Katalin Kovacs Katalin.Kovacs@nottingham.ac.uk
Secretion and assembly of functional mini-cellulosomes from synthetic chromosomal operons in Clostridium acetobutylicum ATCC 824
Kovacs, Katalin; Willson, Benjamin J.; Schwarz, Katrin; Heap, John T.; Jackson, Adam; Bolam, David N.; Winzer, Klaus; Minton, Nigel P.
Benjamin J. Willson firstname.lastname@example.org
Katrin Schwarz Katrin.Schwarz@email@example.com
John T. Heap firstname.lastname@example.org
David N. Bolam
Klaus Winzer email@example.com
Nigel P. Minton firstname.lastname@example.org
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.
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.
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.
|Journal Article Type||Article|
|Publication Date||Aug 20, 2013|
|Journal||Biotechnology for Biofuels|
|Peer Reviewed||Peer Reviewed|
|APA6 Citation||Kovacs, K., Willson, B. J., Schwarz, K., Heap, J. T., Jackson, A., Bolam, D. N., …Minton, N. P. (2013). Secretion and assembly of functional mini-cellulosomes from synthetic chromosomal operons in Clostridium acetobutylicum ATCC 824. Biotechnology for Biofuels, 6(117), doi:10.1186/1754-6834-6-117|
|Copyright Statement||Copyright information regarding this work can be found at the following address: http://eprints.nottingh.../end_user_agreement.pdf|
Copyright information regarding this work can be found at the following address: http://eprints.nottingham.ac.uk/end_user_agreement.pdf
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