistance than the other yeast β‐glucosidases. These results suggest that the F35 H. uvarum, F30 S. cerevisiae and the F6 T. asahii β‐glucosidases may have some potentially applicable values in the fermentation industry. Copyright © 2015 The Institute of Brewing & Distilling     

Mutations in the N‐terminal region of the Schizosaccharomyces pombe glutathione transporter pgt1+ allows functional expression in Saccharomyces cerevisiae

Pgt1p encodes a glutathione transporter in Schizosaccharomyces pombe, orthologous to the Saccharomyces cerevisiae glutathione transporter, Hgt1p. Despite high similarity to Hgt1p, Pgt1p failed to display functionality during heterologous expression in S. cerevisiae. In the present study we employed a genetic strategy to investigate the reason behind the non‐functionality of pgt1⁺ in S. cerevisiae. Functional mutants were isolated after in vitro mutagenesis. Several mutants were obtained and four mutants analysed. Among these, three yielded different point mutations in the N‐terminal region (301–350 bp) of the transporter before the first transmembrane domain, while one mutant contained a deletion of 42 nucleotides within the same region. The mutant pgt1⁺ proteins not only expressed and localized correctly, but displayed high‐affinity glutathione transport capabilities in S. cerevisae. Comparison of wild‐type pgt1⁺ with the functional mutants revealed that a loss in protein expression was responsible for lack of functionality of wild‐type pgt1⁺ in S. cerevisiae. The mRNA levels in wild‐type and mutants were comparable, suggesting that the block was in translation. The formation of a strong stem–loop structure appeared to be responsible for inefficient translation in pgt1⁺ and disruption of these structures in the mutants was probably permitting translation. This was confirmed by making silent mutations in this region of wild‐type pgt1⁺, which led to their functionality in S. cerevisiae. This genetic strategy to relieve functional blocks in expression should greatly facilitate the study of these and other transporters from more intractable genetic organisms in a heterologous expression system. Copyright © 2012 John Wiley & Sons, Ltd.     

Functional display of amylase on yeast surface from Rhizopus oryzae as a novel enzyme delivery method

Fungal amylase has great importance in fermentation industry such as brewing, food fermentation, starch hydrolysis and for improving microbial populations in chicken intestine through feed applications. In the present investigation, alpha amylase cDNA from Rhizopus oryzae was cloned, sequenced, and successfully surface anchored in functional form using Saccharomyces cerevisiae EBY100 as host, yielding enzyme activity of 4.35 (±0.5) U/ml. The surface displayed yeast expressed amylase activity using plate assay, produced glucose and maltose as hydrolysis products using starch as substrate. The targeted and armed yeast with displayed enzyme was evaluated for its characterization at various pH and temperatures. The engineered yeast showed optimal activity at neutral pH and at 50°C incubation temperature. Reducing sugars produced by displayed enzyme were visualized by paper chromatography. The data suggested successful heterologous expression and display of amylase enzyme on yeast cell surface. The displayed system could further be utilized in fermentation industry for improving cost-effectiveness of the process.     

Transforming sugars into fat – lipid biosynthesis using different sugars in Yarrowia lipolytica

In an era of ever‐increasing energy demands, a promising technology is being developed: the use of oleaginous microorganisms such as Yarrowia lipolytica to convert waste materials into biofuels. Here, we constructed two Y. lipolytica strains that displayed both increased lipid accumulation and more efficient use of biomass‐derived sugars, including glucose, fructose, galactose and inulin. The first strain, Y. lipolytica YLZ150, was derived from the French wild‐type strain W29. It had inhibited triacylglycerol mobilization (?tgl4 ) and β‐oxidation (?pox1–6 ), and it overexpressed GPD1 , DGA2 , HXK1 , the native Leloir pathway, SUC2 from Saccharomyces cerevisiae and INU1 from Kluyveromyces marxianus . The second strain, Y. lipolytica Y4779, was derived from the Polish A‐101 strain. It had inhibited β‐oxidation (?mfe2 ) and overexpressed GPD1 , DGA1 , HXK1 , YHT3, SUC2 and INU1 . In the first experiment, strain YLZ150 was batch‐cultured in media containing different hexoses; the highest values for lipid concentration and yield of lipids from the substrate were obtained using fructose (20.3 g dm^?3 and 0.14 g g^?1, respectively). In the second experiment, we grew the two strains in fed‐batch cultures to examine lipid biosynthesis from inulin (a fructose polymer). For Y4779, the lipid concentration was 10.3 g dm^?3 and the yield of lipids from substrate was 0.07 g g^?1; in contrast, for YLZ150, these values were 24 g dm^?3 and 0.16 g g^?1, respectively. The YLZ150 strain is thus able to efficiently exploit glucose, fructose, galactose, sucrose and inulin for lipid biosynthesis. Copyright © 2017 John Wiley & Sons, Ltd.     

