Scaffold protein, cellulose body, and preparation method and application of cellulose body

Abstract

The invention discloses a scaffold protein, a cellulose body, a preparation method of the cellulose body and application of the cellulose body, and the scaffold protein comprises at least one adhesion module; and at least one carbohydrate binding module; wherein the structural domain of the adhesion module is one or more of CL7, IM7, dockerin and cohesin, and the structural domain of the adhesion module is one or more of CL7, IM7, dockerin and cohesin. According to the preparation method of the cellulose body, stable combination between cellulase and a scaffold protein structure and a pichia pastoris industrial host cellulose body with more excellent protein secretion capacity can be integrated, and the cellulose body can be obtained through engineering construction and has the advantage of large capacity.

Description

Technical Field

[0001] The present invention belongs to the field of synthetic biotechnology, and in particular relates to a scaffold protein, a cellulosome, a method for preparing the cellulosome and applications thereof. Background Art

[0002] Low-value agricultural and papermaking waste-derived lignocellulosic biomass is perhaps the most abundant renewable resource in the world. Therefore, the degradation of lignocellulosic biomass is considered valuable in various industrial applications, including feed additives, biofuel production, agricultural waste recycling, etc. However, due to its highly complex structure, efficient degradation of lignocellulose has been very challenging. In the feed industry, the traditional method to improve lignocellulose degradation and feed utilization is to add commercial cellulases.

[0003] However, most of the current cellulase additive products exist in the form of free enzymes, lacking the synergistic effect of structure and function. In order to further improve the efficiency of cellulase, several new strategies have been developed in the existing technology, such as bacterial secretion of cellulase, assembly of cellulosomes in microorganisms, and integrated bioprocessing that integrates enzyme production and cellulose hydrolysis.

[0004] Several studies have shown that assembling multiple cellulases in the form of cellulosomes on the surface of Saccharomyces cerevisiae cells in vivo or in vitro can significantly increase the degradation efficiency of lignocellulose.

[0005] Cellulosomes are scaffolded multienzyme complexes in nature composed of lignocellulose-degrading enzymes that dock with the cohesin domain of a universal scaffold protein through their dockerin domains. Cellulosomes are highly efficient supramolecular enzyme complexes produced primarily by Clostridium thermocellum and other anaerobic cellulolytic bacteria.

[0006] So far, two highly advanced cellulosome engineering strategies have been developed in yeast surface display systems. Specifically, the first engineering strategy employs a whole assembly in which the bacterial cohesin-dockerin pair is replaced by the ultra-high affinity IM7 / CL7 protein pair. In this system, the CL7 tag engineered from the cohesin E7 DNase (CE7) of Escherichia coli retains its ultra-high binding affinity to its inhibitory protein 7 (IM7). The whole surface display structure consists of two or three IM7 modules fused to the surface anchor protein SED1 of Saccharomyces cerevisiae and three basic types of cellulases EGs, CBHs, BGSs fused to the CL7 tag, which binds to IM7.

[0007] The second engineering strategy synthesized the surface display structure on the probiotic yeast Kluyveromyces marxianus, which used the scaffold protein gene (CipA) and the anchor protein gene (OlpB) from C. thermocellum. In addition, the engineering strategy also used type I and type II cohesin-dockerin pairs from C. thermocellum and the anchor domain glycosylphosphatidylinositol (ScGPI) from Saccharomyces cerevisiae. This large final assembly can accommodate up to 63 cellulases and is the largest known synthetic cellulosome.

[0008] Although the above strategies have enhanced cellulase activity on cellulose-based substrates, each of these studies has its own shortcomings. Based on this, the inventors of this case hope to improve the shortcomings of the prior art, such as those that can be overcome through more synergistic and combined strategies.

