A set of isomeric episomal plasmids for systematic examination of mitotic stability in Saccharomyces cerevisiae.

Yeast episomal shuttle vectors (YEp type) are commonly used in fundamental research and biotechnology whenever elevated product levels are desired. Their instability, however, poses an impediment not only in industrial scale fermentation. In order to analyse instability which might be linked to plasmid structure, a series of YEp type plasmids that are identical in size has been assembled, differing only in the overall arrangement of the fragments used. The performance of the eight plasmid isoforms was studied with respect to mitotic stability. While transformation efficiency in two laboratory strains of Saccharomyces cerevisiae does not differ dramatically between the eight plasmids, the plasmids do not, however, perform equally well in terms of segregational stability. Although stable at about 90% plasmid-bearing cells in selective medium, under non-selective conditions, three plasmid forms performed better than the other five with an up to 5.7-fold higher stability as compared with the least favourable isoform. In a subset of four plasmids (including stable and unstable isoforms) copy numbers were determined. Furthermore the functionality of the selection marker was characterized with respect to plasmid-derived relative HIS3 transcript levels. No significant differences in HIS3 transcript levels could be observed between strains carrying any one of the four plasmids. Ruling out copy number and performance of HIS3, the results indicate nevertheless that plasmid architecture has an impact on mitotic segregation in yeast and that construction of an expression vector should take into account that the plasmid backbone itself might already show a more or less favourable arrangement of its segments. © 2017 The Authors. Yeast published by John Wiley & Sons, Ltd.