Multi-enzyme synergistic catalysis carbon sequestration method

Abstract

The invention discloses a multienzyme synergistic catalysis carbon sequestration method, and belongs to the technical field of bioengineering. Virus-like nanoparticles P22 composed of capsid protein (CP) and scaffold protein (SP) are used as a platform for constructing a carboxyl body simulant, carbonic anhydrase (CA) and ribulose-1, 5-diphosphate carboxylase / oxygenase (RuBisCO) are connected to the scaffold protein in a gene fusion mode, the non-covalent self-assembly characteristic of CP and SP is utilized, and the carboxyl body simulant is constructed through the non-covalent self-assembly characteristic of CP and SP. The artificial carboxyl body based on the virus-like particles is constructed by adding the virus-like particles into the carrier to realize intracellular and extracellular recruitment of the cargo protein, the catalytic efficiency of RuBisCO is further improved through multi-enzyme synergistic catalysis, and the artificial carboxyl body can be used for enhancing the CO2 immobilization capability of cells. According to the invention, a simple carboxyl body simulant is constructed from bottom to top, and early evolution benefits of multi-enzyme concerted catalysis realized by co-localization of CA and RuBisCO are deeply understood, so that a new solution idea is provided for better understanding the carbon sequestration function of a carboxyl body.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and relates to a multi-enzyme coordinated catalytic carbon fixation method, and specifically relates to the construction of an artificial carboxysome based on virus-like particles and its application in multi-enzyme coordinated catalytic carbon fixation. Background Art

[0002] Carboxysomes are bacterial microcompartments that encapsulate carbonic anhydrase and ribulose-1,5-bisphosphate carboxylase / oxygenase and, as sites of CO2 fixation, play a crucial role in the CO2 concentrating mechanism of many chemolithoautotrophs and all cyanobacteria. Carboxysomes and other bacterial microcompartments self-assemble through specific protein-protein interactions that are often mediated by conserved protein domains. Through the activity of the CAs, the captured HCO 3- Rapidly dehydrated to CO2, enriched near RuBisCO, and then fixed by the RuBisCO holoenzyme molecule contained in each organelle through the Calvin cycle, fixing one molecule of CO2 and adding it to one molecule of 1,5-bisphosphate ribulose (RUBP) to produce two molecules of 3-phosphoglycerate (3-PGA), which are directly discharged from the carboxysome for use by the cell. In addition to providing a high concentration of CO2, the outer shell acts as a barrier to O2 on the surface, thereby increasing the efficiency of CO2 fixation and isolating competition from O2.

[0003] In order to mimic the self-assembly of natural proteins, scientists have developed different design strategies. Genetic manipulation has been used to improve the catalytic efficiency of Rubisco in green plants. In addition to these biological systems for engineering Rubisco in vivo, scientists have also attached great importance to establishing in vitro methods to enhance the catalytic activity of Rubisco. For example, enzyme immobilization has been used in Rubisco engineering; however, inappropriate enzyme immobilization often leads to conformational changes in the enzyme, resulting in reduced enzyme activity or even inactivation. So the choice of technology should be very cautious. Therefore, improving the carboxylation efficiency of Rubisco remains a challenge that scientists are committed to crossing.

[0004] Among all enzyme immobilization methods, covalent immobilization provides the strongest bond between the enzyme and the support, but it usually involves complex linkers and organic solvents.

[0005] Virus-like particles (VLPs) are self-assembled supramolecular protein nanoparticles that are self-assembled from multiple copies of one (or several different) protein subunits. VLPs can be used as a good platform for loading protein cargo due to their uniform size and shape, multiple attachment sites on the inner and outer surfaces, ease of modification, and biocompatibility. The cargo proteins loaded on VLPs have the characteristics of high copy number, high local concentration, easy tracking, long half-life, and good robustness. Therefore, VLPs have broad application prospects in drug carriers, vaccine development, gene therapy, and synthetic biology. Summary of the invention

[0006] In view of the problem of low catalytic efficiency of RuBisCO mentioned above, the object of the present invention is to provide a multi-enzyme synergistic catalytic carbon fixation method. The present invention selects a virus-like particle P22 protein cage model system, uses SP as a mediator, and utilizes the spontaneous self-assembly characteristics of CP and SP to connect fluorescent proteins CFP and YFP to SP domains to form fusion proteins, so that CFP and YFP are encapsulated on the inner surface of nanoparticles, or different cargo proteins ribulose-1,5-bisphosphate carboxylase / oxygenase (RuBisCO) and carbonic anhydrase (CA) are respectively connected to the SP domain, so that RuBisCO and CA are encapsulated on the inner surface of nanoparticles, ultimately achieving efficient loading of different cargo proteins on the inner surface of P22 nanoparticles.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] In a first aspect, the present invention provides a method for constructing an artificial carboxysome based on a virus-like particle, comprising the following steps:

