Metabolic and Thermodynamic Analysis of C. thermocellum Strains Engineered for High Ethanol Production

Student: Jordan Brown
Mentor: Daniel Amador-Noguez (University of Wisconsin-Madison)

4 strains of the biofuel producing bacteria, C. thermocellum, were engineered with differing pathways for the conversion of phosphoenolpyruvate (PEP) to pyruvate. The strains were analyzed from an integrated metabolomics and thermodynamic point-of-view. Finding the optimal pathway for this conversion is a step towards optimizing the bacteria as a whole to more effectively produce ethanol from cellulosic biomass.

Ethanol is a carbon neutral fuel that can be produced from the microbial conversion of cellulosic biomass. One microorganism that is being engineered to perform this conversion is the aerobic thermophillic bacteria, Clostridium thermocellum. Past efforts that focused solely on metabolomic analyses have been able to increase ethanol production, but only up to a certain amount. Studies show that the overall pathway of this conversion is not as energetically favorable as in other biofuel producing strains. This causes the conversion of cellulose to ethanol to proceed slowly and, in some cases, allows for reactions in the pathway to proceed in reverse. By making the change in free energy (∆G) more negative at key steps in the reaction, the overall reaction will become more spontaneous and proceed at a faster rate. This would increase ethanol production, and drive down the price, allowing ethanol-based biofuels to better compete with fossil fuels at the pump.

One key step in the pathway to be optimized is the conversion of phosphoenolpyruvate (PEP) to pyruvate.  C. thermocellum utilizes the Emben-Meyerhof-Parnas (EMP) pathway, but unlike other bacteria that use this pathway it does not have the enzyme pyruvate kinase (PYK), which catalyzes the direct conversion of PEP to pyruvate. Instead it utilizes two different pathways. One is the direct conversion of PEP to pyruvate by a different enzyme, pyruvate phosphate dikinase (PPDK). This pathway has been experimentally shown not to be able to be the sole pathway in PEP to pyruvate conversion. The other pathway is the malate shunt, which uses PEP carboxylase (PEPCK), malate dihydrogenase (MDH) and malic enzyme (ME)1. This pathway has been shown to be able to be the sole PEP to pyruvate pathway.