The broad goal in our laboratory is to better understand, and then beneficially manipulate, microbial expression systems. While we have carried out some projects involving bacteria, our primary focus is on filamentous fungi. We use a sophisticated set of analytical tools to asses fungal morphology and the physical properties of fungal cell walls. As an important tool in our molecular studies, we use a functional-genomic technique called proteomic analysis. This process involves the identification and quantification of individual proteins from various cellular fractions (e.g., cytoplasm, vacuoles, cell wall). These data are used for differential comparison allowing us to make deductions about cellular mechanisms. We are also developing new ways to use proteomic analysis to study cellular signaling pathways (phosphoproteomics). Recently, we have used all of these tools to better understand a fundamental cellular recycling-pathway called autophagy. And currently, we are carrying out: (i) fundamental research in systems biology, (ii) applied research in cellular engineering and (iii) transnational research seeking to produce next generation antibiotics.
Fundamental Research: Systems Biology
The goal of this project is to develop a new approach for modeling gene regulatory networks. We are testing the hypothesis that initial experimental characterization of a network subset will permit identification of the biomolecular constituents and their connectivity, thus establishing network topology. System wide time-course measurements can then be used to refine this network into a reaction kinetic model capable of making accurate system predictions. The cell wall integrity signaling pathway of the experimentally tractable model fungus Aspergillus nidulans is serving as a model. This pathway responds to cell wall damage by activating repair mechanisms that restore cell integrity. Because protein kinases play a pivotal role in mediating cellular regulatory activities, we are focusing on a subset of kinases and the discovery of their associated substrates to initially assemble a rudimentary network. Subsequently the system will be experimentally perturbed for measuring its dynamic response using a robust transcriptomic, proteomic and phosphoproteomic platform. Using this data, we will take a two-step approach to developing the dynamic system of coupled ordinary differential equations able to describe dynamic behavior of a model gene regulatory network. First, an ensemble approach of approximate models will be tested and refined. In the second step, the ensemble will act as the seed population for use in an evolutionary algorithm to generate a more refined and accurate model. We will then validate the model by iterative comparisons of in silico predictions with experimental results. This project involves collaboration with both the University of Nebraska Lincoln and the University of Connecticut and is sponsored by the National Science Foundation.
Applied Research: Cellular Engineering
Filamentous fungi are capable of producing and secreting proteins in stunning (i.e., > 100 g/L) quantities. Yet for many recombinant proteins productivity is much lower, often due to inadequate protein secretion. The overarching goal in this project is to develop a deeper understanding of protein secretion in filamentous fungi, so strategies can be developed to improve productivity during fungal fermentation. It is hypothesized that fungal protein secretion and hyphal morphology are intimately linked, such that identification of morphological mutants will also select for both hypo- and hyper-secretors. To test this hypothesis, temperature sensitive (Ts) morphological mutants with aberrant protein secretion have been developed. Approximately fifty of these mutants have been subjected to whole genome sequencing (through collaboration with the Joint Genome Institute), to identify the genes and molecular mechanisms responsible for their interesting phenotypes. This combination of a classical genetic screen with modern “-omics” technology will allow rapid identification of gene function. This project involves collaboration with the University of Nebraska Lincoln and the University of Connecticut and is sponsored by the National Science Foundation.
Translational Research: Next Generation Antibiotics
For over 50 years, it is has been common practice in the agricultural industry to add low doses of traditional (i.e., “medically important”) antibiotics to animal feed to improve production efficiency of food animals. While these “antibiotic growth promoters” provide tremendous economic benefit, this practice also leads to antibiotic-resistant bacterial strains which have the potential to infect humans. As a result, the US Food and Drug Administration has implemented measures to stop this practice. While numerous alternatives to medically important antibiotics have been proposed, most have not been commercially successful as they are not adequately effective. As an alternative to medically important antibiotics, the use of antimicrobial proteins (AMP) has great promise, but a significant limitation to their commercial production has been cost. Thus the ability to economically manufacture antimicrobial proteins would represent a significant benefit to the agricultural industry as it would allow farmers to maintain high levels of productivity while not generating antibiotic-resistant strains that pose a danger to humans. This project explores the potential of replacing medically-important antibiotic feed-additives with antimicrobial protein (AMP). Work described here seeks to generate proof-of-concept data, expressing model AMPs, at commercially viable titers, using recombinant filamentous fungi. Eventually these AMPs will be tested in poultry by our collaborators at the USDA. This project involves collaboration with MycoInnovation, LLC and is sponsored by the National Science Foundation.
Fundamental Research: Understand Fungal Autophagy
Autophagy is an important, eukaryotic, cellular pathway which recycles large proteins and even whole organelles. We are working to better understand autophagy in filamentous fungi. We have found a number of autophagy related gene products which appear to mediate, previously unknown, cellular functions. We hypothesize engineering the autophagy pathway may lead to increased productivity in bioprocesses. To understand how to alter the pathway, we make targeted gene deletions and use sophisticated tools to assess the resulting cellular phenotypes. Image shows localization of GFP tagged Atg8 in Aspergillus nidulans after induction of autophagy by addition of rapamycin.
One of Dr. Marten’s favorite articles…