In vitro reconstitution of kalvin cycle systems

Abstract

The invention provides an in-vitro Kalvin circulating system and a reconstruction method thereof. The system comprises carbonic anhydrase (CA), 2-phosphoglycollic acid enzyme (PGPase), formate dehydrogenase (FDH) and polyphosphatase (PKK2). The Kalvin cycle system reconstructed in vitro can help people to further study the reaction sequence of the Kalvin cycle in plants, the biochemical characteristics of related enzymes and the regulation of the enzymes on the Kalvin cycle.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to in vitro reconstruction of the Calvin cycle. Background Art

[0002] Photosynthesis includes two independent stages: light reaction and carbon assimilation reaction. Light reaction converts the light energy absorbed by plants into active chemical energy, while carbon assimilation converts active chemical energy into stable chemical energy. Photosynthetic carbon assimilation reaction, also known as Calvin cycle, is the main carbon fixation pathway for higher plants. It was discovered and completed by Melvin Calvin, Andrew Benson and James Basham of the University of California, Berkeley. The full name of the cycle is Calvin-Benson-Basshamcycle (CBB cycle).

[0003] The Calvin cycle is mainly divided into three stages: carbon fixation, carbon reduction, and substrate regeneration. In the carbon fixation stage, ribulose-1,5-bisphosphate (RuBP) combines with CO2 under the catalysis of ribulose-1,5-bisphosphate carboxylase / oxygenase (Rubisco) to form two molecules of 3-phosphoglyceric acid (3PGA); in the carbon reduction stage, 3PGA is reduced to 3-phosphoglyceraldehyde (G3P) and dihydroxyacetone phosphate (DHAP) under the action of 3-phosphoglycerate kinase (PGK) and 3-phosphoglyceraldehyde dehydrogenase (GAPDH), consuming one ATP and one NADPH at the same time; in the substrate regeneration stage of the cycle, A small part of the two triose phosphates is used to synthesize sucrose and starch, and most of them are converted back into the carbon dioxide acceptor molecule RuBP (ribulose-1,5-bisphosphate) by triose phosphate isomerase (TPI), 1,6-bisphosphate fructose aldolase (FBA), 1,6-bisphosphofructoylase (FBPase), 1,7-bisphospho-sedoheptulase (SBPase), transketolase (TK), 5-phosphate ribulose isomerase (RPE), 5-phosphate ribose isomerase (RPI) and 5-phosphate ribulose kinase (PRK), completing the regeneration of the substrate. Summary of the invention

[0004] The present invention constructs a Calvin cycle system in vitro using enzymes Rubisco, PGK, GAPDH, TPI, FBA, FBPase, SBPase, TK, RPE, RPI and PRK, and uses carbonic anhydrase (CA for short), 2-phosphoglycolic acid enzyme (PGPase for short), formate dehydrogenase (FDH for short) and polyphosphatase (PKK2 for short) to allow the cycle to be carried out in vitro for a long time. The addition of CA increases the concentration of HCO3- in the solution, the addition of PGPase reduces the influence of the cycle byproduct 2PGA (2-phosphoglycolic acid), and the addition of FDH and PKK2 provides reducing power and driving force for the cycle system. The Calvin cycle is highly regulated, and each cycle fixes a CO2 molecule, and six cycles generate a glucose molecule, and the regeneration of the substrate and the output of the intermediate product are always balanced.

[0005] The present invention successfully established a sustainable and efficient in vitro Calvin cycle system, and successfully quantified the system using the fluorescence reaction of the intermediate product of the cycle, erythrose 4-phosphate (abbreviated as E4P). The Calvin cycle system reconstructed in vitro by the present invention can help people further study the reaction sequence of the Calvin cycle in plants and the biochemical properties of the enzymes involved, as well as the regulation of the Calvin cycle by these enzymes.

[0006] Specifically, the present invention provides the following technical solutions:

[0007] On the one hand, the present invention provides an in vitro Calvin cycle system, characterized in that the system includes carbonic anhydrase (CA), 2-phosphoglycolic acid enzyme (PGPase), formate dehydrogenase (FDH) and polyphosphatase (PKK2).

