Sang Duck Jeon

Cellulosome-based, Clostridium-derived multi-functional enzyme complexes for advanced biotechnology tool development: Advances and applications

The cellulosome is one of natures most elegant and elaborate nanomachines and a key biological and biotechnological macromolecule that can be used as a multi-functional protein complex tool. Each protein module in the cellulosome system is potentially useful in an advanced biotechnology application. The high-affinity interactions between the cohesin and dockerin domains can be used in protein-based biosensors to improve both sensitivity and selectivity. The scaffolding protein includes a carbohydrate-binding module (CBM) that attaches strongly to cellulose substrates and facilitates the purification of proteins fused with the dockerin module through a one-step CBM purification method. Although the surface layer homology (SLH) domain of CbpA is not present in other strains, replacement of the cell surface anchoring domain allows a foreign protein to be displayed on the surface of other strains. The development of a hydrolysis enzyme complex is a useful strategy for consolidated bioprocessing (CBP), enabling microorganisms with biomass hydrolysis activity. Thus, the development of various configurations of multi-functional protein complexes for use as tools in whole-cell biocatalyst systems has drawn considerable attention as an attractive strategy for bioprocess applications. This review provides a detailed summary of the current achievements in Clostridium-derived multi-functional complex development and the impact of these complexes in various areas of biotechnology.

Bi-functional cellulases complexes displayed on the cell surface of Corynebacterium glutamicum increase hydrolysis of lignocelluloses at elevated temperature.

Introducing cellulases into Corynebacterium glutamicum leads to the direct degradation of lignocellulosic materials for energy sources. In this study, a cellulase complex containing two cellulolytic enzymes, endoglucanase E (CelE) and β-glucosidase A (BglA), was established to completely degrade cellulose to glucose. The cellulases complexes were displayed on the cell surface of C. glutamicum by using the mechanosensitive channel (Msc) to anchor enzymes in the cytoplasmic membrane. As confirmed by comparison enzyme activities in the cell pellet fraction and supernatant and dual color based immunofluorescence microscopy, the cellulolytic enzymes was successfully associated with the cell surface of C. glutamicum. The displayed cellulases complexes had a synergic effect on the direct conversion of biomass to reducing sugars leading to 3.1- to 6.0-fold increase compared to the conversion by the secreted cellulases complexes. In addition, the displayed cellulases complexes increased the residual activities of cCelE and cBglA at 70°C from 28.3% and 24.3% in the secreted form to 65.1% and 82.8%, respectively. The display of cellulases complexes on the cell surface of C. glutamicum enhances the polysaccharide equivalent and the direct saccharification of low cost biomass via the action of multi-thermostable enzyme complexes.

Cellulosomic profiling produced by Clostridium cellulovorans during growth on different carbon sources explored by the cohesin marker

Clostridium cellulovorans produces large extracellular enzyme complex, called cellulosomes. The diversity of the cellulosomal enzymes, which are secreted by C. cellulovorans that has been cultured on different carbon sources, such as Avicel, xylan, AXP (Avicel-xylan-pectin, 3:1:1) and cellobiose, was explored by two-dimensional gel electrophoresis. To identify the cellulosomal enzymes, we constructed a biomarker using cohesin 6, one of the CbpA cohesins, that was labeled with fluorescence. The major apparent spots were isolated and identified by ESI MS/MS protein sequencing. Fluorescently labeled cohesin clearly showed that the amount of the cellulosomal enzymes was influenced by the available carbon source. EngE, ExgS, EngK, XynB and ManA were most frequently expressed under all conditions. However, EngY was only observed on the AXP culture. We found two novel putative cellulosomal proteins, NC1[GH9] and NC2[GH26], and five unknown proteins, NU1, NU2, NU3, NU4 and NU5. The cohesin biomarker clearly showed different production patterns of the cellulosomal subunits under different culture conditions and revealed novel cellulosomal subunits.

The processive endoglucanase EngZ is active in crystalline cellulose degradation as a cellulosomal subunit of Clostridium cellulovorans.

Clostridium cellulovorans produces an efficient enzyme complex for the degradation of lignocellulosic biomass. In our previous study, we detected and identified protein spots that interacted with a fluorescently labeled cohesin biomarker via two-dimensional gel electrophoresis. One novel, putative cellulosomal protein (referred to as endoglucanase Z) contains a catalytic module from the glycosyl hydrolase family (GH9) and demonstrated higher levels of expression than other cellulosomal cellulases in Avicel-containing cultures. Purified EngZ had optimal activity at pH 7.0, 40°C, and the major hydrolysis product from the cellooligosaccharides was cellobiose. EngZs specific activity toward crystalline cellulose (Avicel and acid-swollen cellulose) was 10-20-fold higher than other cellulosomal cellulase activities. A large percentage of the reducing ends that were produced by this enzyme from acid-swollen cellulose were released as soluble sugar. EngZ has the capability of reducing the viscosity of Avicel at an intermediate-level between exo- and endo-typing cellulases, suggesting that it is a processive endoglucanase. In conclusion, EngZ was highly expressed in cellulolytic systems and demonstrated processive endoglucanase activity, suggesting that it plays a major role in the hydrolysis of crystalline cellulose and acts as a cellulosomal enzyme in C. cellulovorans.

