Engineering nitrogen metabolism in Clostridium thermocellum for cellulosic ethanol production

Clostridium thermocellum is a leading candidate organism for implementing a consolidated bioprocessing (CBP) strategy for biofuel production due to its native ability to rapidly consume cellulose and its existing ethanol production pathway.  C. thermocellum converts cellulose and soluble cellodextrins such as cellobiose to lactate, formate, acetate, H₂, ethanol, amino acids, and other products.  Therefore, metabolic engineering is required to optimize flux to a single product. A mutant strain of C. theromcellum was constructed to remove major side product formation, resulting in C. theromcellum ΔhydG Δldh Δpfl Δpta-ack.  This strain no longer produced formate, acetate and lactate; hydrogen production decreased by four fold; and the ethanol yield doubled compared with the wild type on cellobiose, crystalline cellulose Avicel, and pretreated biomass. While this mutant exhibited higher ethanol yield, amino acids were still produced as end products.  Therefore, genetic analysis of nitrogen metabolism is being investigated to understand the mechanism of ammonium assimilation and to devise strategies to prevent production of secreted amino acids as fermentation products. Progress in altering nitrogen metabolism in C. thermocellum and the impact on product formation will be discussed.