- Open Access
Assembly of cellulases with synthetic protein scaffolds in vitro
© Yu et al.; licensee Springer. 2015
- Received: 18 December 2014
- Accepted: 15 March 2015
- Published: 1 April 2015
Enzymatic cascades in metabolic pathways are spatially organized in such a way as to facilitate the flow of substrates. The construction of artificial cellulase complexes that mimic natural multienzyme assemblies can potentially enhance the capacity for cellulose hydrolysis. In this study, an artificial cellulase complex was constructed by tethering three cellulases to a synthetic protein scaffold.
Three pairs of interacting proteins were selected and characterized. The artificial protein scaffolds were constructed by fusing three interacting proteins. Cellulases were tethered to these synthetic scaffolds in different orders. The optimal assembly resulted in a 1.5-fold higher hydrolysis of cellulose than that achieved by unassembled cellulases.
A novel artificial protein scaffold was constructed and used to assemble three cellulases. The resultant increase in enzymatic activity suggests that this can be used as a strategy for enhancing the biocatalytic capacity of enzyme cascades.
Multienzyme pathways in living systems comprise cascades in which enzymes are tethered together into assemblies that facilitate substrate flow between components, limit the diffusion of intermediate metabolic products, and increase the yield from sequential reactions [1,2]. There have been various attempts to produce multienzyme assemblies in vitro, including by gene fusion, protein or DNA scaffold construction, and chemical modification . Although the simplest way is by enzyme fusion, this often results in loss of enzymatic activity or formation of inclusion bodies , while chemical modification can also impair enzymatic activity . Moreover, the high cost of generating DNA scaffolds makes this approach infeasible on a large scale [6,7].
Protein scaffolds are an attractive strategy for bringing together enzymes. A 77-fold enhancement in product concentration was observed in an assembly of three mevalonate biosynthetic enzymes with a protein scaffold composed of metazoan signaling proteins . In another study, a self-assembled enzyme complex using cellulosome achieved a 13.4-fold increase in reaction rate , while a proliferating cell nuclear antigen-based assembly of P450 with ferredoxin and ferredoxin reductase showed high catalytic activity . The protein scaffolds used in these studies were limited to metazoan signaling proteins and cellulosome components [11-14], which may not be amenable to all types of enzyme cascades. As such, there is an ongoing need for novel and different types of protein scaffolds.
Cellulose is the most abundant renewable resource on Earth and plays a significant role in biofuel production . Cellulose is broken down into oligosaccharides by endoglucanase (EG) and exoglucanase (CBH) before β-glucosidase (BGL) hydrolyzes cellobiose into glucose . EG, CBH, and BGL are enzymes in the cellulose degradation pathway that act synergistically; coexpressing these enzymes improves the efficiency of cellulose degradation . It was hypothesized that a highly ordered assembly containing the three cellulases would enhance their activities and thereby increase cellulose hydrolysis. Therefore, CelccA (EG), CelccE (CBH), and Cel2454 (BGL) were selected as a model system for protein scaffold-mediated assembly strategy.
Strains and medium
Escherichia coli DH5α was used for cloning, and E. coli BL21 (DE3) was used for protein expression. Cells were cultured in Luria-Bertani (LB) medium (10.0 g/l tryptone, 5.0 g/l yeast extract, 10.0 g/l NaCl) supplemented with either 100 mg/l ampicillin or 50 mg/l kanamycin.
Primers used in this work
Plasmid pET28a-IPaA had an expression cassette containing IPa and celCCA at the N and C termini, respectively. A DNA fragment encoding IPa was amplified with the primer pair IPaA-F/IPaA-R and cloned into pET28a-celCCA by Seamless Cloning . Plasmid pET28a-AIPa containing celCCA and IPa at the N and C termini, respectively, was generated with the primer pair AIPa-F/AIPa-R. Plasmid pET28a-IPbE containing IPb and celCCE at the N and C termini, respectively, was generated with primer pair IPbE-F/IPbE-R. Plasmid pET28a-EIPb containing celCCE and IPb at the N and C termini, respectively, was constructed with primer pair EIPb-F/EIPb-R. Plasmid pET28a-IPc4 containing IPc and cel2454 at the N and C termini, respectively, was generated with primer pair IPc4-F/IPc4-R. Plasmid pET28a-4IPc containing cel2454 and IPc at the N and C termini, respectively, was constructed with primer pair 4IPc-F/4IPc-R.
