Heterologous protein production in Saccharomyces cerevisiae with different genotypes and ploidy were investigated. Bovine aprotinin and human insulin analogues precursors (AspB28) were used as model proteins. Both laboratory and industrial strains were studied with different cultivations methods, picturing long-term observation up to 500 generations. Adaptation studies with respect to growth kinetics were investigated and the recombinant gene amplification (copy number, real-time qPCR), its transcription (mRNA level, DNA arrays) and secretion efficiency (target protein conc.) were determined, respectively. Challenging the cells to unlimited growth conditions through repeated shake flask cultivations resulted in a decreased protein burden, as metabolism shifted towards higher specific growth rates, and lower protein yields were found. The secreted protein level increased as a function of its mRNA level until a certain threshold was achieved. A promoter truncation technique on the auxotrophic marker genes was successfully applied in order to adjust the recombinant gene level. Differences among the strain backgrounds were found on target gene amplification potential, adaptation ability, protein folding and secretion efficiency, and processing and degradation of target mRNA. The choice of the expression system, proved to be crucial for protein production stability. The identification of reporter transcription factors highlighted some interesting differences among the genetic backgrounds. Some hypotheses for the decline of protein production in prolonged processes were generated, including down-regulation of glycolytic enzymes, protein recirculation due to improper folding, and the inhibitory activity of the heterologous protein during the secretory pathway. These hypotheses will be discussed during the presentation.