Biomedical subjects

I S Pretorius

Publications and source records attributed to I S Pretorius.

At least 55 records · Page 3Linked to original sources

PubMed1995

Cloning and expression of an Aspergillus kawachii endo-1,4-beta-xylanase gene in Saccharomyces cerevisiae.

First-strand cDNA was prepared from mRNA isolated from Aspergillus kawachii IFO4308 and the beta-xylanase gene (xynC) amplified by using the polymerase chain reaction (PCR) technique. This gene was inserted between the yeast phosphoglycerate kinase (PGK1) gene promoter (PGK1p) and terminator (PGK1T) sequences. The PGK1p-xynC-PGK1T construct (designated XYN3) was cloned into a multicopy episomal plasmid and the XYN3 gene was expressed in Saccharomyces cerevisiae. Functional beta-xylanase (Xyn3) was produced and secreted by the recombinant yeast. Xyn3 was stable between 30 and 50 degrees C, and the optimum temperature and pH were shown to be at 60 degrees C and lower than pH 3, respectively. An autoselective furl::LEU2 XYN3 recombinant strain was developed that allowed beta-xylanase production at a level of 300 nkat/ml in a non-selective complex medium.

Aspergillus↗

PubMed1995

Characterization of a novel alpha-amylase from Lipomyces kononenkoae and expression of its gene (LKA1) in Saccharomyces cerevisiae.

A highly active alpha-amylase (76,250 Da) secreted by the raw starch-degrading yeast Lipomyces kononenkoae strain IGC4052B was purified and characterized. Using high performance liquid chromatography (HPLC), end-product analysis indicated that the L. kononenkoae alpha-amylase acted by endo-hydrolysis on glucose polymers containing alpha-1,4 and alpha-1,6 bonds, producing mainly maltose, maltotriose and maltotetraose. The following NH2-terminal amino acids were determined for the purified enzyme: Asp-Cys-Thr-Thr-Val-Thr-Val-Leu-Ser-Ser-Pro- Glu-Ser-Val-Thr-Gly. The L. kononenkoae alpha-amylase-encoding gene (LKA1), previously cloned as a cDNA fragment, was expressed in Saccharomyces cerevisiae under the control of the PGK1 promoter. The native signal sequence efficiently directed the secretion of the glycosylated protein in S. cerevisiae. De-glycosylation of the enzyme indicated that post-translational glycosylation is different in S. cerevisiae from that in L. kononenkoae. Zymogram analysis indicated that glycosylation of the protein in S. cerevisiae had a negative effect on enzyme activity. Southern-blot analysis revealed that there is only a single LKA1 gene present in the genome of L. kononenkoae.

Amino Acid Sequence↗

PubMed1994

Multiple positive and negative cis-acting elements of the STA2 gene regulate glucoamylase synthesis in Saccharomyces cerevisiae.

Expression of the glucoamylase-encoding gene (STA2) in Saccharomyces cerevisiae was previously shown to be regulated transcriptionally by both positive and negative factors. The objective of this work was to identify the cis-acting elements responsible for STA2 transcriptional activation as well as the transcriptional repressor effects of STA10 and MATa/MAT alpha. We identified two upstream activation regions (UAS). Three repressor regions responsive to STA10-mediated repression were identified, as well as two regions for down-regulation of STA2 expression. MATa/MAT alpha repression appears to effect STA2 expression either downstream from the translational start site or, indirectly, since no functional a1/alpha 2-responsive sequence was identified in the promoter region.

Base Sequence↗

PubMed1994

Expression of the Butyrivibrio fibrisolvens endo-beta-1,4-glucanase gene together with the Erwinia pectate lyase and polygalacturonase genes in Saccharomyces cerevisiae.