[0009] (1) inserting the capsid protein CP gene fragment of virus-like particle P22 into a plasmid vector to construct a plasmid capable of expressing CP in host cells;

[0010] (2) connecting the gene fragment of the scaffold protein SP domain of the virus-like particle P22 with the gene fragment of the target protein through a flexible linker sequence to obtain a recombinant gene fragment capable of expressing the target protein molecule with the scaffold protein SP domain, inserting the fragment into a plasmid vector, and constructing a plasmid capable of expressing the target protein molecule with the scaffold protein SP domain in a host cell; the target proteins are ribulose-1,5-bisphosphate carboxylase / oxygenase (RuBisCO) and carbonic anhydrase (CA);

[0011] (3) The plasmid capable of expressing CP in host cells constructed in step (1) and the target protein molecule with the SP scaffold protein domain constructed in step (2) are co-transfected into host cells. After culturing and distribution-induced protein expression, the capsid protein CP and the target protein connected to the SP domain spontaneously assemble, the host cells are broken, and artificial carboxysomes are obtained by size exclusion chromatography separation.

[0012] Based on the above technical solution, further, the amino acid sequence of the capsid protein CP of the virus-like particle P22 in step (1) is as shown in SEQ ID NO.1.

[0013] Based on the above technical solution, further, the plasmid vector described in step (1) is a plasmid vector with a T7 promoter, the gene fragment is inserted after the T7 promoter of the plasmid vector, and the capsid protein is expressed in large quantities in the host cell after induction by arabinose.

[0014] Based on the above technical solution, further, the plasmid vector described in step (1) is a pBAD vector, and the CP gene fragment is inserted between the NcoI and XhoI sites of the pBAD vector.

[0015] Based on the above technical solution, further, the amino acid sequence of the scaffold protein SP described in step (2) is shown as SEQ ID NO.2.

[0016] Based on the above technical solution, further, in step (2), the amino acid sequence of RuBisCO is shown as positions 1-466 in SEQ ID NO.5; the amino acid sequence of CA is shown as positions 2-261 in SEQ ID NO.6.

[0017] Based on the above technical solution, further, the amino acid sequence encoded by the flexible linker sequence between the gene fragment of the scaffold protein SP domain and the gene fragment of the target protein in step (2) is (GGS) n or (GGGGS) n , n=1-10, preferably (GGGGS)3.

[0018] Based on the above technical scheme, the plasmid vector described in step (2) is a plasmid vector with a T7 promoter, and the recombinant gene fragment is inserted after the T7 promoter of the plasmid vector, and the target protein is expressed in large quantities in the host cell after induction by isopropylthiogalactoside (IPTG), preferably pCDF-Duet-1 and pACYC-Duet-1.

[0019] Based on the above technical scheme, the plasmid vector described in step (2) is the pACYC-Duet-1 plasmid, and the recombinant gene fragment of the target protein molecule with the SP domain is inserted into the NcoI and EcoRI sites and between NdeI and XhoI of the pACYC-Duet-1 plasmid.

[0020] Based on the above technical solution, further, the host cell described in step (3) includes Escherichia coli, yeast and animal cells, preferably Escherichia coli BL21 (DE3).

[0021] Based on the above technical scheme, further, the specific process of distribution induction described in step (3) is: first add 0.1-1mM IPTG to induce the expression of the target protein, and after 3-5h of expression, add arabinose with a final concentration of 1-3% to induce the expression of capsid protein.

[0022] Based on the above technical solution, further, the OD of the host cell in step (3) 600 When the pH reached 0.6-0.8, IPTG was added to induce protein expression, and the IPTG concentration was 0.3 mM.

[0023] In a second aspect, the present invention also provides an artificial carboxysome obtained by the above-mentioned construction method.

[0024] In a third aspect, the present invention provides a multi-enzyme synergistic catalytic carbon fixation method, wherein the above-mentioned artificial carboxylate body is added to a mixture containing 1,5-bisphosphate ribulose (RUBP) and HCO3 - In a buffer solution, 3-phosphoglycerate is synthesized by the synergistic catalysis of ribulose-1,5-bisphosphate carboxylase / oxygenase (RuBisCO) and carbonic anhydrase (CA).

[0025] Based on the above technical solution, further, the buffer solution is composed of 100mM Bicine, 25mM MgCl2, pH 8.0.

