Glycoside hydrolases are a class of enzymes that catalyze the hydrolysis of bonds between carbohydrates. Although there are a number of fully characterized glycoside hydrolases, it remains difficult to assign specific substrates and activities to hypothetical proteins based on primary sequence and secondary structure predictions alone. The objective of this project is to use high
throughput proteomics, comparative genomics, protein expression and enzyme assays to rapidly generate functional data and annotation for secreted glycoside hydrolases. The initial reference organism is the Aspergillus niger ATCC1015 genome released by the Joint Genome Institute (JGI) in April 2006.
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| M. Himmel et al, 200. US Dept of Energy Genome Programs. http://genomics.energy.gov |
The ANL experimental team has used comparative genomics to identify the complete set of glycoside hydrolases in the A. niger genome. Our analysis indicates that A. niger contains at least 236 hydrolase genes, with representatives from 49 families. Targets are prioritized by the PNNL Fungal Biotechnology Team1 through proteomic analysis of A. niger cultures grown on a variety of complex biomass substrates. Bioinformatic analysis of proteins identified from secretomes is used to guide the selection of hydrolase genes for high throughput protein expression. The correlation of the glycoside hydrolases identified through proteomics with the feedstock used for induction will add an additional layer of functional genomic annotation.
A major limitation to studying enzymes is a lack of highly purified, soluble protein. This is exacerbated with targets from eukaryotic species, as many proteins are not expressed or are insoluble in Escherichia coli production systems. At ANL we are using the yeast Pichia pastoris as an expression host to produce high-quality glycoside hydrolases from eukaryotic organisms. Purified glycoside hydrolases generated during this project will be tested for enzyme activity and substrate specificity at PNNL. Resulting data will be used to improve annotation of the purified targets, and can subsequently be used for refinement of structural and functional annotation for this enzyme class in newly sequenced geneomes. In the future we plan to investigate enzymes produced in other fungal species such as Laccaria bicolor and Piromyces sp. 2. Experience gained from this target set can also be applied to the heterologous protein production in Pichia pastoris for enzymes from a variety of eukaryotic organisms. |