[0009] The shortcomings of the above strategy are as follows:

[0010] For the first engineering strategy, its advantage is the use of ultra-high affinity IM7 / CL7 protein pairs in the scaffold protein, resulting in a very stable binding between cellulase and scaffold protein, which is the bottleneck of current cellulosome engineering. However, it only produces an extremely simplified mini-cellulosome, and each structural unit can only accommodate 3 enzymes. Therefore, in addition to the three basic enzyme types, it cannot contain auxiliary enzymes, such as lytic polysaccharide monooxygenase (LPMO) and cellobiose dehydrogenase (CDH), both of which have been shown to improve the hydrolytic activity of cellulases. In addition, the SED1 anchor protein comes from Saccharomyces cerevisiae, rather than Pichia pastoris, which is a more suitable industrial host for yeast surface display systems due to its excellent protein secretion ability.

[0011] For the second engineering strategy, although it successfully synthesized the largest cellulosome to date, capable of accommodating up to 63 enzymes, including all three basic cellulases (EGs, CBHs, BGSs) and two auxiliary enzymes (LPMO and CDH), the anchoring protein OlpB and the cohesin-dockerin pair were both from the genome of Clostridium thermocellum, not from eukaryotic yeast. Studies have shown that the stability of anchoring proteins is species-specific and can affect the overall stability of cellulosomes in yeast surface display systems. In addition, the KD value of the cohesin-dockerin pair has been reported to be 10 -12 to 10 -7 M range, and the KD value is 10 -17 to 10 -14 Compared with the IM7 / CL7 protein pair, the expression of

[0012] In summary, although the prior art has successfully used yeast surface display systems to engineer advanced artificial cellulosomes to increase cellulase activity during lignocellulose degradation, there are still many deficiencies, such as the following:

[0013] The synthesized cellulosome is extremely simplified, with small enzyme capacity per structural unit and lacking auxiliary enzymes used to improve the hydrolytic activity of cellulase during the production process;

[0014] The binding between cellulase and scaffold protein is not stable enough, which is the bottleneck of current cellulosome engineering;

[0015] The host cell protein secretion capacity is limited and its stability is weak, making it unsuitable for industrial and large-scale production.

[0016] In summary, it is desirable to obtain a cellulosome that can be constructed in a surface display system such as Pichia pastoris, and that has the following advantages: (1) a large capacity, capable of accommodating a large number of different types of cellulases (e.g., EG, CBH, BGS, LPMO, and CDH) as a scaffold structure; (2) the need to include, for example, an anchoring protein GCW61 from Pichia pastoris; and (3) having, for example, an ultra-strong cohesin-dockerin pair, such as IM7 / CL7 in the scaffold protein. Summary of the invention

[0017] In view of the above-mentioned deficiencies of current cellulosomes, the present invention provides a scaffold protein, a cellulosome, a method for preparing a cellulosome and its application. The technical solution of the present invention can integrate the stable binding between cellulase and the scaffold protein structure with the cellulosome of the industrial host Pichia pastoris with better protein secretion ability, and the cellulosome can be obtained by engineering construction.

[0018] In order to achieve the above object, the present invention provides a scaffold protein, wherein the scaffold protein comprises:

[0019] at least one adhesion module;

[0020] and at least one carbohydrate binding module;

[0021] Wherein, the structural domain of the adhesion module is one or more of CL7, IM7, dockerin and cohesin.

[0022] Preferably, the number of the adhesion modules is at least 5, preferably 9;

[0023] And / or, there are two carbohydrate binding modules.

[0024] Preferably, the scaffold protein further comprises a second domain connected to the anchoring fusion protein, and the second domain is one or more of I-dockerin, II-dockerin and III-dockerin.

[0025] It should be noted that the applicant of this case took into account that the typical Clostridium thermocellum cellulosome is composed of a central non-enzyme scaffold protein subunit called "cellulosomal integral protein A" (i.e., CipA), which has nine type I cohesins. CipA contains an integral cellulose binding module (CBM) that binds to the cellulose matrix. A cellulosome enzyme contains a type I dockerin, which interacts with the type I cohesin of CipA. The entire scaffold protein and enzyme subunit are attached to the bacterial cell surface through the interaction between the type II dockerins of CipA and the type II cohesins of one of the three surface anchoring proteins SdbA, Orf2p or OlpB, which usually contain seven type II cohesins. The cohesin-dockerin interaction of the cellulosome promotes the spatial proximity between various cellulases (i.e., the proximity effect), while the cellulose binding ability of the CBM enhances the utilization of cellulose (i.e., the targeting effect).

