Pichia Lipidomics

The industrial yeast Pichia pastoris is widely used as an experimental system for the heterologous expression of proteins. Despite the extensive commercial use, fundamental biochemical, cell biological and molecular biological knowledge about this microorganism is largely missing. As a striking example, Pichia pastoris lipids have not been studied much in the past although it is obvious that knowledge about this class of biomolecules is highly important to understand the impact of biological (organelle) membranes on protein targeting and intracellular translocation of heterologously expressed proteins. For this reason, we wish to make use of the knowledge of our laboratory in yeast organelle analysis and lipid research and investigate lipids and biomembranes from Pichia pastoris in some detail. The goal of this study will be establishing the Pichia pastoris lipidome, and constructing Pichia pastoris mutant strains and variants with modified lipid profiles. A stepwise strategy with increasing specificity will lead from fundamental knowledge of Pichia pastoris cellular and organelle lipids to the application of this know-how to protein expression studies using engineered Pichia pastoris strains. First, we will perform lipidome analysis with Pichia pastoris organelle membranes which will include bulk lipids but also less abundant lipid components. Then, we will identify and characterize selected key enzymes of Pichia pastoris lipid metabolisms, and construct strains bearing deletions and/or over-expressions of selected prominent lipid biosynthetic and metabolic enzymes from Pichia pastoris which result in modified organelle lipid profiles. Basic phenotype studies of the novel strains will identify possible bottlenecks linked to lipid metabolism in Pichia pastoris. Finally, we will study expression of selected proteins, especially membrane bound proteins and secretory proteins, in the modified membrane lipid environment as proof of principle. This knowledge and these tools will be made public and available to the Pichia community for application to protein expression studies upon individual request and requirement. Thus, the proposed project will combine fundamental with applied research and add a novel facet to our knowledge of heterologous protein expression in Pichia pastoris.

The industrial yeast Pichia pastoris is widely used as an experimental system for the heterologous expression of proteins. Despite the extensive use of this system, fundamental biochemical, cell biological and molecular biological knowledge about this microorganism is largely missing. As a striking example, Pichia pastoris lipids have not been studied much in the past although it is obvious that knowledge about this class of biomolecules is highly important to understand the impact of biological (organelle) membranes on protein targeting and intracellular translocation of proteins. For this reason, we wished to make use of the knowledge of our laboratory in yeast organelle analysis and lipid research and investigate lipids and biomembranes from Pichia pastoris in some detail. The first goal of this study was to establish the Pichia pastoris lipidome, and to analyze lipids from the different organelles of this yeast in detail. For this purpose previous studies performed in our laboratory were extended and completed. These studies led to important results, such as elucidation of the lipidome from microsomes (endoplasmic reticulum), the plasma membrane and lipid droplets. The second aim of this project was generation of mutants in lipid biosynthesis. Importantly, strains bearing defects in the synthesis and degradation of triacylglycerols were generated. Results of these studies were transferred to an industry related project (ACIB; Austrian Center of Industrial Biotechnology) and applied for studies of protein expression. Information and strains from this TRP project were made public and provided to the Pichia Community. This strategy led to a useful link between fundamental and applied research and became a novel contribution to understand the cell biology of Pichia pastoris.