Basic phenotypic analysis of six novel yeast genes reveals two essential genes and one which affects the growth rate

Phenotypic analysis was performed on six mutants of Saccharomyces cerevisiae deleted in one of the following open reading frames (ORFs), located on chromosome II: YBR254c, YBR255w, YBR257w, YBR258c, YBR259w and YBR266c. Disruption of the ORFs was carried out in the diploid strain FY1679 using the kanMX4 marker flanked by short sequences homologous to the target locus. Tetrad analysis following sporulation of the heterozygous disruptants showed that YBR254c and YBR257w are essential genes. YBR257w was later characterized and renamed POP4, its gene product being involved in 5.8S rRNA and tRNA processing (Chu et al., 1997). The tetrad analysis performed for the heterozygous disruptant for YBR2…

A multidomain xylanase from a Bacillus sp. with a region homologous to thermostabilizing domains of thermophilic enzymes

The gene xynC encoding xylanase C from Bacillus sp. BP-23 was cloned and expressed in Escherichia coli. The nucleotide sequence of a 3538 bp DNA fragment containing xynC gene was determined, revealing an open reading frame of 3258 bp that encodes a protein of 120,567 Da. A comparison of the deduced amino acid sequence of xylanase C with known beta-glycanase sequences showed that the encoded enzyme is a modular protein containing three different domains. The central region of the enzyme is the catalytic domain, which shows high homology to family 10 xylanases. A domain homologous to family IX cellulose-binding domains is located in the C-terminal region of xylanase C, whilst the N-terminal r…

On the fermentative behavior of auxotrophic strains of Saccharomyces cerevisiae

Background: The selection of new yeast strains could lead to improvements in bioethanol production. Here, we have studied the fermentative capacity of different auxotrophic mutants of Saccharomyces cerevisiae, which are routinely used as hosts for the production of heterologous proteins. It has recently been found that these strains exhibit physiological alterations and peculiar sensitivities with respect to the parental prototrophic strains from which they derive. In this work the performance of auxotrophic S. cerevisiae CEN.PK strains was compared to the corresponding prototrophic strain, to S. cerevisiae T5bV, a strain isolated from grape must and to another auxotrophic strain, S. cerevi…

Phenotype traits associated with different alleles at the RPS5 locus in Saccharomyces cerevisiae

The RPS5 gene has been characterised through its ability to reduce invertase production by the SUC5 gene. In this paper we show that RPS5 acts by maintaining low levels of SUC5 mRNA. We also show that RPS5 acts on the SUC1 and SUC4 genes but not on SUC2 and SUC3, which are members of the SUC family. RPS5 also shows a pleiotropic effect on the amount of mitochondrial cytochromes.

Bread making with Saccharomyces cerevisiae CEN.PK113-5D expressing lipase A from Bacillus subtilis: Leavening characterisation and aroma enhancement

Summary Lipase A from Bacillus subtilis was expressed in the yeast Saccharomyces cerevisiae CEN.PK113-5D strain as a cell wall-immobilised enzyme. The recombinant yeast was used in bread making to test the effect of lipase A on the bread properties such as rheological and aromatic properties. The results were compared to the not transformed strain and the commercial baker's yeast. The recombinant strain resulted a good leavening agent comparable to the commercial baker's yeasts provided 1% sucrose was added to the dough. Whereas, during the leavening, the trend of the rheological properties (cohesivness, gumminess and rigidity) differed from the commercial and the nontransformed (NT) strain…

Identification of glucan-mannoprotein complexes in the cell wall of Candida albicans using a monoclonal antibody that reacts with a (1,6)- -glucan epitope

The use of a novel monoclonal antibody (mAb) that reacts with (1,6)-beta-glucan has permitted the study of the different covalent linkages between glucan and mannoproteins in the cell wall of Candida albicans. The mAb JRR1 was originally raised by immunization with Zymolyase extracts from C. albicans cell walls, but it soon became apparent that it reacted with a (1,6)-beta-glucan epitope. By using this antibody, we show the existence of glucan-mannoprotein complexes between the (1,6)-beta-glucan epitope recognized by the antibody and cell wall mannoproteins. The topology of the (1,6)-beta-glucan in the cell wall of C. albicans has also been studied.

