An engineered Pichia pastoris mixed bacterial system for PET plastic degradation

Abstract

The present disclosure provides a scaffold protein, which includes a domain located on the cell surface; one or more carbohydrate binding domains; and one of the one or more polypeptide interaction pairs. The present disclosure also provides an engineered Pichia pastoris mixed bacterial system for PET plastic degradation, the system comprising the scaffold protein. The present disclosure utilizes a cell surface display strategy to allow the scaffold protein-enzyme complex to self-assemble on the cell surface, thereby significantly improving the efficiency of plastic degradation.

Description

Technical Field

[0001] The present disclosure belongs to the biological field, and particularly relates to an engineered Pichia pastoris mixed bacteria system for PET plastic degradation. Background Art

[0002] As a type of pollutant, plastic has a certain impact on the ecosystem. Polyethylene terephthalate (PET) is widely used in textiles, food packaging, and medical protection. In the absence of microbial activity, its degradation rate is negligible, and its half-life ranges from thousands to millions of years, depending on the polymer type and environmental conditions. Biodegradable plastics are a green and clean way to degrade plastics, with the advantages of low cost and environmental friendliness. Its development has greatly broadened the channels for plastic recycling.

[0003] PET polymers are difficult to degrade due to their high degree of polymerization. Cell surface display is of great significance in improving the stability and reaction efficiency of enzymes. Pichia pastoris has the advantages of high-density growth, no endotoxin, and post-translational modification of protein expression. Engineered strains of Pichia pastoris P. pastoris GS115 (His-, Mut+) is a widely used strain for secretory expression of exogenous genes, while the protease-deficient strain P. pastoris SMD1168 (His-, pep4-, Mut+) reduced the degradation of extracellular secretory proteins.

[0004] At present, there are studies on displaying PET hydrolases on the surface of yeast to achieve degradation effects. For example, Chen Z. et al. co-displayed PETase (degrading PET) and HFBI (no degradation effect, used to improve affinity with PET) on the surface of yeast. P. pastorisGS115, achieved biodegradation of PET plastic (total product amount of 0.92umol under reaction conditions of 30℃ for 48 h) (Chen, Z. et al., Nat Commun 13, 7138 (2022)). However, the PET degradation effect shown in previous studies still needs to be improved, and the surface display chassis strain Pichia pastoris GS115 needs to be optimized. In addition, PET can be degraded by PETase to produce terephthalic acid (TPA) and ethylene glycol (EG), while the intermediate products mono(2-hydroxyethyl)terephthalate (MHET) and bis(2-hydroxyethyl) terephthalate (BHET) produced during the degradation process will competitively inhibit the degradation activity of PET degrading enzymes. Therefore, it is necessary to further develop methods to improve the degradation efficiency of PET. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention discloses an engineered Pichia pastoris mixed bacteria system with higher PET degradation efficiency by mixing engineered bacteria that display scaffold proteins on their surfaces and engineered bacteria that secrete and express related enzymes, so that the scaffold protein-multi-enzyme complex self-assembles on the surface of yeast cells.

[0006] According to one aspect of the present disclosure, a scaffold protein is provided, the scaffold protein comprising:

[0007] Domains localized to the cell surface;

[0008] one or more carbohydrate binding domains; and

[0009] One member of one or more polypeptide interaction pairs.

[0010] In some embodiments, the domain localized on the cell surface is a transmembrane or membrane-associated polypeptide. Preferably, the domain localized on the cell surface is selected from an anchor protein. In some embodiments, the domain localized on the cell surface includes, but is not limited to, glycosylated phosphatidylinositol (GPI) anchor protein, PIR protein, lectin and flocculin or any combination, functional variant or fragment thereof. In some embodiments, the domain localized on the cell surface is selected from SED1 protein, Agα1 protein, GCW21 protein, GCW51 protein, GCW61 protein or any combination, functional variant or fragment thereof.

[0011] In some embodiments, the carbohydrate binding domain is selected from a carbohydrate binding module, a hydrophobin (HFB), an anchor peptide, or any combination, functional variant, or fragment thereof. In some embodiments, the carbohydrate binding domain is selected from a CBM, HFB1, HFB4, HFB7, Tachystain TA2, or any combination, functional variant, or fragment thereof.

[0012] In some embodiments, the polypeptide interaction pair is selected from the group consisting of dockerin-cohesin interaction pairs, growth factor and growth factor receptor interaction pairs, cytokine and cytokine receptor interaction pairs, chemokine and chemokine receptor interaction pairs, antibody-ligand interaction pairs, and enzyme-substrate interaction pairs. In some embodiments, the dockerin-cohesin interaction pair is selected from the group consisting of CelK-CipA, CelF-CipC, ScaA-ScaF, Cel44A-ScaA, Ce3B-ScaC, XDoc-ScaE, docCipA-olpB, ScaC-ScaI, ScaH-ScaE, ScaF-ScaJ, ScaG-ScaJ, Cel44A-CipA, Xyn10B-CipA, Xyn11A-CipA, SH3 L -SH3 D or ScaA-ScaB.

[0013] In some embodiments, a plurality of members of the polypeptide interaction pairs are separated by or directly linked to each other by the carbohydrate binding domain.

[0014] In some embodiments, the scaffold protein comprises a first polypeptide interaction pair and a second polypeptide interaction pair. Preferably, the first polypeptide interaction pair is different from the second polypeptide interaction pair, for example, selected from CelK-CipA and CelF-CipC, respectively.

[0015] In some embodiments, the scaffold protein includes, from N-terminus to C-terminus, a member of a first polypeptide interaction pair; a carbohydrate binding domain or a functional variant thereof; a member of a second polypeptide interaction pair; and a domain localized on the cell surface or a functional variant thereof.

[0016] In some embodiments, the scaffold protein includes, from N-terminus to C-terminus, a domain localized on the cell surface or a functional variant thereof; a member of a first polypeptide interaction pair; a carbohydrate binding domain or a functional variant thereof; and a member of a second polypeptide interaction pair.

[0017] In some embodiments, the first polypeptide interaction pair member, the carbohydrate binding domain or a functional variant thereof, the second polypeptide interaction pair member, and / or the cell surface localized domain or a functional variant thereof are directly connected or connected via a linker.

[0018] In some embodiments, the linker is selected from the amino acid sequence of any one of SEQ ID NOs: 10-12.

[0019] In some embodiments, the scaffold protein has an amino acid sequence as described in any one of SEQ ID NOs: 13-16, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity thereto.

[0020] According to another aspect of the present disclosure, a plastic degradation system is provided, which comprises a surface display module and one or more plastic degradation modules; wherein the surface display module comprises cells and a scaffold protein as described in the present disclosure displayed on the cell surface; and the plastic degradation module comprises a plastic degradation domain and the other member of a polypeptide interaction pair.

[0021] In some embodiments, the plastic degradation system includes a first plastic degradation module and a second plastic degradation module, the first plastic degradation module includes the other member of the first polypeptide interaction pair and the first plastic degradation domain; the second plastic degradation module includes the other member of the second polypeptide interaction pair and the second plastic degradation domain; the first plastic degradation domain and the second plastic degradation domain are plastic degradation enzymes.