[0026] In a fourth aspect, the present invention provides a method for constructing an intracellular protein recruitment system based on virus-like particles, comprising the following steps:

[0027] (1) inserting the capsid protein CP gene fragment of virus-like particle P22 into a plasmid vector to construct a plasmid capable of expressing CP in host cells;

[0028] (2) connecting the gene fragment of the scaffold protein SP domain of the virus-like particle P22 with the gene fragment of the target protein through a flexible linker sequence to obtain a recombinant gene fragment capable of expressing the target protein molecule with the scaffold protein SP domain, inserting it into a plasmid vector, and constructing a plasmid capable of expressing the target protein molecule with the scaffold protein SP domain in the host cell; the target protein fluorescent proteins CFP and YFP;

[0029] (3) The plasmid capable of expressing CP in host cells constructed in step (1) and the target protein molecule with the SP scaffold protein domain constructed in step (2) are co-transferred into the host cells. After culturing and distribution to induce protein expression, the capsid protein CP and the target protein connected with the SP domain are spontaneously assembled in the cell, thereby constructing a protein recruitment system in the host cell.

[0030] Based on the above technical solution, further, the amino acid sequence of the capsid protein CP of the virus-like particle P22 in step (1) is as shown in SEQ ID NO.1.

[0031] Based on the above technical solution, further, the plasmid vector described in step (1) is a plasmid vector with a T7 promoter, the gene fragment is inserted after the T7 promoter of the plasmid vector, and the capsid protein is expressed in large quantities in the host cell after induction by arabinose, preferably a pBAD vector.

[0032] Based on the above technical solution, further, the plasmid vector described in step (1) is a pBAD vector, and the CP gene fragment is inserted between the NcoI and XhoI sites of the pBAD vector.

[0033] Based on the above technical solution, further, the amino acid sequence of the scaffold protein SP described in step (2) is shown as SEQ ID NO.2.

[0034] Based on the above technical solution, further, in step (2), the amino acid sequence of the fluorescent protein CFP is shown as positions 14-252 in SEQ ID NO.3; the amino acid sequence of the fluorescent protein YFP is shown as positions 12-248 in SEQ ID NO.4.

[0035] Based on the above technical solution, further, the amino acid sequence encoded by the flexible linker sequence between the gene fragment of the scaffold protein SP domain and the gene fragment of the target protein in step (2) is (GGS) n or (GGGGS) n , n=1-10, preferably (GGGGS)3.

[0036] Based on the above technical scheme, the plasmid vector described in step (2) is a plasmid vector with a T7 promoter, and the recombinant gene fragment is inserted after the T7 promoter of the plasmid vector, and the target protein is expressed in large quantities in the host cell after induction by isopropylthiogalactoside (IPTG), preferably pCDF-Duet-1 and pACYC-Duet-1.

[0037] Based on the above technical solution, the plasmid vector described in step (2) is the pCDF-Duet-1 plasmid, and the recombinant gene fragment of the target protein molecule with the SP domain is inserted into the NcoI and SacI sites and between NdeI and XhoI of the pCDF-Duet-1 plasmid.

[0038] Based on the above technical solution, further, the host cell described in step (3) includes Escherichia coli, yeast and animal cells, preferably Escherichia coli BL21 (DE3).

[0039] Based on the above technical scheme, further, the specific process of distribution induction described in step (3) is: first add 0.1-1mM IPTG to induce the expression of the target protein, and after 3-5h of expression, add arabinose with a final concentration of 1-3% to induce the expression of capsid protein.

[0040] Based on the above technical solution, further, the OD of the host cell in step (3) 600 When the pH reached 0.6-0.8, IPTG was added to induce protein expression, and the IPTG concentration was 0.3 mM.

[0041] The beneficial effects of the present invention compared with the prior art are as follows:

[0042] 1. The present invention constructs an extracellular artificial carboxysome system based on virus-like particles with a stable structure. The system can provide technical support for the fixation of CO2 through multi-enzyme coordinated catalysis in cells.

[0043] 2. The present invention has developed a method based on a viroid-based intracellular protein recruitment system, which can achieve the effect of controllable recruitment of model proteins within cells and can be widely used.

[0044] 3. The present invention loads different cargo proteins on the inner surface of virus-like particles P22, and develops a method for constructing a self-assembly system based on virus-like particles P22. This method provides technical support for constructing artificial multi-enzyme complexes, increasing local enzyme concentrations, promoting substrate transfer effects, and improving enzymatic reaction efficiency.

