1a. Objectives (from AD-416):
To characterize cellulose degradation by consolidated bioprocessing microorganisms, and to use genomic, microarray and in vitro transcription/translation methodologies to identify superior polysaccharide hydrolase enzymes and combinations thereof.
1b. Approach (from AD-416):
We will obtain genomic DNA, mRNA, and protein preparations from C. thermocellum and other CBP bacteria grown on different substrates and/or at different growth rates. The mRNA will be used for microarray analysis of gene expression, and the protein preparations will be used for proteomic identification and quantitation of specific proteins involved in polysaccharide degradation. DNA will be used to PCR-amplify genes of interest (determined from microarray and proteomic experiments) or for site-directed mutagenesis. These DNA preparations will be used in an in vitro transcription/translation system to produce pure proteins for direct analysis of cellulase or other polysaccharide hydrolase activity. For some experiments, protein synthesis will be performed in the presence of wild-type and mutant scaffoldin genes, with the intent of generating novel cellulosomal structures. The resulting complexed and non-complexed proteins will be used in assays of cellulose hydrolysis, based on the cellodextrin-fermenting, cellulose-nondegrading bacterium Thermoanaerobacterium saccharolyticum B6A. Complexed and noncomplexed cellulases will be assayed in different combinations to test for enzyme synergy.
3. Progress Report:
This project relates to Objective 4 of the parent project: Develop technologies to enable commercially viable consolidated bioprocessing (CBP) of lignocellulosic biomass to fuel ethanol and adhesive co-products. Using a novel transcription/translation system developed by the non-ARS cooperator, genomic DNA from several CBP-competent bacteria supplied by an ARS scientist in Madison, WI was used to produce catalytic cores of cellulose enzymes, either attached or not attached to the native scaffoldin protein. Cellulose-degrading activity of individual enzymes and enzyme combinations was quantified using a novel bioassay developed by the ARS scientist. In this bioassay, a cellulose substrate, candidate cellulase enzymes, and the bacterium, Thermoanaerobacterium saccharolyticum B6A, are incubated in a sealed culture vessel at 60 degrees C. Fermentation of the resulting cellulose hydrolysis products (cellodextrins) by the bacterium results in gas production and an increase in headspace pressure, which can be quantified by use of a digital pressure transducer. With this assay, it was demonstrated that combinations of enzymes displayed up to 4.7-fold synergy of hydrolysis of pure cellulose relative to the average of the activities of the individual enzymes. These results reinforced the concept of synergy among enzymes from microbes, which would be useful in the CBP platform of biomass conversion.