Incorporation of Nasutitermes takasagoensis Endoglucanase into Cell Surface-Displayed Minicellulosomes in Pichia pastoris X33
Incorporation of Nasutitermes takasagoensis Endoglucanase into Cell Surface-Displayed Minicellulosomes in Pichia pastoris X33
Journal of Microbiology and Biotechnology. 2014. Sep, 24(9): 1178-1188
Copyright © 2014, The Korean Society For Microbiology And Biotechnology
  • Received : February 18, 2014
  • Accepted : May 20, 2014
  • Published : September 28, 2014
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Jingshen, Ou
Yicheng, Cao

In this study, the yeast Pichia pastoris was genetically modified to assemble minicellulosomes on its cell surface by the heterologous expression of a truncated scaffoldin CipA from Clostridium acetobutylicum . Fluorescence microscopy and western blot analysis confirmed that CipA was targeted to the yeast cell surface and that NtEGD, the Nasutitermes takasagoensis endoglucanase that was fused with dockerin, interacted with CipA on the yeast cell surface, suggesting that the cohesin and dockerin domains and cellulose-binding module of C. acetobutylicum were functional in the yeasts. The enzymatic activities of the cellulases in the minicellulosomes that were displayed on the yeast cell surfaces increased dramatically following interaction with the cohesin-dockerin domains. Additionally, the hydrolysis efficiencies of NtEGD for carboxymethyl cellulose, microcrystal cellulose, and filter paper increased up to 1.4-fold, 2.0-fold, and 3.2-fold, respectively. To the best of our knowledge, this is the first report describing the expression of C. acetobutylicum minicellulosomes in yeast and the incorporation of animal cellulases into cellulosomes. This strategy of heterologous cellulase incorporation lends novel insight into the process of cellulosome assembly. Potentially, the surface display of cellulosomes, such as that reported in this study, may be utilized in the engineering of S. cerevisiae for ethanol production from cellulose and additional future applications.
Natural cellulose degradation is coordinated by a multitude of bacterial and fungal enzymes. Free-state cellulase systems occur in most fungal and other eukaryotic organisms in addition to prokaryotic microorganisms. Most cellulases consist of either a catalytic domain and cellulose-binding module (CBM) or a catalytic domain in a single polypeptide chain. The CBM targets the catalytic module to the cellulose surface and initiates the disruption and degradation of cellulose chains. Different types of free cellulases are thus distributed randomly and interact freely in a synergistic manner [21] . In contrast with the free cellulase systems of aerobic microorganisms, anaerobic bacteria produce relatively small quantities of enzymes to achieve efficient cellulose degradation [2] . Cellulosomes that are produced by certain anaerobic bacteria, such as Clostridium thermocellum and C. cellulyticum , have supermolecular architectures. They are multienzyme complexes that specialize in cellulose degradation and are mainly composed of a pivotal noncatalytic subunit, scaffoldin, which incorporates various enzymatic subunits into the complex via cohesin-dockerin interactions [58] . Cellulosomes are assembled by the tenacious binding of scaffoldin-based cohesin modules to enzyme-borne dockerin domains in a species-specific manner, which ensures the synergistic and orderly binding of enzymes to pertinent substrates through target and proximity effects to facilitate cellulose degradation [1 , 8 , 10 , 26 , 30] . Compared with the free wild-type enzymes, cellulases that are incorporated into cellulosomes have been shown to exhibit approximately 1.5- to 2.6-fold greater activities [32 , 36 , 37] . Therefore, cellulosomes are considered to be more efficient than cellulase in the free state.
