- Open Access
Efficient yeast surface-display of novel complex synthetic cellulosomes
© The Author(s) 2018
- Received: 31 January 2018
- Accepted: 1 August 2018
- Published: 7 August 2018
The self-assembly of cellulosomes on the surface of yeast is a promising strategy for consolidated bioprocessing to convert cellulose into ethanol in one step.
In this study, we developed a novel synthetic cellulosome that anchors to the endogenous yeast cell wall protein a-agglutinin through disulfide bonds. A synthetic scaffoldin ScafAGA3 was constructed using the repeated N-terminus of Aga1p and displayed on the yeast cell surface. Secreted cellulases were then fused with Aga2p to assemble the cellulosome. The display efficiency of the synthetic scaffoldin and the assembly efficiency of each enzyme were much higher than those of the most frequently constructed cellulosome using scaffoldin ScafCipA3 from Clostridium thermocellum. A complex cellulosome with two scaffoldins was also constructed using interactions between the displayed anchoring scaffoldin ScafAGA3 and scaffoldin I ScafCipA3 through disulfide bonds, and the assembly of secreted cellulases to ScafCipA3. The newly designed cellulosomes enabled yeast to directly ferment cellulose into ethanol.
This is the first report on the development of complex multiple-component assembly system through disulfide bonds. This strategy could facilitate the construction of yeast cell factories to express synergistic enzymes for use in biotechnology.
Lignocellulosic bioethanol has been widely proposed as a generate sustainable and environmentally-friendly biofuel to replace or complement bioethanol derived from starch or sugar [1, 2]. Consolidated bioprocessing (CBP), which combines cellulase production, cellulose hydrolysis and fermentation into a single process, is currently considered a promising method for bioethanol production owe to its potential low cost and high efficiency [3, 4]. Saccharomyces cerevisiae, a traditional ethanol producer, is an attractive candidate for the construction of CBP cell factories [5, 6]. However, as S. cerevisiae does not naturally produce cellulases, it cannot degrade cellulose into fermentable sugars.
Heterologous cellulases have been expressed in S. cerevisiae using three approaches: secreting the enzymes to the extracellular space, displaying the enzymes on the cell surface and assembling multienzymatic complexes on the cell surface in a structure named the cellulosome [7–10]. Cellulosomes from anaerobic bacteria contain noncatalytic scaffoldins consisting of one or more cellulose-binding domains (CBD) and repeated cohesin domains that can dock different cellulases, tagged with a dockerin domain, in the presence of Ca2+ ions . The highly ordered structure facilitates enzyme–enzyme synergy and host–enzyme–substrate proximity which allows efficient cellulose hydrolysis [11, 12]. Previously, heterologous cohesin–dockerin pairs from anaerobic bacteria, such as Clostridium thermocellum, C. cellulolyticum and Ruminococcus flavefaciens, were used for the self-assembly of cellulosome on the surface of yeast [13, 14]. Efforts have also been made to improve the functional efficiency of yeast cellulosomes. For example, the display level of scaffoldin was increased by replacing the the display system with that from the galactose induced a-agglutinin to constitutively expressed α-agglutinin . However, the number of cells with efficient display was still relatively low. Other approaches such as increasing the cohesin by using two scaffoldins instead of one could improve the assembly efficiency of cellulases [12, 16]. Despite progress in this field, the direct conversion of cellulose into ethanol using yeast cellulosomes remains challenging, while the limitations including the low scaffoldin display level, the inefficient self-assembly of cellulases on scaffoldin, and the low activity of cellulases.
In the cellulosome, the dockerin domains of the cellulase and cohesin domains of the scaffoldin interact through non-covalent bonds, including significant hydrophobic interactions and complementary hydrogen bonding . While most studies have directly introduced these dockerins and cohesins into S. cerevisiae, a previous study reported using the Z domain protein A and the Fc domain of human IgG as cohesion and dockerin, respectively, to regulate the assembly ratio of cellulosomal cellulases . The Z domain of staphylococcal protein A in Staphylococcus aureus is responsible for binding the Fc-portion of immunoglobulin G (IgG) through non-covalent bonds, which plays an important role in qualitative and quantitative immunology . This study indicated that protein pairs which can interact with each other, other than traditional cellulosomal cohesin–dockerin, could also be used for cellulosome assembly.
