title: Metabolic engineering of Clostridium cellulolyticum for the production of n-butanol from crystalline cellulose.
abstract: BACKGROUND
  Sustainable alternatives for the production of fuels and chemicals are needed to reduce our dependency on fossil resources and to avoid the negative impact of their excessive use on the global climate. Lignocellulosic feedstock from agricultural residues, energy crops and municipal solid waste provides an abundant and carbon-neutral alternative, but it is recalcitrant towards microbial degradation and must therefore undergo extensive pretreatment to release the monomeric sugar units used by biofuel-producing microbes. These pretreatment steps can be reduced by using microbes such as Clostridium cellulolyticum that naturally digest lignocellulose, but this limits the range of biofuels that can be produced. We therefore developed a metabolic engineering approach in C. cellulolyticum to expand its natural product spectrum and to fine tune the engineered metabolic pathways.
  
  
  RESULTS
  Here we report the metabolic engineering of C. cellulolyticum to produce n-butanol, a next-generation biofuel and important chemical feedstock, directly from crystalline cellulose. We introduced the CoA-dependent pathway for n-butanol synthesis from C. acetobutylicum and measured the expression of functional enzymes (using targeted proteomics) and the abundance of metabolic intermediates (by LC-MS/MS) to identify potential bottlenecks in the n-butanol biosynthesis pathway. We achieved yields of 40 and 120 mg/L n-butanol from cellobiose and crystalline cellulose, respectively, after cultivating the bacteria for 6 and 20 days.
  
  
  CONCLUSIONS
  The analysis of enzyme activities and key intracellular metabolites provides a robust framework to determine the metabolic flux through heterologous pathways in C. cellulolyticum, allowing further improvements by fine tuning individual steps to improve the yields of n-butanol.
authors.affiliation: Department of Industrial Biotechnology, Fraunhofer Institute for Molecular Biology and Applied Ecology, Forckenbeckstrasse 6, 52074, Aachen, Germany. sgaida@udel.edu.
authors.forename: Stefan Marcus
authors.initials: SM
authors.lastname: Gaida
authors.affiliation: Department of Industrial Biotechnology, Fraunhofer Institute for Molecular Biology and Applied Ecology, Forckenbeckstrasse 6, 52074, Aachen, Germany. andrea.liedtke@ime.fraunhofer.de.
authors.forename: Andrea
authors.initials: A
authors.lastname: Liedtke
authors.affiliation: Department of Industrial Biotechnology, Fraunhofer Institute for Molecular Biology and Applied Ecology, Forckenbeckstrasse 6, 52074, Aachen, Germany. andreas.jentges@rwth-aachen.de.
authors.forename: Andreas Heinz Wilhelm
authors.initials: AH
authors.lastname: Jentges
authors.affiliation: Department of Industrial Biotechnology, Fraunhofer Institute for Molecular Biology and Applied Ecology, Forckenbeckstrasse 6, 52074, Aachen, Germany. benedikt.engels@jennewein-biotech.de.
authors.forename: Benedikt
authors.initials: B
authors.lastname: Engels
authors.affiliation: Department of Industrial Biotechnology, Fraunhofer Institute for Molecular Biology and Applied Ecology, Forckenbeckstrasse 6, 52074, Aachen, Germany. stefan.jennewein@ime.fraunhofer.de.
authors.forename: Stefan
authors.initials: S
authors.lastname: Jennewein
mesh_terms: D020001:1-Butanol / Q000378:metabolism*; D056804:Biofuels; D002482:Cellulose / Q000378:metabolism*; D046973:Ruminiclostridium cellulolyticum / Q000187:drug effects / Q000378:metabolism*; D008954:Models, Biological
chemical_list: D056804:Biofuels; D020001:1-Butanol; D002482:Cellulose