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 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. Gene expression by continuous culture of the ethanol-producing bacterium, Ruminococcus albus, was compared at the same growth rate during growth on cellulose versus growth on the soluble sugar cellobiose. Expression of a novel set of tryptophan metabolism genes increased dramatically (10-15x) during growth on cellulose. Elucidation of the role of tryptophan and its precursors in regulating expression of cellulolytic gene products may provide a novel means to enhance cellulose degradation capabilities by up-regulating their expression. The results provide a novel strategy to scientists to enhance the bioconversion of cellulosic biomass to ethanol.