Engineering Clostridium thermocellum within the BioEnergy Science Center: recent advances in biofuel production from cellulose

Clostridium thermocellum is a promising organism for the implementation of a consolidated bioprocessing (CBP) strategy for biofuel production due to its native ability to rapidly deconstruct cellulose and ferment the resulting sugars. The BioEnergy Science Center (BESC) is a multi-institutional research consortium with a focus on engineering C. thermocellum to convert cellulosic biomass into fuels such as ethanol and isobutanol. Because the wild type strain ferments the sugars to a mixture of lactate, formate, acetate, H₂, ethanol, amino acids, and other products, metabolic engineering is required to optimize flux to a single product. We are using a combination metabolic modeling, ¹³C fluxomics, enzymology, transcriptomics, proteomics, and genetics to identify new targets for increasing yield and titer. While the wild type ethanol yield is approximately 30% of the theoretical maximum, we have recently engineered C. thermocellum to produce ethanol at yields > 80% and a titers > 25 g/L. We are also engineering C. thermocellum to produce isobutanol from cellulose, where one of the challenges is the lack of a well-characterized thermophilic isobutanol biosynthetic pathway. Using a combinatorial approach to gene expression, we have produced isobutanol at a yield of 41% of the theoretical maximum and titer of 5.4 g/L. Recent progress in understanding the metabolic pathways utilized by C. thermocellum as well as advances in increasing yields and titers of these biofuels will be discussed.