[0008] In some embodiments, the system further comprises ribulose-1,5-bisphosphate carboxylase / oxygenase (Rubisco), 3-phosphoglycerate kinase (PGK), 3-phosphoglyceraldehyde dehydrogenase (GAPDH), triose phosphate isomerase (TPI), 1,6-bisphosphate fructose aldolase (FBA), 1,6-bisphosphofructoylase (FBPase), 1,7-bisphospho-sedoheptulase (SBPase), transketolase (TK), 5-phosphoribulose isomerase (RPE), 5-phosphoribose isomerase (RPI) and 5-phosphoribulose kinase (PRK).

[0009] In some embodiments, the system further includes NADH and ATP to provide reducing power and power.

[0010] In some embodiments, the ribulose-1,5-bisphosphate carboxylase / oxygenase (Rubisco) is a Syn6301-Rubisco enzyme, whose nucleotide sequence is shown in SEQ ID NO:1.

[0011] In some embodiments, the system further comprises HCO3 - and Mg 2+ To ensure the enzymatic activity of Syn6301-Rubisco enzyme.

[0012] In some embodiments, the system also includes formate to regenerate NADH with the help of formate dehydrogenase (FDH).

[0013] In some embodiments, the system further includes PolyP6 to regenerate ATP with the help of polyphosphatase (PKK2).

[0014] In some embodiments, the system further comprises Tris-HCl.

[0015] In some embodiments, the system further comprises a TPP.

[0016] In some embodiments, the system further comprises DTT.

[0017] In some embodiments, the system further includes ribulose-1,5-bisphosphate (RuBP) to initiate the cycle reaction.

[0018] In some embodiments, the pH of the system is 6-10, preferably pH 8.

[0019] In another aspect, the present invention provides a method for reconstructing a Calvin cycle system in vitro, characterized in that the method comprises:

[0020] a. Recombinant expression of ribulose-1,5-bisphosphate carboxylase / oxygenase (Rubisco), 3-phosphoglycerate kinase (PGK), 3-phosphoglyceraldehyde dehydrogenase (GAPDH), triosephosphate isomerase (TPI), fructose-1,6-bisphosphate aldolase (FBA), fructose-1,6-bisphosphate enzyme (FBPase), sedoheptulose-1,7-bisphosphate enzyme (SBPase), transketolase (TK), ribulose-5-phosphate isomerase (RPE), ribose-5-phosphate isomerase (RPI), ribulose-5-phosphate kinase (PRK), carbonic anhydrase (CA), 2-phosphoglycolic acid enzyme (PGPase), formate dehydrogenase (FDH) and polyphosphatase (PKK2) in vitro;

[0021] b. purifying the enzyme expressed in step a;

[0022] c. Add Tris-HCl, NaHCO3, Mg(COOH)2, TPP, PolyP6, ATP, DTT and NADH to the purified enzyme in step b;

[0023] d. Add ribulose-1,5-bisphosphate (RuBP) to start the cycle reaction.

[0024] In some embodiments, the pH of the system is adjusted to 6-10, preferably the pH is adjusted to 8.

[0025] In some embodiments, the nucleotide sequence of the carbonic anhydrase (CA) is as shown in SEQ ID NO:11.

[0026] In some embodiments, the nucleotide sequence of the 2-phosphoglycolic acid enzyme (PGPase) is shown as SEQ ID NO:10.

[0027] In some embodiments, the nucleotide sequence of the formate dehydrogenase (FDH) is as shown in SEQ ID NO:15.

[0028] In some embodiments, the nucleotide sequence of the polyphosphatase (PKK2) is as shown in SEQ ID NO:9.

[0029] In some embodiments, the nucleotide sequence of the 3-phosphoglycerate kinase (PGK) is as shown in SEQ ID NO:14.

[0030] In some embodiments, the nucleotide sequence of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is as shown in SEQ ID NO:13.

[0031] In some embodiments, the nucleotide sequence of the triose phosphate isomerase (TPI) is as shown in SEQ ID NO:12.

[0032] In some embodiments, the nucleotide sequence of the fructose-1,6-bisphosphate aldolase (FBA) is as shown in SEQ ID NO:2.

[0033] In some embodiments, the nucleotide sequence of the fructose-1,6-bisphosphatase (FBPase) is as shown in SEQ ID NO:3.

