Characterizing and utilizing the large peroxisomes of Ogataea parapolymorpha for heterologous protein compartmentalization

Organelles are cellular compartments surrounded by a membrane. Cells of higher organisms use organelles to separate reactions from the rest of the cell. One example is the peroxisome.

Inside this organelle, the cell uses oxygen to generate hydrogen peroxide, which is then used to degrade molecules without damaging other important cellular proteins. It is a good model system because it hosts a variety of reactions. The yeast Ogataea parapolymorpha can create very large peroxisomes, which in this project will be used to compartmentalize a variety of proteins.

The capacity of the peroxisomes to house cargo proteins, and proteins of different types, will be characterized. As a collaboration between U.C. Berkeley and San Francisco State University, graduate and undergraduate students from both institutions will be trained and conduct research as part of this project.

The model yeast, O. parapolymorpha, can produce peroxisomes with high protein cargo capacity. In this project, induction of peroxisome proliferation will be quantified, rewired, and utilized to compartmentalize heterologous proteins. Fluorescence microscopy data will be used to accurately calculate the volume and surface area of the peroxisome.

Shotgun proteomics will be employed to determine the percentage of protein compartmentalized in the peroxisomes with and without induction. Synthetic control over peroxisome proliferation will be gained by engineering transcription factors devoid of native regulation. This will allow achievement of high cargo capacity in the presence of glucose.

Ultimately, the plasticity of the peroxisome for high-density compartmentalization of heterologous cargo proteins will be directly tested using a model protein of commercial interest, human serum albumin. The effect on expression level, growth rate, and the protection of human serum albumin from degradation from proteases will be determined.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.