[0026] Therefore, to design a scaffold protein with a stable connection structure, it is far from enough to set up multiple adhesion modules in order to meet the large capacity. When comprehensively considering the proximity effect and the targeting effect, the applicant of this case designed the scaffold protein described in this case (preferably IM7 / CL7), which can use the ultra-high affinity IM7 / CL7 protein as the cohesin-dockerin pair to give full play to the synergistic effect of structure and function, so that the cellulase and the scaffold protein are very stably combined, solving the bottleneck of the current cellulosome engineering.

[0027] In addition, the present invention provides a cellulose body, comprising:

[0028] The scaffold proteins described above;

[0029] an enzyme, the enzyme being linked to the adhesion module on the scaffold protein;

[0030] And an anchor protein fusion protein, wherein the anchor fusion protein is OlpB-GCW61.

[0031] Preferably, the enzyme is cellulase and / or coenzyme.

[0032] Preferably, the cellulase is selected from one or more of the following:

[0033] EG, CBH and BGS.

[0034] Preferably, the coenzyme is selected from one or more of the following:

[0035] LPMO and CDH.

[0036] In addition, the present invention also discloses a method for preparing the above-mentioned cellulose body, and the preparation method comprises the following steps:

[0037] constructing a first expression strain and a second expression strain respectively;

[0038] The first expression strain and the second expression strain are co-fermented, secreted, expressed, and self-assembled to obtain the desired cellulosome.

[0039] Preferably, the first expression strain expresses a first assembly module protein, wherein the first assembly module protein comprises an enzyme;

[0040] The second expression strain expresses a second assembly module protein and / or a third assembly module protein, wherein the second assembly module protein includes the scaffold protein and the third assembly module protein includes the anchor protein fusion protein.

[0041] Preferably, the first assembly module is one or more of the following five fusion proteases:

[0042] EG-CL7, CBH-CL7, BGS-CL7, LPMO-CL7 and CDH-CL7.

[0043] It should be noted that, in the above scheme, the EG includes the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0044] The CBH comprises the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0045] The BGS comprises the amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0046] The LPMO comprises the amino acid sequence shown in SEQ ID NO: 4, or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0047] The CDH comprises the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0048] The CL7 comprises the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0049] The fusion protease EG-CL7 includes the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0050] The fusion protease CBH-CL7 comprises the amino acid sequence shown in SEQ ID NO: 8, or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0051] The fusion protease BGS-CL7 comprises the amino acid sequence shown in SEQ ID NO: 9, or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0052] The fusion protease LPMO-CL7 comprises the amino acid sequence shown in SEQ ID NO: 10, or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0053] The fusion protease CDH-CL7 comprises the amino acid sequence shown in SEQ ID NO: 11, or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0054] Preferably, the first expression strain or the second expression strain is selected from Saccharomyces cerevisiae, probiotic yeast or Pichia pastoris, and further preferably, the first expression strain or the second expression strain is selected from Pichia pastoris.

[0055] In a third aspect, the present invention proposes the use of the above-mentioned cellulose body as a degradation agent for degrading cellulose.

[0056] Compared with the prior art, the technical solution of the present invention has the following advantages and beneficial effects:

[0057] The scaffold protein, cellulosome, cellulosome preparation method and application thereof of the present invention provide a scaffold structure with large capacity and capable of accommodating a large number of different types of cellulases, design an IM7 / CL7 protein cohesin-dockerin pair with ultra-high affinity, give full play to the synergistic effect of structure and function, so that the cellulase and the scaffold protein are very stably combined, thus solving the bottleneck of current cellulosome engineering; at the same time, based on the species specificity of the anchor protein, the anchor protein GCW61 from eukaryotic yeast is included to improve the overall stability of the cellulosome in the yeast surface display system. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0059] Figure 1 This is a schematic structural diagram of the cellulose body according to one embodiment of the present invention;

[0060] Figure 2 This is a diagram showing the SDS-PAGE electrophoresis expression results of the cellulosome described in the present invention in one embodiment;