[0022] Preferably, the first plastic degradation domain and the second plastic degradation domain are each independently selected from one or more of FAST-PETase enzyme or its mutant, MHETase enzyme or its mutant and LCC or its mutant.

[0023] In some embodiments, the first plastic degradation module and / or the second plastic degradation module are expressed by cells and secreted outside the cells.

[0024] In some embodiments, the plastic degrading enzyme includes one or more of PETase, IsPETase, FAST-PETase, ThermoPETase, DuraPETase, DepoPETase, BhrPETase, TurboPETase, CaPETaseM9, HotPETase, LCC, LCC-A2, ICCG, WCCG, ICCM, WCCM, MHETase, KLMHETase and optiMHETase.

[0025] In some embodiments, the first plastic degradation domain and the second plastic degradation domain are different from each other.

[0026] In some embodiments, the first plastic degradation module and the second plastic degradation module are expressed by the same cell and secreted outside the cell, or are expressed by different cells and secreted outside the cell respectively.

[0027] In some embodiments, the first plastic degradation module and / or the second plastic degradation module has an amino acid sequence as described in any one of SEQ ID NOs: 5-6 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity thereto.

[0028] In some embodiments, the cell is selected from bacteria, fungi, insect cells, plant cells, or animal cells.

[0029] In some embodiments, the cell is selected from Escherichia coli, Bacillus subtilis, or yeast.

[0030] In some embodiments, the cell is selected from Pichia pastoris.

[0031] According to yet another aspect of the present disclosure, an isolated nucleic acid molecule is provided, wherein the nucleic acid molecule encodes the scaffold protein described in the present disclosure, or the system described in the present disclosure.

[0032] According to yet another aspect of the present disclosure, an expression vector is provided, wherein the expression vector comprises the isolated nucleic acid molecule described in the present disclosure.

[0033] In some embodiments, the expression vector is selected from a viral expression vector, a bacterial expression vector or a fungal expression vector.

[0034] In some embodiments, the expression vector is selected from a yeast expression vector, an adenoviral vector, an adeno-associated viral vector (AAV), a retroviral vector or a lentiviral vector.

[0035] According to another aspect of the present disclosure, a host cell is provided, comprising a surface display host cell and / or a secretory expression host cell, wherein the surface display host cell displays the scaffold protein as described in the present disclosure on the cell surface, and the secretory expression host cell secretorily expresses the plastic degradation domain and the other member of the polypeptide interaction pair.

[0036] In some embodiments, the domain localized on the cell surface is a transmembrane or membrane-associated polypeptide. Preferably, the domain localized on the cell surface is selected from an anchor protein. In some embodiments, the domain localized on the cell surface includes, but is not limited to, glycosylated phosphatidylinositol (GPI) anchor protein, PIR protein, lectin and flocculin or any combination, functional variant or fragment thereof. In some embodiments, the domain localized on the cell surface is selected from SED1 protein, Agα1 protein, GCW21 protein, GCW51 protein, GCW61 protein or any combination, functional variant or fragment thereof.

[0037] In some embodiments, the carbohydrate binding domain is selected from a carbohydrate binding module, a hydrophobin (HFB), an anchor peptide, or any combination, functional variant, or fragment thereof. In some embodiments, the carbohydrate binding domain is selected from a CBM, HFB1, HFB4, HFB7, Tachystain TA2, or any combination, functional variant, or fragment thereof.

[0038] In some embodiments, the polypeptide interaction pair is selected from the group consisting of dockerin-cohesin interaction pairs, growth factor and growth factor receptor interaction pairs, cytokine and cytokine receptor interaction pairs, chemokine and chemokine receptor interaction pairs, antibody-ligand interaction pairs, and enzyme-substrate interaction pairs. In some embodiments, the dockerin-cohesin interaction pair is selected from the group consisting of CelK-CipA, CelF-CipC, ScaA-ScaF, Cel44A-ScaA, Ce3B-ScaC, XDoc-ScaE, docCipA-olpB, ScaC-ScaI, ScaH-ScaE, ScaF-ScaJ, ScaG-ScaJ, Cel44A-CipA, Xyn10B-CipA, Xyn11A-CipA, SH3 L -SH3 D or ScaA-ScaB.

[0039] In some embodiments, a plurality of members of the polypeptide interaction pairs are separated by or directly linked to each other by the carbohydrate binding domain.

[0040] In some embodiments, the scaffold protein comprises a first polypeptide interaction pair and a second polypeptide interaction pair. Preferably, the first polypeptide interaction pair is different from the second polypeptide interaction pair, for example, selected from CelK-CipA and CelF-CipC, respectively.

[0041] In some embodiments, the scaffold protein includes, from N-terminus to C-terminus, a member of a first polypeptide interaction pair; a carbohydrate binding domain or a functional variant thereof; a member of a second polypeptide interaction pair; and a domain localized on the cell surface or a functional variant thereof.

[0042] In some embodiments, the scaffold protein includes, from N-terminus to C-terminus, a domain localized on the cell surface or a functional variant thereof; a member of a first polypeptide interaction pair; a carbohydrate binding domain or a functional variant thereof; and a member of a second polypeptide interaction pair.

[0043] In some embodiments, the first polypeptide interaction pair member, the carbohydrate binding domain or a functional variant thereof, the second polypeptide interaction pair member, and / or the cell surface localized domain or a functional variant thereof are directly connected or connected via a linker.

[0044] In some embodiments, the linker is selected from the amino acid sequence of any one of SEQ ID NOs: 10-12.

[0045] In some embodiments, the scaffold protein has an amino acid sequence as described in any one of SEQ ID NOs: 13-16, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity thereto.

[0046] In some embodiments, the secretory expression host cell secretes and expresses the first plastic degradation module and the second plastic degradation module. In some embodiments, the first plastic degradation module and the second plastic degradation module are secreted and expressed by the first secretory expression host cell, or the first plastic degradation module and the second plastic degradation module are secreted and expressed by the second secretory expression host cell and the third secretory expression host cell, respectively.

[0047] In some embodiments, the first plastic degradation module includes another member of the first polypeptide interaction pair and a first plastic degradation domain; the second plastic degradation module includes another member of the second polypeptide interaction pair and a second plastic degradation domain; the first plastic degradation domain and the second plastic degradation domain are plastic degrading enzymes.

[0048] Preferably, the first plastic degradation domain and the second plastic degradation domain are each independently selected from one or more of FAST-PETase enzyme or its mutant, MHETase enzyme or its mutant and LCC or its mutant.

[0049] In some embodiments, the first plastic degradation module and / or the second plastic degradation module are expressed by cells and secreted outside the cells.

[0050] In some embodiments, the plastic degrading enzyme includes one or more of PETase, IsPETase, FAST-PETase, ThermoPETase, DuraPETase, DepoPETase, BhrPETase, TurboPETase, CaPETaseM9, HotPETase, LCC, LCC-A2, ICCG, WCCG, ICCM, WCCM, MHETase, KLMHETase and optiMHETase.

[0051] In some embodiments, the first plastic degradation domain and the second plastic degradation domain are different from each other.

[0052] In some embodiments, the first plastic degradation module and the second plastic degradation module are expressed by the same secretory expression host cell and secreted outside the cell, or are expressed by different secretory expression host cells and secreted outside the cell respectively.