The S. cerevisiae cell adhesion protein a-agglutinin displayed on the cell surface is composed of surface cell wall anchorage subunit Aga1p and cell–cell binding subunit Aga2p, which is linked to Aga1p by the formation of two disulfide bonds [20, 21]. a-Agglutinin has been widely used as the yeast surface display system for anchoring heterologous proteins in numerous applications, including vaccine and antibody development, library screening, bioconversion, and biosorption [22–24]. The precursor of Aga1p contains a secretory signal peptide, a domain rich in threonine and serine residues and the glycosyl phosphatidylinositol (GPI) anchor . The N-terminal 149-residue fragment of Aga1p, named tAga1p, is responsible for the formation of a disulfide-linked complex with Aga2p . Thus, tAga1p and Aga2p could be a potential protein pair for the assembly of protein complexes, similar to the role of cohesin and dockerin.
In this study, we constructed novel synthetic cellulosomes using the Aga1p and Aga2p protein pair display system. The display level of novel scaffoldin ScafAGA containing repeated tAga1p domains was significantly improved compared with the benchmark display level of scaffoldin ScafCipA3 from C. thermocellum. The assembly of cellulases through disulfide bonds was also more efficient than that thought non-covalent bonds. A complex synthetic cellulosome with two scaffoldins, using ScafAGA as anchor scaffoldin II and ScafCipA3 as scaffoldin I, was also successfully constructed. This interaction system of Aga2p and repeated tAga1p domains can provide multiple docking units for heterologous proteins to improve their display efficiency, and therefore facilitate yeast to express synergistic enzyme systems in the future.
Design of synthetic scaffoldins and cellulosomes
Selection of cellulosomal cellulases
Functional assembly of cellulases on synthetic scaffoldins through disulfide bonds
Functional assembly of complex cellulosome through disulfide bonds
Optimization of cellulase activity and scaffoldin length increased ethanol production from PASC
We recently showed that engineering the yeast secretory pathway including protein translocation, protein folding and vesicle trafficking can improve the extracellular activity of cellulases [28, 29]. Therefore, three key components in the secretory pathway including disulfide isomerase Pdi1p, t-SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor proteins) Sso1p and v-SNARE Snc2p were over-expressed to further improve the cellulosomal cellulase activity. Compared with control strain PYX which contained both enzyme expression and empty plasmid pYX242WS, Sso1p overexpression further increased the extracellular activity of Tr-L-dBGL1 by 19.8%, while Snc2p overexpression improved the extracellular activity of PYD1-L-dCBH1 by 45.1% (Fig. 6d, e). Besides, we have previously shown that Pdi1p overexpression enhanced the extracellular activity of Ct-CelA by 17% .
As the number of dockerins in the scaffoldin also affects the assembly efficiency, we created ScafAGA5, which has five tAag1p repeat units. The percentage of stained cells for the ScafAGA5 expressing strain was 55.6%, which was similar to the value for the ScafAGA3 expressing strain (Fig. 6f).
Several studies have shown that displaying cellulosomes on the S. cerevisiae cell surface can enable yeast to produce ethanol from cellulose [12–14, 31]. However, the application of yeast cellulosomes was limited by the low display efficiency of scaffoldins, the length of scaffoldins and the inefficient secretion of cellulases. To improve the performance of yeast cellulosome, the cell surface display was optimized by comparing various yeast cell wall proteins, scaffoldin was complicated by increasing cohesion number and utilizing double-layered scaffoldins, and better catalytic activity was obtained by improving enzyme activity and adjusting the cellulase ratio [12, 14–16]. After this engineering, most of the stains only produced about 1 or 2 g/L ethanol from PASC. Although higher ethanol production (3.5 g/L) was obtained, the cellulases were produced in Escherichia coli and then assembled on the yeast surface in vitro (Tsai et al. ). All these synthetic cellulosomes were assembled through hydrophobic interactions and hydrogen bonds, which have relatively low stability. In contrast, covalent bonds were more stable than these non-covalent bands. To investigate whether covalent bonds could be applied to construct yeast synthetic cellulosomes, using covalent disulfide bonds, which are involved in maintaining correct protein structure and forming multimeric proteins, was tested in this work [32, 33]. Fortunately, yeast cell wall protein a-agglutinin is a double-subunit protein . Based on the a-agglutinin structure, we attempted to design Aga1p and Aga2p as a new cohesin and dockerin pair to assemble the cellulosome through disulfide bonds. Compared with traditional cellulosomes, the synthetic scaffoldins with repeating tAga1 units had a better display efficiency, while the synthetic cellulosomes had higher assembly efficiencies. Although the ethanol production was not as high as that of the previously reported cellulosome in yeast, the covalent bonds mediated cellulosome assembly improved the docking efficiency of the cellulases.
