Cellular Mechanisms of Regulation

Background.

Yeasts are attractive hosts capable to synthesize high amounts of important commercial products. Among these products, recombinant proteins for biopharmaceutical or industrial purposes play a major role. Currently, 20 % of biopharmaceutical proteins and approx. half of industrial enzymes are produced in yeasts and fungal systems. Among them, the methylotrophic yeast Pichia pastoris is commonly employed for the production of functional secreted proteins.

During the past decade, we have developed genome scale systems biology techniques (including genome sequencing, transcriptomics, proteomics, metabolomics, flux analysis, and a genome scale metabolic model) and applied these for strain characterization in addition to conventional cell biology methods. These data were exploited to generate an advanced knowledge-base about the responses of host cells to protein overproduction, and their interrelation to environmental stress conditions typically occurring during biotechnological production processes.

Aims and methods.

Gene expression is regulated at various levels, including epigenetic and transcriptional control of gene expression in response to external signals. So far, most studies on industrially relevant microorganisms focused mainly on differential abundance of transcripts or proteins. However, these expression patterns are mainly the readout of higher level regulatory mechanisms including transcriptional and epigenetic control. Proteins that interact with the DNA are responsible for these higher level control mechanisms. Therefore, this project sets out to analyze cellular regulation mechanisms beyond transcription in the yeast protein production host Pichia pastoris.

In eukaryotic cells, DNA is organized into a chromatin structure, which is formed by repeating nucleosomes. DNA interacting proteins regulate the accessibility of genes. Understanding the organization of these proteins across a genome and how this organization regulates genes is crucial for a systems-level understanding how cells respond to different stimuli or stress. However, work on eukaryotic microorganisms has so far only begun with a few model organisms. In the course of this project, we plan to set up the methodology for assessing various regulation levels (genome wide nucleosome occupancy, gene-specific histone modification) in P. pastoris. Transcriptional regulation e.g. during externally or internally imposed stress may involve nucleosome repositioning, while robust transcriptional activity usually correlates with nucleosome depletion making the DNA accessible for transcriptional regulators and the transcription machinery.

Chromatin accessibility and regulatory active DNA under conditions relevant for protein production will be analyzed by formaldehyde-assisted isolation of regulatory elements followed by next-generation sequencing. For selected genomic regions, differences in chromatin-associated proteins and histone modifications present on the chromatin will be investigated in more detail.