Unique contribution of the cell wall-binding endoglucanase G to the cellulolytic complex in Clostridium cellulovorans.

ABSTRACT The cellulosomes produced by Clostridium cellulovorans are organized by the specific interactions between the cohesins in the scaffolding proteins and the dockerins of the catalytic components. Using a cohesin biomarker, we identified a cellulosomal enzyme which belongs to the glycosyl hydrolase family 5 and has a domain of unknown function 291 (DUF291) with functions similar to those of the surface layer homology domain in C. cellulovorans. The purified endoglucanase G (EngG) had the highest synergistic degree with exoglucanase (ExgS) in the hydrolysis of crystalline cellulose (EngG/ExgS ratio = 3:1; 1.71-fold). To measure the binding affinity of the dockerins in EngG for the cohesins of the main scaffolding protein, a competitive enzyme-linked interaction assay was performed. Competitors, such as ExgS, reduced the percentage of EngG that were bound to the cohesins to less than 20%; the results demonstrated that the cohesins prefer to bind to the common cellulosomal enzymes rather than to EngG. Additionally, in surface plasmon resonance analysis, the dockerin in EngG had a relatively weak affinity (30- to 123-fold) for cohesins compared with the other cellulosomal enzymes. In the cell wall affinity assay, EngG anchored to the cell surfaces of C. cellulovorans using its DUF291 domain. Immunofluorescence microscopy confirmed the cell surface display of the EngG complex. These results indicated that in C. cellulovorans, EngG assemble into both the cellulolytic complex and the cell wall complex to aid in the hydrolysis of cellulose substrates.

Enhanced thermostability of mesophilic endoglucanase Z with a high catalytic activity at active temperatures

This is the first study for therrmostable mutants of mesophilic endoglucanase EngZ from Clostridium cellulovorans using by site-directed mutagenesis. K94R, S365P and their double mutant K94R/S365P had a wide range of active temperatures (30-60 °C). In addition, the optimal temperature of K94R/S365P was increased by 7.5 °C. K94R/S365P retained 78.3% relative activity at 70 °C, while the wild type retained only 5.8%. Especially, K94R/S365P remained 45.1-fold higher activity than the wild type at 70 °C. In addition, K94R/S365P was 3.1-fold higher activity than the wild type at 42.5 °C, which is the optimal temperature of the wild type. K94R/S365P showed also stimulated in 2.5-fold lower concentration of CaCl2 and delayed aggregation temperature in the presence of CaCl2 compared to the wild type. In pH stability, K94R/S365P was not influenced, but the optimum pH was transferred from pH 7 to pH 6. In long-term hydrolysis, K94R/S365P reduced the newly released reducing sugar yields after 12h reaction; however, the yields consistently increased until 72h. Finally, the total reducing sugar of K94R/S365P was 5.0-fold higher than the wild type at 50 °C, pH6. EngZ (K94R/S365P) can support information to develop thermostability of GH9 endoglucanase with a high catalytic efficiency as the potential industrial bioprocess candidate.

An enhanced protein-protein interaction based on enzymatic complex through replacement of the recognition site

Clostridium cellulovorans, produce multi-enzymatic complexes known as cellulosomes, which assemble via the interaction of a dockerin module in the cellulosomal subunit with one of the several cohesin modules in the scaffolding protein, to degrade the plant cell wall polymer. An enhanced cohesin-dockerin interaction was demonstrated by modified certain cellulosomal enzymes with altered amino acid residues at the crucial binding site, 11th and 12th positions in dockerin module. In fluorescence intensity analyses using the cellulosome-based biomarker system, the modified cellulosomal enzymes (EngE SL to AI and EngH SM to AI) showed an increased intensity (1.4- to 2.2-fold) compared with the wild-type proteins. Conversely, modified ExgS (AI to SM) exhibited a reduced intensity (0.6- to 0.7-fold) compared with the wild type. In enzyme-linked and competitive enzyme-linked interaction assays, the some modified protein (EngE SL to AI and EngH SM to AI) showed their increased binding affinity toward the cohesins (Coh2 and Coh9). Surface plasmon resonance analysis quantitatively demonstrated the binding affinity of these two modified proteins toward cohesins showed similar or higher affinity comparing with its with wild type proteins. These results suggest the replacement of amino acid residues in the certain recognition site significantly affects the binding affinity of the cohesin-dockerin interaction.