Disrupting the methionine biosynthetic pathway in Candida guilliermondii: characterization of the MET2 gene as counter‐selectable marker

Candida guilliermondii (teleomorph Meyerozyma guilliermondii) is an ascomycetous species belonging to the fungal CTG clade. This yeast remains actively studied as a result of its moderate clinical importance and most of all for its potential uses in biotechnology. The aim of the present study was to establish a convenient transformation system for C. guilliermondii by developing both a methionine auxotroph recipient strain and a functional MET gene as selection marker. We first disrupted the MET2 and MET15 genes encoding homoserine‐O‐acetyltransferase and O‐acetylserine O‐acetylhomoserine sulphydrylase, respectively. The met2 mutant was shown to be a methionine auxotroph in contrast to met15 which was not. Interestingly, met2 and met15 mutants formed brown colonies when cultured on lead‐containing medium, contrary to the wild‐type strain, which develop as white colonies on this medium. The MET2 wild‐type allele was successfully used to transfer a yellow fluorescent protein (YFP) gene‐expressing vector into the met2 recipient strain. In addition, we showed that the loss of the MET2‐containing YFP‐expressing plasmid can be easily observed on lead‐containing medium. The MET2 wild‐type allele, flanked by two short repeated sequences, was then used to disrupt the LYS2 gene (encoding the α‐aminoadipate reductase) in the C. guilliermondii met2 recipient strain. The resulting lys2 mutants displayed, as expected, auxotrophy for lysine. Unfortunately, all our attempts to pop‐out the MET2 marker (following the recombination of the bordering repeat sequences) from a target lys2 locus were unsuccessful using white/brown colony colour screening. Nevertheless, this MET2 transformation/disruption system represents a new versatile genetic tool for C. guilliermondii. Copyright © 2014 John Wiley & Sons, Ltd.     

Dual functions of Mdt1 in genome maintenance and cell integrity pathways in Saccharomyces cerevisiae

Recent evidence indicates considerable cross‐talk between genome maintenance and cell integrity control pathways. The RNA recognition motif (RRM)‐ and SQ/TQ cluster domain (SCD)‐containing protein Mdt1 is required for repair of 3′‐blocked DNA double‐strand breaks (DSBs) and efficient recombinational maintenance of telomeres in budding yeast. Here we show that deletion of MDT1 (PIN4/YBL051C) leads to severe synthetic sickness in the absence of the genes for the central cell integrity MAP kinases Bck1 and Slt2/Mpk1. Consistent with a cell integrity function, mdt1Δ cells are hypersensitive to the cell wall toxin calcofluor white and the Bck1–Slt2 pathway activator caffeine. An RRM‐deficient mdt1‐RRM0 allele shares the severe bleomycin hypersensitivity, inefficient recombinational telomere maintenance and slt2 synthetic sickness phenotypes, but not the cell wall toxin hypersensitivity with mdt1Δ. However, the mdt1‐RRM(3A) allele, where only the RNA‐binding site is mutated, behaves similarly to the wild‐type, suggesting that the Mdt1 RRM functions as a protein–protein interaction rather than a nucleic acid‐binding module. Surprisingly, in a strain background where double mutants are sick but still viable, bck1Δmdt1Δ and slt2Δmdt1Δ mutants differ in some of their phenotypes, consistent with the emerging concept of flexible signal entry and exit points in the Bck1–Mkk1/2–Slt2 pathway. Overall, the results indicate that Mdt1 has partially separable functions in both cell wall and genome integrity pathways. Copyright © 2009 John Wiley & Sons, Ltd.     

Redirection of Pyruvate Pathway of Lactic Acid Bacteria to Improve Cheese Quality

Metabolic engineering in Lactic acid bacteria (LAB) has focused on changing of pyruvate metabolism to increase production of desired flavor compounds. A constructed mutant strain should contain no foreign DNA and antibiotic resistance genes. Therefore, food grade lactate dehydrogenase (ldh ^d) and diacetyl reductase (dar ^d) mutant strains were created using two plasmid system in this study. Metabolic end products (pyruvate, lactate, formate and acetoin) of these strains in glucose medium and in cheese were determined using HPLC. Created mutant and wild type strains were used as a starter culture in cheese. Compared to the wild type strain, different levels of metabolites were observed in cheese during three weeks of ripening. The ldh ^d strains produced less lactate but high acetoin as a result of gene deletion. Deletion of dar gene decreased the production of acetoin. The dar deficient strains have low diacetyl reductase activity and are able to reduce significant amounts of acetoin but not terminate it completely. Genetic modification made the shift from homolactic to mixed acid fermentation, but the desired compound production hardly improved. The basis of these results and techniques are promising for the further studies.     

Tryptophan accumulation in Saccharomyces cerevisiae under the influence of an artificial yeast TRP gene cluster

Plasmid pME559, carrying all five yeast TRP genes, was constructed. This plasmid is a yeast/Escherichia coli shuttle vector based on pBR322 and 2 μm-DNA sequences derived from plasmid pJDB207. We studied in yeast (i) the stability of the plasmid under selective and non-selective conditions, (ii) expression of all five TRP genes and (iii) tryptophan accumulation in yeast transformants. These studies were conducted in comparison with an earlier construction, pME554, which differs from plasmid pME559 in the expression of the TRP1 gene and which carries the TRP2 wild type instead of the TRP2^fbr mutant allele. For stable maintenance of the plasmids in yeast a selection was necessary. Plasmid pME559 displayed normal expression of all TRP genes, and enzyme levels on average 23-fold higher than in the wild type strain were found. In comparison, the maximal tryptophan flux observed in such a plasmid-carrying strain was about ten-fold higher than the maximal flux capacity in the wild type strain.     