Based on previous studies, synthetic cellulosomes are a combination of multiple independent elements, including cohesin, dockerin and cellulases. Firstly, synthetic cellulosomes were constructed using cellulosomal cellulases which natively contained dockerin. Furthermore, free cellulases, which did not contain native dockerins, were fused to dockerins to successfully assemble cellulosome. Furthermore, immune proteins that interact with each other through non-covalent bonds have also been used as new cohesin–dockerin pair [14, 19]. Accordingly, scaffoldins have been further optimized by increasing the number of cohesion domains or using double-layered scaffoldins . Therefore, we also integrated the new cohesin–dockerin pair into traditional cellulosomes. As shown in the results, scaffoldin consisting of five tAga1 s was displayed on the cell surface and used as the primary scaffoldin, and the traditional scaffoldin composed of three cohesins from C. thermocellum CipA was used as the secondary scaffoldin. The double-layered cellulosome was successfully assembled and applied to ethanol production.
We found that dockerin fusion significantly decreased the extracellular activity of Sf-BGL1, perhaps because the fusion of dockerin affects the structure of the enzyme. In a previous study, a linker between the two protein domains was required for the proteins to function well [34, 35]. Our results showed that a proper linker was required between the dockerin and catalytic domains, which could enhance the catalytic activity significantly. The secretion or display of heterologous proteins is often limited by protein misfolding and inefficient transport [36, 37]. Engineering the secretory pathway (including protein translocation, protein folding, glycosylation and vesicle trafficking) is an effective strategy for improving the production of heterologous proteins in S. cerevisiae . Herein, we strengthened the protein folding and vesicle trafficking, resulting in a significant increase in the secretion of these cellulases. However, the cellulase activities were still relatively low. Therefore, engineering one or two components in the secretory pathway seems to only increase cellulase secretion slightly, with multiple engineering strategies remaining necessary to further increase protein secretion.
The co-incorporation of multiple enzymes in close proximity allows the direct transfer of intermediates among catalytic sites, reducing the diffusion of intermediates and significantly improving the catalytic efficiency . Three dockerin-fused enzymes including AlsS, AlsD, and Bdh1 were assembled onto the scaffoldin, which was displayed on the yeast surface, and this multi-enzyme complex increased the 2,3-butanediol production clearly compared with free enzymes . A cascade of redox enzymes involved in multiple electron-release through fuel oxidation was fused with a dockerin and then attached to the scaffoldin, with the multi-enzyme complex showing enhanced performance compared with traditional biofuel cells . The tAga1–Aga2p pair in our work affords a new approach to the assembly of multi-enzyme complexes. Furthermore, other forms of covalent bonds could be explored to assemble the multi-enzyme complex. For instance, the protein that incorporates an unnatural amino acid (tyrosine analogues Ffact) can react with another active cysteine-containing protein based on the covalent reaction between Ffact and cysteine . The utilization of functional groups of unnatural amino acids may be another potential approach.
In this study, a complex multiple-component assembly system constructed through disulfide bonds was developed for the first time. When applied to synthetic cellulosome construction, this system showed great advantages, such as increasing the display and assembly efficiency of the cellulosomes. This system provides multiple docking units for proteins and is suitable for expressing several synergistic enzymes, and will, therefore, have wide applications in synergistic catalysis.
