Understanding amino acid metabolism and nitrogen assimilation in Clostridium thermocellum

The ability of Clostridium thermocellum to rapidly degrade cellulose and ferment resulting hydrolysis products into ethanol makes it a promising platform organism for cellulosic biofuel production via consolidated bioprocessing.  While native ethanol yields are far below theoretical maximum due to branched fermentation pathways, recent metabolic engineering strategies aimed at deleting competing pathways have improved ethanol yields.  However, more than 4% of carbon is still diverted towards amino acids (primarily valine and alanine) that could otherwise be used for ethanol production.  Here we attempted to reduce secreted amino acid production by (i) reducing valine synthesis and secretion through deletion of the branched chain amino acid (BCAA) aminotransferase and a putative BCAA exporter, respectively, and (ii) decreasing ammonium assimilation through deletion of glutamate dehydrogenase, glutamine synthetase, or glutamate oxoglutarate aminotransferase (GOGAT).  Deletion of the BCAA aminotransferase resulted in an isoleucine, leucine, and valine auxotroph.  While deletion of a putative BCAA exporter reduced secreted valine levels, valine was still measured in supernatants suggesting additional methods for valine secretion.  The latter deletion resulted in a marginal increase in ethanol yields.  Deletion of glutamate dehydrogenase, reduced growth rate and decreased valine production, but had marginal impact on other fermentation products, including ethanol. Deletion of glutamate oxoglutarate aminotransferase (GOGAT) had negligible impacts on fermentation products and secreted amino acids. The impact of enzyme cofactor (NADH, NADPH, or ferredoxin) specificities during ammonium assimilations will be discussed.