[0061] Figure 3 The SDS-PAGE electrophoresis diagram of the non-denaturing PAGE result of the CipA-OlpB-GCW61 scaffold structure and the scaffold-anchored fusion protease of the cellulosome in one embodiment of the present invention;

[0062] Figure 4 This is a verification analysis diagram of the enzyme activity measurement results of the cellulosome according to one embodiment of the present invention;

[0063] Figure 5 is an assembly roadmap of the first expression strain of the cellulosome of the present invention under one embodiment;

[0064] Figure 6 The figure is an assembly roadmap of the second expression strain of the cellulosome of the present invention under one embodiment. DETAILED DESCRIPTION

[0065] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0066] Figure 1 FIG. 1 is a schematic diagram of the structure of the cellulose body according to one embodiment of the present invention. Figure 1 As shown, the present invention uses the scaffold protein of the cellulosome of Clostridium thermocellum as a skeleton to create an efficient, complex and stable Pichia pastoris in vitro cellulosome display structure. The structure includes two assembly modules: assembly module (1) and assembly module (2).

[0067] The assembly module (1) includes a multi-enzyme complex (e.g., EG, CBH, BGS, LPMO, and CDH), which can efficiently degrade plant cellulose. The C-terminus of the enzyme in the multi-enzyme complex is expressed by fusion of linker and dockerin CL7, thereby forming EG-CL7, CBH-CL7, BGS-CL7, LPMO-CL7, and CDH-CL7 fusion proteases.

[0068] The assembly module (2) includes the scaffold protein of Clostridium thermocellum (CipA) and the anchor protein fusion protein (OlpB-GCW61), wherein CipA contains 9 cohesin IM7 domains that can bind to dockerin CL7, 2 CBM domains, and 1 C-terminal Type II dockerin domain (referred to as 2B9C in this case). Figure 1 It can be seen that OlpB-GCW61 is designed to contain 7 Type II cohesin domains that can bind to Type II dockerin.

[0069] It should be noted that the above two assembly modules are constructed in two independent Pichia expression strains (i.e., the first expression strain and the second expression strain), and the final mature cellulosome structure is formed through co-fermentation culture, secretory expression, self-assembly and other processes.

[0070] During the self-assembly process, each cohesin-dockerin component specifically recognizes and binds to each other. Specifically, the CL7 at the C-terminus of EG-CL7, CBH-CL7, BGS-CL7, LPMO-CL7, and CDH-CL7 fusion proteases specifically recognizes and binds to the nine IM7 domains on the scaffold protein CipA to form a diverse multi-enzyme complex, but the number and type of enzymes in this project (the way they are arranged and combined) are random. In addition, the Type II dockerin domain at the C-terminus of CipA also specifically binds to the seven Type II cohesin domains on OlpB-GCW61 to form a complete CipA-OlpB-GCW61 scaffold structure.

[0071] In order to obtain a complete self-assembled cellulose structure, Pichia pastoris strains containing assembly modules (1) and (2) were fermented separately, and then the scaffold structure and the expression of multiple enzymes in the fermentation broth were detected. SDS-PAGE detected the successful expression of five enzymes (EG, CBH, BGS, LPMO and CDH) in the fermentation broth containing assembly module (1), among which CDH-CL7 had the highest expression level (for this result, see Figure 2 Moreover, the results of non-denaturing PAGE showed that in the fermentation broth containing the assembly module (2), there were CipA-OlpB-GCW61 scaffold structures that had completed self-assembly to varying degrees (for this result, see Figure 3 ).

[0072] In order to detect the cellulase activity of the complete cellulosome, the Pichia pastoris strain containing assembly modules (1) and (2) was co-fermented, and the supernatant of assembly module (1) containing the five enzymes was extracted. At the same time, the supernatant of assembly module (2) was removed and the bacterial cells were retained. This step will remove CipA and OlpB-GCW61 that are not bound to the cell surface. The supernatant of assembly module (1) and the bacterial cells of assembly module (2) were then mixed and cultured to obtain the final sample.