Strains and media
Escherichia coli strain Trans2-blue (TransGene Biotech, Beijing, China) was used for plasmid propagation. S. cerevisiae CEN.PK102-5B (MATa; URA3-52, HIS3Δ1, LEU2-3,112) was used as the host for cellulosome construction . E. coli was cultivated in Luria–Bertani (LB) medium (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl) with 100 μg/mL ampicillin. CEN.PK102-5B strain was grown in YPD medium (20 g/L tryptone, 5 g/L yeast extract, 20 g/L glucose). All recombinant yeast strains (Additional file 6: Table S2) were cultivated in SC–SCAA medium as reported previously without uracil, leucine or histidine [29, 44].
Plasmid and strain construction
The primers and plasmids used in this work are shown in Additional file 5: TableS1 and Additional file 6: Table S2. Primers and genes were synthesized by Genewiz (Beijing, China). For cellulosome construction, expression vectors for scaffoldins and cellulases were constructed using the Gibson assembly . Firstly, AGA1 was amplified from the S. cerevisiae genome and cloned into plasmid pJFE3  to construct pJFE3-AGA1. For the construction of a ScafCipA3 expression plasmid, the CipA3 fragment containing the first three cohesins and the CBD of scaffoldin CipA was amplified from C. thermocellum genomic DNA, and inserted into pYD1 to construct pYD1-CipA3. A fragment containing AGA2, CipA3 and V5 tag was obtained by PCR from pYD1-CipA3 and ligated into pIYC04  to form plasmid pIYC04-CipA3–AGA2. Finally, the fragment containing promoter PGK1, AGA2, CipA3, V5 tag and terminator CYC1 was amplified and cloned into pJFE3-AGA1 to generate plasmid ScafCipA3. For construction of novel scaffoldin ScafAGA3, two tAga1p with a CBD from Trichoderma reesei CBH1 were synthesized and inserted into pJFE3 to afford pJFE3-2AGA1. AGA1 without a signal peptide was amplified and inserted into pJFE3-2AGA1 to construct plasmid ScafAGA3. The C-terminal 687 bp of SED1 was amplified and ligated into pJFE3-2AGA1 to afford plasmid 2AGA-SED. The genes encoding exoglucanases from Talaromyces emersonii, H. grisea and C. thermophilum (synthesized by Genewiz), endoglucanases from Trichoderma reesei, C. thermocellum and C. cellulolyticum (amplified from the corresponding genomic DNA) and β-glucosidase (amplified from plasmid pTH-BGL ) were fused with the corresponding dockerin derived from C. thermocellum CelS, CelA, and XynC and ligated into pIYC04. C. thermocellum endoglucanase CelA, T. emersonii exoglucanase CBHI, and S. fibuligera β-glucosidase BGL1 were fused with AGA2 and were also inserted in pIYC04. Linkers derived from T. reesei CBH1, C. cellulovorans EngB and commercial plasmid pYD1 were inserted between the dockerin domain and catalytic domain of the respective cellulosomal cellulases. The recombinant plasmids are described in Additional file 6: Table S2. These plasmids were transformed into S. cerevisiae CEN.PK102-5B and the resulting recombinant strains are shown in Additional file 7: Table S3.
Enzymes activity measurement
The activity of cellobiohydrolase was quantified using p-nitrophenyl-β-d-cellobioside (pNPC) (Sigma, USA) as the substrate as described previously . Enzymes were incubated in 50 mM citrate buffer (pH 4.8) with 2 mM pNPC at 50 °C for 30 min. The reaction was stopped by the addition of 10% sodium carbonate and p-nitrophenol (pNP) released from pNPC was determined at 405 nm. Endoglucanase activity was measured using carboxymethylcellulose sodium salt (CMC-Na; Sigma, USA) as the substrate . Reducing sugars from hydrolyzed CMC-Na were boiled with dinitrosalicylate (DNS) for 10 min and then detected at 540 nm. β-Glucosidase activity was determined as described previously using p-nitrophenyl-β-d-glucopyranoside (pNPG; Sigma, USA) as the substrate . Enzymes were mixed in 50 mM citrate buffer (pH 5.0) with 5 mM pNPG at 50 °C for 30 min. 10% sodium carbonate was added to stop the reaction and pNP released from pNPG was detected at 405 nm. One unit of the enzyme activity was defined as the amount of enzyme that released 1 μmol of product (p-nitrophenol or glucose) from the substrate at 50 °C in 1 min.