Antimicrobial Activity, in silico Molecular Docking, ADMET and DFT Analysis of Secondary Metabolites from Roots of Three Ethiopian Medicinal Plants

Mathewos Anza,1 Milkyas Endale,1 Luz Cardona,2 Diego Cortes,3 Rajalakshmanan Eswaramoorthy,1 Jesus Zueco,4 Hortensia Rico,4 Maria Trelis,5 Belen Abarca2 1Department of Applied Chemistry, School of Applied Natural Science, Adama Science and Technology University, Adama, Ethiopia; 2Department of Organic Chemistry, Faculty of Chemistry, University of Valencia, Burjassot, Spain; 3Department of Pharmacology, Faculty of Pharmacy, University of Valencia, Burjassot, Spain; 4Department of Microbiology and Ecology, Faculty of Pharmacy, University of Valencia, Burjassot, Spain; 5Parasites and Health Research Group, Department of Pharmacy, Pharmaceutical Technology and Parasitology, Faculty of Pharmacy…

Reversion of 7-methylguanosine 5′-phosphate inhibition of mRNA translation by polysomal and soluble factors isolated from Saccharomyces cerevisiae

Abstract Protein fractions that overcome m7GMP inhibition of mRNA translation have been purified from the yeast S. cerevisiae . An active fraction isolated from polysomes contains two polypeptides of 220- and 190-kDa. The active fraction isolated from postribosomal supernatant contains a major polypeptide of 28-kDa and other species of 32-, 24-, 22- and 21-kDa, and sediments in sucrose gradients as a high molecular weight complex of about 200000. This fraction restored yeast mRNA translation in reticulocyte lysates under conditions of yeast and globin mRNA competition; however, this effect was not observed with the 220- and 190-kDa polypeptides from polysomes. Nevertheless, translation of y…

Identification of a mannoprotein present in the inner layer of the cell wall of Saccharomyces cerevisiae.

Cell wall extracts from the double-mutant mnn1 mnn9 strain were used as the immunogen to obtain a monoclonal antibody (MAb), SAC A6, that recognizes a specific mannoprotein--which we have named Icwp--in the walls of cells of Saccharomyces cerevisiae. Icwp runs as a polydisperse band of over 180 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of Zymolyase extracts of cell walls, although an analysis of the secretory pattern of the mannoprotein shows that at the level of secretory vesicles, it behaves like a discrete band of 140 kDa. Immunofluorescence analysis with the MAb showed that Icwp lies at the inner layer of the cell wall, being accessible to the antibody on…

Characterization of a disulphide-bound Pir-cell wall protein (Pir-CWP) ofYarrowia lipolytica

In this work we have studied the disulphide-bound group of cell wall mannoproteins of Yarrowia lipolytica and Candida albicans. In the case of Y. lipolytica, SDS-PAGE analysis of the beta-mercaptoethanol-extracted material from the purified cell walls of the yeast form, showed the presence of a main polypeptide of 45 kDa and some minor bands in the 100-200 kDa range. This pattern of bands is similar to that obtained in identical extracts in Saccharomyces cerevisiae (Moukadiri et al., 1999), and besides, all these bands cross-react with an antibody raised against beta-mercaptoethanol-extracted material from the purified cell walls of S. cerevisiae, suggesting that the 45 kDa band could be th…

Micromón València (Universitat de València)

En Julio de 2017 se creó la red SWI@Spain, auspiciada por el grupo de Docencia y Difusión de la Microbiología (DDM) de la Sociedad Española de Microbiología (SEM), para desarrollar la iniciativa internacional Small World Initiative (SWI) en la península ibérica. En la Universitat de València (UV) se constituyó entonces el grupo de Innovación Docente en Microbiología (IDM) para implementar el proyecto a nivel local. Avalados por el Servei de Formació Permanent i Innovació Educativa (SFPIE) de la UV, el grupo ha llevado a cabo diferentes iniciativas relacionadas con el objetivo fundamental del proyecto: divulgar la problemática actual relacionada con el uso inadecuado de antibióticos, el incr…

Immobilization of <i>Saccharomyces cerevisiae</i> Cells to Protein G-Sepharose by Cell Wall Engineering

In this work, we explored the possibility of using the targeting of a heterologous protein to the cell wall of <i>Saccharomyces cerevisiae</i>, by fusing it to a cell wall protein, to construct yeast strains whose cells display on their surface proteins that bind to a matrix, so as to achieve the immobilization of the whole cells. With this aim, we created a gene fusion that comprises the region responsible for attachment of a cell wall protein to the cell wall, and the IgG binding region of staphylococcal protein A, and expressed it in the <i>mnn1mnn9</i> strain of <i>S. cerevisiae</i>. The surface display of the protein A-Icwp fusion protein was positiv…

A singleFKShomologue inYarrowia lipolyticais essential for viability

The synthesis of β-1,3-glucan, the structural component of the yeast cell wall which gives shape to the cell, occurs at the plasma membrane and is the result of the activity of at least a two-component complex. Fks1p is the catalytic subunit directly responsible for the synthesis of β-1,3-glucan, whilst the second subunit, Rho1p, has a GTP-dependent regulatory role. FKS1 has been characterized in Saccharomyces cerevisiae, where its function is at least partially redundant with that of FKS2/GSC2. FKS homologues have also been identified in several other fungal species, including Candida albicans, Schizosaccharomyces pombe, Aspergillus nidulans, Cryptococcus neoformans and Paracoccidiodes bra…

Implementation of Antibiotic Discovery by Student Crowdsourcing in the Valencian Community Through a Service Learning Strategy

© 2020 Maicas, Fouz, Figàs-Segura, Zueco, Rico, Navarro, Carbó, Segura-García and Biosca.