Clostridium acetobutylicum ATCC 824 is widely used in industrial applications. It is an anaerobic, mesophilic, spore-forming bacterium that contains a cellulosome gene cluster but cannot utilize cellulose to produce acetonebutanol-ethanol solvents [38 , 43] . Previous research into the genetic modification of C. acetobutylicum for the direct use of cellulose for ABE fermentation have been unsuccessful [27 , 34 , 35 , 41] . Notably, the scaffolding protein CipA can be bonded with cellulases by cohesin-dockerin interactions [44] . In fact, the truncated CipA containing a CBM and two cohesin proteins was assembled with a Cel48A endoglucanase from C. acetobutylicum to form a minicellulosome, and its cellulose binding capacity was approximately 10-fold higher than those of C. cellulovorans and C. thermocellum [44] . Cellulosomes from C. thermocellum , C. cellulolyticum , and C. cellulovorans have also been expressed in yeast, but there are no reports describing the heterologous expression of C. acetobutylicum cellulosomes in yeast [20 , 28 , 29 , 38 , 49 , 52 , 55 , 56 , 62] . It has been suggested that the use of noncellulosome cellulases for the design of cellulosome assembly may be biased [6] . For example, some studies have revealed that when free-state cellulase is incorporated into cellulosomes by a dockerin that has been appended to its terminus, the dockerin fusion site affects its enzymatic activity. In some cases, the specific activity of the incorporated cellulase is only slightly enhanced during soluble and amorphous cellulose hydrolysis but is significantly enhanced during Avicel hydrolysis [5 - 7] . When the endoglucanases Cel48 and Cle9 from C. thermocellum were fused with additional CBM or dockerin and assembled to minicellulosomes, Avicel hydrolysis rates in the modified cellulosomes increased, but amorphous cellulose hydrolysis rates decreased. In contrast with the wild-type cellulosomes, Avicel hydrolysis rates of the modified cellulosomes that had been fused with an additional CBM or dockerinappended cellulases decreased [33] . Previous studies have also shown that the glycoside-hydrolase family 9 is a main cellulolytic enzyme family in C. thermocellum , C. cellulovorans , C. josui , and C. cellulolyticum [3 , 4 , 11 - 14 , 17 , 24 , 42 , 45 , 50] . The endo-β-1,4-glucanase NtEG (448 aa in length) from the termite Nasutitermes takasagoensis belongs to glycosidehydrolase family 9 and exhibits a hydrolytic activity of 1,200 units/mg protein for carboxymethyl cellulose [22] . The NtEG can synergize with β-glucosidase to hydrolyze cellulose to glucose [53] . Thus, as the candidate cellulase in the cellulosomes, NtEG may enhance the cellulolytic activities of the designed minicellulosomes that are expressed in yeast.
Recently, several yeast cell-surface display (CSD) systems have been developed and applied [31 , 48 , 57] . Most of the CSD methods for yeast are based on the agglutinin and flocculin models [46] . An endoglucanase that was fused with several CBMs was expressed on yeast cell surfaces and showed some increases in both binding affinity and hydrolysis activity [23] . To date, several studies have successfully used the cell wall proteins Aga1 and Cwp2 to display minicellulosomes on the S. cerevisiae cell surface [29 , 56 , 62] . Previous studies have also revealed that the yeast cell wall protein Flo1p is more efficient than Aga1 [19 , 31 , 61] . Thus, the Flo1p protein was used as the CSD protein in our study. In contrast with S. cerevisiae , the wildtype P. pastoris strains confer several advantages in the production of recombinant heterologous proteins, such as the ease of growth to high cell densities, high levels of protein expression at the intra- or extra-cellular level, and less elaborate hyperglycosylation [9 , 60] . The less elaborate hyperglycosylation of P. pastoris in particular may be beneficial to our study, because the deglycosylation of cellulosomal enzymes enhances cellulosome assembly in yeast [49] .
Our previous study incorporated the Thermomonospora fusca endoglucanase E4 catalytic domain (CAD) and the dockerin of C. acetobutylicum Cel48A into unanchored freestate minicellulosomes, which resulted in the successful enhancement of the E4 CAD hydrolysis rates of CMC and microcrystalline cellulose [39] . To gain further knowledge regarding the use of the CSD system for minicellulosome degradation with N. takasagoensis endoglucanases, the minicellulosomes were anchored on the P. pastoris cell surfaces in this study, which were composed of the following: (i) the scaffoldin CipA and dockerin from C. acetobutylicum , (ii) the endoglucanase NtEG from the termite Nasutitermes takasagoensis , and (iii) the cell wall protein FS domain from S. cerevisiae ( Fig. 1 C). We aimed to explore whether the CipA of C. acetobutylicum is functional in yeast and whether NtEG may operate in cellulosomes following fusion with a dockerin tag. The results that are presented here indicate that the truncated scaffoldin from C. acetobutylicum may be successfully expressed on yeast cell surfaces and assembled with other species’ cellulases by cohesin-dockerin interactions. In addition to comparing the enzyme activities of free-state cellulases with those that had been incorporated into minicellulosomes, we compared designed free-state minicellulosomes that had been anchored onto yeast cell surfaces.