[0053] In some embodiments, the first plastic degradation module and / or the second plastic degradation module has an amino acid sequence as described in any one of SEQ ID NOs: 5-6 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity thereto.

[0054] In some embodiments, the cell is selected from bacteria, fungi, insect cells, plant cells, or animal cells.

[0055] In some embodiments, the cell is selected from Escherichia coli, Bacillus subtilis, or yeast.

[0056] In some embodiments, the cell is selected from Pichia pastoris.

[0057] In some embodiments, the surface display host cell displays a scaffold protein on the cell surface, wherein the scaffold protein comprises operably linked CipA-CBM-CipC-SED1.

[0058] In some embodiments, the secretory expression host cell secretes and expresses CelK-FAST-PETase and CelF-MHETase.

[0059] In some specific embodiments, the genes of the scaffold protein and the plastic degradation module are inserted into the Pichia genome to obtain a Pichia engineered strain that can stably express the relevant proteins. By mixing engineered bacteria that display the scaffold protein on the surface and engineered bacteria that secrete and express the enzyme, the scaffold protein and enzyme complex are self-assembled on the surface of the Pichia cells, and an engineered mixed bacteria system with higher PET degradation efficiency is constructed.

[0060] According to another aspect of the present disclosure, provided is the use of the scaffold protein, the plastic degradation system, the nucleic acid molecule, the expression vector or the host cell described in the present disclosure in plastic degradation.

[0061] In some embodiments, the plastic degradation is PET degradation.

[0062] According to another aspect of the present disclosure, a plastic degradation method is provided, wherein the plastic degradation method uses the host cell described in the present disclosure to degrade plastic.

[0063] In some embodiments, the first plastic degradation module and the second plastic degradation module are secretory expressed by a first secretory expression host cell, and in the host cell, the content ratio of the surface display host cell to the first secretory expression host cell is 1:(1-10), for example, 1:1, 1:5, 1:10 or any value therebetween.

[0064] In some embodiments, the first plastic degradation module and the second plastic degradation module are secreted and expressed by a second secretory expression host cell and a third secretory expression host cell, respectively. In the host cells, the content ratio of the surface display host cell to the second secretory expression host cell and the third secretory host cell is 1:(1-10):(1-10), for example, 1:1:1, 1:5:5, 1:10:10 or any value therebetween.

[0065] In some embodiments, the plastic is PET plastic.

[0066] The present invention discloses a scaffold protein-multi-enzyme complex designed by coupling a scaffold protein with two PET degradation-related enzymes; a genetically engineered strain is constructed to achieve surface display of the scaffold protein and secretory expression of the enzyme; and a cell surface display strategy is utilized to enable the scaffold protein-multi-enzyme complex to self-assemble on the cell surface, thereby achieving improved PET plastic degradation efficiency.

[0067] The present invention provides a modular design idea for biodegradable plastics by coupling a scaffold protein-multienzyme complex with two PET degradation-related enzymes through a scaffold protein. A living cell biodegradation system is further constructed to achieve efficient degradation of PET plastics. The plastic degradation system disclosed in the present invention has the characteristics of exogenous gene integration into chromosomes, no need for resistance drugs to maintain expression, controllable components, low degradation reaction cost, and convenient large-scale fermentation production, which is easy to achieve industrialization. In addition, the scaffold protein and the enzyme are located in different strains, which is conducive to adjusting the ratio of the scaffold protein to the enzyme, making the PET degradation reaction system flexible and controllable. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 A schematic diagram of the design of the engineered yeast mixed bacteria system is shown.

[0069] Figure 2 The figure shows the levels of cell surface displayed scaffold proteins of different anchoring proteins GCW61, GCW51, SED1, and Aga1 analyzed by flow cytometry, where the Control group is a control without anchoring protein.

[0070] Figure 3 The surface display effect of the engineering bacteria co-culture system expressing FAST-PETase and MHETase respectively in different types of Pichia pastoris strains GS115, SMD1168, and KM71 was shown using flow cytometry. The control group was P. pastoris SMD1168 His4::CipA-CBM-CipC-SED1 Mixed with the corresponding purified protein FAST-PETase or MHETase. The scaffold protein (Scaf.) has a V5 tag, and MHETase (M) and FAST (F) both have a His tag.

[0071] Figure 4A-4D The results of co-culture of surface display engineered bacteria with single secretion expression engineered bacteria or double secretion expression engineered bacteria in different cell mass ratios (1:1, 1:5, 1:10 or 1:1:1, 1:5:5, 1:10:10) were shown, where the control group was P. pastoris SMD1168 His4::CipA-CBM-CipC-SED1 Wild type P. pastoris SMD1168 was mixed and cultured at an initial cell-to-cell ratio of 1:10. Among them, the scaffold protein (Scaf.) was tagged with V5, and MHETase (M) and FAST (F) were both tagged with His.

[0072] Figure 5A-Figure 5B The surface display of enzyme-scaffold protein complex in the mixed system of engineered strains in BMMY medium every 24 hours is shown by flow cytometry. The control group is wild type. P. pastoris SMD1168. Among them, the scaffold protein (Scaf.) has a V5 tag, and MHETase (M) and FAST (F) both have a His tag.

[0073] Figure 6A-6B The figure shows the surface display of enzyme-scaffold protein complex in the mixed system of engineered strains in BMMY medium containing PET plastic substrate every 24 hours using flow cytometry. The control group is wild type. P. pastoris SMD1168. Among them, the scaffold protein (Scaf.) carries a V5 tag, and MHETase (M) and FAST (F) both carry a His tag.

[0074] Figure 7 The figure shows the surface display effects of scaffold proteins, enzymes and multi-enzyme complexes detected by flow cytometry after 96 hours of induction expression; among them, the scaffold protein (Scaf.) has a V5 tag, MHETase (M) has a His tag, and FAST (F) has a Myc tag; the numbers-letters represent the strain type number-expression protein information.

[0075] Figure 8 The surface display effects of scaffold proteins, enzymes and multi-enzyme complexes were detected by laser confocal microscopy after 96 hours of induction expression; among them, the scaffold protein (Scaf.) has a V5 tag, MHETase (M) has a His tag, and FAST (F) has a Myc tag; the numbers-letters represent the strain type number-expression protein information.

[0076] Fig. 9 The total reaction product content of each treatment group after 96 hours of induction expression in the scaffold protein surface display bacteria: single secretion / double secretion expression engineering bacteria = 1:10 / 1:10:10 mixed bacteria system and degradation in BMMY at 30°C for 48 hours is shown.

[0077] Fig.10 The total reaction product content of each treatment group after 96 hours of induction expression in the scaffold protein surface display bacteria: single secretion / double secretion expression engineering bacteria = 1:10 / 1:10:10 mixed bacteria system and degradation in Glycine-NaOH at 30°C for 48 hours is shown.

[0078] Fig.11 The total reaction product content of each treatment group after 96 hours of induction expression in the mixed bacterial system of scaffold protein surface display bacteria: single secretion / double secretion expression engineering bacteria = 1:5 / 1:5:5 and degradation in Glycine-NaOH at 30°C for 48 hours was shown.