[0045] 4. The virus-like particles (VLPs) used in the present invention can spontaneously self-assemble to form caged proteins, and are therefore suitable as protein recruitment scaffolds for constructing artificial carboxysomes, with the following advantages: (1) The self-assembly of P22 coat protein into a 58 nm capsid depends on the interaction between the coat protein and the helix-turn-helix motif on the scaffold protein (SP), while the rest of the scaffold protein can be significantly truncated, with little or no effect on the assembly; (2) Compared with other virus-like particles, it has a larger inner cavity and less steric hindrance, and can provide a large encapsulation capacity, capable of encapsulating 20 kDa to 180 kDa cargo; (3) P22 has a robust supramolecular protein cage structure and is structurally stable. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the embodiments of the present invention, the drawings related to the embodiments are briefly introduced below.

[0047] Figure 1 Schematic diagram of the self-assembly of the artificial carboxysome system based on virus-like particles of the present invention.

[0048] Figure 2 The expression profile of CP, RuBisCO-SP and CA-SP fusion proteins in P22-RuBisCO-CA VLP was tested by SDS-PAGE protein electrophoresis. Band 1 is P22-RuBisCO-CA VLP, in which the theoretical molecular weight of CP is 46.7 kDa, the theoretical molecular weight of RuBisCOsp is 70.7 kDa, and that of CAsp is 50.7 kDa. M is a marker.

[0049] Figure 3 Gel chromatogram of the complex assembled between CP and RuBisCO-SP and CA-SP in vitro.

[0050] Figure 4 Transmission electron microscopy image of the complex assembled from CP and RuBisCOsp-CAsp.

[0051] Figure 5 Dynamic light scattering detection analysis diagram of virus-like particles P22 before and after assembly with RuBisCO and CA protein molecules.

[0052] Figure 6 This is a diagram showing the results of multi-enzyme synergistic catalytic carbon fixation based on artificial carboxysomes.

[0053] Figure 7 This is the result of laser scanning confocal microscopy observation of the assembly of intracellular CP, CFP-SP and YFP-SP.

[0054] Figure 8 The CFP fluorescence lifetime graphs in the experimental group and the control group were detected for the fluorescence lifetime imaging experiment. DETAILED DESCRIPTION

[0055] For ease of understanding of the present invention, the present invention is described in detail below in conjunction with embodiment. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive, but embodiments of the present invention are not limited thereto.

[0056] Obviously, the embodiments described below are only partial embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative work and all of them fall into the protection scope of the present invention.

[0057] The experimental methods in the following examples where specific conditions are not specified are generally carried out under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

[0058] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0059] The present invention is further described below through specific examples, but is not intended to limit the protection scope of the present invention.

[0060] Example 1: Wild-type virus-like particle P22 WT And the construction of engineering strains of capsid protein CP and target protein molecules YFP-SP, CFP-SP, RuBisCO-SP, CA-SP

[0061] All construction processes in this embodiment are based on the basic principle of polymerase chain reaction (PCR).

[0062] This embodiment selects virus-like particles P22, which is a mature protein cage model system, which has been used as a nanomaterial in cargo encapsulation, molecular presentation, and as a model for understanding the physical aspects of infectious phage structure and function. The assembly of VLP occurs through the copolymerization of 420 capsid proteins (CP) and 100 to 300 internalized scaffold proteins (SP), with a larger inner cavity, smaller steric hindrance, and can provide a large encapsulation amount, while having structural stability, good biocompatibility and other characteristics, and there is no potential pathogenic risk and other advantages, and has the potential to be used as a cargo protein loading platform. The present invention functionally modifies the inner surface of P22, uses SP as a mediator, and utilizes the characteristics of spontaneous self-assembly of CP and SP to connect fluorescent proteins CFP and YFP to the SP domain through flexible links to form fusion proteins, so that CFP and YFP are encapsulated on the inner surface of nanoparticles. Different cargo proteins RuBisCO and CA are also connected to the SP domain through flexible links, and finally realize the efficient loading of different cargo proteins on the inner surface of P22 nanoparticles.