The Role of Age and Gender in Perceived Vulnerability to Infectious Diseases

Background: The study of the immune system has been approached using two separate paths, the biological immune system and the behavioral immune system. Recently, Gangestad and Grebe proposed a unique integrated compensatory immune system, where both systems work together and one of them could compensate for the other when necessary. However, few studies have confirmed the existence of this integrated compensatory immune system. Our study represents an attempt to explore the existence of this unique immune system, investigating if the behavioral immune system variables increase when the biological immune system weakens with age. Material and Methods. The cross-sectional design study was made…

Translational fusion to the Pir4 cell wall protein as a general and efficient method for cell surface immobilization or growth medium secretion of recombinant antigens and enzymes in Saccharomyces cerevisiae

A comparative study of the incorporation of a 1,6-beta-glucan and an O-glycosylated protein epitope into the cell wall of Candida albicans.

The topological distribution of two epitopes in the cell wall of Candida albicans, the kinetics of their incorporation into the regenerating protoplast wall, and the effect of different antibiotics upon their incorporation and localization have been studied. To do so, two monoclonal antibodies that react against an O-glycosylated mannoprotein (1B12) and against a 1,6-beta-glucan epitope (JRR1) were used. The results show that the JRR1 epitope is localized in an internal layer of the cell wall, in contrast to the 1B12 epitope, which is superficial, and that the incorporation of the JRR1 epitope into walls of regenerating protoplasts precedes that of the 1B12 epitope. The JRR1 epitope is norm…

Temporal aspects of the O-glycosylation of Saccharomyces cerevisiae mannoproteins

Abstract Cleavage of the O-glycosyl bonds of Saccharomyces cerevisiae cell wall mannoproteins by β-elimination resulted in the release of about 8% of the carbohydrate in the form of mannose and other low molecular weight oligomannosaccharides (mannose to mannopentaose), leaving 92% mannose still covalently linked to the peptide, and suggesting that this alkali-resistant fraction was N-glycosidically linked. At the non-permissive temperature, S. cerevisiae sec mutants accumulated in the cytoplasm mannoproteins with different degrees of O- and N-glycosylation. The glycoproteins of mutant sec 20-1 contained 60% of the carbohydrate linked by N-bonds, the remainder being O-glycosidically linked.…

RCS1, a gene involved in controlling cell size inSaccharomyces cerevisiae

Cloning and sequencing of RCS1, Saccharomyces cerevisiae gene whose product seems to be involved in timing the budding event of the cell cycle, is described. A haploid strain in which the 3'-terminal region of the chromosomal copy of the gene has been disrupted produces cells that are, on average, twice the size of cells of the parental strain. The critical size for budding in the mutant is similarly increased, and the disruption mutation is dominant in a diploid heterozygous for the RCS1 gene. Spores from this diploid have a reduced ability to germinate, the effect being more pronounced in the spores carrying the disrupted copy of RCS1. However, disrupted cells recover from alpha-factor tr…

RHO1(YlRHO1) is a non-essential gene inYarrowia lipolyticaand complementsrho1Δlethality inSaccharomyces cerevisiae

The synthesis of beta-1,3-glucan, the structural component of the yeast cell wall that gives shape to the cell, occurs at the plasma membrane and is the result of the activity of at least a two-component complex. Fks1p is the catalytic subunit directly responsible for the synthesis of beta-1,3-glucan, whilst the second subunit, Rho1p, has a GTP-dependent regulatory role (Yamochi et al., 1994). RHO1 has been characterized in Saccharomyces cerevisiae (Yamochi et al., 1994), and in several other fungal species. In this work, we have used degenerate oligonucleotides derived from the conserved regions of Rho1ps to isolate the RHO1 gene of Yarrowia lipolytica. The gene isolated in this way, which…

Isolation of theMNN9gene ofYarrowia lipolytica(YlMNN9) and phenotype analysis of a mutantylmnn9Δ strain

In this work we describe the isolation of the Yarrowia lipolytica homologue of Saccharomyces cerevisiae MNN9 gene, which we have named YlMNN9, and the phenotype analysis of a Y. lipolytica strain containing the disrupted YlMNN9 allele. YlMNN9 was cloned using degenerate consensus oligonucleotides to generate specific probes that were in turn used to screen mini-gene libraries. The gene is defined by a 1014 bp ORF predicted to encode a protein 337 amino acids long that shares significant homology with the Mnn9ps of S. cerevisiae, Candida albicans and Hansenula polymorpha, including a putative N-terminal transmembrane domain. Disruption of YlMNN9 leads to phenotypes such as resistance to sodi…

Characterization of a glycosylphosphatidylinositol-bound cell-wall protein (GPI-CWP) in Yarrowia lipolytica.