Recombinant Saccharomyces cerevisiae strains capable of simultaneous secretion of bacterial glucanase and pectinase enzymes have been developed. The Butyrivibrio fibrrisolvens endo-beta-1,4-glucanase gene (end1), the Erwinia chrysanthemi pectate lyase gene (pelE) and E. carotovora polygalacturonase gene (peh1) were each inserted between a yeast expression-secretion cassette and yeast gene terminator, and cloned into yeast-centromeric shuttle vectors. Transcription initiation signals present in the expression-secretion cassette were derived from the yeast alcohol dehydrogenase gene promoter (ADC1P), whereas the transcription termination signals were derived from the yeast tryptophan synthase gene terminator (TRP5T). Secretion of glucanase and pectinases was directed by the signal sequence of the yeast mating pheromone alpha-factor (MF alpha 1S). These YCplac111-based constructs, designated END1, PEL5, AND PEH1, respectively, were transformed into S. cerevisiae. The END1, PEL5 and PEH1 constructs were co-expressed in laboratory strains of S. cerevisiae as well as in wine and distillers' yeasts. DNA-RNA hybridization analysis showed the presence of END1, PEL5 and PEH1 transcripts. Carboxymethylcellulose and polypectate agarose assays revealed the production of biologically active endo-beta-1,4-glucanase, pectate lyase and polygalacturonase by the S. cerevisiae transformants. Interestingly, although the same expression-secretion cassette was used in all three constructs, time-course assays indicated that the pectinases were secreted before the glucanase. It is tempting to speculate that the bulkiness of the END1-encoded protein and the five alternating repeats of Pro-Asp-Pro-Thr(Gln)-Pro-Val-Asp within the glucanase moiety could be involved in the delayed secretion of the glucanase.

Amino Acid Sequence↗

PubMed1993

Co-expression of an Erwinia chrysanthemi pectate lyase-encoding gene (pelE) and an E. carotovora polygalacturonase-encoding gene (peh1) in Saccharomyces cerevisiae.

A pectate lyase (PL)-encoding gene (pelE) from Erwinia chrysanthemi and a polygalacturonase (PG)-encoding gene (peh1) from E. carotovora were each inserted between a novel yeast expression-secretion cassette and a yeast gene terminator, and cloned separately into a yeast-centromeric shuttle vector (YCp50), generating recombinant plasmids pAMS12 and pAMS13. Transcription initiation signals present in the expression-secretion cassette were derived from the yeast alcohol dehydrogenase gene promoter (ADC1P), whereas the transcription termination signals were derived from the yeast tryptophan synthase gene terminator (TRP5T). Secretion of PL and PG was directed by the signal sequence of the yeast mating pheromone alpha-factor (MF alpha 1s). A pectinase cassette comprising ADC1P-MF alpha 1s-pelE-TRP5T and ADC1P-MF alpha 1s-peh1-TRP5T was subcloned into YCp50, generating plasmid pAMS14. Subsequently, the dominant selectable Geneticin G418-resistance (GtR) marker, APH1, inserted between the yeast uridine diphosphoglucose 4-epimerase gene promoter (GAL10P) and yeast orotidine-5'-phosphate carboxylase gene terminator (URA3T), was cloned into pAMS14, resulting in plasmid pAMS15. Plasmids pAMS12, pAMS13 and pAMS14 were transformed into a laboratory strain of Saccharomyces cerevisiae, whereas pAMS15 was stably introduced into two commercial wine yeast strains. DNA-DNA and DNA-RNA hybridization analyses revealed the presence of these plasmids, and the pelE and peh1 transcripts in the yeast transformants, respectively. A polypectate agarose assay indicated the extracellular production of biologically active PL and PG by the S. cerevisiae transformants and confirmed that co-expression of the pelE and peh1 genes synergistically enhanced pectate degradation.

Bacterial Proteins↗

PubMed1993

Regional sequence homologies in starch-degrading enzymes.

The enzymatic hydrolysis of starch, consisting of linear (amylose) and branched (amylopectin) glucose polymers, is catalyzed by alpha-, beta- and glucoamylases (gamma-amylases), cyclodextrinases, alpha-glucosidases, and debranching enzymes. Saccharomyces cerevisiae cannot utilize starch. Our laboratory has previously co-expressed the Bacillus amyloliquefaciens alpha-amylase (AMY) and the Saccharomyces diastaticus glucoamylase (STA2) genes in S. cerevisiae. A gene encoding a debranching enzyme (pullulanase) from Klebsiella pneumoniae ATCC15050 was cloned and its nucleotide sequence determined. This gene will be co-expressed with the alpha- and gamma-amylase to produce an amylolytic S. cerevisiae strain. Extensive data base comparisons of the K. pneumoniae pullulanase amino-acid sequence with the amino-acid sequences of other debranching enzymes and alpha-, beta- and gamma-amylases (from bacteria, yeasts, higher fungi and higher eukaryotes), indicated that these debranching enzymes have amino-acid regions similar to those found in alpha-amylases. The conserved regions in alpha-amylases comprise key residues that are implicated in substrate binding, catalysis, and calcium binding and are as follows. Region 1: DVVINH; region 2: GFRLDAAKH and region 4: FVDNHD. When comparing conserved regions, no similarity could be detected between debranching enzymes and beta- and gamma-amylases.