[0073] After crude extraction and concentration of the sample, it was incubated with a cellulose substrate (sodium carboxymethyl cellulose, CMC for short) and then the cellulase activity of the cellulosome was determined by detecting the amount of reducing sugar released. Figure 4 .

[0074] like Figure 4 As shown, the final test results were consistent with expectations, and the negative control wild-type Pichia pastoris (i.e. Figure 4 ) Because it has almost no ability to degrade cellulose, the amount of reducing sugar released is very low, which is different from the assembly module (2) containing only CipA and OlpB-GCW61 without cellulase activity (i.e. Figure 4 The corresponding bar graph of "module (2)" in the figure is close to the level of ). However, the assembly module (1) containing the multi-enzyme complex (i.e. Figure 4 The amount of reducing sugar produced by "module (1)" in the figure is significantly higher than that of the wild-type control and assembly module (2). However, when assembly modules (1) and (2) are co-cultured to form a complete cellulosome structure (i.e. Figure 4 After the assembly of the multienzyme complex with module (1), the amount of reducing sugar released was significantly higher than that of the multienzyme complex with module (1).

[0075] Experimental example

[0076] 1. Design and synthesis of cellulosome structures:

[0077] The inventors of this case selected three types of fungal cellulases, including EG enzyme from Trichodermareesei, a completely synthetic CBH enzyme, β-glucosidase (BGS) from the animal rumen anaerobic fungus Neocallimastix patriciarum, cellulase enhancing enzyme (LPMO) from Thermoascus aurantiacus and electron donor (CDH) from Thermothelomyces thermophila.

[0078] In order to introduce a dockerin-cohesin pair with stronger affinity, dockerin CL7 from the IM7 / CL7 pair of Colicin E7DNase (CE7) from E. coli was connected to the five enzymes through a linker and fused for expression. CL7 was fused to the C-terminus of the enzyme, reducing the effect of its fusion expression at the N-terminus on the enzyme activity and affinity for binding to IM7.

[0079] The obtained amino acid sequences refer to SEQ ID NO:1-13, wherein the EG includes the amino acid sequence shown in SEQ ID NO:1; the CBH includes the amino acid sequence shown in SEQ ID NO:2; the BGS includes the amino acid sequence shown in SEQ ID NO:3; the LPMO includes the amino acid sequence shown in SEQ ID NO:4; the CDH includes the amino acid sequence shown in SEQ ID NO:5; the CL7 includes the amino acid sequence shown in SEQ ID NO:6; the fusion protease EG-CL7 includes the amino acid sequence shown in SEQ ID NO:7; the fusion protease CBH-CL7 includes the amino acid sequence shown in SEQ ID NO:8; the fusion protease BGS-CL7 includes the amino acid sequence shown in SEQ ID NO:9; the fusion protease LPMO-CL7 includes the amino acid sequence shown in SEQ ID NO:10; the fusion protease CDH-CL7 includes the amino acid sequence shown in SEQ ID NO:11. The IM7 includes the amino acid sequence shown in SEQ ID NO:12. The CBM includes the amino acid sequence shown in SEQ ID NO:13.

[0080] The corresponding coding sequences can be found in SEQ ID NOs: 14 to 20. Specifically, the coding sequence of EG-CL7 is shown in SEQ ID NO: 14, the coding sequence of CBH-CL7 is shown in SEQ ID NO: 15, the coding sequence of BGS-CL7 is shown in SEQ ID NO: 16, the coding sequence of LPMO-CL7 is shown in SEQ ID NO: 17, and the coding sequence of CDH-CL7 is shown in SEQ ID NO: 18. The coding sequence of CipA2B9C-IM7 is shown in SEQ ID NO: 19. The coding sequence of OlpB-GCW61 is shown in SEQ ID NO: 20.

