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.
This project provides data on potential synergy among cellulase enzymes for biofuels production applications that supports sub-objective 4B of the in-house parent project. A bioassay for enzymatic cellulose degradation was developed that combines a cellulose substrate, candidate cellulase enzymes, and the bacterium, Thermoanaerobacterium saccharolyticum B6A, in a sealed culture vessel. Fermentation of the resulting cellulose hydrolysis products (cellodextrins) by the bacterium resulted in gas (H2 + CO2) production and a consequent increase in headspace pressure, which can be quantified by use of a digital pressure transducer. Using this assay, combinations of candidate enzymes (produced by an in vitro transcription/translation system) displayed up to 4.7-fold synergy of hydrolysis of pure cellulose relative to the average of the activities of the individual enzymes. Activites were monitored by bi-weekly telephone calls and e-mails with the cooperator.