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.

Figure 1

Schematic representation of CipA variants integrated into the genome of C. acetobutylicum (a) and their integration sites into the chromosome without (b) and with (c) additional promoter (P). All 11 CipA variants (CipA2-CipA full length) carry a C-terminal Flag tag. Encoded genes are: thl, thiolase; ermB, erythromycin ribosomal methylase B, cipA* variant, CAC2872 predicted open reading frame and atpB, F0F1 ATP synthase subunit A.

Figure 2

SDS-PAGE and western blot analysis of all synthetic CipA variants. Supernatants of CipA-expressing recombinant C. acetobutylicum strains were mixed with Avicel (crystalline cellulose) and proteins harbouring a carbohydrate binding module (CBM), including the CipA variants, were pulled down. (a) SDS-PAGE analysis and Simply blue staining (Invitrogen) of the Avicel bound culture supernatants of the first six CipA variants (CipA2-CipA7) (b) Western blot analysis of the Avicel bound CipA scaffold variants (CipA2-CipA7) using ANTI-FLAG M2 monoclonal antibody-horseradish peroxidise conjugate (Sigma). Samples were concentrated 100 fold. (c) Western blot analysis of the Avicel bound CipA scaffold variants (CipA6-CipA full length) using the same antibody. These samples were concentrated 150 fold. Expected molecular masses are indicated by red arrows. M, ColorPlus Prestained Protein Ladder (10–230 kDa; New England Biolabs), Flag, N-Flag-Bap control protein (50 kDa, 50 ng/lane; Sigma).

Figure 3

Western blot analysis of the three full length CipA variants. Supernatants of CipA-expressing recombinant C. acetobutylicum strains (CipA_BB2, CipA_DNA2.0 and CipAnative) were TCA precipitated and concentrated 200-fold. ANTI-FLAG M2 monoclonal antibody-horseradish peroxidise conjugate (Sigma) was used to detect the Flag-tagged CipA proteins. Expected molecular masses are indicated by red arrows. M, ColorPlus Prestained Protein Ladder (10–230 kDa; New England Biolabs), Flag, N-Flag-Bap control protein (50 kDa, 50 ng/lane; Sigma).

Figure 4

Expression of CipA scaffoldin variants driven by an additional promoter. Supernatants of CipA-expressing recombinant C. acetobutylicum strains (CipA2, CipA3, thlCipA2, thlCipA3, CipA_BB2, CipA_DNA2.0, thl_CipA_BB2, thl_CipA_DNA2.0) were TCA precipitated and concentrated 100-fold. ANTI-FLAG M2 monoclonal antibody-horseradish peroxidise conjugate (Sigma) was used to detect the Flag-tagged CipA variants. M, ColorPlus Prestained Protein Ladder (10–230 kDa; New England Biolabs). The N-Flag-Bap control protein (50 kDa, 50 μg/lane; Sigma) was used to estimate the amount of secreted CipA variants. Expected molecular masses are indicated by arrows.

Figure 5

Expression of C. thermocellum cellulases in C. acetobutylicum. (a) Recombinant C. acetobutylicum strains expressing the Cel8A, Cel9B and Cel9K cellulases were grown until late stationary phase (OD600 > 3) and their supernatants TCA precipitated, concentrated 100-fold and analysed by SDS-PAGE. (b) The recombinant strain expressing the Cel48S was grown in buffered 2xYTG until early exponential phase (OD600 of 0.4[1] and 0.8[2]), concentrated 100-fold and separated by SDS-PAGE. All four cellulases were detected using ANTI-FLAG M2 monoclonal antibody-horseradish peroxidise conjugate (Sigma). Expected molecular masses are indicated by arrows. M, ColorPlus Prestained Protein Ladder (10–230 kDa; New England Biolabs), Flag, N-Flag-Bap control protein (50 kDa, 50 ng/lane; Sigma).

Figure 6

Synthetic operons inserted into the C. acetobutylicum chromosome. Encoded genes are: thl, thiolase; ermB, erythromycin ribosomal methylase B, C. thermocellum derived cellulases cel9B and cel8A, mini-scaffoldin cipA3. All operons are preceded by the chromosomal thl promoter and the celllulosomal components by an additional synthetic thlOID promoter and followed by a transcriptional terminator (from Clostridium pasteurianum ferredoxin gene).

Figure 7

Western blot analysis of cellulosomal operons secreted by the recombinant C. acetobutylicum strains. The wild type (wt, C. acetobutylicum ATCC 824) and the recombinant strains (thlOIDCel9B, thlOIDCel8A, thlOIDCel9B_CipA3, thlOIDCel8A_CipA3, thlOIDCel9B_Cel8A_CipA3, thlOIDCipA3) were grown in buffered 2xYTG (pH 7 using 40 mM MOPS) until early exponential phase (OD600 0.8), concentrated 50-fold and separated by SDS-PAGE. All heterologous proteins were detected using ANTI-FLAG M2 monoclonal antibody-horseradish peroxidise conjugate (Sigma). M, ColorPlus Prestained Protein Ladder (10–230 kDa; New England Biolabs); Flag, N-Flag-Bap control protein (50 kDa, 50 ng/lane; Sigma). Expected molecular masses are indicated by arrows.

Figure 8

Native PAGE and western blot analysis of cellulosomal operons assembled in vivo by the recombinant C. acetobutylicum strains. The culture supernatants of the recombinant strains (thlOIDCel9B, thlOIDCel8A, thlOIDCel9B_CipA3, thlOIDCel8A_CipA3, thlOIDCel9B_Cel8A_CipA3, thlOIDCipA3) were concentrated 70-fold by ultracentrifugation and 10 μl of the concentrated samples loaded onto a native PAGE Tris-acetate gel. The presence of the cellulosomal subunits and the in vivo assembly of the mini-cellulosomes was detected using ANTI-FLAG M2 monoclonal antibody-horseradish peroxidise conjugate (Sigma). The presence of multiple bands in the samples expressing mini-cellulosomes (thlOIDCel9B_CipA3, thlOIDCel8A_CipA3 and thlOIDCel9B_Cel8A_CipA3) likely represents unsaturated complexes with cellulases binding different cohesin domains. These samples were loaded at two different concentrations: 10 and 5 μl (½) of the original samples.