[0081] The original scaffold protein skeleton is derived from the CipA gene (CP000568) of C. thermocellum ATCC 27405. CipA contains 9 type I cohesin domains that can bind to type I dockerin, 2 CBM domains, and 1 C-terminal Type II dockerin domain. In order to introduce a dockerin-cohesin pair with stronger affinity, IM7 in the IM7 / CL7 pair derived from e. coli Colicin E7 DNase (abbreviated as CE7) is used to replace the 9 type I cohesins in the original sequence of CipA. In this way, the scaffold protein obtained after the transformation is CipA2B9C-IM7 (its amino acid sequence is shown in SEQ ID NO: 21.) This scaffold protein can form a specific and tight binding with 5 CL7 fusion enzymes.

[0082] Then, using OlpB as the skeleton, a Pichia pastoris cell surface scaffold protein was designed.

[0083] Seven type II cohesin proteins of OlpB were selected, and the original anchoring domain (also known as SLH) was replaced with the Pichia pastoris-specific anchoring domain GCW61 to improve the specificity and stability of the scaffold structure in the surface display system. OlpB contains long tandem repeats (TRs) with a highly repetitive TPSDEP amino acid sequence. Considering the repetitiveness of the OlpB DNA sequence, the TR length was reduced, and the seven cohesins were directly fused with the trimmed TR and GCW61 domains. In addition, the codon randomization of the cohesin repeat sequence was performed to avoid the limitation of DNA synthesis. The amino acid sequence of the modified OlpB-GCW61 is shown in SEQ ID NO:22. In order to improve the translation efficiency, all the expressed coding sequences in the present invention were codon-optimized for the yeast host, and the optimized DNA sequence was synthesized by Shanghai Sangon Biotechnology Technology Service Co., Ltd.

[0084] 2. Construction of Pichia pastoris expression vector

[0085] All synthesized DNA sequences contained EcoRI at the 5' end and NotI at the 3' end, so all sequences were cloned from the storage vector pUC57ria into the Pichia pastoris methanol-inducible expression vector pPIC9k by EcoRI / NotI digestion and ligation. The obtained expression vectors were named: pPIC9k-EG; pPIC9k-CBH; pPIC9k-BGS; pPIC9k-LPMO and pPIC9k-CDH. The five vectors pPIC9k-EG, pPIC9k-CBH, pPIC9k-BGS, pPIC9k-LPMO and pPIC9k-CDH were sequentially integrated into the genome of Pichia pastoris GS115 by homologous recombination to form a strain (i.e., the first expression strain) containing the GS115: (pAOX1-EG)-(pAOX1-CBH)-(pAOX1-BGS)-(pAOX1-LPMO)-(pAOX1-CDH) assembly module (1). The assembly structure is shown in Figure 5 .

[0086] At the same time, the two vectors pPIC9k-CipA and pPIC9k-OlpB-GCW61 were sequentially integrated into the genome of Pichia pastoris GS115 by homologous recombination to form an assembly module (2) strain (i.e., the second expression strain) containing GS115: (pAOX1-CipA)-(pAOX1-OlpB-GCW61). The assembly structure is shown in Figure 6 .

[0087] 3. Preparation of competent yeast and vector genetic transformation

[0088] (1) Pichia pastoris GS115 (purchased from Invitrogen, catalog number C18100) was streaked and picked in 5 mL YPD at 27°C, 220 rpm, overnight;

[0089] (2)OD 600 Controlled at 1.8-2.0, yeast culture time at 12h-18h, take the fermentation liquid and centrifuge at 500g, 4℃;

[0090] (3) Add 2 mL of ddH2O and centrifuge at 500 g, repeat twice;

[0091] (4) Add 0.5 mL 1 M LiCl, 0.5 mL 10*TE (100 mM Tris 7.5, 10 mM EDTA), 4 mL ddH2O, 27°C, 220 rpm, shake for 1 h;

[0092] (5) Add 125 μL DTT (1 M), shake at 27°C, 220 rpm, for 30 min;

[0093] (6) Centrifuge at 500 g, remove the supernatant, and wash twice with 2 mL of ddH2O;

[0094] (7) Add 1 mL of 1 M sorbitol, resuspend, and place on ice for 5 min;