[0063] The amino acid sequence of the capsid protein CP of the virus-like particle P22 is shown in SEQ ID NO.1, and the amino acid sequence of the scaffold protein SP is shown in SEQ ID NO.2. Fluorescent proteins include CFP and YFP, cargo proteins include RuBisCO and CA, the amino acid sequence of CFP-SP is shown in SEQ ID NO.3, the amino acid sequence of YFP-SP is shown in SEQ ID NO.4, the amino acid sequence of RuBisCO-SP is shown in SEQ ID NO.5, the amino acid sequence of CA-SP is shown in SEQ ID NO.6, the amino acid sequence of CFP is shown in SEQ ID NO.3 at positions 14-252, the linker sequence is (GGGGS)3, and the amino acid sequence of SP is SEQ ID NO.3 at positions 268-430; the amino acid sequence of YFP is shown in SEQ ID NO.4 at positions 12-248, the linker sequence is (GGGGS)3, and the amino acid sequence of SP is SEQ ID NO.4 at positions 264-426; the amino acid sequence of RuBisCO is shown in SEQ ID NO.5 at positions 1-466, the linker sequence is (GGGGS)3, and the amino acid sequence of SP is SEQ ID NO.5, positions 482-644; the amino acid sequence of CA is shown in SEQ ID NO.6, positions 2-261, the linker sequence is (GGGGS)3, and the amino acid sequence of SP is SEQ ID NO.6, positions 277-439.

[0064] In order to construct pBAD-CP, the vector carrying the target gene CP was used as a template and CP-F / R as primers. The DNA fragment of CP was obtained by PCR amplification and inserted between the NcoI and XhoI sites of the pBAD plasmid by homologous recombination. Gibson ligation and transformation were performed. After obtaining the positive plasmid, sequencing was verified and the accurately sequenced plasmid was stored for future use.

[0065] In order to construct PCDF-Duet-1-CP-SP, the vector carrying the target gene CP was used as a template and SP-VF / VR was used as primers to obtain the vector fragment containing CP through PCR amplification; then the vector carrying the target gene SP was used as a template and SP-F / R was used as primers to obtain the DNA fragment of SP through PCR amplification; the two fragments obtained above were purified by PCR products, Gibson ligated and transformed, and the positive plasmid was obtained and sequenced for verification, and the accurately sequenced plasmid was stored for future use.

[0066] The genes of protein molecules YFP-SP and RuBisCO-SP were respectively inserted into the NcoI and SacI sites of the plasmid pCDF-Duet-1 and the NcoI and EcoRI sites of PACYC-Duet-1 by homologous recombination. At the same time, the CFP-SP and CA-SP genes were respectively inserted into the NdeI and XhoI sites of the pCDF-Duet-1-YFP-SP plasmid and the NdeI and XhoI sites of PACYC-Duet-1-RuBisCO-SP by homologous recombination. Gibson ligation and transformation were performed to obtain positive plasmids pCDF-Duet-1-YFPsp-CFPsp and pACYC-Duet-1-RuBisCOsp-CAsp, which were then sequenced and verified. The accurately sequenced plasmids were stored for future use.

[0067] The obtained plasmids were then introduced into Escherichia coli cells BL21 (DE3), thereby obtaining engineering strains that can express coat protein CP and scaffold protein SP, engineering strains that can express coat protein CP and protein molecules YFP-SP / CFP-SP, and engineering strains that can express coat protein CP and RuBisCO-SP / CA-SP.

[0068] All plasmids were verified by sequencing and then transformed into E. coli BL21 (DE3) for recombinant protein expression. The primers used in this example are shown in Table 1, and the corresponding PCR amplification system is shown in Table 2.

[0069] Table 1. Main primers used in designing fusion plasmids in this example

[0070]

[0071] Table 2. PCR system and homologous recombination system

[0072]

[0073] Example 2: Expression, purification and characterization of constructed artificial carboxysomes

[0074] (1) Recombinant plasmid transformation and expression: The obtained protein recombinant plasmid pCDF-Duet-1-CP-SP was introduced into E. coli BL21 (DE3) by heat shock transformation method, and the obtained positive clone strain was amplified and cultured at 37°C and 220 rpm for 6 h. Similarly, pBAD-CP and pACYC-Duet-1-RuBisCOsp-CAsp were introduced into E. coli BL21 (DE3) by heat shock transformation method, and the obtained positive clone strain was amplified and cultured at 37°C and 220 rpm. The distribution induction method was used, and when the OD of the E. coli fermentation broth reached 600 When it reaches 0.6-0.8, add IPTG (final concentration is 0.3mM) to induce cargo protein expression for 4 hours, then add 2% arabinose to induce capsid protein expression, and continue expression for 2 hours. Centrifuge the E. coli fermentation broth after a total of 6 hours of induction culture at 12000rpm, collect the bacteria, and discard the supernatant. Resuspend the bacteria in 20ml binding buffer (1.6MNaCl, 50mM Tris-HCl, pH=8.0) and use a high-pressure homogenizer to break it for more than 5 minutes. Centrifuge the broken cell resuspension at 12000rpm for 10 minutes, collect the supernatant, and filter it with a 0.22μm filter membrane. Use elution buffer (1.6M NaCl, 50mM Tris-HCl, pH=8.0) size exclusion chromatography (SEC) to separate and purify the target protein. Finally, collect the protein, concentrate and package it, add 10% glycerol and store it at -80℃ for later use;