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Design of a yeast cell surface system to assemble minicellulosomes. (A) The construction of the pPFSCipA plasmid for the expression of the fusion protein FSCipA. (B) The construction of the pGND plasmid for the expression of the protein NtEGD. (C) Cell surface-displayed minicellulosome schema using CipA. CipA: C. acetobutylicum scaffoldin. CBM: cellulose-binding module; Coh: cohesin; FS: N-terminal Flo1P flocculation domain.
Materials and Methods
- Strains, Plasmids, and Media
All yeast and bacterial strains that were used in this study and their relevant genotypes are listed in Table 1 . Escherichia coli cells were cultivated in Luria-Bertani broth (10 g peptone/l, 5 g yeast extract/l, and 5 g NaCl/l) at 37℃. Ampicillin for selecting and proliferating the transformants was added in a final concentration of 50 mg/l. Pichia pastoris X33 cells were cultivated at 30℃ in either YPD medium (containing 20 g peptone/l, 10 g yeast extract/l and 20 g glucose/l) or BMMY medium (containing 10 g yeast extract/l, 20 g peptone/l, 13.4 g yeast nitrogen base/l, 100 mM potassium phosphate buffer (pH 6.0), and 10 ml methanol/l). C. acetobutylicum was cultured anaerobically in 2× YTG medium (containing 16 g peptone/l, 10 g yeast extract/l, 4 g NaCl/l, and 5g glucose/l, pH 5.8). Solid media containing 2% agar were used for the transformant selection and maintenance. The X33-B2-E2 strain expressing the NtEG and CBM fusion protein B2-E2 was constructed in our laboratory. MiniCipA and E4SD proteins were expressed and purified from E. coli in our previous study [39] .
Microbial strains and plasmids used in this study.
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ATCCa, American Type Culture Collection.
- Cloning Strategy and Plasmid Construction
Standard procedures for the isolation and manipulation of DNA were used throughout this study [47] . Restriction enzymes, T4 DNA ligase (Thermo Fisher Scientific Inc.) and KOD Plus DNA polymerase (Toyobo Co. Ltd, Shanghai, China) were also used to carry out these procedures.
To express portions of the C. acetobutylicum cellulosome on the cell surface of P. pastoris , the truncated CipA was cloned from C. acetobutylicum , and the lectin-like anchor flocculation protein (Flo1p) from S. cerevisiae that facilitated GPI-mediated anchoring on the yeast cell wall was cloned from the plasmid pKFS [48] . CipA was cloned from the C. acetobutylicum CipA scaffoldin gene, which encodes an N-terminal CBM, four hydrophilic domains, and two cohesin modules. The CipA expression cassette was inserted under the control of the P. pastoris AOX promoter and terminator sequences in the vector pPFSCipA. In this study, the N-terminal CBM was fused to the yeast N-terminal flocculation functional domain FS.
The PCR primers that were used in this study to amplify the fragments of several genes are listed in Table 2 . The plasmid DNAs that were used as templates to amplify the respective gene fragments are listed in Table 1 . All of the PCR-generated fragments were sequenced by Invitrogen Co. Ltd. (Guangzhou, China).
Primers used to amplify gene fragments.
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aRE, restriction enzyme site. Restriction enzyme sites included in primer sequences for cloning purposes are indicated in bold.
To construct the yeast CSD vector pPFS, the FS fragment from pKFS was digested by Bgl II and Eco RI and cloned into pGAPZα to generate pPFS with the N-terminal functional flocculation domain FS. The 2,451 bp fragment of the C. acetobutylicum CipA was amplified by PCR from the C. acetobutylicum ATCC824 genome with the primers CipAF/CipAR and then cloned into pPFS between the Kpn I and Not I sites. Finally, pPFSCipA, containing the AOX P -FS-CipA-myc-His 6 -AOX T expression cassette, was constructed ( Fig. 1 A).