[0079] Fig.12The total reaction product content of each treatment group after 96 hours of induction expression in the mixed bacterial system of scaffold protein surface display bacteria: single secretion / double secretion expression engineering bacteria = 1:1 / 1:1:1 and degradation in Glycine-NaOH at 30°C for 48 hours was shown.

[0080] Fig.13 The crystallinity test results of the PET plastic substrate in Example 3 are shown. DETAILED DESCRIPTION

[0081] The present invention discloses a scaffold protein CipA-CBM-CipC-SED1 containing a carbohydrate binding module (CBM), which is connected to FAST-PETase fused with CelK and MHETase fused with CelF. By co-culturing different engineered Pichia pastoris, the cell surface self-assembly of the multi-enzyme complex is achieved, thereby effectively biodegrading PET plastics, which has far-reaching application prospects.

[0082] In some embodiments, see Figure 1 The present invention provides a component-regulatable Pichia pastoris genetic engineering strain mixed bacterial system, the system includes a scaffold protein gene inserted P. pastoris His::CipA-CBM-CipC-SED1 , insert single enzyme gene P. pastoris His::FAST and P. pastoris His::MHETase , or including insertion of a scaffold protein gene P. pastoris His::CipA-CBM-CipC-SED1 and insertion of the dual enzyme gene P. pastori His::FAST-MHETase The mixed bacteria system disclosed in the present invention can produce a scaffold protein-multi-enzyme complex to achieve PET plastic degradation. The system has low fermentation cost, flexible and controllable components, convenient large-scale production, and easy industrialization.

[0083] In order to make the purpose, technical scheme and advantages of the present disclosure more clear, the present disclosure is further described in detail below in conjunction with the accompanying drawings and embodiments. The specific embodiments described herein are only used to explain the present disclosure and are not intended to constitute any limitation to the present disclosure. In addition, in the following description, the description of well-known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0084] Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly used in the field to which this disclosure belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0085] Unless the context clearly dictates otherwise, the expressions "a", "an" and "an" as used herein include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0086] The term "about" as used herein refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0087] As used herein, the term "plastic" is used to refer to any article or product comprising at least one polymer, such as plastic sheets, tubes, rods, profiles, shapes, films, blocks, fibers, etc. Preferably, the plastic is a manufactured product, such as rigid or flexible packaging (bottles, trays, cups, etc.), agricultural films, bags and sacks, disposable items, etc., carpet waste, fabrics, textiles, etc. The plastic article may contain additional substances or additives, such as plasticizers, minerals, organic fillers or dyes. In the context of the present disclosure, the plastic may contain a mixture of semi-crystalline and / or amorphous polymers and / or additives.

[0088] As used herein, the terms "polyethylene terephthalate (PET)" or "PET and derivatives" include high molecular weight polymers produced by condensing ethylene glycol with dimethyl terephthalate or terephthalic acid, regardless of how prepared. In addition, these terms mean the well-known polyethylene terephthalate polymers modified by including small amounts, such as less than about 20% by weight of the polyethylene terephthalate polymer, of comonomers or other well-known modifiers. Such comonomers or modifiers include aromatic and aliphatic diols and polyols; aromatic and aliphatic carboxylic acids; or a single molecule containing both carboxyl and alcohol functions. Examples of diols include 1,4-butanediol, cyclohexanedimethanol, diethylene glycol, and / or 1,3-propylene glycol. Examples of dicarboxylic acids include isophthalic acid, adipic acid, 2,6-naphthalenedicarboxylic acid, and p-hydroxybenzoic acid. Small amounts of chain branching agents and / or chain terminators may also be used. The chain branching agent includes, for example, a polyfunctional acid and / or a polyfunctional alcohol, such as trimethylolpropane and pentaerythritol. Chain terminators include monofunctional alcohols and / or monofunctional carboxylic acids, such as stearic acid and benzoic acid. Mixtures of the chain branching and chain terminators may also be used. In some embodiments, PET and derivatives include, but are not limited to, PET powder, PET bottles, PET films, PET fibers, and the like.

[0089] The term "degradation" as used herein includes depolymerization, which refers to the process of converting a polymer into its final monomer and / or degrading into smaller molecules (such as monomers and / or oligomers and / or any degradation products). The term "degradation" more generally describes the cleavage of a polymer chain by at least one enzyme in an enzyme, thereby producing a shorter polymer chain. For example, PET can be degraded by PETase, and the intermediate products mono(2-hydroxyethyl) terephthalate (MHET) and ethylene terephthalate (bis(2-hydroxyethyl) terephthalate, BHET) are produced during the degradation process, and the final products are terephthalic acid (TPA) and ethylene glycol (EG), wherein the intermediate products MHET and BHET compete to inhibit the degradation activity of the PET degrading enzyme. In some embodiments, the enzyme used to degrade plastics includes one or more of a FAST-PETase enzyme or a mutant thereof, a MHETase enzyme or a mutant thereof, and a LCC or a mutant thereof.

[0090] As used herein, the terms "polypeptide interaction pair", "protein interaction pair", "polypeptide interaction pair" or "protein interaction pair" refer to highly specific physical contacts established between two or more polypeptide molecules or protein molecules due to interactions including electrostatic forces, hydrogen bonding and / or hydrophobic effects. Many are physical contacts of molecular associations that occur in cells or living organisms under specific biomolecular environments. Any polypeptide interaction pair involved in the present disclosure is a known or suspected protein. In the case of growth factors and growth factor receptors (e.g., epidermal growth factor EGF / epidermal growth factor receptor EGFR), such interacting proteins may be composed of SH2 of banham proteins or fragments thereof (Src homology domain 2), such as phospholipase C (PLC) or Src homology domain 2 containing transforming protein 1 (SHC1). In the case of serine / threonine kinase receptors, such as TGFβ, activin, BMP receptors, such interacting proteins may be Smad proteins or fragments thereof. In the case of cytokine receptors, such as interferon-α / β or interferon-γ receptors, such interacting proteins may be signal transducers and activators of transcription (STAT) proteins, such as, but not limited to, Stat1, Stat2; Janus kinase (JAK) proteins Jak1, Jak2 or Tykk2 or fragments thereof. Other possible polypeptide interactions include antibody-ligands, enzyme-substrate dimerization proteins, members of signal cascade transduction, and other protein pairs known in the art. In the case of the dockerin-cohesin interaction pair, such interacting proteins may be selected from CelK-CipA, CelF-CipC, ScaA-ScaF, Cel44A-ScaA, Ce3B-ScaC, XDoc-ScaE, docCipA-olpB, ScaC-ScaI, ScaH-ScaE, ScaF-ScaJ, ScaG-ScaJ, Cel44A-CipA, Xyn10B-CipA, Xyn11A-CipA, SH3 L -SH3, ScaA-ScaB or fragments thereof. In some embodiments, the polypeptide interaction pair is CelK-CipA. In other embodiments, the protein interaction pair is CelF-CipC.

