Browse
Submit Data
Databases
API
Help

Dataset Information

22 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Molecular cloning and expression in Saccharomyces cerevisiae of two Aspergillus nidulans xylanase genes.

ABSTRACT: Two Aspergillus nidulans genes, xlnA and xlnB, encoding the X22 and X24 xylanases from this fungus, respectively, have been cloned and sequenced. Their cDNAs have been expressed in a laboratory Saccharomyces cerevisiae strain under the control of a constitutive yeast promoter, resulting in the construction of recombinant xylanolytic yeast strains.

SUBMITTER: Perez-Gonzalez JA

PROVIDER: S-EPMC167998 | biostudies-other | 1996 Jun

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Publications

Molecular cloning and expression in Saccharomyces cerevisiae of two Aspergillus nidulans xylanase genes.

Pérez-Gonzalez J A JA De Graaff L H LH Visser J J Ramón D D

Applied and environmental microbiology 19960601 6

Two Aspergillus nidulans genes, xlnA and xlnB, encoding the X22 and X24 xylanases from this fungus, respectively, have been cloned and sequenced. Their cDNAs have been expressed in a laboratory Saccharomyces cerevisiae strain under the control of a constitutive yeast promoter, resulting in the construction of recombinant xylanolytic yeast strains. ...[more]

PMID: 8787417

Similar Datasets

Opposite patterns of expression of two Aspergillus nidulans xylanase genes with respect to ambient pH.

Project description:The Aspergillus nidulans xylanase genes xlnA and xlnB are subject to regulation by ambient pH via the zinc finger transcription factor PacC. In the presence of D-xylose, xlnA is expressed under conditions of alkaline ambient pH while xlnB is expressed at acidic ambient pH. These data have been confirmed for acidity- and alkalinity-mimicking A. nidulans mutants.

| S-EPMC107024 | biostudies-literature

Cloning, Expression, and Characterization of an GHF 11 Xylanase from Aspergillus niger XZ-3S.

Project description:A xylanase gene (xynZF-2) from the Aspergillus niger XZ-3S was cloned and expressed in Escherichia coli. The coding region of the gene was separated by only one intron with the 68 bp in length. It encoded 225 amino acid residues of a protein with a calculated molecular weight of 24.04 kDa plus a signal peptide of 18 amino acids. The amino acid sequence of the xynZF-2 gene had a high similarity with those of family 11 of glycosyl hydrolases reported from other microorganisms. The mature peptide encoding cDNA was subcloned into pET-28a(+) expression vector. The resultant recombinant plasmid pET-28a-xynZF-2 was transformed into E. coli BL21(DE3), and finally the recombinant strain BL21/xynZF-2 was obtained. A maximum activity of 42.33 U/mg was gained from cellular of E. coli BL21/xynZF-2 induced by IPTG. The optimum temperature and pH for recombinant enzyme which has a good stability in alkaline conditions were 40 °C and 5.0, respectively. Fe(3+) had an active effect on the enzyme obviously.

| S-EPMC3516665 | biostudies-literature

Molecular cloning and expression in Saccharomyces cerevisiae of a laccase gene from the ascomycete Melanocarpus albomyces.

Project description:The lac1 gene encoding an extracellular laccase was isolated from the thermophilic fungus Melanocarpus albomyces. This gene has five introns, and it encodes a protein consisting of 623 amino acids. The deduced amino acid sequence of the laccase was shown to have high homology with laccases from other ascomycetes. In addition to removal of a putative 22-amino-acid signal sequence and a 28-residue propeptide, maturation of the translation product of lac1 was shown to involve cleavage of a C-terminal 14-amino-acid extension. M. albomyces lac1 cDNA was expressed in Saccharomyces cerevisiae under the inducible GAL1 promoter. Extremely low production was obtained with the expression construct containing laccase cDNA with its own signal and propeptide sequences. The activity levels were significantly improved by replacing these sequences with the prepro sequence of the S. cerevisiae alpha-factor gene. The role of the C-terminal extension in laccase production in S. cerevisiae was also studied. Laccase production was increased sixfold with the modified cDNA that had a stop codon after the native processing site at the C terminus.

| S-EPMC321277 | biostudies-literature

Expression and function of an Hac1-regulated multi-copy xylanase gene in Saccharomyces cerevisiae.