The dockerin fragments of Cel48A were amplified from the C. acetobutylicum ATCC824 genome by PCR. The NtEG (GenBank Accession No. AB013272.2) fragments were amplified from pBluescript-NtEG [53] . The NtEG and dockerin fragments that had been generated by PCR were linked by overlapping extension, thereby generating NtEGD fragments, which were then digested with Kpn I and Not I and cloned into pGAPZαA to obtain the vector pGND containing the GAP P -α-factor-NtEGD-myc-His6-AOX T expression cassette ( Fig. 1 B).
All of the bacterial transformations and DNA isolations were carried out according to standard protocols [47] . All of the yeasts were transformed via electrotransformation under the following conditions: 1.5 KV, 50 μF, and 200 Ω.
- Immunofluorescence Microscopy
Immunofluorescence microscopy was carried out according to a previous study [48] . Immunostaining was performed as follows: the parent yeast X33 and transformant X33-FSCipA were cultivated for 96 h at 30℃ in BMMY medium. Cultivated cells were centrifuged and washed twice in ice-cold water and resuspended at 4℃ in PBS (pH 7.4, containing 1 mg/ml BSA). The primary antibody (anti-myc mouse mAb; Abmart, Shanghai, China) against the myc-tag of the FSCipA fusion protein, which was diluted 1:800, was added to the cells, which were then slowly agitated on a shaker at room temperature for 2 h. Next, the cells were washed with and again resuspended in PBS (containing 1% BSA), and the second antibody (goat anti-mouse IgG-dylight-488; Earthox LLC., CA, USA) was added at a dilution of 1:500 and incubated for 1 h at room temperature. Subsequently, the cells were washed three times with PBS (pH 7.4) and examined at 1,000× magnification using fluorescence microscopy.
- Cellulose-Binding Capacity of Cell-Wall-Targeted CipA Protein in P. pastoris
The cellulose-binding capacity as mediated by the CipA CBM was used to visually detect the expression of the CipA protein on the cell wall of P. pastoris . The X33-ND cells and the mixture of X33-ND/X33-FSCipA were both cultured for 96 h at 30℃ in selective medium containing Whatman 1 MM filter paper (cut to 2 × 1 cm in size). Then, the filter papers were removed carefully and rinsed three times with excess 50 mM HEPES buffer (pH 7.0) before microscopic examination [29] . A total of eight fields each for X33-ND and X33-ND/X33-FSCipA were examined to rule out cellulose-binding artifacts.
- Cell Wall Isolation and Protein Extraction
The cell wall protein extractions were carried out as described previously [48] . Briefly, cells were harvested by centrifugation at 5,000 × g and washed three times with pre-chilled 10 mM Tris-HCl (pH 7.8) containing 1 mM PMSF. The cells, buffer, and acidwashed glass beads were mixed at 1:2:1 (wet w/v/w) and agitated vigorously with incubation on ice for 1 min between each step. The cell walls were collected by centrifugation at 10,000 × g and washed three times with the same buffer. Cell walls from 10 9 cells were treated with 0.2 U laminarinase in 50 mM sodium acetate buffer (pH 5.4) and incubated overnight at 37℃. Then, the extracts were separated by centrifugation at 10,000 × g .
- Western Blot Detection
For the detection of the heterologously expressed FSCipA, the cell-wall-associated protein fractions that had been isolated from X33 and X33-FSCipA cells were analyzed using western blots. The primary antibody (anti-myc mouse mAb; Abmart, Shanghai, China) against the myc-tag of the FSCipA fusion protein and the secondary goat anti-mouse IgG antibody (1:2,000 dilution ratio), which was conjugated with horseradish peroxidase, were used and the protein bands were visualized with enhanced chemiluminescencedetection reagents.
