The importance of glycosylation for microbial evolution

Protein glycosylation is an important property of eukaryotic cells with manifold functions. Even in yeast cells, gene deletion mutants associated with protein glycosylation show various defects in protein quality control, cell proliferation and stress resistance. As such, the cellular protein glycosylation pathway is an important part of the regulatory machinery in yeast cells. Bacteria and yeasts have been used for numerous studies on molecular evolution and long-term adaptation, providing basic insights into the adaptive changes and flexibility of gene regulatory networks during environmental specialization. Nevertheless, specifically for eukaryotic microbes, the implications of long-term stress adaptation in context with compromised regulatory networks are not well characterized. In order to increase our understanding of the evolution of yeasts in stressful environments, with special emphasis on the importance of fully functional protein glycosylation, the evolvability of the biotechnologically relevant yeast Pichia pastoris will be analyzed. By parallel laboratory evolution of wild-type and glycosylation-mutant P. pastoris cells in stress and control environments, insights into the implications of protein glycosylation for their evolutionary potential will be acquired. An in-depth systems wide analysis of the adapted populations and the resulting insights into the molecular evolution of this non-conventional yeast will greatly extend our knowledge on the dynamics of regulatory networks and adaptation strategies of microbial cells. Additionally, the obtained data will provide valuable information for the establishment of in silico cellular models for future biotechnological research and lead to increased knowledge of fungal physiology.

During the project "The importance of glycosylation for microbial evolution" the effects of deficits of the cellular glycosylation machinery during evolution on a laboratory scale were investigated. The industrial yeast Pichia pastoris was selected as a model organism. A suitable experimental setup was successfully established to study the long-term effects of glycosylation defects in various control and stress conditions. Subsequently, Pichia pastoris populations were cultivated for several hundred generations and investigated with different methods with respect to growth differences and changes on the genomic level. From the analysis of the data important findings with respect to the molecular biology of Pichia pastoris and yeasts in general were made. The results support the initial project hypothesis that the impairment of cellular glycosylation influences the adaptation to certain environmental conditions. Glycosylation mutant populations showed a reduced adaptive potential than the control populations. Furthermore, genomic mutations that were specific for certain environmental conditions, as well as specific for the glycosylation-deficient mutant could be identified by genome analysis. Several mutations that play a potential role in the adaptation to specific growth environments were identified. A high degree of recursion, with respect to the genetic targets in specific environmental conditions, was observed. The results indicated strain-specific incompatibility of these mutations. Among the genetically and environmentally linked mutational targets, species-specific transcription factors with an obvious key role in the environmental adaptation of Pichia pastoris could be identified. Furthermore, data comparison with similar studies performed for other yeast species led to important insights regarding common trends and differences with organisms such as the baker's yeast, Saccharomyces cerevisiae. Finally, genes with an important role for efficient growth on methanol as sole carbon source were identified. The applicability of certain Pichia pastoris strains, selected by environmental long-term adaptation, for the production of different recombinant proteins was also evaluated. It was found that some strains showed higher recombinant protein productivity in small and large scale production scenarios with different nutrient feeding strategies. In summary, it has been shown that the concept of the laboratory evolution can be successfully applied for Pichia pastoris. The data obtained from such experiments can lead to significant novel insights regarding the molecular biology of this yeast species and may be applied for the genotype-specific design of novel concepts for yeast-based biotechnological process engineering.