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).
Results and discussion
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.
- Ricca E, Brucher B, Schrittwieser JH (2011) Multi-enzymatic cascade reactions: overview and perspectives. Advan Synthesis Catalysis 353(13):2239–2262View ArticleGoogle Scholar
- Conrado RJ, Varner JD, DeLisa MP (2008) Engineering the spatial organization of metabolic enzymes: mimicking nature’s synergy. Curr Opin Biotechnol 19(5):492–499View ArticleGoogle Scholar
- Schoffelen S, van Hest JCM (2012) Multi-enzyme systems: bringing enzymes together in vitro. Soft Matter 8(6):1736–1746View ArticleGoogle Scholar
- Adlakha N, Sawant S, Anil A, Lali A, Yazdani SS (2012) Specific fusion of beta-1,4-endoglucanase and beta-1,4-glucosidase enhances cellulolytic activity and helps in channeling of intermediates. Appl Environ Microbiol 78(20):7447–7454View ArticleGoogle Scholar
- Schoffelen S, Beekwilder J, Debets MF, Bosch D, van Hest JC (2013) Construction of a multifunctional enzyme complex via the strain-promoted azide-alkyne cycloaddition. Bioconjug Chem 24(6):987–996View ArticleGoogle Scholar
- Fu J, Liu M, Liu Y, Woodbury NW, Yan H (2012) Interenzyme substrate diffusion for an enzyme cascade organized on spatially addressable DNA nanostructures. J Am Chem Soc 134(12):5516–5519View ArticleGoogle Scholar
- Sun Q, Madan B, Tsai SL, DeLisa MP, Chen W (2014) Creation of artificial cellulosomes on DNA scaffolds by zinc finger protein-guided assembly for efficient cellulose hydrolysis. Chem Commun (Camb) 50(12):1423–1425View ArticleGoogle Scholar
- Dueber JE, Wu GC, Malmirchegini GR, Moon TS, Petzold CJ, Ullal AV, Prather KL, Keasling JD (2009) Synthetic protein scaffolds provide modular control over metabolic flux. Nat Biotechnol 27(8):753–759View ArticleGoogle Scholar
- You C, Myung S, Zhang YH (2012) Facilitated substrate channeling in a self-assembled trifunctional enzyme complex. Angew Chem Int Ed Engl 51(35):8787–8790View ArticleGoogle Scholar
- Hirakawa H, Nagamune T (2010) Molecular assembly of P450 with ferredoxin and ferredoxin reductase by fusion to PCNA. ChemBioChem 11(11):1517–1520View ArticleGoogle Scholar
- Liu F, Banta S, Chen W (2013) Functional assembly of a multi-enzyme methanol oxidation cascade on a surface-displayed trifunctional scaffold for enhanced NADH production. Chem Commun (Camb) 49(36):3766–3768View ArticleGoogle Scholar
- Tsai SL, DaSilva NA, Chen W (2013) Functional display of complex cellulosomes on the yeast surface via adaptive assembly. ACS Synth Biol 2(1):14–21View ArticleGoogle Scholar
- Borne R, Bayer EA, Pages S, Perret S, Fierobe HP (2013) Unraveling enzyme discrimination during cellulosome assembly independent of cohesin-dockerin affinity. FEBS J 280(22):5764–5779View ArticleGoogle Scholar
- Fan LH, Zhang ZJ, Yu XY, Xue YX, Tan TW (2012) Self-surface assembly of cellulosomes with two miniscaffoldins on Saccharomyces cerevisiae for cellulosic ethanol production. Proc Natl Acad Sci U S A 109(33):13260–13265View ArticleGoogle Scholar
- Geddes CC, Nieves IU, Ingram LO (2011) Advances in ethanol production. Curr Opin Biotechnol 22(3):312–319View ArticleGoogle Scholar
