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.     

Identification and characterization of a drug‐sensitive strain enables puromycin‐based translational assays in Saccharomyces cerevisiae

Puromycin is an aminonucleoside antibiotic with structural similarity to aminoacyl tRNA. This structure allows the drug to bind the ribosomal A site and incorporate into nascent polypeptides, causing chain termination, ribosomal subunit dissociation and widespread translational arrest at high concentrations. In contrast, at sufficiently low concentrations, puromycin incorporates primarily at the C‐terminus of proteins. While a number of techniques utilize puromycin incorporation as a tool for probing translational activity in vivo, these methods cannot be applied in yeasts that are insensitive to puromycin. Here, we describe a mutant strain of the yeast Saccharomyces cerevisiae that is sensitive to puromycin and characterize the cellular response to the drug. Puromycin inhibits the growth of yeast cells mutant for erg6?, pdr1? and pdr3? (EPP) on both solid and liquid media. Puromycin also induces the aggregation of the cytoplasmic processing body component Edc3 in the mutant strain. We establish that puromycin is rapidly incorporated into yeast proteins and test the effects of puromycin on translation in vivo. This study establishes the EPP strain as a valuable tool for implementing puromycin‐based assays in yeast, which will enable new avenues of inquiry into protein production and maturation. Copyright © 2014 John Wiley & Sons, Ltd.     

Characterization of Fusarium verticillioides strains by PCR‐RFLP analysis of the intergenic spacer region of the rDNA

Thirty‐three Fusarium verticillioides strains from diverse origins and hosts have been analysed for fumonisin production and characterized in order (i) to detect the variability present in this species and (ii) to discriminate among isolates. The method used was a combination of polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) generated by restriction endonucleases applied to the IGS region (intergenic spacer of rDNA). All the F. verticillioides strains associated with crops produced fumonisins B₁ and B₂ except those isolated from banana. Analysis of the IGS region by PCR‐RFLP proved to be useful to detect variability within F. verticillioides and allowed discrimination of two related groups of isolates belonging to distinct lineages differing in fumonisin production and host preferences: the fumonisin‐producing group associated with cereals and the fumonisin non‐producing group associated with banana. The method used facilitates early detection and characterization of F. verticillioides strains required to control both types of pathogens and to evaluate plant exposure to the toxin, quality of the raw material to be processed and the potential fumonisin contamination in order to prevent fumonisins entering the food chain. Copyright © 2005 Society of Chemical Industry     

NY‐ESO‐1 protein glycosylated by yeast induces enhanced immune responses

Vaccine strategies that target dendritic cells to elicit potent cellular immunity are the subject of intense research. Here we report that the genetically engineered yeast Saccharomyces cerevisiae, expressing the full‐length tumour‐associated antigen NY‐ESO‐1, is a versatile host for protein production. Exposing dendritic cells (DCs) to soluble NY‐ESO‐1 protein linked to the yeast a‐agglutinin 2 protein (Aga2p) protein resulted in protein uptake, processing and MHC class I cross‐presentation of NY‐ESO‐1‐derived peptides. The process of antigen uptake and cross‐presentation was dependent on the glycosylation pattern of NY‐ESO‐1‐Aga2p protein and the presence of accessible mannose receptors. In addition, NY‐ESO‐1‐Aga2p protein uptake by dendritic cells resulted in recognition by HLA‐DP4 NY‐ESO‐1‐specific CD4⁺ T cells, indicating MHC class II presentation. Finally, vaccination of mice with yeast‐derived NY‐ESO‐1‐Aga2p protein led to an enhanced humoral and cellular immune response, when compared to the bacterially expressed NY‐ESO‐1 protein. Together, these data demonstrate that yeast‐derived full‐length NY‐ESO‐1‐Aga2p protein is processed and presented efficiently by MHC class I and II complexes and warrants clinical trials to determine the potential value of S. cerevisiae as a host for cancer vaccine development. Copyright © 2010 John Wiley & Sons, Ltd.     

Application of polymerase chain reaction–restriction fragment length polymorphism analysis and lab‐on‐a‐chip capillary electrophoresis for the specific identification of game and domestic meats

BACKGROUND: The objective of this study was to adapt and improve previously published polymerase chain reaction–restriction fragment length polymorphism (PCR‐RFLP) methods aimed at the identification of game and domestic meats, by replacing the gel electrophoretic steps for DNA fragment analysis by a chip‐based capillary electrophoresis system. RESULTS: PCR amplification of a mitochondrial 12S rRNA gene fragment and subsequent digestion of the amplicons with either MseI or a combination of MboII, BslI, and ApoI endonucleases generated characteristic PCR‐RFLP profiles that allowed discrimination among ten relevant game and domestic meat species. The Agilent 2100 Bioanalyzer utilises capillary electrophoresis on a microchip device that is capable of rapidly sizing DNA fragments, offering a valuable recent development for the analysis of complex DNA banding patterns. CONCLUSION: Results obtained in this work indicated that banding resolution on the system was sensitive, with detection of some small DNA fragments that were not observed with the published conventional PCR‐RFLP gel‐based method. Therefore, the new faster and easy handling procedure provides an additional powerful tool that can be employed for meat speciation. Copyright © 2009 Society of Chemical Industry     