[0091] Carbohydrate-binding modules (CBMs) are non-catalytic domains of glycoside hydrolases and other proteins that recognize and bind to polysaccharides. CBMs are often found in fungal and bacterial proteins that contain glycoside hydrolases that degrade insoluble polysaccharides. However, CBMs have also been identified in proteins that do not contain glycoside hydrolase domains but are involved in the degradation of insoluble polysaccharides such as cellulose. CBMs are divided into different families based on the similarity of their protein sequences. Different CBMs can specifically recognize cellulose, chitin, glucan, xylan, inulin, mannan, galactan, starch or glycogen based on their different substrate binding specificities. In addition, some CBMs can bind to polysaccharides on the cell surface.

[0092] Hydrophobins (HFB) are a group of highly functional, low molecular weight proteins that can self-assemble at the interface to form amphipathic protein films, thereby changing the hydrophilicity and hydrophobicity of the interface. Hydrophobins are non-toxic and non-immunogenic.

[0093] The term "PETase" used in this article refers to a PET plastic depolymerase excavated from Ideonella sakaiensis. Based on the engineering modification of PETase, the following enzymes have been developed: ThermoPETase, IsPETase, DuraPETase, FastPETase, DepoPETase, BhrPETase, TurboPETase, CaPETase M9 and a series of PET plastic depolymerases including HotPETase.

[0094] The term "LCC" used in this article refers to a cutinase found in compost, called LCC (leaf-branch compost cutinase), which can cut off the ester group and decompose PET into terephthalic acid and ethylene glycol. However, the rate at which LCC degrades PET is too low. The genetically modified LCC can efficiently degrade polyethylene terephthalate (PET), such as LCC-A2, ICCG, ICCM, WCCG ​​and WCCM.

[0095] The term "CipA" used in this article refers to the scaffolding protein CipA of the cellulosome of Clostridium thermocellum, which is a hydrophobic protein that easily aggregates to form protein crystal inclusions (PCIs) under conditions of large-scale protein expression. Its formation mechanism is similar to that of general inclusion bodies. Secondly, CipA can act as a scaffold to assemble the co-expressed fusion proteins into subcellular structures without affecting the activity of the proteins.

[0096] The term "CipC" as used herein is a scaffolding protein that is organized similarly to the scaffolding protein of Clostridirmcellulovorans. It is 160,000 in size and is composed of a series of oval modules connected like a pearl necklace. Its N-terminus has a CBM3a module, followed by an X2 module of unknown function and seven hydrophobic adhesion protein modules. Its C-terminus contains a second X2 module of unknown function, followed by an eighth adhesion protein module, but does not contain any docking protein module, which may be involved in the process of anchoring to the cell surface, similar to the scaffolding protein CipA of the cellulosome of Clostridium thermocellum.

[0097] The term "CelF" as used herein is a dockerin (anchoring protein module) of the cellulase CelF that can specifically bind to CipC. The enzyme is 80,000 Da in size, of which the anchoring protein module is 13,000 Da in size and is present in Clostridium cellulosum ( Clostridium cellulolyticum ) in the genomic DNA and together with the scaffolding proteins form the cellulosome. The genes encoding the enzyme and anchoring protein module are located between CipC (the gene encoding the cellulosome scaffolding protein) and CelC (encoding endoglucanase C), and the corresponding enzymes belong to the cellulase L family (glycosyl hydrolase family).

[0098] The term "CelK" as used herein is a dockerin (anchoring protein module) of the cellulase CelK that specifically binds to CipA. The enzyme has a size of 98,000 Da, of which the anchoring protein module has a size of 11,000 Da, and is a member of the family Clostridium thermocellum ( C. thermocellum ) is the main component of the cellulosome. The enzyme domain consists of the IV family cellulose binding domain (CBD), the 9 family glycosyl hydrolase domain and the dockerin domain.

[0099] The term "anchor protein" as used herein means a protein that can be anchored to a cell membrane. Anchor proteins that can be used herein include, but are not limited to, GPI proteins, PIR proteins, lectin systems, and flocculin systems, or any combination, functional variants, or fragments thereof. In some embodiments, the anchor protein can be SED1 protein, Agα1 protein, GCW21 protein, GCW51 protein, GCW61 protein, or any combination, functional variants, or fragments thereof.

[0100] The term "mutant" as used herein refers to a protein referred to in the present disclosure, wherein one or more amino acids are added, and / or substituted, and / or removed (deleted), and / or incorporated (inserted) into the N-terminus, and / or C-terminus, and / or the native amino acid sequence within the protein.

[0101] The term "active fragment" used herein has the same or similar meaning as that conventionally understood by those skilled in the art, and refers to that the amino acid sequence of the fragment is a part of the amino acid sequence of the complete protein or polypeptide, but the fragment has the same or similar function or activity as the complete protein or polypeptide. Specifically, in the present disclosure, "active fragment" means any amino acid fragment obtained from any enzyme described in the present disclosure that has the activity of the enzyme.

[0102] As used herein, the term "replacement" or "substitution" of an amino acid may refer to a conservative amino acid substitution, wherein an amino acid residue is substituted with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, if an amino acid in a polypeptide is substituted with another amino acid from the same side chain family, the substitution is considered conservative. In another aspect, a string of amino acids can be conservatively replaced with a structurally similar string that differs in the order and / or composition of side chain family members.

[0103] "Percent sequence identity" or "percent identity" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by the sequences over the comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matching position is any position where the same nucleotide or amino acid is present in both the target sequence and the reference sequence. Gaps present in the target sequence are not counted because gaps are not nucleotides or amino acids. Likewise, gaps present in the reference sequence are not counted because target sequence nucleotides or amino acids are counted and nucleotides or amino acids from the reference sequence are not counted.

[0104] The sequence identity percentage can be calculated by the following process: determine the number of positions where the same amino acid residue or nucleic acid base occurs in both sequences to obtain the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the sequence identity percentage. The comparison of sequences and the determination of the sequence identity percentage between the two sequences can be completed using software that is easy to use and download online. Suitable software programs are available from various sources for the comparison of protein and nucleotide sequences. A suitable program for determining the sequence identity percentage is bl2seq, which is part of the BLAST program suite available from the BLAST website (blast.ncbi.nlm.nih.gov) of the National Center for Biotechnology Information of the U.S. government. Bl2seq uses BLASTN or BLASTP algorithms to compare between two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water or Matcher, part of the EMBOSS suite of bioinformatics programs and also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk / Tools / psa.

[0105] The term "operably connected" as used herein refers to the connection of at least a first element and a second element so that the constituent elements are in a relationship that allows them to function in their intended manner. For example, if a regulatory sequence (e.g., a promoter sequence) and a coding sequence are connected in a manner that allows the expression of the coding sequence to be controlled by the regulatory sequence, then the nucleic acid regulatory sequence is "operably connected" to the nucleic acid coding sequence. In some embodiments, the regulatory sequence that is "operably connected" is covalently bound directly or indirectly to the coding sequence (e.g., in a single nucleic acid molecule). In some embodiments, the regulatory sequence controls the expression of the coding sequence in a trans manner, and it is not a requirement for the regulatory sequence to be included in the same nucleic acid as the coding sequence to be operably connected.

[0106] An expression vector may contain a nucleic acid as described herein in a form suitable for expressing the nucleic acid in a host cell, which means that the expression vector includes one or more regulatory elements, which may be selected based on the host cell used for expression, and which are operably linked to the nucleic acid sequence to be expressed. Within an expression vector, "operably linked" is intended to link the nucleotide sequence of interest to the regulatory elements in a manner that allows the nucleotide sequence to be expressed (e.g., in an in vitro transcription / translation system or when the vector is introduced into a host cell, in a host cell).

