Ruben Hohnholz

• Yeast, in particular Saccharomyces cerevisiae, is a frequently used host for the production of numerous nutritional and pharmaceutical compounds. Cells can be transformed in order to produce recombinant products with the foreign genetic information being placed on hybrid multi-copy plasmids (YEp-type) due to the anticipated gene dosage effect. These plasmids contain sequences of the yeast endogenous 2µm plasmid encompassing its origin of replication. Expression vectors based on the 2μm plasmid are the only vectors in S. cerevisiae which satisfy two major biotechnological requirements: segregational stability and high copy number. Under non-selective conditions, those plasmids show, however, a lower stability compared to the authentic 2µm plasmid. Stability can be divided into structural and segregational stability. Both were analyzed in detail for a set of isomeric YEp-type plasmids created in this work. Efforts were made to investigate plasmid-based recombination events that on the one hand allow the plasmid to amplify by employing 2µm-encoded proteins and on the other hand affect the stability of the plasmid. The correct structure might be compromised by the innate amplification mechanism of YEp-type plasmids, adapting a similar mechanism as the endogenous 2µm. This work identified multiple structures that likely represent amplification intermediates where YEp–type plasmid and 2µm fusion products were found. This thesis work shows that the architecture of functional elements of a basic multicopy model plasmid has a huge impact on the segregational stability with differences of up to 5.7-fold between the eight isoforms created. The addition of a synthetic gene coding for a yEGFP3 reporter protein to a particularly stable model plasmid in all possible positions and orientations has an influence on both structural and segregational stability. One particularly favorable and one unfavorable isoform were identified.