Browsing by Subject "Aspergillus nidulans"
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Item Altered secretion patterns and cell wall organization caused by loss of PodB function in the filamentous fungus Aspergillus nidulans(Nature, 2018-07-30) Boppidi, Karthik R.; Ribeiro, Liliane FragaCosta; Iambamrung, Sirasa; Nelson, Sidney M.; Wang, Yan; Momany, Michelle; Richardson, Elizabeth A.; Lincoln, Stephen; Srivastava, Ranjan; Harris, Steven D.; Marten, Mark R.Filamentous fungi are widely used in the production of a variety of industrially relevant enzymes and proteins as they have the unique ability to secrete tremendous amounts of proteins. However, the secretory pathways in filamentous fungi are not completely understood. Here, we investigated the role of a mutation in the POlarity Defective (podB) gene on growth, protein secretion, and cell wall organization in Aspergillus nidulans using a temperature sensitive (Ts) mutant. At restrictive temperature, the mutation resulted in lack of biomass accumulation, but led to a significant increase in specific protein productivity. Proteomic analysis of the secretome showed that the relative abundance of 584 (out of 747 identified) proteins was altered due to the mutation. Of these, 517 were secreted at higher levels. Other phenotypic differences observed in the mutant include up-regulation of unfolded protein response (UPR), deformation of Golgi apparatus and uneven cell wall thickness. Furthermore, proteomic analysis of cell wall components in the mutant revealed the presence of intracellular proteins in higher abundance accompanied by lower levels of most cell wall proteins. Taken together, results from this study suggest the importance of PodB as a target when engineering fungal strains for enhanced secretion of valuable biomolecules.Item Insights into the protein secretory pathway in filamentous fungi using both random and gene-specific mutational approaches(2018-01-01) Boppidi, Karthik; Marten, Mark R; Chemical, Biochemical & Environmental Engineering; Engineering, Chemical and BiochemicalFilamentous 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 (28oC) this mutant behaves similarly to its isogenic parent. However, during higher temperature growth (42oC) 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.Item PHOSPHOPROTEOMIC AND TRANSCRIPTOMIC-DRIVEN STUDY OF THE CELL WALL INTEGRITY SIGNALING PATHWAY (CWIS) IN THE MODEL FUNGUS, ASPERGILLUS NIDULANS(2020-01-20) Chelius, Cynthia Louise; Marten, Mark R; Chemical, Biochemical & Environmental Engineering; Engineering, Chemical and BiochemicalFilamentous fungi are industrially relevant and medically significant. In both cases the cell wall plays a critical role in protecting fungi from physical and environmental stress. To increase our general understanding of cell-wall repair, we focused on the model species Aspergillus nidulans studying the regulation of biosynthetic proteins responsible for wall maintenance and repair via the cell wall integrity signaling pathway (CWIS). The CWIS contains a mitogen-activated protein kinase (MAPK) cascade which becomes activated upon physical or environmental stress. MpkA, the final kinase in this cascade, controls expression of some key wall biosynthesis genes (?-1,3-glucan synthases). However, the transcription of many other wall related genes (e.g., ?-1-3-glucan and chitin synthase genes) is regulated in an unknown, MpkA-independent, manner. To better understand the various cellular roles MpkA plays during steady-state growth, we compared global changes in protein phosphorylation and gene expression between an mpkA deletion mutant (?mpkA) and its isogenic parent. We found strong evidence suggesting MpkA is involved in maintaining cell-wall strength, branching regulation, and response to iron starvation. Next, we sought to characterize the CWIS pathway and its downstream effectors. Because cellular signaling is a dynamic process, we used a multi-omic approach employing quantitative, label-free, mass spectrometry (short timeframe; 10 min) to assess protein phosphorylation and RNA-sequencing (long timeframe; 120 min) to assess gene-expression levels. To differentiate statistically-significant dynamic behavior from noise, a multivariate adaptive regression splines (MARS) model was applied to both data sets. Overall, we identified 794 phosphorylation sites and 1800 genes dynamically phosphorylated and expressed, respectively, upon cell wall perturbation. Several of these phosphorylation sites belong to kinases, and 12 of these kinases were tested for their involvement in CWIS using deletion strains to determine cell-wall strength and micafungin sensitivity. Combining -omic data with characterization studies reveals putative, new connections between the CWIS pathway and calcium signaling, the high osmolarity-glycerol (HOG) pathway, and the septation initiation network (SIN). This multi-omic experiment was replicated in the ?mpkA strain to develop hypotheses about which signaling events are MpkA-independent. This work demonstrates the capacity of a multi-omics approach to study signaling networks.