[0107] Examples and drawings are provided below to help understand the present disclosure. However, it should be understood that these examples and drawings are only used to illustrate the present disclosure and do not constitute any limitation. The actual scope of protection of the present disclosure is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present disclosure.

[0108] Example 1 Establishment of engineered bacteria and mixed bacteria system

[0109] (1) Construction of engineered bacteria

[0110] In the mixed bacteria system disclosed in the present invention, the genetically engineered bacteria include surface display engineering bacteria that display scaffold proteins on the surface, single secretion expression engineering bacteria that secrete and express a single enzyme FAST-PETase or MHETase, and double secretion expression engineering bacteria that secrete and express two enzymes. The engineering bacteria used in this embodiment are shown in Table 1 below.

[0111] Table 1

[0112]

[0113] The specific steps for constructing engineering bacteria are as follows:

[0114] For the engineered bacteria with only a single exogenous gene inserted, the linear exogenous genes CipA-CBM-CipC-SED1 (CipA-linker1-CBM-linker2-CipC-GS linker-SED1), CipA-CBM-CipC-GCW51 (CipA-linker1-CBM-linker2-CipC-GS linker-GCW51), CipA-CBM-CipC-GCW61 (CipA-linker1-CBM-linker2-CipC-GS linker-GCW61), CipA-CBM-CipC-Aga1 (CipA-linker1-CBM-linker2-CipC-GS linker-GCW61), linker-Aga1), FAST (FAST-PETase-CelK) or MHETase (MHETase-CelF) were connected with the linear pPIC9K expression vector (PCR amplified using primers pPIC9K-F / pPIC9K-R) by seamless cloning to form a single gene expression vector. The single gene expression vector was linearized by SacI and then transformed into P. pastoris GS115 (CC200, Coolbo), P. pastoris KM71 (CC110, Coolbo), P. pastoris SMD1168 (CC120, Coolebob), using histidine-deficient medium (SC-his, Solebol) and G418 antibiotic selection to obtain gene-inserted engineered bacteria P. pastoris SMD1168 His::CipA-CBM-CipC-SED1 , P. pastoris SMD1168 His::CipA-CBM-CipC-GCW51 , P. pastoris SMD1168 His::CipA-CBM-CipC-GCW61 , P. pastoris SMD1168 His::CipA-CBM-CipC-Aga1 , P. pastoris SMD1168 His::FAST , P. pastoris SMD1168 His::MHETase , P. pastoris GS115 His::CipA-CBM-CipC-SED1 , P. pastoris GS115 His::FAST , P. pastoris GS115 His::MHETase , P. pastoris KM71 His::CipA-CBM-CipC-SED1 , P. pastoris KM71 His::FAST , P. pastoris KM71 His::MHETase .

[0115] For the engineered bacteria with both FAST and MHETase inserted, the linearized exogenous gene FAST was connected to the linearized pPICZC expression vector (PCR amplified using primers pPICZC-F / pPICZC-R) by seamless cloning to form a dual-gene expression vector. P. pastoris SMD1168 His::MHETase , Zeocin was used to screen for engineered bacteria with multiple copies of double genes inserted P. pastoris SMD1168 His::FAST-MHETase .

[0116] For detection of the copy number of exogenous gene insertion, P. pastoris SMD1168 His4::CipA-CBM-CipC-SED1 , P. pastoris SMD1168 His4::CipA-CBM-CipC-GCW51 , P. pastoris SMD1168 His4::CipA-CBM-CipC-GCW61 , P. pastoris SMD1168 His4::CipA-CBM-CipC-Aga1 , P. pastoris SMD1168 His4::FAST , P. pastoris SMD1168 His4::MHETase , P. pastoris SMD1168 His4::FAST-MHETaseThe genomic DNA was used as a template, and the primers qC-F / qC-R, qF-F / qF-R, and qM-F / qM-R were used to amplify the genes CipA-CBM-CipC, FAST, and MHETase, respectively; the primers qGAPDH-F / qGAPDH-R were used to amplify the housekeeping gene GAPDH (as an internal reference) to obtain the copy number of the exogenous protein inserted into the Pichia pastoris genome.

[0117] The sequence information involved in the construction of the engineered bacteria is shown in Table 2 (amino acid sequence) and Table 3 (DNA sequence), and the primer sequences used for the detection of the copy number of the exogenous gene insertion are shown in Table 4. The results of the copy number of the exogenous gene insertion in the engineered Pichia pastoris are shown in Table 5.

[0118] Table 2

[0119]

[0120] Table 3

[0121]

[0122] Table 4

[0123]

[0124] Table 5

[0125]

[0126] (2) Mixed bacteria system

[0127] The surface display engineered bacteria were mixed with two single secretion expression engineered bacteria and one double secretion expression engineered bacteria to construct a mixed bacteria PET degradation system containing three different engineered yeasts or two different engineered yeasts.

[0128] The specific degradation system construction method is as follows:

[0129] The surface display engineered bacteria and secretory expression engineered bacteria were pre-cultured separately in BMGY medium (1% yeast extract, 2% peptone, 100 mM pH 6.0 potassium hydrogen phosphate buffer, 1.34% amino acid-free yeast nitrogen source, 1% glycerol) at 30°C for 12 h and the OD of the pre-cultured products was detected. 600 According to the OD of the pre-culture product 600 Adjust the amount of each component added in the induced expression system, and the final system contains OD 600 =0.2 P. pastoris SMD1168 His::CipA-CBM-CipC-SED1 OD 600 =0.2 or 1.0 or 2.0 P. pastoris SMD1168 His::MHETaseOD 600 =0.2 or 1.0 or 2.0 P. pastoris SMD1168 His::FAST and / or OD 600 =0.2 or 1.0 or 2.0 P. pastoris SMD1168 His::FAST-MHETase (See Table 6 below for mixed bacteria system information).

[0130] Table 6 Construction of a mixed bacterial system of Pichia pastoris to achieve PET degradation

[0131]

[0132] Through similar steps as above, respectively use P. pastoris SMD1168 His4::CipA-CBM-CipC-GCW51 , P. pastoris SMD1168 His4::CipA-CBM-CipC-GCW61 and P. pastoris SMD1168 His4::CipA-CBM-CipC-Aga1 As a backbone protein display bacteria, P. pastoris SMD1168 His4::MHETase , P. pastoris SMD1168 His4::FAST , P. pastoris SMD1168 His4::FAST-MHETase Construct a mixed bacteria system.

[0133] In addition, we constructed P. pastoris GS115 His::CipA-CBM-CipC-SED1 , P. pastoris GS115 His::MHETase , P. pastoris GS115 His::FAST Mixed bacteria system, and P. pastoris KM71 His::CipA-CBM-CipC-SED1 , P. pastoris KM71 His::MHETase ,and P. pastoris KM71 His::FAST Mixed bacteria system.