For the detection of the cohesin-dockerin interactions, the supernatants of the X33-ND cultures containing the enzyme NtEGD were concentrated by ultrafilter membranes. The X33 and X33-FSCipA cells were centrifuged and washed with 0.1 M sodium acetate buffer (pH 5.8) containing 5 mM CaCl 2 . The NtEGD was added to both the X33 (native control) and X33-FSCipA washed cells, which were then incubated for 1 h at room temperature. Subsequently, they were centrifuged and washed three times with 0.1 M sodium acetate buffer (pH 5.8) containing 5mM CaCl 2 and resuspended in 0.25 M EDTA buffer to facilitate the chelation of Ca 2+ and elution of the NtEGD proteins from the cellulosomes [25] . The EDTA-eluted fractions were dialyzed and concentrated by ultrafiltration. The X33-ND supernatants (used as control) and elution fraction were subjected to western blots as described above.
- Enzyme Activity Assays
To compare the hydrolysis activities of NtEGD in both the freestate and organized minicellulosomes, the X33-FSCipA cells were grown for 120 h at 30℃ in BMMY medium to facilitate the expression of CipA on the cell surfaces, and the X33-ND cells were grown for 50 h at 30℃ in YPD medium to allow for the secretion of the NtEGD proteins. The X33-FSCipA cell cultures were centrifuged at 3,000 × g for 5min, washed, and resuspended in sodium acetate buffer (pH 5.8) to a final absorbance of 10 at 600 nm. The X33-ND cultures were centrifuged at 5,000 × g for 10 min, and the NtEGD in the supernatants was concentrated using 10k Amicon Ultra-15 filters (Merck Millipore Ltd. Co., Cork, IRL) and then mixed with the X33-FSCipA cells to a final absorbance of 5 at 600 nm. The mixtures were incubated for 1 h at room temperature to allow for the assembly of cell surface minicellulosomes. The CipA and E4SD proteins were added to final concentrations of 0.6 μM as described previously [39] .
The recombinant enzymes and minicellulosomes were incubated with 1% (w/v) sodium carboxymethyl cellulose for 30 min and 1% (w/v) microcrystal cellulose or filter paper, respectively, for 4 h in 0.1 M sodium acetate-10 mM Ca 2+ buffer (pH 5 .8) at 37℃. Reduced sugars were detected using the dinitrosalicylic acid reagent [18] and calculated as glucose equivalents. One unit of enzyme activity was defined as the amount of enzyme producing one μmol of reduced sugar in glucose equivalents per hour.
- Immunofluorescence Microscopy of X33-FSCipA Cells
CipA in the X33-FSCipA cells was detected using immunofluorescence microscopy. The cells were cultivated for 120 h in BMMY medium. Green fluorescence was observed around the edges of the X33-FSCipA cells ( Fig. 2 B). In contrast, no green fluorescence was observed on the control cells ( Fig. 2 A). These results indicated that the fusion protein FSCipA was expressed and anchored on the surfaces of the X33-FSCipA cells.
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Immunofluorescent detection of the fusion protein FSCipA at 1000× magnification. (A) The cells of the parent Pichia pastoris X33 without the fusion protein FSCipA construct as a control. (B) The cells of P. pastoris X33-FSCipA expressing the FSCipA fusion protein. Cells were labeled with the primary antibody (mouse anti-Myc mAb) and the goat antimouse IgG-dylight-488 as the secondary antibody. The left column depicts the light microscopy image, and the right column shows the fluorescent image.
- Targeting of CipA on Yeast Cell Surface
To localize the fusion protein FSCipA, the glucanaseextracted fractions were subjected to SDS-PAGE, blotted onto a PVDF membrane, and then immunostained as described above. In this study, we used the Protease Inhibitor Cocktail Set I (Merck KGaA, Darmstadt, Germany) and PMSF, and did not observe any differential effects between the two.
FSCipA was detected in the glucanase-extracted fraction of X33-FSCipA ( Fig. 3 A, lane 1), indicating that the fusion protein was attached to the cell wall. The molecular mass of FSCipA was higher than 260 kDa ( Fig. 3 A, lane 1) and was thus larger than the calculated value of 208 kDa. This was most likely due to the glycosylation of the protein, which has been described previously [51] .
