Alexander W.M. Strasser

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 bakers yeastSaccharomyces 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.

High-level expression of foreign genes in Hansenula polymorpha

The methylotrophic yeast Hansenula polymorpha belongs to a limited number of non-Saccharomyces yeast species used as hosts for heterologous gene expression. It has successfully been applied for the production of hormones, antigens and enzymes. The system excells by mitotically stable recombinant strains, high productivity and faithful processing of the produced polypeptides. The favourable characteristics of this microorganism for protein production at an industrial scale are described in the following article focusing on some recent representative examples.

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

SummaryHigh 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 α-amylase gene from S. occidentalis secreted about five times more α-amylase than cells without additional copies of the α-amylase gene. Both the chromosomal copy and the plasmid-carried copies of the α-amylase gene were repressed in the presence of glucose. This transformation system provides a possibility to improve starch degradation by S. occidentalis.

Striking structural and functional similarities suggest that intestinal sucrase-isomaltase, human lysosomal α-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 α‐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 α‐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.

Strain and process development for the production of human cytokines in Hansenula polymorpha

The early status of strain development for the production of interleukin (IL)-6, IL-8, IL-10, and interferon (IFN) gamma is described. The general approach to generating such strains was to amplify gene sequences encoding the mature forms of the various cytokines by PCR from commercially available cDNA sources. The design of the amplificates allowed an in-frame fusion to an MFalpha1 leader segment contained in two basic expression vectors, pFPMT121-MFalpha1 and pTPSMT-MFalpha1. The two vectors differ in that one harbors the methanol-inducible FMD promoter and the other the constitutive TPS1 promoter as control elements for heterologous gene expression. The most advanced process development example is that of IFNalpha-2a. Here, the MOX promoter derived from another key gene of methanol metabolism is used for expression control. The successful development of a production process for Hansenula polymorpha-derived IFNalpha-2a is summarized. This was achieved by combining genetic engineering of suitable production strains with improved processing capabilities for the secreted cytokine, and by purification procedures from cultures grown in yeast extract-peptone-glycerol-based media.

Improvement of baker's and brewer's yeast by gene technology

Abstract Polysaccharides such as starch, cellulose or hemicellulose which constitute the largest part of plant biomass are not fermentable by the yeast Saccharomyces cerevisiae. At the moment the conversion of such compounds depends on the addition of enzymes (e.g. α‐amylase, glucoamylase, xylose isomerase, endoglucanase exoglucanase and β‐glucanase) prior to fermentation. Therefore it is of great commercial interest to create new yeast strains which are able to convert polysaccharides of plant biomass directly into fermentable sugars. In a first step we have constructed an amylolytic S. cerevisiae strain which is able to grow on starch as a sole carbon source. This strain was transformed with DNA harbouring the genes for a‐amylase and glucoamylase from Schwanniomyces occidentalis. Both genes are expressed and the gene products are secreted. Since the two S. occidentalis enzymes are especially suitable in brewing, we thus constructed new yeast strains which could be desirable for the production of low car...