Consolidated bioprocessing of cellulose to isobutanol using Clostridium thermocellum

Consolidated bioprocessing (CBP) has the potential to reduce bioluel or biochemical production costs by processing cellulose hydrolysis and fermentation simultaneously without the addition of pre manufactured cellulases. In particular, Clostridium thertnocellum is a promising thermophilic CBP host because of its high cellulose decomposition rate. Here we report the engineering of C. thermocellum to produce isobutanol. Metabolic engineering for isobutanol production in C thermocellurn is hampered by enzyme toxicity during cloning, time consuming pathway engineering procedures, and slow turnaround in production tests. In this work, we first cloned essential isobutanol pathway genes under different promoters to create various plasmid constructs in Escherichiu coli. Then, these constructs were transformed and tested in C. thermocellurn. Among these engineered strains, the best isobutanol producer was selected and the production conditions were optimized. We confirmed the expression of the overexpressed genes by their mRNA quantities. We also determined that both the native ketoisovalerate oxidoreductase (KOR) and the heterologous ketoisovalerate decarboxylase (MVO) expressed were responsible for isobutanol production. We further found that the plasmid was integrated into the chromosome by single crossover. The resulting strain was stable without antibiotic selection pressure. This strain produced 5.4 g/L of isobutanol horn cellulose in minimal medium at 50 C within 75 h, Coffesponding to 41% of theoretical yield. (C) 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.