Troubleshooting Metabolism in a Thermophilic Cellulolytic Bacterium Using a Cell-Free System

The thermophilic cellulolytic bacterium, Clostridium thermocellum, is a promising candidate for biofuel production from cellulose. Recent work has focused on improving the ethanol production abilities of this organism, resulting in an ethanol yield of 80% of the theoretical maximum and titer of 27 g/L. However, for commercial application, the titer needs to be further improved. To understand the factors that limit ethanol titer in C. thermocellum, we developed a cell-free extract reaction (CFER) system and demonstrated that cellobiose could be converted to ethanol in-vitro. We performed a rigorous evaluation of quenching methods to stop enzymatic conversion without degrading metabolites. With then proceeded to perturb metabolism in two different ways, by either adding intermediate metabolites, or by adding mesophilic enzymes. By looking at the time-varying concentrations of 22 metabolites, we were able to identify a key limitation in the existing ethanol production pathway, and develop several new hypotheses for metabolic engineering strategies.