초록
<P><B>Abstract</B></P> <P> <I>Clostridium thermocellum</I> rapidly deconstructs cellulose and ferments resulting hydrolysis products into ethanol and other products, and is thus a promising platform organism for the development of cellulosic biofuel production via consolidated bioprocessing. While recent metabolic engineering strategies have targeted eliminating canonical fermentation products (acetate, lactate, formate, and H<SUB>2</SUB>), <I>C. thermocellum</I> also secretes amino acids, which has limited ethanol yields in engineered strains to approximately 70% of the theoretical maximum. To investigate approaches to decrease amino acid secretion, we attempted to reduce ammonium assimilation by deleting the Type I glutamine synthetase (<I>glnA</I>) in an essentially wild type strain of <I>C. thermocellum</I>. Deletion of <I>glnA</I> reduced levels of secreted valine and total amino acids by 53% and 44% respectively, and increased ethanol yields by 53%. RNA-seq analysis revealed that genes encoding the RNF-complex were more highly expressed in Δ<I>glnA</I> and may have a role in improving NADH-availability for ethanol production. While a significant up-regulation of genes involved in nitrogen assimilation and urea uptake suggested that deletion of <I>glnA</I> induces a nitrogen starvation response, metabolomic analysis showed an increase in intracellular glutamine levels indicative of nitrogen-rich conditions. We propose that deletion of <I>glnA</I> causes deregulation of nitrogen metabolism, leading to overexpression of nitrogen metabolism genes and, in turn, elevated glutamine levels. Here we demonstrate that perturbation of nitrogen assimilation is a promising strategy to redirect flux from the production of nitrogenous compounds toward biofuels in <I>C. thermocellum</I>.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Type I glutamine synthetase (<I>glnA</I>) was deleted to decrease amino acid secretion. </LI> <LI> Ethanol yields increased by 53% and secreted amino acids decreased by 44%. </LI> <LI> Nitrogen uptake and assimilation and RNF complex encoding genes were upregulated. </LI> <LI> Nitrogen metabolism was deregulated. </LI> </UL> </P>