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Western blot assay results. (A) Western blot detection of the FSCipA fusion protein. Lane 1, the glucanase extraction of strain X33-FSCipA; lane 2, the glucanase extraction of the parent strain X33 (used as a control). (B) Western blot detection of cohesin and dockerin interaction. Lane 1, the elution fraction of NtEGD mixed with X33 cells; lane2, the elution fraction of NtEGD mixed with X33-FSCipA cells; lane 3, the X33-ND supernatant as a control.
To further detect the localization and function of the CipA protein on the cell surface of P. pastoris , a microscopic inspection of its cellulose-binding capacity was performed. This binding was mediated by the CBM of the CipA protein anchor on the cell surface. The CBM of the C. acetobutylicum CipA has a higher affinity for Avicel than those of C. cellulovorans and C. thermocellum [44] . Thus, when CipA is displayed on the P. pastoris X33-FSCipA cell surface, the yeast cell may tightly attach to cellulose fibers. As shown in Fig. 4 , the attachment of the yeast cells to the filter paper could be clearly detected for the co-cultured strains X33-ND/X33-FSCipA ( Figs. 4 C- 4 D), but almost none of the cells were attached to the cellulose fibers in the single strain X33-ND culture ( Figs. 4 A- 4 B) because the surface of the parent P. pastoris cell lacked CBM. These results further suggest that CipA was targeted to and acted on the cell surfaces of the yeasts displaying the designed minicellulosomes.
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Microscopic images (at 1,000× magnification) of (A)~(B) the Whatman No. 1 filter paper after culturing with the P. pastoris X33-ND strain expressing NtEGD, and (C)~(D) the expression of the FSCipA protein in the P. pastoris X33-FSCipA and X33-ND co-culture stains. The attachment of the yeast cells to the filter paper was clearly visible in the P. pastoris X33-FSCipA and X33-ND co-culture stains. Solid arrows indicate cellulose fiber; dashed-line arrows indicate yeast cells.
- Functional NtEGD Fusion Expression and Cohesin-Dockerin Interactions in Displayed Minicellulosomes
The cohesin-dockerin interaction was calcium-dependent. If Ca 2+ had been chelated by EDTA in the system, then the cohesin-dockerin complex would disassemble [1 , 8 , 10 , 26 , 30] . To determine whether the dockerin in NtEGD and the cohesin in FSCipA interact, the X33-ND supernatants were mixed with the parent yeast X33 and with the transformant X33-FSCipA for 1 h at room temperature with slow shaking. The mixtures were washed and eluted as previously reported [25] , and the two eluted fractions and X33-ND supernatants (used as controls) were subjected to western blot analyses ( Fig. 3 B). NtEGD was detected in the X33-FSCipA eluted fraction ( Fig. 3 B, lane 2) but not in the eluted fraction of the blank control ( Fig. 3 B, lane 1, X33 cells). These results indicate that the cohesin of CipA interacted with the dockerin of NtEGD in the yeast expressing FSCipA and that the NtEGD was able to be grafted.
- Hydrolysis Activities of Cellulases Both in Free State and Incorporated Into Minicellulosomes in the Degradation of Three Types of Celluloses
To compare the degradation capabilities of the free cellulases with those of the cellulases that were organized into minicellulosomes, hydrolysis efficiencies in parallel hydrolysis reaction conditions were measured. These reactions were set up using three types of cellulose substrates; namely, carboxymethyl cellulose (CMC), microcrystalline cellulose (MC), and filter paper (FP). In these reactions, NtEGD was mixed with free-state miniCipA, X33-FSCipA cells, or NtEGD as a control.
As shown in Fig. 5 A, there was only unanchored miniCipA in the free state and anchored miniCipA in the X33-FSCipA cells, and no cellulases were involved; thus, no significant cellulose hydrolysis was measured.
