Consolidated Bioprocessing of Cellulose to an Advanced Biofuel Using a Cellulolytic Thermophile
Consolidated bioprocessing (CBP) has the potential to reduce biofuel and biochemical production costs by processing cellulose hydrolysis and fermentation simultaneously, without the addition of premanufactured cellulases and other hydrolytic enzymes. In particular, Clostridium thermocellum is a promising thermophilic CBP host because of its high cellulose decomposition rate. Toward this end, researchers at the Department of Energy’s BioEnergy Science Center (BESC) researchers engineered C. thermocellum to produce isobutanol, an advanced biofuel. Metabolic engineering for isobutanol production in C. thermocellum is hampered by enzyme toxicity during cloning, time-consuming pathway engineering procedures, and slow turnaround in production tests. Engineering of the isobutanol pathway into C. thermocellum was facilitated by first cloning plasmids into Escherichia coli before transforming these constructs into C. thermocellum for testing and optimization. Among these engineered strains, the best isobutanol producer was selected. Interestingly, both the native ketoisovalerate oxidoreductase (KOR) and the heterologous ketoisovalerate decarboxylase (KIVD) were expressed and found to be responsible for isobutanol production. A single crossover integration of the plasmid into the chromosome resulted in a stable strain not requiring antibiotic selection. This strain produced 5.4 g/L of isobutanol from cellulose in minimal medium at 50°C within 75 hours, corresponding to 41% of theoretical yield. While there is significant room for further optimization, this initial engineering of a cellulolytic thermophile to produce an advanced biofuel demonstrates the potential of this strategy to help create a sustainable and commercially viable biofuel.
Lin, P. P., L. Mi, A. H. Morioka, K. M. Yoshino, S. Konishi , S. C. Xu , B. A. Papanek, L. A. Riley, A. M. Guss, and J. C. Liao. 2015. “Consolidated Bioprocessing of Cellulose to Isobutanol Using Clostridium thermocellum,” Metabolic Engineering 31, 44-52. DOI:10.1016/j.ymben.2015.07.001.