[0034] In some embodiments, the nucleotide sequence of the 1,7-bisphospho-sedoheptulose enzyme (SBPase) is shown as SEQ ID NO:5.

[0035] In some embodiments, the nucleotide sequence of the transketolase (TK) is as shown in SEQ ID NO:4.

[0036] In some embodiments, the nucleotide sequence of the ribulose-5-phosphate isomerase (RPE) is as shown in SEQ ID NO:7.

[0037] In some embodiments, the nucleotide sequence of the 5-phosphoribosyl isomerase (RPI) is as shown in SEQ ID NO:6.

[0038] In some embodiments, the nucleotide sequence of 5-phosphoribulose kinase (PRK) is as shown in SEQ ID NO:8.

[0039] On the other hand, the present invention provides the use of the system as described above or the system reconstructed by the method as described above in the study of Calvin cycle, for example, in the study of Calvin cycle carbon fixation efficiency and key enzymes of Calvin cycle.

[0040] definition

[0041] PolyP6: Sodium hexametaphoshpate, molecular formula is Na6P6O 18 , CAS: 10124-56-8, involved in the enzymatic reaction of PKK2.

[0042] TPP: Thiamine pyrophosphate, molecular formula is C 12 H 19 ClN4O7P2S, CAS: 154-87-0, participates in the enzymatic reactions of RPE.

[0043] DTT: Dithiothreitol, molecular formula is C4H 10 O2S2, CAS: 3483-12-3, acts as a reducing agent to prevent intra- or intermolecular disulfide bonds formed between cysteines in proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Schematic diagram of the extracorporeal Calvin cycle.

[0045] Figure 2 The fluorescence analysis results of the derivatives are shown. A: Standard curve of E4P concentration and fluorescence value of Schiff base derivatives. B: Fluorescence analysis of the reaction products of Calvin cycle intermediate metabolites and cofactors (1 mM) with DABA.

[0046] Figure 3 The results of continuous detection of E4P accumulation in the in vitro Calvin cycle are shown. DETAILED DESCRIPTION

[0047] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0048] Example 1 Gene cloning and expression vector construction of enzymes required for in vitro reconstruction of the Calvin cycle

[0049] The invention uses the cDNA of wheat or Arabidopsis thaliana or Chlamydomonas or Escherichia coli as a template, designs specific primers and uses Phusion II DNA Polymerase (Thermo Scientific) to perform PCR amplification on the cDNA. Syn6301-Rubisco (ribulose-1,5-bisphosphate carboxylase / oxygenase), FBA (fructose-1,6-bisphosphate aldolase), FBPase (fructose-1,6-bisphosphate enzyme), TK (transketolase), SBPase (sedoheptulase-1,7-bisphosphate), RPI (ribose-5-phosphoribosyl isomerase), RPE (ribulose-5-phosphoribosyl isomerase), PRK* (ribulose-5-phosphoribosyl kinase), PKK2* (polyphosphatase), PGPase (2-phosphoglycolic acid enzyme), and CA (carbonic anhydrase) were constructed into the expression vector pET28a (Mei5bio), and PGK (3-phosphoglycerate kinase), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), and TPI (triosephosphate isomerase) were constructed into the expression vector pHUE (An Efficient System for High-Level Expression and Easy Purification of Authentic Recombinant Proteins, AM Catanzariti et al., Protein Science, Volume 13, Issue 5, p.1331-1339), FDH (formate dehydrogenase) was constructed into the expression vector pET22b (Biomed). The PCR product and the vector after restriction digestion were constructed using a one-step cloning kit (Novizan). The vector construction system was 5×CEⅡ buffer 4μL, linearized cloning vector Y (Y=vector length bp×0.02) ng, gene fragment X (gene length bp×0.04) ng, ExnaseⅡ2μL, water was added to 20μL, reacted at 37℃ for 30min and immediately placed on ice for cooling, then 100μL E.coli DH5α competent cells were added, transformed using the heat shock method, and evenly spread on the corresponding vector resistance plate with a sterile coating rod. The pET28a vector was kanamycin resistant, and the pHUE vector and pET22b vector were ampicillin resistant, and cultured at 37℃ overnight. On the second day, single clones were taken for colony PCR screening and sequencing, and the single clones with correct sequences were expanded and preserved.

