EFFECT OF GROWTH-RATE AND LIMITING FACTOR ON CELLULOSOMAL GENE EXPRESSION IN STEADY-STATE CLOSTRIDIUM THERMOCELLUM CULTURES

Clostridium thermocellum is an anaerobic, thermophilic, soil bacterium that utilizes crystalline cellulose efficiently and ferments the cellodextrins to ethanol. Therefore, it could potentially play a part in a consolidated bioprocess, allowing a relatively low-cost conversion of biomass to biofuel. The hallmark of the cellulose degradation system is the cellulosome, an extracellular, multi-enzyme complex, which can anchor up to 63 catalytic subunits at a time (mainly cellulases and hemicellulases). There are approximately 70 genes that encode these subunits. Interestingly, works by our group and others revealed that several cellulosomal genes are up-regulated during slow growth rates in continuous cultures under carbon limitation. Yet, it is possible that this phenomenon is derived from the carbon limitation itself and not caused by the growth rate. We studied the influence of growth rate and the limiting factor on C. thermocellum transcriptome via RNA-seq and real-time RT-PCR, on RNA extracted from carbon or nitrogen limited continuous cultures, cultivated at growth rates ranging between 0.2 hr^-1 and 0.05 hr^-1. RNA-seq revealed that C. thermocellum cultures limited on carbon vs. nitrogen source have a significantly different gene expression pattern in all growth rates. This manifests in the Urease system, which showed a 1000-fold increase in expression in nitrogen limited cultures. Although the cellulosomal genes did not significantly differ in the total transcriptome of either carbon or nitrogen limited cultures, an in-depth RT-PCR analysis revealed a significant 10-fold up regulation of the cellulosomal’s scaffoldin gene, the anchoring proteins and the processive cellulase cel48S in slow vs. fast growing carbon limited cultures and not in the nitrogen limited ones. These results could impact the fermentation process of C. thermocellum and consequently the conversion of biomass to bioethanol.