Project description:Saccharomyces cerevisiae-based expression systems, which rely on safe, food-grade strains, are low cost, simple to operate, and can be used for large-scale fermentation. However, low levels of foreign protein expression by S. cerevisiae have limited their widespread application. The ability of the endoplasmic reticulum (ER) to fold and process foreign proteins is an important factor restricting the expression of foreign proteins. In the current study, the effects of transcription factor Hac1p, which is involved in the unfolded protein response pathway, on S. cerevisiae-based expression of xylanase gene xynB from Aspergillus niger were examined. Overlap extension polymerase chain reaction (PCR), rDNA integration and droplet digital PCR technology were used to generate a S. cerevisiae strain (S8) containing eight copies of xynB, allowing high-yield secretory expression of xylanase. The effects of subsequent overexpression of HAC1 in strain S8 on the expression of genes associated with protein folding in the ER were then examined using the GeXP system. Results confirmed the constitutive secretory expression of the multiple copies of xynB following rDNA-based integration of the expression cassette, with a maximum xylanase yield of 325 U/mL. However, overexpression of HAC1 further improved xylanase production by strain S8, resulting in a yield of 381 U/mL.

| S-EPMC7363925 | biostudies-literature

Molecular cloning and transcriptional regulation of the Aspergillus nidulans xlnD gene encoding a beta-xylosidase.

Project description:The xlnD gene encoding the 85-kDa beta-xylosidase was cloned from Aspergillus nidulans. The deduced primary structure of the protein exhibits considerable similarity to the primary structures of the Aspergillus niger and Trichoderma reesei beta-xylosidases and some similarity to the primary structures of the class 3 beta-glucosidases. xlnD is regulated at the transcriptional level; it is induced by xylan and D-xylose and is repressed by D-glucose. Glucose repression is mediated by the product of the creA gene. Although several binding sites for the pH regulatory protein PacC were found in the upstream regulatory region, it was not clear from a Northern analysis whether PacC is involved in transcriptional regulation of xlnD.

| S-EPMC106163 | biostudies-literature

Expression of an Aspergillus niger phytase gene (phyA) in Saccharomyces cerevisiae.

Project description:Phytase improves the bioavailability of phytate phosphorus in plant foods to humans and animals and reduces phosphorus pollution of animal waste. Our objectives were to express an Aspergillus niger phytase gene (phyA) in Saccharomyces cerevisiae and to determine the effects of glycosylation on the phytase's activity and thermostability. A 1.4-kb DNA fragment containing the coding region of the phyA gene was inserted into the expression vector pYES2 and was expressed in S. cerevisiae as an active, extracellular phytase. The yield of total extracellular phytase activity was affected by the signal peptide and the medium composition. The expressed phytase had two pH optima (2 to 2.5 and 5 to 5.5) and a temperature optimum between 55 and 60 degrees C, and it cross-reacted with a rabbit polyclonal antibody against the wild-type enzyme. Due to the heavy glycosylation, the expressed phytase had a molecular size of approximately 120 kDa and appeared to be more thermostable than the commercial enzyme. Deglycosylation of the phytase resulted in losses of 9% of its activity and 40% of its thermostability. The recombinant phytase was effective in hydrolyzing phytate phosphorus from corn or soybean meal in vitro. In conclusion, the phyA gene was expressed as an active, extracellular phytase in S. cerevisiae, and its thermostability was affected by glycosylation.

| S-EPMC91276 | biostudies-literature

Degradation of xylan to D-xylose by recombinant Saccharomyces cerevisiae coexpressing the Aspergillus niger beta-xylosidase (xlnD) and the Trichoderma reesei xylanase II (xyn2) genes.

Project description:The beta-xylosidase-encoding xlnD gene of Aspergillus niger 90196 was amplified by the PCR technique from first-strand cDNA synthesized on mRNA isolated from the fungus. The nucleotide sequence of the cDNA fragment was verified to contain a 2,412-bp open reading frame that encodes a 804-amino-acid propeptide. The 778-amino-acid mature protein, with a putative molecular mass of 85.1 kDa, was fused in frame with the Saccharomyces cerevisiae mating factor alpha1 signal peptide (MFalpha1(s)) to ensure correct posttranslational processing in yeast. The fusion protein was designated Xlo2. The recombinant beta-xylosidase showed optimum activity at 60 degrees C and pH 3.2 and optimum stability at 50 degrees C. The K(i(app)) value for D-xylose and xylobiose for the recombinant beta-xylosidase was determined to be 8.33 and 6.41 mM, respectively. The XLO2 fusion gene and the XYN2 beta-xylanase gene from Trichoderma reesei, located on URA3-based multicopy shuttle vectors, were successfully expressed and coexpressed in the yeast Saccharomyces cerevisiae under the control of the alcohol dehydrogenase II gene (ADH2) promoter and terminator. These recombinant S. cerevisiae strains produced 1,577 nkat/ml of beta-xylanase activity when expressing only the beta-xylanase and 860 nkat/ml when coexpressing the beta-xylanase with the beta-xylosidase. The maximum beta-xylosidase activity was 5.3 nkat/ml when expressed on its own and 3.5 nkat/ml when coexpressed with the beta-xylanase. Coproduction of the beta-xylanase and beta-xylosidase enabled S. cerevisiae to degrade birchwood xylan to D-xylose.

