The Cellulase System of Acidothermus cellulolyticusEPA Grant Number: U915305
Title: The Cellulase System of Acidothermus cellulolyticus
Investigators: Hennegan, Kevin P.
Institution: University of Colorado at Boulder
EPA Project Officer: Just, Theodore J.
Project Period: January 1, 1998 through January 1, 2001
Project Amount: $25,637
RFA: STAR Graduate Fellowships (1998) RFA Text | Recipients Lists
Research Category: Fellowship - Molecular Biology/Genetics , Biology/Life Sciences , Academic Fellowships
The objective of this research project is to identify and characterize novel components of the cellulase system of Acidothermus cellulolyticus. These proteins will be tested for their suitability in a plant-based expression system for industrial enzymes.
The thermophilic actinomycete A. cellulolyticus has provided one of the most promising biomass-conversion cellulases discovered to date. This enzyme, E1, is an endoglucanase that possesses many desirable qualities. Furthermore, our laboratory has shown that it is possible to express significant amounts of this protein in transgenic Arabidopsis thaliana plants. The results are encouraging because these plants show no gross phenotypic abnormality, although E1 retains its enzymatic activity (Ziegler and Danna, submitted). To provide a complete cellulase system for the efficient conversion of cellulose to glucose, two other classes of cellulases will be required—an exoglucanase and a beta-glucosidase. A. cellulolyticus may possess genes encoding both of these enzyme classes. To identify these genes, a plasmid expression library will be constructed in Escherichia coli from the A. cellulolyticus genome. The library will be screened on agar plates containing fluorescent derivatives of the substrates for beta-glucosidase or exoglucanase, and bacterial colonies that fluoresce under ultraviolet (UV) illumination will be selected for further investigation. Plasmid DNA will be prepared from these colonies, and will be sequenced for subsequent analysis. These clones also will be tested on other substrates to confirm the enzymatic activity. Once the genes have been positively identified, the encoded proteins will be expressed at high levels for biochemical analysis. These proteins may prove useful for our plant expression strategy; one promising strategy involves targeting the transgenic proteins to the plant cell vacuole for storage.