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Hydrolysis activities of the free-state cellulases, cell-free minicellulosomes, and yeast cell surface-displayed minicellulosomes on CMC, microcrystal cellulose (MC), and filter paper (FP) degradation. (A) MiniCipA protein and X33-FSCipA cells; (B) NtEGD mixed with X33FSCipA without Ca2+, and B2-E2, in which the dockerin of NtEGD was replaced by CBM, mixed with X33-FSCipA with Ca2+; (C) and (D) E4SD mixed with free-state miniCipA and X33-FSCipA cells, respectively; (E) NtEGD mixed with free-state miniCipA and X33-FSCipA cells to form minicellulosomes, respectively; and (F) β-glucosidase added with NtEGD and its cellulosome. B2-E2: NtEG fused with a CBM; NtEGD: endoglucanase NtEG fused with a dockerin; E4SD: endoglucanase E4 CAD fused with a dockerin; X33-FSCipA: the P. pastoris X33-FSCipA cell.
Even when NtEGD was mixed with X33-FSCipA in reaction buffers lacking Ca 2+ , only slight enhancements in CMC hydrolysis were observed ( Fig. 5 B). Because the cohesin-dockerin interaction is calcium dependent [1 , 8 , 10 , 26 , 30] , if the reaction buffer lacks Ca 2+ , they will not associate, and no cellulosomes will be assembled. Similar observations were also reported in our previous study (data not published), in which minicellulosomes could not be organized in X33-FSCipA cells that had been mixed with the enzyme B2-E2 because the dockerin of NtEGD was replaced by CBM.
Furthermore, the E4SD enzymes were also incorporated into minicellulosomes, both in the unanchored (free) and anchored states. Compared with the free-state E4SD, the hydrolysis efficiencies of E4SD for the CMC, MC, and FP were enhanced up to levels of approximately 1.3-fold, 3.0-fold, and 1.8-fold, respectively, when minicellulosomes were in the unanchored state. With the cell-surfacelocalized minicellulosomes, the hydrolysis efficiencies of E4SD for CMC, MC, and FP degradation were significantly enhanced up to levels of approximately 3.5-fold, 4.7-fold, and 3.0-fold, respectively ( Figs. 5 C and 5 D).
When the minicellulosomes only involved the enzyme NtEGD, the hydrolysis efficiencies in the free state were enhanced up to levels of approximately 1.2-fold, 1.7-fold, and 2.4-fold for CMC, MC, and FP, respectively, and those that were anchored to the cell surfaces were enhanced up to levels of approximately 1.4-fold, 2.0-fold, and 3.2-fold, respectively, in comparison with those of the free-state NtEGD ( Fig. 5 E). These results indicate that the cellsurface-anchored minicellulosomes are capable of enhancing the hydrolytic abilities of cellulases, which are incorporated into minicellulosomes by cohesin-dockerin interactions.
When β-glucosidase was added to the reaction system involving NtEGD ( Fig. 5 F), the hydrolytic activities of the free-state NtEGD enzymes and both the unanchored and anchored minicellulosomes proportionally increased during CMC and MC hydrolysis, while significant enhancement was also observed during filter paper hydrolysis.
Therefore, the cohesin-dockerin interaction between NtEGD and CipA, which facilitates the assembly of the minicellulosomes, could significantly enhance the hydrolytic capacities ( Figs. 5 B and 5 E). These enhancements of the efficiency of hydrolysis may be aided by the targeting and proximity effects of the cellulosomes [15] .
Cohesin and dockerin, which are the crucial building blocks of cellulosomes, are typically static protein modules that retain their biological properties when they are expressed independently [16] . This essential property makes it possible to graft fungal or bacterial cellulases or other protein modules in the free state in vivo for the successful incorporation of minicellulosomes into other fungal or bacterial species [32] and to engineer a reversible high-affinity system for efficient protein purification [25] .