[0050] Note: * is the gene synthesized by Shanghai Jierui Company after codon optimization of PRK gene sequence of Synechococcus elongatus PCC 7942 and PKK2 gene sequence of Corynebacterium glutamicum ATCC 13032.

[0051] Vector 1: pET28a

[0052] Insert tandem gene fragments: Syn6301-Rubisco (X03220.1), FBA (XM_020291696.1), FBPase (AK44885.1), TK (XM_020304017.1), SBPase (XM_020342414.1), RPI (AK448630.1), RPE (AK456944.1), RPK (Synthesized), PKK2 (Synthesized), PGPase (NP_417844.1), CA (NP_414668.1) (each gene has an independent promoter and terminator)

[0053] Vector 2: pHUE

[0054] Insert tandem gene fragments: TPI (NM_001335732.1), GAPDH (MF477938.1), PGK (X15233.1) (each gene has an independent promoter and terminator)

[0055] Vector 3: pET22b

[0056] Insert tandem gene fragment: FDH (XM_002493126.1) (each gene has an independent promoter and terminator)

[0057] Example 2 Protein purification of enzymes required for in vitro reconstruction of the Calvin cycle

[0058] The correctly sequenced monoclonal colonies were inoculated into 20 ml LB liquid medium (LB medium containing 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl) containing the corresponding antibiotics, and cultured overnight at 37°C with shaking at 220 rpm. The next day, 20 ml of E. coli culture was transferred to 1 L of liquid LB medium containing the corresponding antibiotics at a ratio of 1:50, and cultured with shaking until the OD600 of the culture was about 0.4-0.6, and a final concentration of 0.2 mM IPTG was added. Except for FBA-pET28a and SBPase-pET28a, which were induced at 28°C for 5 hours, the remaining vectors were induced at 16°C for 18 hours and then centrifuged at 4000 rpm to collect the cells.

[0059] The cells were resuspended in Ni-NTA lysis buffer (20 mM Tris-HCL, 500 mM NaCl, 10 mM imidazole, pH 8.0), ultrasonically disrupted on ice, and centrifuged at 13,000 rpm for 30 min at 4°C. The supernatant was collected, filtered through a 0.22 μm filter membrane, and added to a Ni-NTA affinity chromatography column (equilibrated in advance with 8 volumes of Ni-NTA lysis buffer), then washed with 8 volumes of Ni-NTA wash buffer I (20 mM Tris-HCL, 500 mM NaCl, 20 mM imidazole, pH 8.0) and Ni-NTA wash buffer II (20 mM Tris-HCL, 500 mM NaCl, 60 mM imidazole, pH 8.0), and finally washed with 8 volumes of Ni-NTA elution buffer (20 mM Tris-HCL, 500 mM NaCl, 250 mM imidazole, pH 8.0) to elute the target protein, and the eluate was dialyzed in dialysis buffer (20mM Tris-HCL, 20mM NaCl, pH 8.0) at 4°C overnight, where PGK, GAPDH and TPI were cut with enzyme 67 to remove the fusion tags. Finally, the protein was concentrated with an ultrafiltration tube, glycerol was added to a final concentration of 20%, and stored in aliquots at -80°C.

[0060] Example 3 Activity detection of enzymes required for in vitro reconstruction of Calvin cycle

[0061] The enzymatic activities of 11 core enzymes were detected in vitro. For the enzymes PGK, TPI, RPE, PRK and Syn6301-Rubisco, the detection system was prepared according to the cascade reaction of each enzyme, and then the change of the absorbance value of NADPH in the system at 340nm was measured; for the enzymes GAPDH, FBA, FBPase, SBPase and TK, the detection system was also prepared according to the cascade reaction of each enzyme, but the absorbance value of NADPH in the system was changed to + The absorbance change at 340nm; for enzyme RPI, according to its characteristics, the absorbance change of the direct catalytic substrate 5-phosphoribose in its catalytic reaction is detected. 5-phosphoribose has a weak absorption at 290nm. The enzyme activity reaction system is 100μL, and the buffer solution contains 100mM Tris-HCl (pH 8.0) and 20mM DTT, as well as the driving force (ATP or ADP) and reducing power (NAD + or NADH), the reaction was started after adding the substrate corresponding to each enzyme, and the absorbance was measured.