[0095] (8) Take 100 μL+ linear DNA (greater than 5 μg) for electroporation, with the metal sheet of the electroporation cup facing the two electrodes and the protrusions on the side of the electroporation cup facing outward. After adding DNA, it needs to be placed on ice for 5 minutes (electroporation shock conditions: voltage: 1500 V, resistance: 400 Ω, capacitance: 25 μF, pulse time: 10 mS, one shock);

[0096] (9) Add 1 mL of 1 M sorbitol and wash into a 1.5 mL EP tube;

[0097] (10) Centrifuge at 500 g, remove the supernatant, add 1 mL YPD, shake at 27 °C, 220 rpm, for 3 h;

[0098] (11) Centrifuge at 500 g, add 0.1 mL YPD, and coat zeocin-resistant plates;

[0099] (12) Place the plate at 27-30°C and culture for 2-3 days. Transformants will grow on the plate. The resulting transformants are the desired first expression strain and second expression strain.

[0100] 4. Transformant identification and test expression

[0101] (1) Use a high-resistance plate to screen transformants, wait for a single colony to grow on the YPD plate, pick a single colony, and perform PCR colony selection verification. According to the PCR verification results, the desired transformants (i.e., the first expression strain and the second expression strain) are obtained.

[0102] The obtained transformant was transferred into a small triangular flask containing 50 ml of BMGY and cultured at 30°C, 200 rpm until OD 600 =1-1.5;

[0103] (2) Transfer the culture system in the small triangular flask into a 100 ml centrifuge tube and centrifuge at 3000 g for 5 min to collect the bacteria;

[0104] (3) Resuspend the precipitate with BMMY to OD 600 =0.3 (sampling volume is 100-200ml);

[0105] (4) Transfer to a 500 ml large triangular flask, start induction expression culture at 30 °C, 200 rpm, take out 1 ml at intervals, freeze it quickly and store it at -80 °C, and add methanol to a final concentration of 0.5%. Sampling time points: 0 h, 12 h, 24 h, 48 h, 72 h, 96 h;

[0106] (5) Centrifuge the samples at each time point for 1 min to obtain the supernatant. Take 30 μl of the supernatant, add 10 μl of loading buffer, heat for 10 min, centrifuge (13000 rpm, 5 min), take 15 μl and run SDS-PAGE. The results are shown in Figure 2 as well as Figure 3 .

[0107] 5. Fermentation and Self-assembly

[0108] To confirm the assembly of the entire cellulose complex on the cell surface, the (pAOX1-CipA)-(pAOX1-OlpB-GCW61) assembly module (2) strain and the GS115:(pAOX1-EG)-(pAOX1-CBH)-(pAOX1-BGS)-(pAOX1-LPMO)-(pAOX1-CDH) assembly module (1) strain were cultured at 30°C and 300 rpm for 48 h, respectively.

[0109] Preparation of sample 1: The culture product of the assembly module (1) was centrifuged at 4°C, 3000 rpm for 30 minutes to separate the supernatant, which was concentrated 50 times using Vivaspin 20 (purchased from Merck Millipore) to obtain sample 1.

[0110] Preparation of sample 2: The culture product of assembly module (2) was centrifuged at 3000 rpm at 4°C, the precipitate (cells) was collected, and washed twice with YP medium (1% yeast extract, 2% peptone and 10 mM CaCl2) to remove proteins not bound to the cell surface;

[0111] Then, the precipitate (cells) of assembly module (2) was suspended in the concentrated supernatant of assembly module (1) (the preparation process of which is shown in Sample 1). Then, 10 mM CaCl2 was added and static culture was performed at 37°C for 3 hours to induce the formation of a complete cellulose structure. Then, the precipitate (cells) was collected by centrifugation at 3000 rpm at 4°C and washed twice with YP medium to remove free proteins. Then, the precipitate (OD 600 =20)) were suspended in YP medium supplemented with 0.04% Tween80 for downstream use (i.e., subsequent cellulase activity assay).

[0112] 6. Cellulase Activity Assay

[0113] The cellulase activity was determined using the national standard (NY / T912-2020) method. The cellulase activity was determined using carboxymethyl cellulose CMC (Sigma, C5678) as a substrate.