[0075] (2) Extracellular characterization of artificial carboxysomes: The molecular weight of the obtained protein monomer was detected by SDS-PAGE electrophoresis. The electrophoresis results showed that the molecular weight of the target band was consistent with the theoretical molecular weight ( Figure 2 ); The molecular weight of the obtained protein was detected by gel chromatography, and the peak position showed that the molecular weight of the target band was consistent with the theoretical molecular weight, indicating that the virus-like particle P22 successfully self-assembled ( Figure 3 ); by transmission electron microscopy (TEM) ( Figure 4) Characterization of the complex assembled by CP and RuBisCOsp-CAsp showed that the virus-like particles P22-RuBisCO-CA showed a stable cage structure with uniform particle size, which was consistent with the theoretical structure. The particle size of P22-RuBisCO-CA was measured by dynamic light scattering technology, and the results showed that the uniformity of P22-RuBisCO-CA particles was very good ( Figure 5 );

[0076] Example 3: Multi-enzyme synergistic catalysis of carbon fixation based on artificial carboxysomes

[0077] (1) Recombinant plasmid transformation and expression: The obtained protein recombinant plasmid pACYC-Duet-1-RuBisCOsp-CAsp was introduced into E. coli BL21 (DE3) by heat shock transformation method. The obtained positive clone strain was amplified and cultured at 37°C and 220rpm for 6h. The E. coli fermentation broth after 6h of induction culture was centrifuged at 12000rpm, the cells were collected, and the supernatant was discarded. The cells were resuspended in 20ml binding buffer (1.6M NaCl, 50mM Tris-HCl, pH=8.0) and disrupted by a high-pressure homogenizer for more than 5min. The cell resuspension after disruption was centrifuged at 12000rpm for 10min, the supernatant was collected, and filtered with a 0.22μm filter membrane. The target proteins were separated and purified by size exclusion chromatography (SEC) using elution buffer (1.6M NaCl, 50mM Tris-HCl, pH=8.0) to obtain RuBisCOsp and CAsp proteins, respectively. Finally, the protein was collected, concentrated, packaged, and stored at -80°C with 10% glycerol for later use;

[0078] (2) Multi-enzyme synergistic catalytic carbon fixation: In order to explore the multi-enzyme synergistic carbon fixation effect of CA and RuBisCO in artificial carboxysomes, we established a CO2 fixation reaction system outside the cell.

[0079] Free RuBisCOsp and CAsp were set as the control group, and P22 VLP co-encapsulating RuBisCOsp and CAsp (P22-RuBisCO-CA VLP) was set as the experimental group.

[0080] First, 50mM NaHCO3, 52nM RuBisCOsp, and 14nM CAsp (the experimental group added P22VLPs co-encapsulating RuBisCOsp and CAsp) were added to the buffer (100mM Bicine, 25mM MgCl2, pH 8.0), and the mixture was left to stand at room temperature (25°C) for 10 minutes to activate RuBisCO and make it active. Then, 300μM substrate RUBP was added to start the reaction, and 200μL formic acid was added 6 minutes later to terminate the reaction. After overnight freeze-drying, the sample was re-dissolved in the same volume of buffer, filtered through a 0.22μm organic filter membrane, and the 3-phosphoglyceric acid (3-PGA) content was quantitatively analyzed by LC-MS.

[0081] Figure 6 The result of the extracellular multi-enzyme synergistic catalytic carbon fixation based on artificial carboxysomes is Figure 6 It can be seen that the experimental results show that compared with the free enzyme system, the 3-PGA obtained by the reaction system of P22-RuBisCO-CA VLP (artificial carboxysome) has a significant increase. This not only proves that CA in the artificial carboxysome successfully enriches CO2 at a high concentration around RuBisCO, increases the catalytic activity of RuBisCO, thereby improving the CO2 fixation efficiency, but also verifies the successful construction of the artificial carboxysome.

[0082] Example 4: The assembly method is applied to Escherichia coli cells to explore its assembly effect in the cells, comprising the following steps:

[0083] (1) The YFP gene carrying SP (YFP-SP) and the CFP gene carrying SP (CFP-SP) were inserted between the NcoI and SacI sites and between the NdeI and XhoI sites of the pCDF-Duet-1 plasmid, respectively, to construct a plasmid that can express YFP-SP / CFP-SP.

[0084] (2) The CP gene was inserted between the NcoI and XhoI sites of the vector pBAD to construct a plasmid that can express the virus-like particle capsid protein CP.