[0062] The above absorbance values ​​were measured using an ultraviolet spectrophotometer, and the relevant characteristic values ​​were calculated using the nonlinear fitting software Graphpadprism, such as Figure 1As shown. CA, FDH, PKK2 and PGPase were used as auxiliary enzymes, but their enzymatic activity characteristics were not tested. They were quantified by adding mass in the system. NADPH and NAD + The calculation formula of enzyme activity by coupling method is as follows:

[0063]

[0064] The RPI enzyme activity calculation formula is as follows:

[0065]

[0066] Conc. (mg / mL) = final concentration of the enzyme tested;

[0067] 6.22 = extinction coefficient of β-NADPH at 340 nm;

[0068] 0.072 = absorbance coefficient of 5-phosphoribose at 290 nm;

[0069] 1U is defined as the amount of enzyme required to catalyze the conversion of 1 micromole of substrate per minute (1U = 1 μmol / min).

[0070] Table 1 Relevant characteristics of the core enzymes of the Calvin cycle in vitro

[0071]

[0072] ND means no data. Km and Vmax were calculated by nonlinear fitting using Graphpad prism software.

[0073] Example 4 In vitro Calvin cycle reconstruction

[0074] 4.1 In vitro Calvin cycle reconstruction system

[0075] Generally, light-activated protons entering the thylakoid cavity will cause pH changes in the chloroplast matrix and thylakoid cavity. The pH of the matrix will generally rise to about 8.0. Therefore, the present invention chooses to control the pH value of the reconstructed system at 8.0, and add NADH and ATP to provide reducing power and power. According to the special properties of the cyanobacterial Syn6301-Rubisco enzyme, a sufficient amount of HCO3 is added to the system. - and Mg 2+ To ensure that the enzyme activity is fully exerted. At the same time, bicarbonate and CA are added to the system to regenerate CO2, FDH and formate are added to regenerate NADH, PolyP6 (Aladdin, CAS No. 10124-56-8) and PKK2 are added to regenerate ATP, and PGPase is added to reduce the inhibitory effect of 2-phosphoglyceric acid (2PGA) on the reaction. Figure 1 shown.

[0076] The final in vitro Calvin cycle reconstruction system contained: 100mM Tris-HCl, 40mM NaHCO3, 10mM Mg(COOH)2 (Sigma-Aldrich, CAS No. 6150-82-9), 1mM TPP (Aladdin, CAS No. 154-87-0), 10mM PolyP6, 1mM ATP (Sigma-Aldrich, CAS No. 34369-07-8), 20mM DTT (Amresco, CAS No. 3483-12-3), 1mM NADH (Sigma-Aldrich, CAS No. 606-68-8), 1U / mL Syn6301-Rubisco, 1U / mL PGK, 1U / mL GAPDH, 1U / mL TPI, 1U / mL FBA, 1U / mL FBPase, 1U / mL SBPase, 1U / mL TK, 1U / mL RPE, 1U / mL RPI, 1U / mL PRK, 0.24 mg / ml CA, 0.24 mg / ml PGPase, 0.32 mg / ml FDH, 0.08 mg / ml PKK2. Finally, RuBP (ribulose-1,5-bisphosphate) (Sigma-Aldrich, CAS No. 24218-00-6) with a final concentration of 0.25 mM was added at 30°C to start the cycle reaction.

[0077] 4.2 Quantitative detection of the in vitro Calvin cycle system

[0078] The present invention uses a detection method for erythrose 4-phosphate (this detection method has been patented, and the patent application publication number is CN 114136936 A) to quantitatively detect the in vitro Calvin cycle system. Erythrose 4-phosphate (abbreviated as E4P) is an intermediate product of the Calvin cycle, and its concentration will accumulate over time, and its aldehyde group can react with the amino group of 3,5-diaminobenzoic acid (abbreviated as DABA) (Aladdin, CAS No. 618-56-4) to generate a Schiff base derivative. At low concentrations, the fluorescence value of this derivative is proportional to the concentration of E4P, such as Figure 2 As shown in A. And compared with the fluorescence values ​​of E4P and DABA derivatives, the fluorescence values ​​of the remaining derivatives are very low, such as Figure 2 As shown in B, the effect is almost negligible, so it is a very sensitive and feasible method for detecting circulating intermediates.