The structure and composition of the cell wall of yeast has so far been studied mainly in Saccharomyces cerevisiae. It is basically made up of three components: beta-glucans, chitin and mannose-containing glycoproteins, also called mannoproteins. Most covalently bound cell-wall mannoproteins belong to the so-called glycosylphosphatidylinositol cell-wall protein (GPI-CWP) family, cell-wall proteins that are bound through the remnant of a GPI residue to 1,6-beta-glucan. The non-conventional yeast Yarrowia lipolytica shares Generally Regarded As Safe (GRAS) status with S. cerevisiae, has some industrial applications and is increasingly being proposed as a host for the production of recombinant…

YJL159w does encode Pir2/Hsp150

In this paper we compare the sequence of the gene HSP150/PIR2, independently determined by two different groups, with that present in the yeast database as YJL159w, determined within the Yeast Sequencing Project. Although YJL159w is believed to encode Hsp150/Pir2, there are important differences between the amino acid sequence coded by this ORF and that of HSP150/PIR2. To find out if this divergence is due to strain polymorphism or to a possible sequencing error, we have analysed the diverging zone of this ORF in three strains and have found it entirely consistent with the sequence reported as HSP150/PIR2, concluding that the divergence is probably due to a sequencing error in YJL159w. Copy…

Performance of the auxotrophic Saccharomyces cerevisiae BY4741 as host for the production of IL-1β in aerated fed-batch reactor: role of ACA supplementation, strain viability, and maintenance energy

Abstract Background Saccharomyces cerevisiae BY4741 is an auxotrophic commonly used strain. In this work it has been used as host for the expression and secretion of human interleukin-1β (IL1β), using the cell wall protein Pir4 as fusion partner. To achieve high cell density and, consequently, high product yield, BY4741 [PIR4-IL1β] was cultured in an aerated fed-batch reactor, using a defined mineral medium supplemented with casamino acids as ACA (auxotrophy-complementing amino acid) source. Also the S. cerevisiae mutant BY4741 Δyca1 [PIR4-IL1β], carrying the deletion of the YCA1 gene coding for a caspase-like protein involved in the apoptotic response, was cultured in aerated fed-batch rea…

Identification of Two Mannoproteins Released from Cell Walls of a Saccharomyces cerevisiae mnn1 mnn9 Double Mutant by Reducing Agents

The cell wall of Saccharomyces cerevisiae represents some 30% of the total weight of the cell and is made up of β-glucans, mannose-containing glycoproteins (mannoproteins), and small amounts of chitin (9, 15). The mannoproteins can be divided into three groups according to the linkages that bind them to the structure of the cell wall: (i) noncovalently bound, (ii) covalently bound to the structural glucan, and (iii) disulfide bound to other proteins that are themselves covalently bound to the structural glucan of the cell wall (8). Our work has focused on the disulfide-bound mannoproteins, probably the least well known of the three groups mentioned above. Previous work (25) showed that trea…

Contribution of polyadenylate sequences to the translational efficiency of globin messenger RNAs

mRNAs from reticulocyte polysomes were fractionated by chromatography on poly(U)-Sepharose and thermal elution. The molar ratio of alpha- to beta-globin mRNA was found to be 2:1 and 1:1 respectively in short- and long-poly(A) size classes. Translational analyses indicated that the globin mRNAs containing long poly(A) tracts (with a mean length of about 70 nucleotides) directed protein synthesis with higher rates than did mRNA containing short poly(A) tracts (15-35 nucleotides). Experiments performed with sub-saturating mRNA concentrations showed that the digestion with RNAase H induced a decrease in the translational capacity of both globin mRNAs and an increase in the alpha- to beta-globin…

Efficient expression of a Paenibacillus barcinonensis endoglucanase in Saccharomyces cerevisiae.

Abstract The endoglucanase coded by celA (GenBank Access No. Y12512) from Paenibacillus barcinonensis, an enzyme with good characteristics for application on paper manufacture from agricultural fibers, was expressed in Saccharomyces cerevisiae by using different domains of the cell wall protein Pir4 as translational fusion partners, to achieve either secretion or cell wall retention of the recombinant enzyme. Given the presence of five potential N-glycosylation sites in the amino acid sequence coded by celA, the effect of glycosylation on the enzymatic activity of the recombinant enzyme was investigated by expressing the recombinant fusion proteins in both, standard and glycosylation-defici…