Presenter: Donald A. Comfort
Advisor(s): Robert M. Kelly
Author(s): Donald A. Comfort and Robert M. Kelly
Graduate Program: Chemical and Biomolecular Engineering

Title: Identifying and Characterizing Hyperthermophilic Glycosyl Hydrolases Functional Genomics and Bioinformatic Approaches

Abstract: There is substantial interest in identifying genes encoding novel biocatalysts within genome sequences, with the ultimate goal of incorporating them into technologically important processes and applications. Of particular interest are thermostabile biocatalysts, such as those produced by hyperthermophilic organisms, which grow optimally at >80C. Heterotrophic members of this group utilize carbohydrates for growth and energy requirements and thus have been examined for carbohydrate active enzymes, including glycosyl hydrolases. 

One key problem in identifying technologically promising biocatalysts in microbial genome sequences is that approximately 40% of the open reading frames (ORFs) are annotated as hypothetical proteins. To address this problem, we are using a functional genomics approach combined with bioinformatic analysis to investigation carbohydrate utilization by hyperthermophilic organisms. Pyrococcus furiosus, a hyperthermophilic heterotroph that grows optimally at 98-100C, was grown on a number of different sugars. RNA was extracted from the biomass generated and interrogated using a whole genome cDNA microarray for hints to carbohydrate processing mechanisms and identification of genes encoding glycosyl hydrolases. Using these techniques we have determined that PF0870 encodes a b-amylase, which hydrolyzes maltooligosaccharides with no apparent transglycosylation activity. In addition, the gene encoded by PF0132 was found to respond to pullulan as a growth substrate for P. furiosus. Because this gene could not be recombinantly produced, the native form was purified from P. furiosus biomass, confirmed to be PF0132 by proteomics analysis, and shown to define a new family of glycosyl hydrolases, based upon hydrophobic cluster analysis of the primary amino acid sequence. Both PF0870 and PF0132 were thus found to encode glycosyl hyrolases that could prove to be useful in the processing of starch-based biomass for the production of biohydrogen or bioethanol. These examples illustrate the power and importance of functional genomics approaches for biocatalyst discovery and characterization.