Oxylipin Associated Co‐Flocculation in Yeasts

According to the lectin‐theory, the yeast Schizosaccharomyces pombe lacks the specific receptors (α‐mannans) necessary to facilitate co‐flocculation with Saccharomyces cerevisiae species. In this study we demonstrate oxylipin associated co‐flocculation between Sacch. cerevisiae and S. pombe strains using differential cell staining, immunofluoresence and ultrastructural studies. Using a 3‐hydroxy (OH) oxylipin specific antibody coupled to a fluorescing compound, 3‐OH oxylipins were found to be present on the cell surfaces of Sacch. cerevisiae and S. pombe. The presence of 3‐OH oxylipins was confirmed using gas chromatography‐mass spectrometry. Strikingly, when acetylsalicylic acid (aspirin), a 3‐OH oxylipins inhibitor, was added to Sacch. cerevisiae which was then mixed with S. pombe strains grown in complex media, co‐flocculation was significantly inhibited. We conclude that aspirin‐sensitive 3‐OH 8:0 is probably involved in co‐flocculation.     

Scale‐up batch fermentation of bioethanol production from the dry powder of Jerusalem artichoke (Helianthus tuberosus L.) tubers by recombinant Saccharomyces cerevisiae

This study is a first exploration of industrial bioethanol production from the dry powder of Jerusalem artichoke (Helianthus tuberosus L.) tubers by recombinant S. cerevisiae 6525. Scale‐up fermentation of bioethanol production from the dry powder of Jerusalem artichoke tubers by recombinant Saccharomyces cerevisiae 6525 was carried out at the scale of 50 and 500 L agitating fermentors. For the 50 L fermentor, 85.67 g/L of ethanol was obtained within 72 h of fermentation, and the ethanol yield was 90.1%. For the 500 L fermentor, the highest ethanol concentration of 77.00 g/L was achieved at 84 h, and the ethanol yield was 81.01%. These results indicated that a relatively high yield of ethanol could also be obtained from a scaled‐up fermentation of Jerusalem artichoke powder. Thus, it may be feasible to use a Jerusalem artichoke tuber feedstock to carry out ethanol fermentations using the recombinant S. cerevisiae 6525 for industrial production. Copyright © 2016 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.     

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.     

Characterization of Osmotolerant Yeasts and Yeast‐Like Molds from Apple Orchards and Apple Juice Processing Plants in China and Investigation of Their Spoilage Potential

Yeasts and yeast‐like fungal isolates were recovered from apple orchards and apple juice processing plants located in the Shaanxi province of China. The strains were evaluated for osmotolerance by growing them in 50% (w/v) glucose. Of the strains tested, 66 were positive for osmotolerance and were subsequently identified by 26S or 5.8S‐ITS ribosomal RNA (rRNA) gene sequencing. Physiological tests and RAPD‐PCR analysis were performed to reveal the polymorphism of isolates belonging to the same species. Further, the spoilage potential of the 66 isolates was determining by evaluating their growth in 50% to 70% (w/v) glucose and measuring gas generation in 50% (w/v) glucose. Thirteen osmotolerant isolates representing 9 species were obtained from 10 apple orchards and 53 target isolates representing 19 species were recovered from 2 apple juice processing plants. In total, members of 14 genera and 23 species of osmotolerant isolates including yeast‐like molds were recovered from all sources. The commonly recovered osmotolerant isolates belonged to Kluyveromyces marxianus, Hanseniaspora uvarum, Saccharomyces cerevisiae, Zygosaccharomyces rouxii, Candida tropicalis, and Pichia kudriavzevii. The polymorphism of isolates belonging to the same species was limited to 1 to 3 biotypes. The majority of species were capable of growing within a range of glucose concentration, similar to sugar concentrations found in apple juice products with a lag phase from 96 to 192 h. Overall, Z. rouxii was particularly the most tolerant to high glucose concentration with the shortest lag phase of 48 h in 70% (w/v) glucose and the fastest gas generation rate in 50% (w/v) glucose.     

Transport and metabolism of fumaric acid in Saccharomyces cerevisiae in aerobic glucose‐limited chemostat culture

Currently, research is being focused on the industrial‐scale production of fumaric acid and other relevant organic acids from renewable feedstocks via fermentation, preferably at low pH for better product recovery. However, at low pH a large fraction of the extracellular acid is present in the undissociated form, which is lipophilic and can diffuse into the cell. There have been no studies done on the impact of high extracellular concentrations of fumaric acid under aerobic conditions in S. cerevisiae, which is a relevant issue to study for industrial‐scale production. In this work we studied the uptake and metabolism of fumaric acid in S. cerevisiae in glucose‐limited chemostat cultures at a cultivation pH of 3.0 (pH < pK). Steady states were achieved with different extracellular levels of fumaric acid, obtained by adding different amounts of fumaric acid to the feed medium. The experiments were carried out with the wild‐type S. cerevisiae CEN.PK 113‐7D and an engineered S. cerevisiae ADIS 244 expressing a heterologous dicarboxylic acid transporter (DCT‐02) from Aspergillus niger, to examine whether it would be capable of exporting fumaric acid. We observed that fumaric acid entered the cells most likely via passive diffusion of the undissociated form. Approximately two‐thirds of the fumaric acid in the feed was metabolized together with glucose. From metabolic flux analysis, an increased ATP dissipation was observed only at high intracellular concentrations of fumarate, possibly due to the export of fumarate via an ABC transporter. The implications of our results for the industrial‐scale production of fumaric acid are discussed. Copyright © 2015 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