title: Consolidated bioprocessing of cellulose to isobutanol using Clostridium thermocellum.
abstract: 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 work, 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. This strain produced 5.4 g/L of isobutanol from cellulose in minimal medium at 50(o)C within 75 h, corresponding to 41% of theoretical yield.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, CA 90095, USA.
authors.forename: Paul P
authors.initials: PP
authors.lastname: Lin
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, CA 90095, USA.
authors.forename: Luo
authors.initials: L
authors.lastname: Mi
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, CA 90095, USA.
authors.forename: Amy H
authors.initials: AH
authors.lastname: Morioka
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, CA 90095, USA.
authors.forename: Kouki M
authors.initials: KM
authors.lastname: Yoshino
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, CA 90095, USA.
authors.forename: Sawako
authors.initials: S
authors.lastname: Konishi
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, CA 90095, USA.
authors.forename: Sharon C
authors.initials: SC
authors.lastname: Xu
authors.affiliation: Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA; Bredesen Center for Interdisciplinary Research and Graduate Education, University of Tennessee, Knoxville, TN 37996, USA.
authors.forename: Beth A
authors.initials: BA
authors.lastname: Papanek
authors.affiliation: Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA; BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA.
authors.forename: Lauren A
authors.initials: LA
authors.lastname: Riley
authors.affiliation: Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA; Bredesen Center for Interdisciplinary Research and Graduate Education, University of Tennessee, Knoxville, TN 37996, USA; BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA.
authors.forename: Adam M
authors.initials: AM
authors.lastname: Guss
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, CA 90095, USA; UCLA-DOE Institute of Genomics and Proteomics, USA. Electronic address: liaoj@seas.ucla.edu.
authors.forename: James C
authors.initials: JC
authors.lastname: Liao
mesh_terms: D000440:Butanols / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism*; D048013:Acetivibrio thermocellus / Q000235:genetics / Q000378:metabolism*; D060847:Metabolic Engineering; D014633:Valine / Q000096:biosynthesis
chemical_list: D000440:Butanols; C040507:isobutyl alcohol; D002482:Cellulose; D014633:Valine
keywords: Biofuel; Butanol; Clostridium thermocellum; Consolidated bioprocessing