[0114] Brief description of the determination method: Perform two tests in parallel. According to the sampling volume recommended by the national standard method, accurately transfer an appropriate amount of the sample and place it in a 100mL volumetric flask. Use acetic acid sodium acetate buffer solution (weigh 23.14g of sodium acetate trihydrate, dissolve in water, add 1.70mL of glacial acetic acid, dilute to 2000mL with water, adjust the pH to 5.5 with acetic acid solution (measure 0.60mL of glacial acetic acid, dilute with water, make up to 100mL, shake well) or sodium acetate solution (weigh 1.36g of sodium acetate trihydrate, dissolve in water and dilute to 100mL, shake well) and make up to the scale, shake well. Dilute with acetic acid sodium acetate buffer solution. If the pH of the enzyme solution deviates from 5.5 after dilution, first adjust the pH to 5.5 with acetic acid solution or sodium acetate solution, and then make up to the volume with acetic acid sodium acetate buffer solution so that the concentration of the enzyme solution to be tested is 0.04U / mL~0.06U / mL. 100 μL assay reaction, take 40 μL concentrated supernatant, add 60 μL buffer (0.4% [wt / vol] substrate CMC and 50 mM sodium acetate, pH 5), incubate at 40°C for 6 h. After incubation, add 250 μL precipitation solution (containing sodium acetate trihydrate, zinc acetate and ethanol, pH 5.0), centrifuge at 8000 rpm for 10 min, and measure absorbance at 590 nm.

[0115] The results showed that the assembly module (1) expressing multiple cellulases released significantly more reducing sugars than the control, proving that it has strong cellulase activity. However, the assembly module (2) expressing only the scaffold structure did not release any significant reducing sugars or cellulase activity compared to the control. Figure 4 In addition, the complete cellulosome structure formed by the assembly module (1) and the assembly module (2) has higher cellulase activity than the assembly module (1) alone, proving that the cellulosome assembly is successful and further enhances the enzyme activity through the synergistic action of multiple enzymes ( Figure 4 ).

[0116] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by any technician familiar with the art within the technical scope disclosed in the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. A scaffold protein, characterized in that The scaffold protein comprises: at least one adhesion module; and at least one carbohydrate binding module; Wherein, the structural domain of the adhesion module is one or more of CL7, IM7, dockerin and cohesin.

2. The scaffold protein according to claim 1, characterized in that The number of the adhesion modules is at least 5; And / or, there are two carbohydrate binding modules.

3. The scaffold protein according to claim 1 or 2, characterized in that The scaffold protein further comprises a second domain connected to the anchoring fusion protein, and the second domain is one or more of I-dockerin, II-dockerin and III-dockerin.

4. A cellulose body, characterized in that The cellulosic body comprises: The scaffold protein according to any one of claims 1 to 3; an enzyme, the enzyme being linked to the adhesion module on the scaffold protein; And an anchor protein fusion protein, wherein the anchor fusion protein is OlpB-GCW61.

5. The cellulose body according to claim 4, characterized in that The enzyme is cellulase and / or coenzyme.

6. The cellulose body according to claim 5, characterized in that The cellulase is selected from one or more of the following: EG, CBH and BGS; And / or, the coenzyme is selected from one or more of the following: LPMO and CDH.

7. A method for preparing the cellulose body according to any one of claims 4 to 6, characterized in that: The preparation method comprises the following steps: constructing a first expression strain and a second expression strain respectively; The first expression strain and the second expression strain are co-fermented, secreted, expressed, and self-assembled to obtain the desired cellulosome.

8. The preparation method according to claim 7, characterized in that: The first expression strain expresses a first assembly module protein, wherein the first assembly module protein comprises an enzyme; The second expression strain expresses a second assembly module protein and / or a third assembly module protein, wherein the second assembly module protein includes the scaffold protein and the third assembly module protein includes the anchor protein fusion protein.

9. The preparation method according to claim 8, characterized in that: The first assembly module is one of the following five fusion proteases: EG-CL7, CBH-CL7, BGS-CL7, LPMO-CL7 and CDH-CL7.

10. Use of the cellulosic body according to any one of claims 4 to 6 as a degrading agent for degrading cellulose.

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