Understanding nitrogen assimilation in Clostridium thermocellum for bioengineering

The ability of Clostridium thermocellum to rapidly deconstruct 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, approximately 10% of carbon is still diverted towards excreted amino acids (primarily valine and alanine) that could otherwise be used for ethanol production. Currently, however, little is known about nitrogen assimilation in C. thermocellum or most other clostridia.  Here we studied nitrogen assimilation in C. thermocellum by systematically deleting genes involved in nitrogen assimilation including glutamate dehydrogenase, glutamate oxoglutarate aminotransferase (GOGAT), and one of four glutamine synthetases.  Deletion of glutamate dehydrogenase resulted in decreased growth rates and final cell densities, demonstrating that it is a key enzyme used in nitrogen assimilation.  Deletion of glutamine synthetase induced a nitrogen starvation response in the presence of excess nitrogen, increasing transcription of urea uptake and metabolism and nitrogen uptake.  Gene expression and metabolomic analysis in these mutants are being further used to gain insight into metabolic flux, nitrogen metabolism, and gene regulation in C. thermocellum. Some strains with deleted nitrogen assimilation genes resulted in decreased amino acid secretion and increased ethanol yield, suggesting that this is a promising approach for further increasing cellulosic ethanol production.