[0085] (3) The two constructed plasmids were introduced into Escherichia coli BL21 (DE3) cells to obtain an engineered strain Strain1 that can express the corresponding proteins under the induction of IPTG and arabinose, thereby obtaining a protein assembly system constructed in the cell.

[0086] (4) The strain Strain 1 was cultured in 10 mL LB medium containing 50 mg / L ampicillin or 50 mg / L streptomycin sulfate until the OD 600When the pH value reached 0.6-0.8, it was divided into two parts. One part was added with 0.3 mM IPTG and cultured at 37 °C for 6 h to induce the expression of fluorescent protein. The other part was cultured at 37 °C for 4 h to induce the expression of fluorescent protein, and then 2% arabinose was added and cultured at 37 °C for 2 h to induce the expression of capsid protein.

[0087] (5) E. coli expressing only fluorescent protein was used as the control group, and E. coli expressing both capsid protein and fluorescent protein was used as the experimental group. 1 μL of bacterial solution was taken from each group to prepare samples, and the assembly effect of intracellular virus-like particles was observed using a laser scanning confocal microscope. If the virus-like particle P22 successfully recruited fluorescent protein molecules, the fluorescent protein would gather at both ends of the cell. If P22 did not recruit fluorescent protein molecules, the fluorescent protein would diffuse in the cytoplasm.

[0088] Figure 7 Laser scanning confocal microscopy was used to observe the fluorescence co-localization of the capsid protein CP and protein molecule YFP-SP / CFP-SP of the intracellular virus-like particles P22 in the experimental group. The fluorescence of YFP and CFP mainly gathered at the two ends of E. coli, which was consistent with the theory. Laser scanning confocal microscopy was used to observe the assembly effect of YFP-SP / CFP-SP in the control group without capsid protein CP encapsulation. Figure 7 It can be seen that YFP and CFP are evenly dispersed in the cells of E. coli without aggregation, which is consistent with the theory.

[0089] Then, 2 μL of bacterial solution was taken for sample preparation, and the fluorescence lifetime of CFP protein was detected by fluorescence lifetime imaging system under excitation of 430 nm using 500 nm ± 7.5 nm filter. Since the emission wavelength of CFP partially overlaps with the excitation wavelength of YFP, when the distance between the two is less than 10 nm, the energy of CFP is transferred to YFP to make it emit light, so Film Fret is used to detect the energy transfer phenomenon between CFP and YFP by fluorescent protein.

[0090] Figure 8 The fluorescence lifetime imaging experiment was used to detect the fluorescence lifetime of CFP. The experimental results showed that the fluorescence lifetime of CFP in the experimental group was significantly lower than that in the control group, indicating that the Fret phenomenon occurred, thereby proving that P22 successfully recruited cargo proteins with SP tags, while the YFP-SP / CFP-SP in the control group was evenly dispersed throughout the E. coli cells without aggregation. At the same time, the results further showed that the YFP-SP / CFP-SP in the experimental group was assembled together by the virus-like particles P22 and aggregated at both ends of the E. coli, indicating that the assembly method of the present invention also has a good assembly effect in E. coli cells.

[0091] Finally, it should be noted that although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be based on the definition of the claims.

Claims

1. A method for constructing an artificial carboxysome based on virus-like particles, characterized in that: The following steps are involved: (1) inserting the capsid protein CP gene fragment of virus-like particle P22 into a plasmid vector to construct a plasmid capable of expressing CP in host cells; (2) connecting the gene fragment of the scaffold protein SP domain of the virus-like particle P22 with the gene fragment of the target protein through a flexible linker sequence to obtain a recombinant gene fragment capable of expressing the target protein molecule with the scaffold protein SP domain, inserting the fragment into a plasmid vector, and constructing a plasmid capable of expressing the target protein molecule with the scaffold protein SP domain in a host cell; the target proteins are ribulose-1,5-bisphosphate carboxylase / oxygenase (RuBisCO) and carbonic anhydrase (CA); (3) The plasmid capable of expressing CP in host cells constructed in step (1) and the target protein molecule with the SP scaffold protein domain constructed in step (2) are co-transfected into host cells. After culturing and distribution-induced protein expression, the capsid protein CP and the target protein connected to the SP domain spontaneously assemble, the host cells are broken, and artificial carboxysomes are obtained by size exclusion chromatography separation.

2. The construction method according to claim 1, characterized in that: The amino acid sequence of the capsid protein CP of the virus-like particle P22 in step (1) is shown in SEQ ID NO.1; the plasmid vector is a plasmid vector with a T7 promoter, the gene fragment is inserted after the T7 promoter of the plasmid vector, and the capsid protein is expressed in large quantities in the host cell after induction by arabinose.