[0079] After the cycle is started, 10 μl of the reaction solution is diluted with 90 μl of double distilled water at 20 minutes, 40 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 24 hours, 48 ​​hours, 72 hours and 96 hours. Then add 50% trichloroacetic acid in the same volume as the diluent to terminate the reaction. After placing on ice for 5 minutes, centrifuge at 13000 r / min at 4°C for 10 minutes. Take 150 μl of the supernatant and add an equal volume of 10 mM DABA. After reacting at 100°C for 15 minutes, cool on ice for 10 minutes. Take 80 μl of the cooled reaction solution into a black 96-well plate (repeated three times) and detect the fluorescence value (excitation light 360 / 40nm; emission light 528 / 20nm; optical element: top; gain: automatic adjustment). Each detection needs to be freshly prepared and used E4P standard solution as calibration.

[0080] result

[0081] The results of long-term E4P concentration detection in vitro Calvin cycle are as follows Figure 3 As shown, within the first 48 hours, the concentration of E4P in the system will continue to accumulate as time goes by, which shows that the system constructed by the present invention is sustainable in vitro. And the accumulation of E4P reaches a maximum of about 3mM on the second day, and then gradually decays. It is speculated that the decay may be caused by the instability of some enzymes in the subsequent system in vitro. If the stability of the enzyme is enhanced, the accumulation of E4P may be further increased.

[0082] The Calvin cycle in vivo is affected by the cell environment, so its action process is much more complicated and difficult to study than in vitro. Therefore, if the process of Calvin cycle is reconstructed and simulated in vitro, the process of Calvin cycle in vitro can be studied, which reduces the influence of many cell environment factors and is simpler and more intuitive. At the same time, the research results of Calvin cycle in vitro can also be verified in vivo, thus providing new ideas for the study of Calvin cycle rate in vivo.

[0083] One of the important effects of the Calvin cycle on the environment is the fixation of carbon dioxide. Research on improving the carbon fixation efficiency of the Calvin cycle in vivo has been ongoing. The Calvin cycle in vitro also requires carbon fixation. Improving the carbon fixation efficiency of the Calvin cycle in vitro has a fundamental and reference significance for improving the carbon fixation efficiency of the Calvin cycle in vivo.

[0084] sequence

[0085] SEQ ID NO:1Syn6301-Rubisco nucleotide sequence

[0086]

[0087] SEQ ID NO: 2FBA nucleotide sequence

[0088]

[0089] SEQ ID NO:3FBPase nucleotide sequence

[0090]

[0091] SEQ ID NO:4TK nucleotide sequence

[0092]

[0093]

[0094] SEQ ID NO:6 RPI nucleotide sequence

[0095] ATGGGCAGCGCCGCCTCGCCGCCGCGGGCCCTCGACGCGGCGACGCAGGAGGACCTCAAGCGCGTCTCCGCGCACCGCGCCGTCGACATGGTGGAGTCCGGCATGACGCTGGGGCTCGGCACCGGCTCCACGGCCGCGCACGCGCTCGACCGCCTCGGGGCCCTCCTCCGCGCCGGCGCGCTGCGCGCGGTCGCCGGGGTGCCCACCTCCCTCAAGACGGAGGCGCACGCCGCGCGCGTCGGGATCCCCATGCTCGCGCTCGCCGACGCCGCCGAGATCCACCTCTCCATCGACGGCGCCGACGAGGTCGACCCGGACCTCAACCTCGTCAAGGGCCGCGGCGGCTCGCTCCTCCGCGAGAAGATGATCGAGGGCGCCGGCGCCCGCTTCGTCGTCATCGTCGACGAGTCCAAGCTCGTCCCCCGCCTCGGCTGCACGGGCTCCGTGCCCGTCGAGGTCGTCCCCTTCGGCAGCGCCTACACGCTCGGCCTCATCCGCAAGGTGTTCGACAAATTGCCGGGCTTCCACGCCAGGCTCAGGACCGTCAAGTCCAAGGCCGGCGACGGCCAGGAGGAGCTCTTTCTCACCGACAACGGCAACCACATCGTCGAGATGTTCTTCGAGGACGGCATACACGGCAACCTGCGCGACATAAGCGACAGCCTGCTGCGCATCACGGGCGTCGTCGAGCACGGCATGTTCCTCGGCATGGCCACCAAGGTGATCGTCGCCAAGAAGGACGGCACCGTGGCGGTCCTCAGCAAGAAGTAG