Immunofluorescence assay and FACS analysis
Saccharomyces cerevisiae CEN.PK 102-5B with different phenotypes (Additional file 7: Table S3) were cultured for 24 h for analysis. To detect the assembly efficiency of traditional dockerin and cohesin, secreted cellulases with dockerin in the supernatant were concentrated about tenfold and incubated with strains expressing scaffoldin ScafCipA3 at an OD600 of 2, 5 and 15. The secreted cellulases incubated with ScafCipA3 expressing strains without concentration were used as controls. Cells were harvested and washed twice with phosphate-buffered saline solution (PBS, pH 7.0). The cells were then suspended in PBS containing 1 mg/mL bovine serum albumin to an OD600 of 1.0. Monoclonal mouse antibodies including anti-V5-FITC antibody (Invitrogen) for scaffoldin, anti-DDDDK tag antibody (DyLight 488; Abcam, UK) for BGL1 and CBHI, and anti-Myc tag antibody (fluorescein isothiocyanate conjugated [FITC]; Abcam, UK) for CelA were used for immunofluorescence microscopy and FACS analyses. The antibodies were mixed with the cell suspension at 25 °C for 1 h with 1:500 dilution. Cells were harvested and washed twice with PBS after staining. Images were taken using immunofluorescence microscopy (Olympus, Japan) and flow cytometry analysis (FACS) was performed using a FACSCanto II system (BD FACSCanto II, USA).
Recombinant strains expressed exoglucanase, endoglucanase, β-glucosidase and different scaffoldins including ScafCipA3, ScafAGA3, both ScafAGA3 and ScafCipA3, and both ScafAGA5 and ScafCipA3, respectively. These strains were first pre-cultured in SC-SCAA medium at 30 °C for 48 h. The scaffoldin-displaying strains were incubated with the supernatant of cellulase-expressing strains at OD600 ratios of 1:1:1:1 or 1:1.25:1.25:0.5 for 4 h to allow cellulosome assembly. For ScafCipA3, the reaction was supplemented with 10 mM CaCl2. Cells with cellulosomes were washed twice and cultivated in YP medium with 1% phosphoric-acid-swollen cellulose (PASC) to an OD600 of 50. PASC was prepared from Avicel microcrystalline cellulose (Sheng Gong, China) as described previously . Fermentation was performed anaerobically in 100-mL flasks containing 40 mL of culture. Samples (1 mL) were taken out periodically and the ethanol concentration was analyzed by HPLC using an Aminex HPX-87 H column (Bio-Rad, Richmond, CA) with 5 mmol/L H2SO4 as the mobile phase at a flow rate of 0.6 mL/min, at 45 °C. Peaks were detected using a RID-10A refractive index detector (Shimadzu, Kyoto, Japan).
HT and JH designed the experiments. HT, JW and SW conducted the experiments. HT, JH, DP, YS and XB prepared and edited the manuscript. All authors read and approved the final manuscript.
We thank Jing Zhu and Zhifeng Li from the Analysis and Testing Center of SKLMT (State Key Laboratory of Microbial Technology, Shandong University) for their assistance with flow cytometry and gene expression analyses. We thank Simon Partridge, Ph.D., from Liwen Bianji, Edanz Editing China (http://www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
All data generated or analyzed during this study are included in this published article and in additional files.
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Ethics approval and consent to participate
This work was supported by the National Natural Science Foundation of China (31711530155, 31470219 and 31300037), Natural Science Foundation of Shandong Province (ZR2017ZB0210), the Key R&D Program of Shandong Province (2017GSF21110), State Key Laboratory of Microbial Technology, Young Scholars Program of Shandong University and Key Laboratory of Chemical Biology (Ministry of Education) Open Projects Fund.
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