Gerd Gellissen

Background: Yeasts provide attractive expression platforms in combining ease of genetic manipulation and fermentation of a microbial organism with the capability to secrete and to modify proteins according to a general eukaryotic scheme. However, early restriction to a single yeast platform can result in costly and time-consuming failures. It is therefore advisable to assess several selected systems in parallel for the capability to produce a particular protein in desired amounts and quality. A suitable vector must contain a targeting sequence, a promoter element and a selection marker that function in all selected organisms. These criteria are fulfilled by a wide-range integrative yeast expression vector (CoMed™) system based on A. adeninivorans- and H. polymorpha- derived elements that can be introduced in a modular way. Results: The vector system and a selection of modular elements for vector design are presented. Individual single vector constructs were used to transform a range of yeast species. Various successful examples are described. A vector with a combination of an rDNA sequence for genomic targeting, the E. coli- derived hph gene for selection and the A. adeninivorans-derived TEF1 promoter for expression control of a GFP (green fluorescent protein) gene was employed in a first example to transform eight different species including Hansenula polymorpha, Arxula adeninivorans and others. In a second example, a vector for the secretion of IL-6 was constructed, now using an A. adeninivorans-derived LEU2 gene for selection of recombinants in a range of auxotrophic hosts. In this example, differences in precursor processing were observed: only in A. adeninivorans processing of a MFα1/IL-6 fusion was performed in a faithful way. Conclusion: rDNA targeting provides a tool to co-integrate up to 3 different expression plasmids by a single transformation step. Thus, a versatile system is at hand that allows a comparative assessment of newly introduced metabolic pathways in several organisms or a comparative co-expression of bottleneck genes in cases where production or secretion of a certain product is impaired.

Hansenula polymorpha – Strengthening the connection between fundamental research and its biotechnological applications.

The yeast Arxula adeninivorans is characterized by a temperature-dependent dimorphism. A. adeninivorans grows as budding cells at temperatures up to 42 degrees C, but forms mycelia at higher temperatures. A strong correlation exists between morphological status and iron uptake, achieved by two transport systems that differ in iron affinity. In the presence of high Fe(II) concentrations (>2 microm), budding cells accumulate iron concentrations up to seven-fold higher than those observed in mycelia, while at low Fe(II) concentrations (<2 microm), both cell types accumulate similar amounts of iron. The copper-dependent Fe(II) oxidase Afet3p, composed of 615 amino acids, is a component of the high-affinity iron transport system. This protein shares a high degree of homology with other yeast iron transport proteins, namely Fet3p of Saccharomyces cerevisiae, Cafet3p of Candida albicans and Pfet3p of Pichia pastoris. Expression of the AFET3 gene is found to be strongly dependent on iron concentration but independent of the morphological stage; however, cell morphology was found to influence post-translational modifications of the gene product. O-glycosylation was observed in budding cells only, whereas N-glycosylation occurred in both cell types. The N-glycosylated 103 kDa glycoprotein matures into the 108.5 kDa form, further characterized by serine phosphorylation. Both N-glycosylation and phosphorylation occur at low iron concentrations (< or =5 microm). The mature Afet3p of 108.5 kDa is uniformly distributed within the plasma membrane in cells of both morphological stages.