| S-EPMC93337 | biostudies-literature

Improvement of homologous GH10 xylanase production by deletion of genes with predicted function in the Aspergillus nidulans secretion pathway.

Project description:Filamentous fungi are important cell factories for large-scale enzyme production. However, production levels are often low, and this limitation has stimulated research focusing on the manipulation of genes with predicted function in the protein secretory pathway. This pathway is the major route for the delivery of proteins to the cell exterior, and a positive relationship between the production of recombinant enzymes and the unfolded protein response (UPR) pathway has been observed. In this study, Aspergillus nidulans was exposed to UPR-inducing chemicals and differentially expressed genes were identified by RNA-seq. Twelve target genes were deleted in A. nidulans recombinant strains producing homologous and heterologous GH10 xylanases. The knockout of pbnA (glycosyltransferase), ydjA (Hsp40 co-chaperone), trxA (thioredoxin) and cypA (cyclophilin) improved the production of the homologous xylanase by 78, 171, 105 and 125% respectively. Interestingly, these deletions decreased the overall protein secretion, suggesting that the production of the homologous xylanase was specifically altered. However, the production of the heterologous xylanase and the secretion of total proteins were not altered by deleting the same genes. Considering the results, this approach demonstrated the possibility of rationally increase the production of a homologous enzyme, indicating that trxA, cypA, ydjA and pbnA are involved in protein production by A. nidulans.

| S-EPMC7264891 | biostudies-literature

New multi-marker strains and complementing genes for Aspergillus nidulans molecular biology.

Project description:Technical advances in Aspergillus nidulans enable relatively easy deletion of genomic sequences, insertion of sequences into the genome and alteration of genomic sequences. To extend the power of this system we wished to create strains with several selectable markers in a common genetic background to facilitate multiple, sequential transformations. We have developed an approach, using the recycling of the pyrG selectable marker, that has allowed us to create new deletions of the biA, pabaA, choA, and lysB genes. We have deleted these genes in a strain that carries the commonly used pyrG89, riboB2, and pyroA4 mutations as well as a deletion of the sterigmatocystin gene cluster and a deletion of the nkuA gene, which greatly reduces heterologous integration of transforming sequences. The new deletions are fully, easily and cheaply supplementable. We have created a strain that carries seven selectable markers as well as strains that carry subsets of these markers. We have identified the homologous genes from Aspergillus terreus, cloned them and used them as selectable markers to transform our new strains. The newly created strains transform well and the new deletion alleles appear to be complemented fully by the A. terreus genes. In addition, we have used deep sequencing data to determine the sequence alterations of the venerable and frequently used pyrG89, riboB2 and pyroA4 alleles and we have reannotated the choA gene.

| S-EPMC5803425 | biostudies-literature

High copy and stable expression of the xylanase XynHB in Saccharomyces cerevisiae by rDNA-mediated integration.

Project description:Xylanase is a widely-used additive in baking industry for enhancing dough and bread quality. Several xylanases used in baking industry were expressed in different systems, but their expression in antibiotic free vector system is highly essential and safe. In the present study, an alternative rDNA-mediated technology was developed to increase the copy number of target gene by integrating it into Saccharomyces cerevisiae genome. A xylanase-encoding gene xynHB from Bacillus sp. was cloned into pHBM367H and integrated into S. cerevisiae genome through rDNA-mediated recombination. Exogenous XynHB expressed by recombinant S. cerevisiae strain A13 exhibited higher degradation activity towards xylan than other transformants. The real-time PCR analysis on A13 genome revealed the presence of 13.64 copies of xynHB gene. Though no antibiotics have been used, the genetic stability and the xylanase activity of xynHB remained stable up to 1,011 generations of cultivation. S. cerevisiae strain A13 expressing xylanase reduced the required kneading time and increased the height and diameter of the dough size, which would be safe and effective in baking industry as no antibiotics-resistance risk. The new effective rDNA-mediated technology without using antibiotics here provides a way to clone other food related industrial enzymes for applications.

| S-EPMC5562786 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data