Amino Acid Sequence↗

PubMed1993

A note on the primary structure and expression of an Erwinia carotovora polygalacturonase-encoding gene (peh1) in Escherichia coli and Saccharomyces cerevisiae.

A 1209-base pair (bp) DNA fragment containing the endopolygalacturonase-encoding gene (peh1) from Erwinia carotovora subsp. carotovora was amplified by the polymerase chain reaction (PCR) technique and expressed in Escherichia coli. The nucleotide sequence of the PCR product was determined and found to be highly homologous to the primary structures of other polygalacturonase-encoding genes. The peh1 DNA fragment encoding the mature polygalacturonase was inserted between two different yeast expression-secretion cassettes and a yeast gene terminator, generating recombinant yeast-integrating shuttle plasmids pAMS10 and pAMS11. These YIp5-derived plasmids were transformed and stably integrated into the genome of a laboratory strain of Saccharomyces cerevisiae. Transcription initiation signals present in these expression-secretion cassettes were derived from the yeast alcohol dehydrogenase (ADC1P) or mating pheromone alpha-factor (MF alpha 1P) gene promoters. The transcription termination signals were derived from the yeast tryptophan synthase gene terminator (TRP5T). Secretion of polygalacturonase was directed by the signal sequence of the yeast mating pheromone alpha-factor (MF alpha 1S). Northern blot analysis revealed the presence of peh1 mRNA in the yeast transformants and a polypectate agarose test was used to monitor polygalacturonase production.

Amino Acid Sequence↗

PubMed1993

Expression and secretion of Bacillus amyloliquefaciens alpha-amylase by using the yeast pheromone alpha-factor promoter and leader sequence in Saccharomyces cerevisiae.

Replacement of the regulatory and secretory signals of the alpha-amylase gene (AMY) from Bacillus amylolique-faciens with the complete yeast pheromone alpha-factor prepro region (MF alpha 1p) resulted in increased levels of extracellular alpha-amylase production in Saccharomyces cerevisiae. However, the removal of the (Glu-Ala)2 peptide from the MF alpha 1 spacer region (Lys-Arg-Glu-Ala-Glu-Ala) yielded decreased levels of extracellular alpha-amylase.

Bacillus↗

PubMed1992

Synthesis and secretion of an Erwinia chrysanthemi pectate lyase in Saccharomyces cerevisiae regulated by different combinations of bacterial and yeast promoter and signal sequences.

Nine different expression-secretion cassettes, comprising novel combinations of yeast and bacterial gene promoters and secretion signal sequences, were constructed and evaluated. A pectate lyase-encoding gene (pelE) from Erwinia chrysanthemi was inserted between each one of these expression-secretion cassettes and a yeast gene terminator, generating recombinant yeast-integrating shuttle plasmids pAMS1 through pAMS9. These YIp5-derived plasmids were transformed and stably integrated into the genome of a laboratory strain of Saccharomyces cerevisiae, and the pectate lyase production was monitored. Transcription initiation signals for pelE expression were derived from the yeast alcohol dehydrogenase (ADC1P), the yeast mating pheromone alpha-factor (MF alpha 1P) and the Bacillus amyloliquefaciens alpha-amylase (AMYP) gene promoters. The transcription termination signals were derived from the yeast tryptophan synthase gene terminator (TRP5T). Secretion of pectate lyase (PLe) was directed by the signal sequences of the yeast mating pheromone alpha-factor (MF alpha 1S), B. amyloliquefaciens alpha-amylase (AMYS) and Er. chrysanthemi pectate lyase (pelES). The ADC1P-MF alpha 1S expression-secretion system proved to be the most efficient control cassette for the expression of pelE and the secretion of PLe in S. cerevisiae.