In the present study, an animal cellulase, NtEG, was incorporated into minicellulosomes. This selected endoglucanase, which is secreted in the free state by N. takasagoensis , contains only a GH-9 catalytic module, exhibits high enzymatic activity, and can synergize with β-glucosidase to hydrolyze cellulose to soluble sugars [54] . The complexes that are generated by the incorporation of NtEG into minicellulosomes onto yeast cell surfaces may enhance the hydrolysis efficiencies of NtEGD for CMC, MC, and FP up to 1.4-fold, 2.0-fold, and 3.2-fold, respectively, compared with those of free-state NtEGD. When non-cellulosomic β-glucosidase was added to the NtEGD reaction system, the hydrolysis efficiencies of NtEGD in the free state and in both the unanchored and cell-surface-anchored minicellulosomes for CMC and MC degradation increased proportionally. However, the enhancement of the hydrolytic activities of NtEGD by minicellulosomes in this study was lower than that reported by You et al . [63] . This may have been due to their usage of three cellulases for cellulosome assembly in Bacillus subtilis , including the endoglucanase Cel5, the processive endoglucanase Cel9, and the cellobiohydrolase Cel48. In the present study, we used only one enzyme, which worked as both an exoglucanase and endoglucanase, and the β-glucosidase was not native to cellulosomes. Further studies are needed to clarify the precise reasons for these differences.
As previously reported, these enhancements in activity levels may be attributed not only to target but also to proximity effects [15] . The incorporation of cellulases from different species into cellulosomes by the appending of dockerin tags to their termini may decrease hydrolytic activities for soluble cellulose but increase hydrolytic activities for microcrystalline cellulose [5 , 7 , 59] . In this study, the CipA of C. acetobutylicum had similarly enhanced hydrolytic activities for GH9 cellulases ( Fig. 5 ) in both the cell-free state and CSD minicellulosomes, suggesting that the C. acetobutylicum CipA exerted superior synergistic effects for cellulases, such as the scaffoldin of other Clostridia.
Although the cohesin-dockerin interaction was corrected in this study, the expression of NtEGD was still very low in yeast, which has been reported previously [22] . This finding may be attributed to the yeast feedback mechanism of repression under secretion stress [40] . To improve the endoglucanase expression levels in yeast CSD system, a-agglutinin [56 , 62] and cell wall protein 2 (Cwp2) [29] were used as anchor proteins to display the cellulosomes on the S. cerevisiae cell surface. Although endoglucanase expression levels were generally enhanced, the yeast CSD system still resulted in low expression levels. Cellulase expression and cell growth were inhibited by the co-expression of two cellulases in S. cerevisiae . In particular, the efficiency of CBHII expression in yeast has been shown to decrease 160-fold when it is co-expressed with three cellulases, which may dramatically decrease the cellulose hydrolysis activities of cellulosomes on the yeast cell surface [62] . The selection of a cellulase that possesses exo/endoglucanase abilities may represent a novel and effective expression strategy. Compared with the E4 CAD, NtEG showed equal abilities to hydrolyze both CMC and microcrystalline cellulose in our study. Furthermore, it could also be synergized with β-glucosidase to hydrolyze cellulose to soluble sugars [54] . Consequently, our strategy may allow for the improvement of protein expression in yeast minicellulosomes. Although the possibility of a bi-enzyme modular minicellulosome for cellulose hydrolysis was explored in this study, the low expression levels of endoglucanase in this yeast CSD system still hinder the development of effective minicellulosome design strategies in these organisms. Thus, further efforts are necessary to resolve these issues.
In this study, the truncated scaffoldin of C. acetobutylicum was able to be expressed on the yeast cell surfaces and assemble with other species’ cellulases by cohesin-dockerin interactions, similar to other clostridial cellulosomes. In addition, the incorporation of the endo-β-1,4-glucanase NtEG into surface-displayed minicellulosomes demonstrated that this incorporation strategy allows for the addition of advantageous cellulases with favorable catalytic activities from different species into designed cellulosomes by an appended dockerin tag. To the best of our knowledge, this is the first report describing the expression of C. acetobutylicum minicellulosomes in yeast and the incorporation of animal cellulases into cellulosomes. These findings with regard to heterologous cellulase incorporation provide novel insight into the process of cellulosome assembly. Potentially, the surface-displayed cellulosomes that were described in this study may be utilized to develop a novel method for the engineering of S. cerevisiae to produce ethanol from cellulose. They may also allow for the future development of further novel applications in yeast.
We are grateful to Professor Ying Lin for providing the plasmid pKFS and to Gaku Tokuda for providing the plasmid pBluescript-NtEG.
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