Plasmid pET21a-ScafBAC had an expression cassette containing IPA flanked by IPB and IPC at the N and C termini, respectively. The DNA fragments encoding IPB, IPA, and IPC were amplified with the primer pairs ScafBAC-F1/IPB-R, ScafBAC-F2/IPA-R, and ScafBAC-F3/ScafBAC-R, respectively. The three fragments were cloned into pET21a by Seamless Cloning. Plasmid pET21a-ScafABC was constructed using primer pairs ScafABC-F1/IPA-R, ScafABC-F2/IPB-R, and ScafABC-F3/ScafABC-R, and plasmid pET21a-ScafBCA was generated using primer pairs ScafBAC-F1/IPB-R, ScafABC-F3/IPC-R, and ScafBCA-F/ScafBCA-R.
Protein expression and purification
Recombinant proteins were precultured overnight at 37°C in LB medium supplemented with appropriate antibiotics. The cultures were inoculated in fresh LB medium containing antibiotics and incubated at 37°C until the optical density at 600 nm reached 0.8. The cultures were then cooled to 18°C, and isopropyl-β-d-thiogalactopyranoside was added to a final concentration of 0.1 mM. After 20 h, cells were harvested by centrifugation for 10 min at 8,000 rpm and 4°C, resuspended in 20 mM phosphate-buffered saline (PBS; pH 7.0), and disrupted by sonication on ice. Cellular debris was removed by centrifugation for 40 min at 11,000 rpm. Proteins were purified using a HisTrapFF column (GE Healthcare, Waukesha, WI, USA), and protein concentration was determined by the Bradford method.
Binding affinities between proteins were measured by biolayer interferometry (Octet QKe; Fortebio, Menlo Park, CA, USA), which detects changes in mass (protein density) on a biosensor; changes in the reflected interference wave pattern between the sample and an internal reference layer result in a phase shift that can be followed in real time in both kinetic and quantitative modes . All experiments were performed in kinetic buffer (20 mM PBS, pH 7.0; 1 mg/ml bovine serum albumin (BSA), and 0.02% Tween 20). One of the proteins (1 μM) was biotinylated by incubating with 2 μl biotinyl N-hydroxysuccinimide ester for 1 h at room temperature, with excess biotin removed using a desalting column. The biotinylated protein was loaded onto the streptavidin biosensor by incubating for 240 s. The immobilized protein was equilibrated with kinetic buffer for 180 s, and the corresponding protein (1 μM) was associated to the biotinylated protein by incubating for 800 s. Dissociation was measured for 800 s in kinetic buffer. For each assay, a control experiment was carried out using BSA. Binding affinity was independent of which protein was loaded onto the streptavidin biosensor [23-26].
The BGL activity was measured by incubating 135 μl of 2.5 mM p-nitrophenyl β-d-glucopyranoside solution in 20 mM sodium phosphate buffer (pH 7.0) with 7.5 μg of pure enzyme solution at 37°C for 30 min. The reaction was terminated by adding 70 μl of 0.4 M Na2CO3 and the absorbance at 420 nm was measured. One unit of enzyme was defined as the activity producing 1 μmol of p-nitrophenol per min under the assay conditions. EG/CBH activity was measured by incubating 90 μl of 1.5% (wt/vol) carboxymethyl cellulose (CMC) in 20 mM sodium phosphate buffer (pH 7.0) with 5 μg of pure enzyme solution at 37°C for 30 min. A 100-μl volume of sample was mixed with 150 μl of 3,5-dinitrosalicylic acid reagent, and after boiling for 10 min, the absorbance at 540 nm was measured. One unit of enzymatic activity was defined as the amount of enzyme required to produce a 1 μmol reduction sugar per min under the assay conditions.
The activity of free or assembled enzymes was assayed in the presence of 0.75% (wt/vol) CMC at 37°C in 20 mM sodium phosphate buffer (pH 7.0). The reduction sugars were measured as described above. Glucose concentration was determined using an SBA biosensor analyzer (Biology Institute of Shandong Academy of Sciences, Jinan, China).
Selection and characterization of interacting proteins
Fusion of cellulases and interacting proteins
Construction of scaffolds and their effect on assembled enzyme complexes
Characterization of the tri-enzyme complex assembled by ScafBAC
An artificial tri-enzyme complex was constructed by assembling three cellulases with a novel protein scaffold composed of interacting proteins. The effect of the order of cellulase within the scaffolds on the catalytic efficiency was determined. Moreover, the complex had higher catalytic activity than the individual components. These results suggest that this novel protein scaffold can serve as a powerful tool for facilitating multienzyme cascades.
This work supported by National Special Fund for State Key Laboratory of Bioreactor Engineering (2060204), and ‘the Fundamental Research Funds for the Central Universities’, People’s Republic of China.
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