Alcohol Dehydrogenase↗

PubMed1992

The value of electrophoretic fingerprinting and karyotyping in wine yeast breeding programmes.

Electrophoretic banding patterns of total soluble cell proteins, DNA restriction fragments and chromosomal DNA were used to characterise ten strains of Saccharomyces cerevisiae used for commercial production of wine. These fingerprinting procedures provided unique profiles for all the different yeast strains and can therefore be used to identify and control industrial strains. Furthermore, the protein profiles, restriction fragments banding patterns and electrophoretic karyotyping by contour clamped homogeneous electric field electrophoresis (CHEF), were valuable to differentiate hybrid and parental strains in yeast breeding programmes. Hybrid strains, with desirable oenological properties, were obtained by mass spore-cell mating between a heterothallic killer yeast and two homothallic sensitive strains and all were shown to have unique DNA fingerprints and electrophoretic karyotypes.

DNA Fingerprinting↗

PubMed1991

Co-expression of a Saccharomyces diastaticus glucoamylase-encoding gene and a Bacillus amyloliquefaciens alpha-amylase-encoding gene in Saccharomyces cerevisiae.

A glucoamylase-encoding gene (STA2) from Saccharomyces diastaticus and an alpha-amylase-encoding gene (AMY) from Bacillus amyloliquefaciens were cloned separately into a yeast-integrating shuttle vector (YIp5), generating recombinant plasmids pSP1 and pSP2, respectively. The STA2 and AMY genes were jointly cloned into YIp5, generating plasmid pSP3. Subsequently, the dominant selectable marker APH1, encoding resistance to Geneticin G418 (GtR), was cloned into pSP3, resulting in pSP4. For enhanced expression of GtR, the APH1 gene was fused to the GAL10 promoter and terminated by the URA3 terminator, resulting in pSP5. Plasmid pSP5 was converted to a circular minichromosome (pSP6) by the addition of the ARS1 and CEN4 sequences. Laboratory strains of Saccharomyces cerevisiae transformed with plasmids pSP1 through pSP6, stably produced and secreted glucoamylase and/or alpha-amylase. Brewers' and distillers' yeast transformed with pSP6 were also capable of secreting amylolytic enzymes. Yeast transformants containing pSP1, pSP2 and pSP3 assimilated soluble starch with an efficiency of 69%, 84% and 93%, respectively. The major starch hydrolysis products produced by crude amylolytic enzymes found in the culture broths of the pSP1-, pSP2- and pSP3-containing transformants, were glucose, glucose and maltose (1:1), and glucose and maltose (3:1), respectively. These results confirmed that co-expression of the STA2 and AMY genes synergistically enhanced starch degradation.

Bacillus↗

PubMed1991

Primary structure and regulation of a glucoamylase-encoding gene (STA2) in Saccharomyces diastaticus.

We have determined the complete nucleotide (nt) sequence of a 5070-bp DNA fragment containing a glucoamylase-encoding gene (STA2) from Saccharomyces diastaticus. The 5' transcription start points for STA1, STA2 and STA3 were determined by primer extension of their respective mRNAs using reverse transcriptase. The sequence data show one major open reading frame (ORF) of 767 amino acids encoding GAII with a calculated Mr of 82,514. The 5' region in the ORF contains two ATG sequences within 30 nt of each other. The upstream region of STA2 was amplified by the polymerase chain reaction (PCR) and fused to the Escherichia coli lacZ gene. Some of the PCR products contained mutations in ATG1 and/or ATG2. Results indicated that both ATG1 and ATG2 encode functional translation start codons, but ATG2 was shown to encode the stronger initiator. The upstream region of STA2 contains a canonical sequence that is homologous to known sites of repression by the MATa/MAT alpha-encoded repressor. Also, consensus RAP1 (Repressor-Activator Protein 1)-binding sites are located in the 5' upstream region and within the coding region of STA2.

Amino Acid Sequence↗

PubMed1991

The glucoamylase multigene family in Saccharomyces cerevisiae var. diastaticus: an overview.

