GlyR3 regulation in <i>Clostridium thermocellum</i>

Bio-ethanol from cellulosic biomass is a promising candidate as a liquid transportation fuel because of its high-energy content and the abundance of cellulose. Consolidated bioprocessing (CBP) helps to reduce the traditional 2-step process of bio-ethanol production into single-step to improve cost efficiency. A bacterium, Clostridium thermocellum, has a multi-enzyme complex for hydrolyzing cellulase, called the Cellulosome that enables the organism to have high rates of cellulose utilization. However, the ethanol yield of C. thermocellum needs to be improved in order to make consolidated bioprocessing with C. thermocellum commercially viable. It is essential to understand the regulation of carbohydrate-degrading enzyme activity to apply metabolic engineering, synthetic biology, and molecular biology techniques to strain improvement. GlyR3 is a protein that regulates the activity of carbohydrate active proteins in C. thermocellum. Recent studies have described how GlyR3 regulates the celC operon, which includes two carbohydrate-active enzymes (Newcomb, Chen, & Wu, 2007a). In this dissertation I investigate additional regulatory targets of the GlyR3 protein, which is a LacI family protein. First, it will be shown that GlyR3 regulates a gene downstream of celC operon, manB, explaining that GlyR3 not only induces its own expression under presence of laminaribiose, but in the case of manB, repress expression. Bioinformatics tools are then used to find putative GlyR3 binding sites in the whole genome in C. thermocellum. Electrophoretic mobility shift assay (EMSA) can show direct binding between GlyR3 and DNA sequences of interest. Second, we are going to show that GlyR3 regulates genes that are located far from the celC operon. Reverse transcript (RT) –PCR and mRNA sequencing will be used to show in vivo changes in expression of genes in the whole genome resulting from changing levels of GlyR3 expression resulting from the addition of laminaribiose. This research reveals two distinct binding motifs for GlyR3, a celC-type and a manB-type, which are used to identify additional operons potentially regulated by GlyR3 and demonstrates a global regulatory role for GlyR3 in C. thermocellum.