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A W Strasser

Publications and source records attributed to A W Strasser.

9 recordsLinked to original sources
PubMed1992

Heterologous protein production in yeast.

The exploitation of recombinant DNA technology to engineer expression systems for heterologous proteins represented a major task within the field of biotechnology during the last decade. Yeasts attracted the attention of molecular biologists because of properties most favourable for their use as hosts in heterologous protein production. Yeasts follow the general eukaryotic posttranslational modification pattern of expressed polypeptides, exhibit the ability to secrete heterologous proteins and benefit from an established fermentation technology. Aside from the baker's yeast Saccharomyces cerevisiae, an increasing number of alternative non-Saccharomyces yeast species are used as expression systems in basic research and for an industrial application. In the following review a selection from the different yeast systems is described and compared.

Gene Expression↗
PubMed1991

Striking structural and functional similarities suggest that intestinal sucrase-isomaltase, human lysosomal alpha-glucosidase and Schwanniomyces occidentalis glucoamylase are derived from a common ancestral gene.

Sequence comparison of the primary structure of the yeast Schwanniomyces occidentalis glucoamylase (GAM) with GAMs in different microorganisms did not reveal significant similarities. By contrast, striking similarities were, surprisingly, found with 3 mammalian secretory and integral membrane proteins: the 2 subunits of intestinal brush border sucrase-isomaltase and human lysosomal alpha-glucosidase. The similarities among these proteins are found as clusters of up to 8 amino acids and distributed all over the protein sequences. The major sequence differences are found in the N-terminal regions accounting, probably, for the different cellular locations of these proteins. The high level of similarities between sucrase, isomaltase, Sch. occidentalis GAM and human lysosomal alpha-glucosidase suggest that these proteins are derived from the same ancestral gene. To our knowledge, this is the first report that describes similarities between a yeast secretory protein and mammalian secretory and integral membrane proteins.

Amino Acid Sequence↗
PubMed1991

Reciprocal communication between the lyase and synthase active sites of the tryptophan synthase bienzyme complex.

It is important to understand how the cleavage of indoleglycerol phosphate, which is catalyzed by the alpha subunits in the alpha 2 beta 2 bienzyme complex of tryptophan synthase, is modulated by the presence of L-serine in the beta subunits. Steady-state kinetic data, including the dependence of kcat on pH, allowed values to be assigned to each of the eight rate constants of the minimal catalytic mechanism. An ionizing group having an apparent pK value near 7.5 must be protonated for activity. The alpha active site ligands indolepropanol phosphate, glyceraldehyde 3-phosphate, and glycerol 3-phosphate increase both the affinity and the molar absorbance of L-serine and L-tryptophan bound to the beta active site. These effects prove that the alpha sites communicate with the beta sites over a distance of 30 A. 6-Nitroindole readily condenses with glyceraldehyde 3-phosphate, but not with L-serine. The turnover numbers for 6-nitroindoleglycerol phosphate and 6-nitroindole increased about 10-fold in both directions in the presence of L-serine bound to the beta 2 subunits. These data prove that the alpha and beta active sites communicate reciprocally and explain why the turnover number for the physiological reaction of indoleglycerol phosphate with L-serine greatly exceeds that of the cleavage reaction of indoleglycerol phosphate.

Binding Sites↗
PubMed1991

Heterologous gene expression in Hansenula polymorpha: efficient secretion of glucoamylase.

We have introduced the glucoamylase gene (GAM1) from Schwanniomyces occidentalis into the genome of the methylotrophic yeast Hansenula polymorpha to study the potential of this organism as a host for high-level expression of a heterologous gene encoding a secretory protein. Transformants of H. polymorpha containing GAM1 under control of the formate dehydrogenase (FMD) promoter are stable and efficiently secrete an active glucoamylase that is faithfully processed and modified. Yields of up to 1.4g/l of active enzyme were obtained at cell densities of 100-130 grams dry weight per liter.

Amino Acid Sequence↗
PubMed1990

Cloning of the Schwanniomyces occidentalis glucoamylase gene (GAM1) and its expression in Saccharomyces cerevisiae.

The Schwanniomyces occidentalis glucoamylase (GAM)-encoding gene (GAM1) was isolated from a lambda Charon4A genomic library using synthetic oligodeoxynucleotides as probes. GAM1 contains an ORF of 2874 nucleotides (nt) coding for 958 amino acids. S1 mapping revealed that the transcript has only a very short 5'-untranslated leader of 8-12 nt. Disruption and displacement of the GAM1 gene in Sc. occidentalis resulted in loss of the ability to grow on starch efficiently. The gam1 strains still exhibit low GAM activity suggesting that at least a second weakly expressed GAM-encoding gene (GAM2) is present in Sc. occidentalis. Expression of the Sc. occidentalis GAM1 gene in Saccharomyces cerevisiae was achieved after promoter exchange. S. cerevisiae cells transformed with centromere plasmids carrying the GAM1 gene fused to promoters of different S. cerevisiae genes, namely GAL1, PDC1 and ADH1, efficiently secrete GAM and are able to grow with soluble starch as a sole carbon source. The essential enzymatic properties of the GAMs secreted from S. cerevisiae and Sc. occidentalis are identical, although the modifications of the proteins are different.

Amino Acid Sequence↗
PubMed1989

Analysis of the alpha-amylase gene of Schwanniomyces occidentalis and the secretion of its gene product in transformants of different yeast genera.

We have cloned and characterized the alpha-amylase gene (AMY1) of the yeast Schwanniomyces occidentalis. A cosmid gene library of S. occidentalis DNA was screened in Saccharomyces cerevisiae for alpha-amylase secretion. The positive clone contained a DNA fragment harbouring an open reading frame of 1536 nucleotides coding for a 512-amino-acid polypeptide with a calculated Mr of 56,500. The deduced amino acid sequence reveals significant similarity to the sequence of the Saccharomycopsis fibuligera and Aspergillus oryzae alpha-amylases. The AMY l gene was found to be expressed from its original promoter in S. cerevisiae, Kluyveromyces lactis and Schizo-saccharomyces pombe leading to an active secreted gene product and thus enabling the different yeast transformants to grow on starch as a sole carbon source.

Amino Acid Sequence↗
PubMed1989

Regulated overproduction of alpha-amylase by transformation of the amylolytic yeast Schwanniomyces occidentalis.

High frequency transformation of a Schwanniomyces occidentalis mutant defective in the last step of tryptophan synthesis was achieved with plasmids containing the tryptophan synthetase gene (TRP5) of Saccharomyces cerevisiae and an autonomous replication sequence from S. occidentalis, which we called "SwARS1". The SwARS1 fragment is also functional in S. cerevisiae. The average copy number of the plasmids in both yeast species was 5-10 per cell under selective conditions. S. occidentalis cells that were transformed with an autonomously replicating plasmid carrying the cloned alpha-amylase gene from S. occidentalis secreted about five times more alpha-amylase than cells without additional copies of the alpha-amylase gene. Both the chromosomal copy and the plasmid-carried copies of the alpha-amylase gene were repressed in the presence of glucose. This transformation system provides a possibility to improve starch degradation by S. occidentalis.

Blotting, Southern↗