Saccharomyces cerevisiae has been used widely both as a model system for unraveling the biochemical, genetic, and molecular details of gene expression and the secretion process, and as a host for the production of heterologous proteins of biotechnological interest. The potential of starch as a renewable biological resource has stimulated research into amylolytic enzymes and the broadening of the substrate range of S. cerevisiae. The enzymatic hydrolysis of starch, consisting of linear (amylose) and branched glucose polymers (amylopectin), is catalyzed by alpha- and beta-amylases, glucoamylases, and debranching enzymes, e.g., pullulanases. Starch utilization in the yeast S. cerevisiae var. diastaticus depends on the expression of the three unlinked genes, STA1 (chr. IV), STA2 (chr. II), and STA3 (chr. XIV), each encoding one of the extracellular glycosylated glucoamylases isozymes GAI, GAII, or GAIII, respectively. The restriction endonuclease maps of STA1, STA2, and STA3 are identical. These genes are absent in S. cerevisiae, but a related gene, SGA1, encoding an intracellular, sporulation-specific glucoamylase (SGA), is present. SGA1 is homologous to the middle and 3' regions of the STA genes, but lacks a 5' sequence that encodes the domain for secretion of the extracellular glucoamylases. The STA genes are positively regulated by the presence of three GAM genes. In addition to positive regulation, the STA genes are regulated negatively at three levels. Whereas strains of S. diastaticus are capable of expressing the STA genes, most strains of S. cerevisiae contain STA10, whose presence represses the expression of the STA genes in an undefined manner. The STA genes are also repressed in diploid cells, presumably by the MATa/MAT alpha-encoded repressor. STA gene expression is reduced in liquid synthetic media, it is carbon catabolite repressed by glucose, and is inhibited in petite mutants.

Base Sequence↗

PubMed1988

Expression of a Bacillus alpha-amylase gene in yeast.

A recombinant plasmid, pSR11.3, containing the alpha-amylase gene (AMY) of Bacillus amyloliquefaciens was characterized and expressed in Bacillus subtilis. A 2.3 kilobase BamHI-BglII fragment carrying AMY was cloned into pBR322 (pEL322) and in both orientations into a multi-copy Escherichia coli-yeast shuttle vector YEp13 (pAM13) and expressed in E. coli HB101 and various Saccharomyces stains. We report on the successful secretion of an active bacterial enzyme in yeast without using yeast promoter and secretory signals. Enzyme production in B. subtilis 1A297(pSR11.3), E. coli HB101(pEL322) and Saccharomyces JM2773-15B(pAM13) transformants was measured as 125, 22 and 123 U/ml, respectively. The molecular weight of the purified alpha-amylase secreted by B. subtilis 1A297(pSR11.3) and Saccharomyces JM2773-15B-(pAM13) was estimated to be 55 kDa. The pH and temperature optima for the alpha-amylase activities of the transformants were 6.5 to 8.0 and 50 to 65 degrees C, respectively. Amylose hydrolysis profiles of the alpha-amylases secreted by B. subtilis 1A297(pSR11.3) and Saccharomyces JM2773-15B(pAM13) indicate effective meso-thermostable hydrolytic enzymes with maltotriose and maltose, respectively, as major end products.

Bacillus subtilis↗

PubMed1988

Localization of yeast glucoamylase genes by PFGE and OFAGE.

Chromosomes of two closely related yeast strains, the amylolytic Saccharomyces diastaticus and the non-amylolytic Saccharomyces cerevisiae, were resolved by pulsed field gel electrophoresis (PFGE) and orthological field alteration gel electrophoresis (OFAGE). Electrophoretic karyotypes of these two strains are identical. Sixteen cloned Saccharomyces genes of known chromosomal location were used to identify individual chromosomes by Southern hybridization analyses. The Southern blots were reprobed with a cloned fragment of the STA2 glucoamylase gene of S. diastaticus. STA2 exhibits homology to STA1 and STA3 as well as the sporulation-specific glucoamylase (SGA) gene from both Saccharomyces strains. The three unlinked, homologous genes, STA1 (DEX2, MAL5), STA2 (DEX1) and STA3 (DEX3) encoding the extracellular glucoamylase isozymes GAI, GAII and GAIII in S. diastaticus were then assigned to chromosomes IV, II and XIV, respectively. The SGA gene, encoding an intracellular glucoamylase in both S. diastaticus and S. cerevisiae, was assigned to chromosome IX. Electrophoretic mapping of the STA and SGA genes is at present the only way to localize these genes, since glucoamylase repressor gene(s) (STA10, INH1 and/or IST2) are present in most laboratory strains of S. cerevisiae and the SGA phenotype is only detectable during sporulation.

