Physiology of Saccharomyces cerevisiae Producing Recombinant Insulin in Continuous Culture

Doctoral thesis, 2012

The yeast Saccharomyces cerevisiae has widely been used as a host for the production of heterologous proteins. High-level production of heterologous proteins is likely to impose a metabolic burden on the host cell and can thus affect various aspects of cellular physiology. Different target proteins often do not result in similar secretion yields, underlining the dependency of secretion efficiency on the physicochemical properties of the protein of interest. Two human insulin analogue precursors (IAPs) were used as model secretory proteins. The IAPs had minor differences in their amino acid sequences, yet pictured more than 7-fold difference in their secretion yields. Global transcriptome analysis carried out in aerobic glucose-limited chemostat experiments pinpointed distinct steps during the protein maturation pathway to be differentially regulated, and indicated an increased degradation of the IAP with the low secretion yield. The use of auxotrophic strains for improved IAP production was examined. The incremental truncation of the promoter of the auxotrophic marker gene URA3 led to higher plasmid copy numbers, and illustrated that the modification of the level of the recombinant gene dosage via the degree of promoter truncation can be a strong tool for optimizing the IAP productivity. The dynamic character of adaptive responses of S. cerevisiae towards a change in their nutrient access was studied based on global gene expression analysis, scrutinizing the impact of restricted supply of phosphate on the physiological state of IAP-expressing cells. The gradual decrease of the phosphate supply resulted in a step-wise modulated phenotypic response, thereby alternating the specific productivity and the secretory flux. A data-driven approach was applied to study the secretory IAP production in S. cerevisiae in prolonged chemostat cultures (80 generations), with the aim to explore the metabolic adaptation of the cells towards the burden of IAP production. Time-course analysis of global transcriptome and targeted metabolome analysis indicated at metabolic re-modeling of the recombinant cells based on augmented negative selection pressure on glycolytic overcapacity, changes in amino acid and central carbon metabolism, and mitochondrial dysfunction to account for decreased cellular expression efficiency in long-term chemostat cultures.