(324g) Characterization and Engineering of Non-Model Fungal and Algal Systems for Bioproduction, Biodegradation, and Biomaterials Applications

Biological systems present attractive alternatives for traditional industrial processes in the search for new and more environmentally friendly technologies. While there has been massive progress in the bioengineering of primarily well-studied, model microbial systems, the enormous number of severely understudied (or simply unstudied) microorganisms presents a vast source of novel engineering functionalities and applications. This talk will cover a selection of our efforts to characterize and engineer two such groups of understudied microorganisms with significant biotechnological potential: the diatom algae and the anaerobic gut fungi.

Diatoms are unicellular, eukaryotic algae recognized for their extraordinary ability to synthesize cell walls made almost entirely of hydrated silica at ambient temperatures and pressures. These cell wall structures typically contain a high degree of intricate micro- and nano-scale patterning. As such, there is increasing interest in the biosynthetic potential of diatoms for producing advanced silica-based materials, with applications ranging from catalyst support to drug delivery to micro-optics. However, the engineering of diatom-derived materials is in its infancy, in part due to an incomplete understanding of the mechanisms underpinning siliceous cell wall synthesis. To address this lack of knowledge, we are investigating the genetic and molecular mechanisms of diatom silicification, with the ultimate goal of more precisely controlling and engineering this process. ‘Omics approaches applied across both model and non-model diatoms with a variety of silica morphologies are enabling us to identify novel genes influencing the intracellular silica-deposition process that leads to cell wall formation. In one example, we have used a comparative genomics approach incorporating protein structural predictions to identify both new members of previously established silicification-associated protein families as well as potentially novel genes involved in diatom silica formation. Additionally, we are using untargeted proteomics on actively forming silica structures to identify further proteins underlying the mechanisms of silica deposition and pattern formation. Lastly, a small but growing set of genetic tools for use in diatoms will enable us to directly probe candidate silica-associated genes to assess their role in cell wall formation. Through these and other approaches we are working to reveal genetic targets and control mechanisms to engineer diatom silicification for the production of advanced biomaterials.

Like the diatoms, the anaerobic gut fungi are a significantly understudied group of microorganisms that are increasingly recognized for their biosynthetic and biodegradative abilities. These fungi, which are obligate anaerobes found predominantly in the digestive tracts of herbivores, possess an expansive array of uncharacterized carbohydrate-active enzymes, indicating a high-degree of potential for applications involving the processing and conversion of lignocellulosic material. Furthermore, as these fungi natively exist in a competitive microbial environment, they are believed to possess diverse secondary metabolites with potential for therapeutic applications. However, our ability to fully understand and access the biotechnological potential of these fungi is severely hampered by an almost complete lack of genetic tools. To overcome these restrictions, we are actively developing tools and techniques for the genetic manipulation of these fungi. A critical first step in this regard has been to develop methods for introducing exogenous DNA into the fungal cells for stable or transient expression. Our group is investigating multiple mechanisms to carry out this genetic transformation, though this work will focus on our efforts to develop a bacterially-mediated transformation protocol. Agrobacterium-mediated transformation is well established in many aerobic fungi, though successful use of this system in anaerobic conditions has not been reported and is one focus of our investigation that has yielded promising results. Additionally, we are studying the potential of bacteria-to-fungi transformation using bacterial plasmid-conjugation systems, as has recently been reported in both yeast and several species of algae. The ongoing development of these systems into functional transformation tools in anaerobic gut fungi will significantly expand the scope of studies that can be performed on these organisms and will usher in the development of engineered fungal strains to address pressing biotechnological applications.