Abstract
BackgroundConsolidated 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.ResultsWe 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.ConclusionsWe 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.
Highlights
Consolidated bioprocessing (CBP) is reliant on the simultaneous enzyme production, saccharification of biomass, and fermentation of released sugars into valuable products such as butanol
Some strains of C. acetobutylicum can grow on cellobiose and amorphous cellulose [11,12], they are unable to grow on crystalline cellulose [13], as the small amount of cellulosome produced appears to be non-functional in this organism
Expression of C. thermocellum derived CipA scaffoldin variants in C. acetobutylicum driven by the host’s chromosomal thiolase promoter CipA is the primary scaffoldin of C. thermocellum and is responsible for the integration of the enzymes into the complex [15,22]
Summary
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, unable to grow on crystalline cellulose 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. Consolidated bioprocessing (CBP) is one such strategy, potentially allowing effective conversion of cheap lignocellulosic materials into high value industrial products [1] This process requires simultaneous enzyme production, include the production of 38.1 g/l ethanol from 92.1 g/l Avicel via a co-culture of modified Clostridium thermocellum and Thermoanaerobacterium saccharolyticum [4]; the ‘recombinant’ approach has recently generated strains of Saccharomyces cerevisiae [5] and Kluyveromyces marxianus [6] able to ferment hot water treated rice straw and Avicel respectively, final ethanol titres were still low. The introduction of a functional cellulosome is one potential approach to engineering C. acetobutylicum to be a suitable CBP organism
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