PhD Dissertation Defense - Karthik Boppidi

Insights into the protein secretory pathway in filamentous fungi using both random and gene-specific mutational approaches

Karthik R. Boppidi

Filamentous fungi are the “workhorses” of the biotechnology industry, used to produce a vast array of products, including small molecules, therapeutics, and enzymes. While fungi are able to express and secrete stunning levels of homologous protein (> 100 g/L), heterologous expression is often much lower, and it has been suggested this may be due to inadequate protein secretion.  While much is known regarding fungal proteins secretion, there are surprisingly large gaps in this knowledge.  For example, deep insight into the basic secretion machinery has been obtained from studies using the yeast Saccharomyces cerevisiae, yet it is clear filamentous fungi are much more complex. To better understand fungal protein secretion, we used Aspergillus nidulans as a model and studied the protein secretory pathway using both the random and targeted mutagenesis approaches.

In our first approach, we used random mutagenesis to create thousands of high secreting, temperature-sensitive mutants. Mutants were screened for increased secretion of two native enzymes, and the six highest secreting strains were subjected to whole genome sequencing.  Through this exercise we identified numerous genes, previously recognized as playing a role in various different biological functions, which may also be playing a role in A. nidulans protein secretion. Furthermore, the results obtained this study may be used to accelerate the development of industrial strains with high secretion. 

In addition to our random mutagenesis approach, we used a targeted approach to study the secretory pathway.  Specifically, we evaluated the role of PodB protein, previously identified as being involved in retrograde vesicle transport in the Golgi apparatus. To carry out this study, we employed a temperature sensitive podB mutant strain.  During growth at low temperature (28^oC) this mutant behaves similarly to its isogenic parent. However, during higher temperature growth (42^oC) this mutant manifests significantly different phenotype.  Most importantly, when compared to its isogenic parent (i.e., control strain), we observed a 15-fold increase in specific protein secretion in the podB mutant. Proteomic analysis of the secretome, revealed elevated levels of cytoplasmic proteins and reduced levels of secretory proteases and GPI-anchored proteins. Other phenotypic differences observed in the mutant include upregulation of the unfolded protein response and an uneven and weaker cell wall. Further analysis of the proteome of the cell wall, using quantitative proteomics, revealed the presence of intracellular proteins in higher abundance accompanied by lower levels of most cell wall proteins. Taken together, these data suggest the podB mutation may be useful for increasing secretion of heterologous proteins.