[0134] Example 2 Expression and assembly of scaffold protein-enzyme complex

[0135] (1) Expression of scaffold protein-enzyme complex

[0136] After the engineered bacteria constructed in Example 1 were mixed according to Table 6, they were transferred to the induction expression medium BMMY (1% yeast extract, 2% peptone, 100 mM pH 6.0 potassium hydrogen phosphate buffer, 1.34% amino acid-free yeast nitrogen source, 1% methanol) to achieve the expression of the corresponding protein and the self-assembly of the scaffold protein-multienzyme complex. The expression was carried out at 30°C, and 0.5% methanol was added to the culture medium every 24 hours for 96 hours.

[0137] (2) Characterization of the self-assembly and display of scaffold protein-PET degrading enzyme on the surface of Pichia pastoris cells using flow cytometry

[0138] After induction of expression, take 50 μL of the diluted OD 600 = 0.1 engineered bacteria / mixed bacteria, centrifuge at 4000 g for 1 min at 4°C to collect the cells and resuspend them in 200 μL DPBS (D6502, Macklin) to wash the cells. Repeat the washing step 3 times. Wash and dilute the yeast cells with DPBS to OD 600 =0.1, take 25 μL of diluted cells and 0.1 μL of corresponding antibodies (V5, His, Myc antibodies, see Table 7 below for information on antibodies and their conjugates) and incubate on ice in the dark for 45 min. After incubation, centrifuge at 4000 g for 10 min at 4°C to remove the supernatant, add 200 μL DPBS and resuspend to wash the cells, and repeat the washing step three times. After washing, resuspend the cells in 1 mL DPBS, and flow cytometry was used to characterize the self-assembly and display of scaffold protein-PET degrading enzyme on the surface of Pichia pastoris cells.

[0139] Table 7 Antibody information for surface display detection of scaffold protein-PET degradation multi-enzyme complex

[0140]

[0141] Figure 2 The engineering bacteria were analyzed using flow cytometry. P. pastoris SMD1168 His::CipA-CBM-CipC-SED1 , P. pastoris SMD1168 His4::CipA-CBM-CipC-GCW51 , P. pastoris SMD1168 His4::CipA-CBM-CipC-GCW61 and P. pastoris SMD1168 His4::CipA-CBM-CipC-Aga1 The levels of scaffold proteins GCW61, GCW51, SED1, and Aga1 were displayed on the cell surface. The control group was cells without scaffold proteins inserted. P. pastoris Results for SMD1168.

[0142] Figure 3 The surface display effects of different types of Pichia pastoris strains GS115, SMD1168, and KM71 expressing the scaffold protein CipA-CBM-CipC-SED1 and the engineered bacteria expressing FAST-PETase and MHETase were shown by flow cytometry analysis. P. pastoris SMD1168 His4::CipA-CBM-CipC-SED1 The results after mixing with the corresponding purified protein FAST-PETase or MHETase. Figure 3 It can be seen that the three Pichia pastoris GS115, SMD1168, and KM71 can all express scaffold protein, FAST-PETase, and MHETase, among which the expression level of SMD1168 is higher than that of Pichia pastoris GS115 and KM71.

[0143] Example 3. Effects of co-culture of mixed bacterial systems in different proportions on the expression of scaffold proteins and FAST-PETase and MHETase

[0144] This example examines the results of co-culturing a mixed bacterial system formed by scaffold protein surface display engineering bacteria and single secretion expression engineering bacteria or double secretion expression engineering bacteria in different cell mass ratios.

[0145] The surface display engineered bacteria and secretory expression engineered bacteria were pre-cultured in BMGY medium at 30°C for 12 h and the OD of the pre-cultured products was detected. 600 According to the OD of the pre-culture product 600 Adjust the amount of each component added in the induced expression system. The mixed bacterial system contains OD 600 =0.2 P. pastoris SMD1168 His::CipA-CBM-CipC-SED1 OD 600 =0.2 or 1.0 or 2.0 P. pastoris SMD1168 His::MHETase OD 600 =0.2 or 1.0 or 2.0 P. pastoris SMD1168 His::FAST See Table 8 below for details:

[0146] Table 8

[0147]

[0148] The culture samples were taken at 24 h, 48 h, 72 h and 96 h, and flow cytometry analysis was performed according to Example 2. The results are shown in FIG. Figure 4A-4D As shown. Figure 4A-4DIt can be seen that the mixed bacterial system of scaffold protein surface display engineering bacteria and single secretion expression engineering bacteria in a ratio of 1:1 / 1:5 / 1:10 or 1:1:1 / 1:5:5 / 1:10:10 can achieve better co-expression of scaffold protein and enzymes FASTase and MHETase.

[0149] Furthermore, the scaffold protein surface display engineering bacteria and the single secretion expression engineering bacteria were mixed in a ratio of 1:1 or 1:1:1 and cultured in BMMY medium at 30°C. The culture samples were taken at 24h, 48h, 72h and 96h, respectively, and flow analysis was performed in the manner of Example 2. The results are shown in Figure 5A-Figure 5B In. From Figure 5A-Figure 5B It can be seen that the mixed bacteria system disclosed in the present invention has the stability to maintain the surface display of the scaffold protein-enzyme FASTase / MHETase for a long time.

[0150] PET plastic substrate (Xingxiang New Materials, 200 mesh, final concentration 1w / v% (0.1g / 10mL)) was added to the mixed bacterial system of scaffold protein surface display engineering bacteria and dual secretion expression engineering bacteria at a ratio of 1:1 or with two single secretion expression engineering bacteria at a ratio of 1:1:1, and then cultured in BMMY medium at 30°C. The culture was taken for 24h, 48h, 72h and 96h, respectively, and flow analysis was performed in the manner of Example 2. The results are shown in Figure 6A-6B In. From Figure 6A-6B It can be seen that in the BMMY culture medium containing PET plastic substrate, the surface display efficiency of the enzyme-scaffold protein complex in the mixed system is reduced.

[0151] Among them, the crystallinity test of PET plastic substrate was carried out using a differential scanning calorimeter (DSC 8000, PerkinElmer). PET was heated from 30°C to 300°C at a heating rate of 30°C / min, kept at 300°C for 1 min, and then cooled to 0°C within 5 min at the same rate of 30°C / min. The theoretical melting heat value (ΔHm°) of 100% crystalline PET is 140.1 J / g. The percentage of crystallinity was calculated using the melting heat (ΔHm) and cold crystallization (ΔHc) values, and the calculation formula is as follows:

[0152]

[0153] The test results are shown in Fig.13 The crystallinity of the PET plastic powder detected was 40.82%, which is a high-crystallinity plastic substrate (crystallinity>30%) and is difficult to degrade. This further illustrates that the engineered strain mixed bacterial system constructed in this patent has good PET degradation ability.

[0154] Example 4. Surface display of enzyme-scaffold protein complexes by engineered bacteria expressing scaffold proteins and single-secretion or double-secretion expression

[0155] This example detects the results of surface display of scaffold proteins and enzymes FASTase / MHETase in mixed bacterial systems of scaffold protein surface display engineering bacteria, single secretion expression engineering bacteria and / or double secretion expression engineering bacteria.