- Rizk M, Antranikian G, Elleuche S (2012) End-to-end gene fusions and their impact on the production of multifunctional biomass degrading enzymes. Biochem Biophys Res Commun 428(1):1–5View ArticleGoogle Scholar
- Liu M, Yu H (2012) Co-production of a whole cellulase system in Escherichia coli. Biochem Eng J 69:204–210View ArticleGoogle Scholar
- Levchenko A, Hu P, Janga SC, Babu M, Díaz-Mejía JJ, Butland G, Yang W, Pogoutse O, Guo X, Phanse S, Wong P, Chandran S, Christopoulos C, Nazarians-Armavil A, Nasseri NK, Musso G, Ali M, Nazemof N, Eroukova V, Golshani A, Paccanaro A, Greenblatt JF, Moreno-Hagelsieb G, Emili A (2009) Global functional atlas of Escherichia coli encompassing previously uncharacterized proteins. PLoS Biol 7(4):e1000096Google Scholar
- Fierobe HP, Gaudin C, Belaich A, Loutfi M, Faure E, Bagnara C, Baty D, Belaich JP (1991) Characterization of Endoglucanase A from Clostridium cellulolyticum. J Bacteriol 173(24):7956–7962Google Scholar
- Gaudin C, Belaich A, Champ S, Belaich JP (2000) CelE, a multidomain cellulase from Clostridium cellulolyticum: a key enzyme in the cellulosome. J Bacteriol 182:1910–1915View ArticleGoogle Scholar
- You C, Zhang XZ, Zhang YH (2012) Simple cloning via direct transformation of PCR product (DNA multimer) to Escherichia coli and Bacillus subtilis. Appl Environ Microbiol 78(5):1593–1595View ArticleGoogle Scholar
- Naik S, Kumru OS, Cullom M, Telikepalli SN, Lindboe E, Roop TL, Joshi SB, Amin D, Gao P, Middaugh CR, Volkin DB, Fisher MT (2014) Probing structurally altered and aggregated states of therapeutically relevant proteins using GroEL coupled to bio-layer interferometry. Protein Sci 23(10):1461–1478View ArticleGoogle Scholar
- Fierobe HP, Mingardon F, Mechaly A, Belaich A, Rincon MT, Pages S, Lamed R, Tardif C, Belaich JP, Bayer EA (2005) Action of designer cellulosomes on homogeneous versus complex substrates: controlled incorporation of three distinct enzymes into a defined trifunctional scaffolding. J Biol Chem 280(16):16325–16334View ArticleGoogle Scholar
- Wei ZH, Chen H, Zhang C, Ye BC (2014) FRET-based system for probing protein-protein interactions between sigma(R) and RsrA from Streptomyces coelicolor in response to the redox environment. PloS One 9(3):e92330View ArticleGoogle Scholar
- Prischi F, Konarev PV, Iannuzzi C, Pastore C, Adinolfi S, Martin SR, Svergun DI, Pastore A (2010) Structural bases for the interaction of frataxin with the central components of iron-sulphur cluster assembly. Nat Commun 1(95):1–10Google Scholar
- Maun HR, Wen X, Lingel A, de Sauvage FJ, Lazarus RA, Scales SJ, Hymowitz SG (2010) Hedgehog pathway antagonist 5E1 binds hedgehog at the pseudo-active site. J Biol Chem 285(34):26570–26580View ArticleGoogle Scholar
- Lu P, Feng MG (2008) Bifunctional enhancement of a beta-glucanase-xylanase fusion enzyme by optimization of peptide linkers. Appl Microbiol Biotechnol 79(4):579–587View ArticleGoogle Scholar
- Smith MC, Scaglione KM, Assimon VA, Patury S, Thompson AD, Dickey CA, Southworth DR, Paulson HL, Gestwicki JE, Zuiderweg ER (2013) The E3 ubiquitin ligase CHIP and the molecular chaperone Hsc70 form a dynamic, tethered complex. Biochemistry 52(32):5354–5364View ArticleGoogle Scholar
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