초록
<P><B>Abstract</B></P> <P> <I>Clostridium thermocellum</I> is an anaerobic, Gram-positive, thermophilic bacterium that has generated great interest due to its ability to ferment lignocellulosic biomass to ethanol. However, ethanol production is low due to the complex and poorly understood branched metabolism of <I>C. thermocellum</I>, and in some cases overflow metabolism as well. In this work, we developed a predictive stoichiometric metabolic model for <I>C. thermocellum</I> which incorporates the current state of understanding, with particular attention to cofactor specificity in the atypical glycolytic enzymes and the complex energy, redox, and fermentative pathways with the goal of aiding metabolic engineering efforts. We validated the model’s capability to encompass experimentally observed phenotypes for the parent strain and derived mutants designed for significant perturbation of redox and energy pathways. Metabolic flux distributions revealed significant alterations in key metabolic branch points (e.g., phosphoenol pyruvate, pyruvate, acetyl-CoA, and cofactor nodes) in engineered strains for channeling electron and carbon fluxes for enhanced ethanol synthesis, with the best performing strain doubling ethanol yield and titer compared to the parent strain. <I>In silico</I> predictions of a redox-imbalanced genotype incapable of growth were confirmed <I>in vivo</I>, and a mutant strain was used as a platform to probe redox bottlenecks in the central metabolism that hinder efficient ethanol production. The results highlight the robustness of the redox metabolism of <I>C. thermocellum</I> and the necessity of streamlined electron flux from reduced ferredoxin to NAD(P)H for high ethanol production. The model was further used to design a metabolic engineering strategy to phenotypically constrain <I>C. thermocellum</I> to achieve high ethanol yields while requiring minimal genetic manipulations. The model can be applied to design <I>C. thermocellum</I> as a platform microbe for consolidated bioprocessing to produce ethanol and other reduced metabolites.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Constructed a <I>C. thermocellum</I> metabolic model for flux quantification and rational strain design. </LI> <LI> Characterized <I>C. thermocellum</I> mutants to validate the model and elucidate their metabolisms. </LI> <LI> Demonstrated <I>C. thermocellum</I> encompasses a robust redox metabolism. </LI> <LI> Elucidated redox bottlenecks hindering efficient ethanol production in <I>C. thermocelllum.</I> </LI> <LI> Used elementary mode analysis to design an optimal strain for enhanced ethanol production. </LI> </UL> </P>