Cellulolytic bacteria have the potential to perform lignocellulose hydrolysis and fermentation simultaneously. The metabolic pathways of these bacteria, therefore, require more comprehensive and quantitative understanding. Using isotope tracer, gas chromatography-mass spectrometry, and metabolic flux modeling, we decipher the metabolic network of Clostridium thermocellum, a model cellulolytic bacterium which represents as an attractive platform for conversion of lignocellulose to dedicated products. We uncover that the Embden–Meyerhof–Parnas (EMP) pathway is the predominant glycolytic route whereas the Entner–Doudoroff (ED) pathway and oxidative pentose phosphate pathway are inactive. We also observe that C. thermocellum's TCA cycle is initiated by both Si- and Re-citrate synthase, and it is disconnected between 2-oxoglutarate and oxaloacetate in the oxidative direction; C. thermocellum uses a citramalate shunt to synthesize isoleucine; and both the one-carbon pathway and the malate shunt are highly active in this bacterium. To gain a quantitative understanding, we further formulate a fluxome map to quantify the metabolic fluxes through central metabolic pathways. This work represents the first global in vivo investigation of the principal carbon metabolism of C. thermocellum. Our results elucidate the unique structure of metabolic network in this cellulolytic bacterium and demonstrate the capability of isotope-assisted metabolite studies in understanding microbial metabolism of industrial interests.
