Cellulose hydrolysis and metabolism in the mesophilic, cellulolytic bacterium, Clostridium termitidis CT1112
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Consolidated bioprocessing (CBP) provides a cost effective cellulose processing strategy, in which enzyme production, substrate hydrolysis, and fermentation of sugars to ethanol are all carried out in a single step by microorganisms. For industrial-scale bioethanol production, CBP-enabling microbes must be able to both efficiently degrade lignocellulosic material to fermentable sugars and synthesize bioethanol with high yields. Microbes with these properties have so far not been identified. Developing naturally occurring cellulolytic isolates with CBP-relevant properties requires a comprehensive understanding of their lignocellulosic hydrolysis mechanism and metabolism. In my quest to find a suitable organism for potential use in CBP, I took to investigate the under-characterized anaerobic bacterium, Clostridium termitidis strain CT1112. C. termitidis produces fermentative hydrogen and ethanol from a variety of lignocellulose derived substrates. I sought to investigate the metabolism of C. termitidis on different substrates and the mechanisms of substrate hydrolysis using a combination of microscopy, comparative bioinformatics, and ‘Omic (transcriptomic and proteomic) analyses. Comparative bioinformatics analyses revealed higher numbers of genes encoding carbohydrate active enzymes (CAZymes) with the potential to hydrolyze a wide-range of carbohydrates, and ‘Omic analyses were used to quantify the levels of expression of CAZymes, including endoglucanases, exoglucanases, hemicellulases and cellulosomal components. While cellulases and cellulosome components were highly expressed on cellulose, xylanases and glucosidases were predominantly expressed on pentoses, and chitinases (as well as cellobiose phosphorylases) were significantly up-regulated on cellobiose. In addition to growth on xylan, the simultaneous consumption of two important lignocellulose constituents, cellobiose and xylose was also observed. The ability to metabolize both hexose and pentose sugars is a highly desirable feature of CBP-relevant organisms. Metabolic profiles in association with ‘Omics analyses showed that hexoses and pentoses are consumed via the Embden-Meyerhof-Parnas and Pentose-Phosphate pathways, respectively, and that the genome content and expression profiles dictate end-product synthesis patterns. Genes and gene-products of enzymes in central metabolism and end-product synthesis were detected in high abundance under all substrate conditions, regardless of the amounts of end-products synthesized. The capabilities described thus far, identifies C. termitidis as a strain of interest for CBP. Further studies are, however, required for its development in to an industry-ready strain for biofuel production.