title: Secretion and assembly of functional mini-cellulosomes from synthetic chromosomal operons in Clostridium acetobutylicum ATCC 824.
abstract: BACKGROUND
  Consolidated bioprocessing (CBP) is reliant on the simultaneous enzyme production, saccharification of biomass, and fermentation of released sugars into valuable products such as butanol. Clostridial species that produce butanol are, however, unable to grow on crystalline cellulose. In contrast, those saccharolytic species that produce predominantly ethanol, such as Clostridium thermocellum and Clostridium cellulolyticum, degrade crystalline cellulose with high efficiency due to their possession of a multienzyme complex termed the cellulosome. This has led to studies directed at endowing butanol-producing species with the genetic potential to produce a cellulosome, albeit by localising the necessary transgenes to unstable autonomous plasmids. Here we have explored the potential of our previously described Allele-Coupled Exchange (ACE) technology for creating strains of the butanol producing species Clostridium acetobutylicum in which the genes encoding the various cellulosome components are stably integrated into the genome.
  
  
  RESULTS
  We used BioBrick2 (BB2) standardised parts to assemble a range of synthetic genes encoding C. thermocellum cellulosomal scaffoldin proteins (CipA variants) and glycoside hydrolases (GHs, Cel8A, Cel9B, Cel48S and Cel9K) as well as synthetic cellulosomal operons that direct the synthesis of Cel8A, Cel9B and a truncated form of CipA. All synthetic genes and operons were integrated into the C. acetobutylicum genome using the recently developed ACE technology. Heterologous protein expression levels and mini-cellulosome self-assembly were assayed by western blot and native PAGE analysis.
  
  
  CONCLUSIONS
  We demonstrate the successful expression, secretion and self-assembly of cellulosomal subunits by the recombinant C. acetobutylicum strains, providing a platform for the construction of novel cellulosomes.
authors: Clostridia Research Group, BBSRC Sustainable BioEnergy Centre, School of Life Sciences, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham, NG7 2RD, UK.
authors: Katalin
authors: K
authors: Kovács
authors: Clostridia Research Group, BBSRC Sustainable BioEnergy Centre, School of Life Sciences, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham, NG7 2RD, UK.
authors: Benjamin J
authors: BJ
authors: Willson
authors: Clostridia Research Group, BBSRC Sustainable BioEnergy Centre, School of Life Sciences, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham, NG7 2RD, UK.
authors: Katrin
authors: K
authors: Schwarz
authors: Clostridia Research Group, BBSRC Sustainable BioEnergy Centre, School of Life Sciences, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham, NG7 2RD, UK.|Current address: Centre for Synthetic Biology and Innovation, Division of Molecular Biosciences, Imperial College London, South Kensington Campus, London, SW7 2AZ, UK.
authors: John T
authors: JT
authors: Heap
authors: Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK.
authors: Adam
authors: A
authors: Jackson
authors: Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK.
authors: David N
authors: DN
authors: Bolam
authors: Clostridia Research Group, BBSRC Sustainable BioEnergy Centre, School of Life Sciences, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham, NG7 2RD, UK.
authors: Klaus
authors: K
authors: Winzer
authors: Clostridia Research Group, BBSRC Sustainable BioEnergy Centre, School of Life Sciences, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham, NG7 2RD, UK.
authors: Nigel P
authors: NP
authors: Minton
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