Most,but not all,yeast strains in the deletion library contain the [PIN+] prion

The yeast deletion library is a collection of over 5100 single gene deletions that has been widely used by the yeast community. The presence of a non‐Mendelian element, such as a prion, within this library could affect the outcome of many large‐scale genomic studies. We previously showed that the deletion library parent strain contained the [PIN⁺] prion. [PIN⁺] is the misfolded infectious prion form of the Rnq1 protein that displays distinct fluorescent foci in the presence of RNQ1–GFP and exists in different physical conformations, called variants. Here, we show that over 97% of the library deletion strains are [PIN⁺]. Of the 141 remaining strains that have completely (58) or partially (83) lost [PIN⁺], 139 deletions were able to efficiently maintain three different [PIN⁺] variants despite extensive growth and storage at 4 °C. One strain, cue2Δ, displayed an alteration in the RNQ1–GFP fluorescent shape, but the Rnq1p prion aggregate shows no biochemical differences from the wild‐type. Only strains containing a deletion of either HSP104 or RNQ1 are unable to maintain [PIN⁺], indicating that 5153 non‐essential genes are not required for [PIN⁺] propagation. Copyright © 2009 John Wiley & Sons, Ltd.     

Construction of a Single PEP4 Allele Deletion in Saccharomyces carlsbergensis and a Preliminary Evaluation of Its Brewing Performance

The secretion of proteinase A (encoded by PEP4) from brewer's yeast is detrimental to the foam stability of unpasteurized beer. The aim of this study was to construct mutants of the allopolyploid Saccharomyces carlsbergensis strain TT, which were partially or completely deficient in proteinase A activity. Allelic PEP4 genes were consecutively disrupted by using the Cre‐loxP recombination system combined with PCR‐mediated gene disruption. A single PEP4 deletion mutant TT‐M was successfully constructed. However, no viable mutant could be obtained when the second allelic PEP4 gene was deleted. The brewing performances of the parent strain and the modified strain were compared on a 100 L pilot fermenter scale. Proteinase A activity in fermented wort brewed with mutant strain TT‐M was significantly lower (p<0.05) than that of the parent strain TT, whereas no significant difference on either maltose or maltotriose assimilation (p>0.05) was found. The mutant TT‐M remained genetically stable, as shown by diagnostic PCR, after re‐streaking for 20 generations. The flavor and taste of the final fermented wort, brewed with the mutant strain TT‐M, was evaluated by the Tsingtao expert sensory panel, and found to be comparable to that of the parent strain and exhibited no distinct defects. The flavor component profiles of these two finished products were also comparable. The study demonstrated allelic genes in polyploid industrial yeasts could be efficiently and consecutively deleted by the retractive primer disruption strategy, and the mutant of Saccharomyces carlsbergensis partially deficient in proteinase A contributed to an improvement in foam stability.     

GABA shunt mediates thermotolerance in Saccharomyces cerevisiae by reducing reactive oxygen production

The GABA shunt pathway involves three enzymes, glutamate decarboxylase (GAD), GABA aminotransferase (GAT) and succinate semialdehyde dehydrogenase (SSADH). These enzymes act in concert to convert glutamate (α‐ketoglutarate) to succinate. Deletion mutations in each of these genes in Saccharomyces cerevisiae resulted in growth defects at 45°C. Double and triple mutation constructs were compared for thermotolerance with the wild‐type and single mutant strains. Although wild‐type and all mutant strains were highly susceptible to brief heat stress at 50°C, a non‐lethal 30 min at 40°C temperature pretreatment induced tolerance of the wild‐type and all of the mutants to 50°C. The mutant strains collectively exhibited similar susceptibility at 45°C to the induced 50°C treatments. Intracellular reactive oxygen intermediate (ROI) accumulation was measured in wild‐type and each of the mutant strains. ROI accumulation in each of the mutants and in various stress conditions was correlated to heat susceptibility of the mutant strains. The addition of ROI scavenger N‐tert‐butyl‐α‐phenylnitrone (PBN) enhanced survival of the mutants and strongly inhibited the accumulation of ROI, but did not have significant effect on the wild‐type. Measurement of intracellular GABA, glutamate and α‐ketoglutarate during lethal heat exposure at 45°C showed higher levels of accumulation of GABA and α‐ketoglutarate in the uga1 and uga2 mutants, while glutamate accumulated at higher level in the gad1 mutant. These results suggest that the GABA shunt pathway plays a crucial role in protecting yeast cells from heat damage by restricting ROI production involving the flux of carbon from α‐ketoglutarate to succinate during heat stress. Copyright © 2013 John Wiley & Sons, Ltd.