[0096] SEQ ID NO:7 RPE nucleotide sequence

[0097] ATGCTCTCGTCGGACTTCGCCAACCTCGCCTCGGAGGCCGAGCGCATGGTCCGCCTCGGCGCCGACTGGCTCCACATGGACGTCATGGATGGGCACTTCGTTCCTAATTTAACTATTGGAGCTCCAGTGATTGAGAGCTTGAGGAAGCACACAAAGGCATATTTGGACTGCCATCTCATGGTCACAAATCCTTCCGATTATGTAGAGGCATTTGGAAAAGCTGGTGCCTCAGGATTCACATTCCATATAGAAGTAGCGAGGGATAACTGGAAAGAGCTCACCCAAAGCATCAAAGCAAAGGGCATGCGGCCTGGTGTATCATTGAAGCCTGGTACTCCTGTGGAGGATGTTTTCCCCCTGGTGGAAGCAGAAACCCCTGTAGAGTTGGTTCTCGTGATGACGGTCGAGCCTGGCTTTGGTGGCCAGAAGTTCATGCCGGAGATGATGGATAAGGTGCGTACGCTGAGGAAGAAGTACCCGTCCCTCGACATTGAGGTCGACGGTGGCCTGGGTCCTTCCACCATCGACGCCGCCGCGTCGGCCGGCGCCAACTGCATCGTCGCCGGGAGCTCCGTGTTCGGCGCGCCCGACCCCGGAGAGGTCATCTCGGCGCTGCGCAAGAGCGTGGAGGCGTCGCAGATCAAGAGCTGA

[0098]

[0099] SEQ ID NO:9 PKK2 nucleotide sequence

[0100] ATGGTTGGCAAACTGCCTATCATGGCCGAAACCAATGAAAATGATCTGCCAGTTATCGATTTAGCACAGATCGAAGGCTATGTTGTGGATGATAGTGATGAAGATGATCCGGTGCTGTTACGTCCGGATGGCACCCCTATCGAAACCTGGCGTGAAGATTTTCCTTATGAAGAACGTGTGACACGCGAAGATTATGAAAAAGTTAAACGTAGTCTACAAATCGAACTGTTAAAATGGCAGAATTGGACCAAAGAAACGGGTCAGCGTCATATCATCCTGTTTGAAGGCCGCGATGCAGCGGGTAAAGGTGGCACCATCAAACGCTTTAATGAACATCTGAATCCTCGCGGCGCCCGTACCGTTGCCTTAGAAAAACCTAGCCCTCGTGAATCTACAAGCTGGTATTTTCAGCGCTATATTCAGCATTTTCCGGCCGCAGGCGAAATTGTGTTTTTTGATCGCTCTTGGTATAATCGTAGCGGTGTGGAGAGAGTTATGGGCTTTTGTACGGAATCACAGCACGCGGAATTTCTGCGCGAAGTTCCAATGCTGGAAAATATGATTCTGGGCTCAGGTATCTCACTGACCAAATTTTGGTTTAGTGTGACCCGCAAAGAACAGCGTACACGCTTTGCAATTCGTCAGGTTGATCCAGTTCGCCAGTGGAAACTGTCTCCAATGGATTTAGCCTCTTTAGATCGTTGGGATGATTATACACGTGCTAAAGAAGAACAGTTTCGCTATACCGATACCGATGAATCTCCTTGGATTACCATCAAATCTAATGATAAAAAACGTGCTCGCATTAATGCCATGCGTTATGTGCTGAGTAAATTTGATTATACGGATAAAGATTATGAACTGGTGGGCGAACCTGATCCTAAAGTGGTTCTGCGCGGTCGCGATCAGATCGGCGATTGA