[0156] The surface display engineered bacteria and secretory expression engineered bacteria were pre-cultured in BMGY medium at 30°C for 12 h and the OD of the pre-cultured products was detected. 600 According to the OD of the pre-culture product 600 Adjust the amount of each component added in the induced expression system. The mixed bacterial system contains OD 600 =0.2 P. pastoris SMD1168 His::CipA-CBM-CipC-SED1 OD 600 =0.2 P. pastoris SMD1168 His::MHETase OD 600 =0.2 P. pastoris SMD1168 His::FAST and / or OD 600 =0.2 P. pastoris SMD1168 His::FAST-MHETase See Table 9 below for details:

[0157] Table 9

[0158]

[0159] The scaffold protein (Scaf.) contains a V5 tag, MHETase (M) contains a His tag, and FAST (F) contains a Myc tag. After 96 hours of induction, the surface display effects of the scaffold protein, enzyme, and multi-enzyme complex were detected using flow cytometry and laser confocal microscopy. The results are shown in Figure 7 and Figure 8 In. From Figure 7 and Figure 8 It can be seen that in the mixed bacteria degradation system, FAST-PETase and MHETase are secreted into the culture medium, and by combining with the surface-displayed scaffold protein Scaf., the self-assembly of the scaffold protein-multi-enzyme complex is achieved.

[0160] Example 5. Effect of the scaffold protein-multi-enzyme complex plastic degradation system disclosed herein on degrading plastics

[0161] According to the method of Example 3, a mixed bacteria system as shown in Table 10 is obtained.

[0162] Table 10

[0163]

[0164] PET powder (200 mesh, final concentration 1w / v%) was added to the mixed bacteria system in Table 10 above, and induced expression in BMGY medium at 30°C. After 96 hours of induction expression, the supernatant and precipitate of the mixed bacteria system were separated by centrifugation after degradation at 30°C for 48 hours. The supernatant of the reaction system was taken and heated at 95°C for 10 minutes to terminate the reaction. The content of degradation reaction products in the supernatant filtered through a 0.22 μm filter membrane was detected by high performance liquid chromatography (HPLC). The PET degradation reaction results of the mixed bacteria system with scaffold protein surface display bacteria: single secretion / double secretion expression engineering bacteria = 1:10 / 1:10:10 are shown in Fig. 9 In. From Fig. 9 The HPLC results showed that the degradation of PET plastic while inducing expression inhibited the surface display of the complex and thus the degradation efficiency.

[0165] The mixed bacterial system in Table 10 was induced to express in BMGY medium at 30°C. 600 When the pH value reached 20.0, the culture product was subjected to degradation reaction in 300 μL of Glycine-NaOH mixed solution (pH 7.0) containing PET powder (200 mesh, 5 mg / mL). The degradation reaction was carried out at 30°C for 48 h. After the degradation reaction at 30°C for 48 h, the supernatant and precipitate of the mixed bacterial system were separated by centrifugation. The supernatant of the reaction system was taken and heated at 95°C for 10 min to terminate the reaction. The content of degradation reaction products in the supernatant filtered through a 0.22 μm filter membrane was detected by high performance liquid chromatography (HPLC). The results are shown in Figure 10-12 middle.

[0166] Fig.10 This is the PET degradation reaction result of the mixed bacteria system of scaffold protein surface display bacteria: single secretion / double secretion expression engineering bacteria = 1:10 / 1:10:10.

[0167] Fig.11 This is the PET degradation reaction result of the mixed bacteria system of scaffold protein surface display bacteria: single secretion / double secretion expression engineering bacteria = 1:5 / 1:5:5.

[0168] Fig.12 This is the PET degradation reaction result of the mixed bacteria system of scaffold protein surface display bacteria: single secretion / double secretion expression engineering bacteria = 1:1 / 1:1:1.

[0169] Figure 10-12The HPLC results showed that both the double-bacteria and triple-bacteria mixed bacteria systems can achieve efficient degradation of PET plastics, and the higher the ratio of enzyme-secreting bacteria: scaffold protein surface-displaying bacteria in the mixed bacteria system, the higher the degradation efficiency of PET plastics by the mixed bacteria system.

[0170] In summary, both the dual-bacteria and triple-bacteria mixed bacterial systems can produce scaffold protein-multienzyme complexes displayed on the cell surface to achieve PET plastic degradation. The dual-bacteria system has a significantly higher degradation efficiency for PET plastic than the triple-bacteria system.

[0171] The technical solution of the present disclosure is not limited to the above-mentioned specific embodiments, and all technical variations made according to the technical solution of the present disclosure fall within the protection scope of the present disclosure.

Claims

1. A scaffold protein, characterized in that The scaffold protein comprises: Domains localized to the cell surface; one or more carbohydrate binding domains; and One member of one or more peptide interaction pairs, Wherein, the amino acid sequence of the scaffold protein is shown in any one of SEQ ID NOs: 13-16.

2. Plastic degradation system, characterized in that: The degradation system comprises a surface display module and one or more plastic degradation modules; wherein, The surface display module comprises a cell and the scaffold protein according to claim 1 displayed on the surface of the cell; The plastic degradation module includes a plastic degradation domain and the other member of a polypeptide interaction pair, and the plastic degradation system includes a first plastic degradation module and a second plastic degradation module. The amino acid sequence of the first plastic degradation module and / or the second plastic degradation module is as shown in any one of SEQ ID NOs: 5-6.

3. The system according to claim 2, characterized in that The first plastic degradation module and the second plastic degradation module are different from each other; and / or The first plastic degradation module and the second plastic degradation module are expressed by the same cell and secreted outside the cell, or are expressed by different cells and secreted outside the cell respectively.

4. The system according to claim 2 or 3, characterized in that: The cell is selected from Pichia pastoris.

5. An isolated nucleic acid molecule, characterized in that The nucleic acid molecule encodes the scaffold protein according to claim 1, or the system according to any one of claims 2-4. An expression vector comprising the nucleic acid molecule of claim 5 .

7. A host cell, characterized in that The host cell includes a surface display host cell and / or a secretory expression host cell, wherein the surface display host cell displays the scaffold protein according to claim 1 on the cell surface; The secretory expression host cell secretes and expresses the plastic degradation domain and the other member of the polypeptide interaction pair.

8. The host cell according to claim 7, characterized in that The secretory expression host cell secretes and expresses the first plastic degradation module and the second plastic degradation module, the first plastic degradation module and the second plastic degradation module are secreted and expressed by the first secretory expression host cell, or the first plastic degradation module and the second plastic degradation module are secreted and expressed by the second secretory expression host cell and the third secretory expression host cell respectively, the first plastic degradation module and the second plastic degradation module are as defined in any one of claims 2 or 3; and / or The host cell is selected from Pichia pastoris.

9. Use of the scaffold protein of claim 1, the system of any one of claims 2 to 4, the nucleic acid molecule of claim 5, the expression vector of claim 6 or the host cell of claim 7 or 8 in plastic degradation.

10. A plastic degradation method, characterized in that: The plastic degradation method uses the host cell described in claim 7 or 8 to degrade plastic.

11. The method according to claim 10, characterized in that In the host cells, the content ratio of the surface display host cells to the first secretory expression host cells is 1:(1-10); or In the host cells, the content ratio of the surface display host cells to the second secretory expression host cells and the third secretory host cells is 1:(1-10):(1-10).

12. The method according to claim 10, characterized in that The plastic is PET plastic.

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