Consolidated bioprocessing of cellulose to isobutanol using Clostridium thermocellum

Journal Article · Fri Jul 10 00:00:00 EDT 2015 · Metabolic Engineering

DOI:https://doi.org/10.1016/j.ymben.2015.07.001· OSTI ID:1265626

Lin, Paul P. ^[1]; Mi, Luo ^[1]; Morioka, Amy H. ^[1]; Yoshino, Kouki M. ^[1]; Konishi, Sawako ^[1]; Xu, Sharon C. ^[1]; Papanek, Beth A. ^[2]; Riley, Lauren A. ^[3]; Guss, Adam M. ^[4]; Liao, James C. ^[5]

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1. Univ. of California, Los Angeles, CA (United States). Dept. of Chemical and Biomolecular Engineering
2. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division; Univ. of Tennessee, Knoxville, TN (United States). Bredesen Center for Interdisciplinary Research and Graduate Education
3. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division; Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). BioEnergy Science Center
4. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division; Univ. of Tennessee, Knoxville, TN (United States). Bredesen Center for Interdisciplinary Research and Graduate Education; Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). BioEnergy Science Center
5. Univ. of California, Los Angeles, CA (United States). Dept. of Chemical and Biomolecular Engineering; Univ. of California, Los Angeles, CA (United States). UCLA-DOE Inst. of Genomics and Proteomics

Consolidated bioprocessing (CBP) has the potential to reduce biofuel or biochemical production costs by processing cellulose hydrolysis and fermentation simultaneously without the addition of pre-manufactured cellulases. In particular, Clostridium thermocellum 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. thermocellum is hampered by enzyme toxicity during cloning, time-consuming pathway engineering procedures, and slow turnaround in production tests. In this paper, we first cloned essential isobutanol pathway genes under different promoters to create various plasmid constructs in Escherichia coli. Then, these constructs were transformed and tested in C. thermocellum. 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 (KIVD) 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. Finally, this strain produced 5.4 g/L of isobutanol from cellulose in minimal medium at 50 °C within 75 h, corresponding to 41% of theoretical yield.

• Research Organization: Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). BioEnergy Science Center (BESC)
• Sponsoring Organization: USDOE; National Science Foundation (NSF)
• DOE Contract Number: AC05-00OR22725
• OSTI ID: 1265626
• Journal Information: Metabolic Engineering, Vol. 31, Issue C; ISSN 1096-7176
• Publisher: Elsevier
• Country of Publication: United States
• Language: English

Related Subjects

09 BIOMASS FUELS
59 BASIC BIOLOGICAL SCIENCES
biofuel
consolidated bioprocessing
Clostridium thermocellum
butanol