Chromosome Mapping↗

PubMed1987

Plasmids in Leuconostoc oenos.

A new procedure was used to isolate 11 plasmids from eight Leuconostoc oenos strains. Plasmid DNA was not detected in 34 other strains of this species. Plasmid sizes ranged from 2.47 to 4.61 kilobase pairs. This is the first report of extrachromosomal elements in L. oenos.

DNA, Bacterial↗

PubMed1986

Molecular cloning and characterization of the STA2 glucoamylase gene of Saccharomyces diastaticus.

The Saccharomyces diastaticus structural gene STA2, encoding an exracellular glucoamylase (1,4-alpha-D-glucan glycohydrolase, EC 3.2.1.3.), has been cloned by complementation of a stao strain. A genomic library was initially constructed from a STA2 yeast strain in the yeast Escherichia coli shuttle cosmid vector pYCl. The Sta+ complementing function was further delimited to an 8.3 kb BglII fragment whose restriction map was found to be similar to related genomic regions of STA1 and STA3. Fusions of several DNA fragments derived from the 8.3 kb BglII fragment with a truncated E. coli beta-galactosidase gene resulted in two overlapping fragments that could direct the production of large fusion proteins in E. coli. These fusion proteins were immunoprecipitable by anti-glucoamylase II antibodies, confirming that the Sta+ complementing fusion was due to the expression of a gene that coded for a yeast glucoamylase. Measurements of the STA1, STA2 and STA3 RNA transcripts by RNA-DNA hybridization using an internal fragment of the cloned STA2 gene as the probe indicated that a common transcript of 2.5 kb is produced by each of the STA genes. Integrative disruption of the STA2 gene through homologous recombination was achieved by transforming a STA2 yeast strain to Sta- using an in vitro constructed donor DNA fragment that has the URA3 gene inserted within the coding region of the cloned glucoamylase gene. This was confirmed by tetrad analysis of crosses between strains carrying a disrupted STA2 and a functional STA2. Southern blot analysis using BamHI digested genomic DNA from 15 tetrads demonstrated consistent co-segregation and Mendelian inheritance of the Sta- phenotype with STA2::URA3. These data further confirm that the cloned DNA that showed Sta+ complementing activity carries a functional STA2 gene that encodes the yeast extracellular glucoamylase II.

Cloning, Molecular↗

PubMed1986

Identification and physical characterization of yeast glucoamylase structural genes.

Each one of at least three unlinked STA loci (STA1, STA2 and STA3), in the genome of Saccharomyces diastaticus controls starch hydrolysis by coding for an extracellular glucoamylase. Cloned STA2 sequences were used as hybridization probes to investigate the physical structure of the family of STA genes in the genomes of different Saccharomyces strains. Sta+ strains, each carrying a single genetically defined STA locus, were crossed with a Sta- strain and the segregation behavior of the functional locus (i.e. Sta+) and sequences homologous to a cloned STA2 glucoamylase structural gene at that locus were analyzed. The results indicate that in all strains examined there is a multiplicity of sequences that are homologous to STA2 DNA but that only the functional STA loci contain extensive 5' and 3' homology to each other and can be identified as residing on unique fragments of DNA; that all laboratory yeast strains examined contain extensive regions of the glucoamylase gene sequences at or closely linked to the STA1 chromosomal position; that the STA1 locus contains two distinct glucoamylase gene sequences that are closely linked to each other; and that all laboratory strains examined also contain another ubiquitous sequence that is not allelic to STA1 and is nonfunctional (Sta-), but has retained extensive sequence homology to the 5' end of the cloned STA2 gene. It was also determined that the DEX genes (which control dextrin hydrolysis in S. diastaticus), MAL5 (a gene once thought to control maltose metabolism in yeast) and the STA genes are allelic to each other in the following manner: STA1 and DEX2, STA1 and MAL5, and STA2 and DEX1 and STA3 and DEX3.

Cloning, Molecular↗