Glycosylation and Microbial Evolution II

Glyco-engineering is a promising approach in order to provide humanized protein glycosylation patterns despite the production of proteins in microbial cells. In this respect, important advances with the industrial yeast Pichia pastoris have been made in recent years. Since glyco-engineering of yeasts depends on the knockout of certain endogenous glycosyltransferases, this engineering approaches come at the cost of severe growth defects such as increased apoptosis rates and reduced growth rates. In a previous study, an experimental evolution approach led to the identification of the fitness and genome evolution trajectories of wildtype and glycosylation-deficient P. pastoris populations that were experimentally evolved in different glucose-based control and salt stress media. Both, species- but also genotype-dependent evolutionary paths were identified and were furthermore associated with improved growth performance. In this context mutations of a species- specific transcription factor involved in minimal medium adaptation and an intriguing environment-genotype correlation of mutations related to osmotic stress signaling were observed. This previous results highlight highly species-specific mutational targets upon laboratory evolution of P. pastoris that are substantially distinct from the model yeast Saccharomyces cerevisiae. Furthermore it shows how engineering- based cellular disturbances lead to alternative evolutionary paths and subsequently the necessity of alternative engineering strategies for glyco-engineered cells to improve microbial growth. Within this project the attempt will be made to further characterize the long-term consequences of environmental adaptation on an evolutionary scale by subjecting different P. pastoris strains to experimental evolution during the exposure to additional biotechnologically relevant stress factors, more specifically heat and oxidative stress. The hypothesis, that the exposure to both stresses leads to compatible evolutionary solutions and cross-protection, will be tested. Our previous results also indicated a significant fitness burden of recombinant production during short-term growth. Thus, it will also be analyzed to which extent recombinant production forces diverging evolutionary paths in both, wildtype and glycosylation-deficient strain backgrounds. Since rational model-based engineering approaches rely on a detailed understanding of the molecular biology and regulatory features of the microbial host cell, this project aims at improving our understanding of P. pastoris-specific and glycosylation deficiency-induced traits. By the application of a well-established laboratory evolution approach it will also contribute to an improved understanding of evolutionary processes on a molecular level.