3. The construction method according to claim 1, characterized in that: The amino acid sequence of the scaffold protein SP described in step (2) is shown in SEQ ID NO.2; the amino acid sequence of RuBisCO is shown in positions 1-466 of SEQ ID NO.5; the amino acid sequence of CA is shown in positions 2-261 of SEQ ID NO.6; the amino acid sequence encoded by the flexible linker sequence between the gene fragment of the scaffold protein SP domain and the gene fragment of the target protein is (GGS) n or (GGGGS) n , n=1-10, preferably (GGGGS)3; the plasmid vector is a plasmid vector with a T7 promoter, the recombinant gene fragment is inserted after the T7 promoter of the plasmid vector, and the target protein is expressed in large quantities in the host cell after induction by isopropylthiogalactoside (IPTG), preferably pCDF-Duet-1 and pACYC-Duet-1.

4. The construction method according to claim 1, characterized in that: The host cells described in step (3) include Escherichia coli, yeast and animal cells, preferably Escherichia coli BL21 (DE3); the specific process of the distribution induction is: first add 0.1-1mM IPTG to induce the expression of the target protein, after expressing for 3 to 5 hours, add arabinose with a final concentration of 1 to 3% to induce the expression of the capsid protein.

5. The artificial carboxysome obtained by the construction method according to any one of claims 1 to 4.

6. A multi-enzyme synergistic catalytic carbon fixation method, characterized in that: The artificial carboxysome according to claim 5 is added to a mixture containing 1,5-bisphosphate ribulose (RUBP) and HCO3 - In a buffer solution, 3-phosphoglycerate is synthesized by the synergistic catalysis of ribulose-1,5-bisphosphate carboxylase / oxygenase (RuBisCO) and carbonic anhydrase (CA).

7. A method for constructing an intracellular protein recruitment system based on virus-like particles, characterized in that: The following steps are involved: (1) inserting the capsid protein CP gene fragment of virus-like particle P22 into a plasmid vector to construct a plasmid capable of expressing CP in host cells; (2) connecting the gene fragment of the scaffold protein SP domain of the virus-like particle P22 with the gene fragment of the target protein through a flexible linker sequence to obtain a recombinant gene fragment capable of expressing the target protein molecule with the scaffold protein SP domain, inserting it into a plasmid vector, and constructing a plasmid capable of expressing the target protein molecule with the scaffold protein SP domain in a host cell; the target protein fluorescent proteins CFP and YFP; (3) The plasmid capable of expressing CP in host cells constructed in step (1) and the target protein molecule with the SP scaffold protein domain constructed in step (2) are co-transferred into the host cells. After culturing and distribution to induce protein expression, the capsid protein CP and the target protein connected with the SP domain are spontaneously assembled in the cell, thereby constructing a protein recruitment system in the host cell.

8. The construction method according to claim 7, characterized in that: The amino acid sequence of the capsid protein CP of the virus-like particle P22 in step (1) is shown in SEQ ID NO.1; the plasmid vector is a plasmid vector with a T7 promoter, the gene fragment is inserted after the T7 promoter of the plasmid vector, and the capsid protein is expressed in large quantities in the host cell after induction by arabinose.

9. The construction method according to claim 7, characterized in that: The amino acid sequence of the scaffold protein SP in step (2) is shown in SEQ ID NO.2; the amino acid sequence of the fluorescent protein CFP is shown in SEQ ID NO.3 at positions 14-252; the amino acid sequence of the fluorescent protein YFP is shown in SEQ ID NO.4 at positions 12-248; the amino acid sequence encoded by the flexible linker sequence between the gene fragment of the scaffold protein SP domain and the gene fragment of the target protein is (GGS) n or (GGGGS) n , n=1-10, preferably (GGGGS)3; the plasmid vector is a plasmid vector with a T7 promoter, the recombinant gene fragment is inserted after the T7 promoter of the plasmid vector, and the target protein is expressed in large quantities in the host cell after induction by isopropylthiogalactoside (IPTG), preferably pCDF-Duet-1 and pACYC-Duet-1.

10. The construction method according to claim 7, characterized in that: The host cells described in step (3) include Escherichia coli, yeast and animal cells, preferably Escherichia coli BL21 (DE3); the specific process of the distribution induction is: first add 0.1-1mM IPTG to induce the expression of the target protein, after expressing for 3 to 5 hours, add arabinose with a final concentration of 1 to 3% to induce the expression of the capsid protein.

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