[0101] SEQ ID NO:10 PGPase nucleotide sequence

[0102] ATGAATAAGTTTGAAGATATTCGCGGCGTCGCTTTTGATCTTGATGGTACGCTGGTCGACAGTGCTCCTGGTCTTGCTGCTGCGGTAGATATGGCGCTGTATGCGCTGGAGTTGCCCGTCGCAGGTGAAGAACGCGTTATTACCTGGATTGGTAACGGCGCAGATGTTCTGATGGAGCGCGCATTGACCTGGGCGCGTCAGGAACGTGCGACTCAGCGTAAAACAATGGGTAAACCGCCCGTTGATGACGACATTCCGGCAGAAGAACAGGTACGTATTCTGCGTAAACTGTTCGATCGCTACTATGGCGAGGTTGCCGAAGAGGGGACGTTTTTGTTCCCGCACGTTGCCGATACGTTGGGCGCGTTGCAGGCTAAAGGCCTGCCGCTAGGCCTGGTCACCAACAAACCGACGCCGTTCGTCGCGCCGCTGCTCGAAGCCTTAGATATCGCCAAATACTTCAGCGTGGTGATTGGTGGTGATGATGTGCAAAACAAAAAACCGCATCCGGACCCGCTGTTACTGGTGGCTGAGCGGATGGGAATTGCCCCACAACAGATGCTGTTTGTCGGCGACTCACGCAATGATATTCAGGCGGCAAAAGCGGCAGGTTGCCCATCAGTTGGCTTAACCTACGGATATAACTACGGCGAGGCTATCGATCTCAGCCAGCCTGATGTAATTTATCAGTCTATAAATGACCTTCTGCCCGCATTAGGGCTTCCGCATAGCGAAAATCAGGAATCGAAAAATGACTAA

[0103] SEQ ID NO:11 CA nucleotide sequence

[0104] ATGAAAGACATAGATACACTCATCAGCAACAATGCACTATGGTCAAAAATGCTGGTGGAAGAGGATCCCGGGTTTTTTGAGAAACTGGCACAAGCGCAAAAACCGCGCTTTCTATGGATTGGATGTTCCGACAGTCGCGTTCCTGCAGAACGTTTAACCGGTCTTGAGCCGGGCGAACTCTTTGTTCACCGTAATGTTGCTAACCTGGTCATTCACACTGACCTGAACTGCCTTTCCGTGGTTCAGTATGCAGTGGATGTACTCGAAGTTGAACACATTATTATCTGTGGCCACTACGGTTGCGGCGGCGTACAAGCCGCAGTTGAAAACCCGGAACTGGGGCTTATCAACAACTGGCTGCTGCATATCCGCGATATCTGGTTCAAACATAGCTCATTGCTCGGCGAAATGCCGCAAGAGCGCCGTCTGGATACCTTGTGTGAACTGAACGTCATGGAACAGGTGTATAACCTGGGCCACTCCACCATTATGCAATCAGCGTGGAAACGCGGGCAGAAAGTTACCATTCACGGCTGGGCCTACGGCATTCACGACGGCTTGCTGCGTGATCTGGATGTTACCGCCACCAACCGCGAAACCCTTGAGCAACGTTACCGTCACGGGATTTCCAACCTCAAGCTGAAACACGCCAACCACAAATAA

[0105] SEQ ID NO:12 TPI nucleotide sequence

[0106] ATGGCAGCTACCTCTCTCACTGCCCCTCCTTCTTTCTCCGGTCTCCGCCGCATTTCTCCCAAGCTCGACGCTGCCGCCGTCTCCTCCCACCAATCCTTCTTCCACCGCGTCAATTCCTCTACCCGTCTCGTTTCTTCCTCTTCTTCTTCTCATCGCTCCCCCAGAGGTGTTGTTGCCATGGCTGGATCCGGAAAGTTTTTCGTTGGAGGAAACTGGAAGTGTAACGGGACTAAGGACTCCATCGCCAAGCTTATCTCCGATCTCAACAGTGCAACCTTGGAAGCAGATGTAGATGTTGTTGTGTCACCTCCATTTGTCTACATCGACCAGGTCAAATCCTCGTTGACAGACCGTATTGACATATCAGGTCAGAACTCTTGGGTTGGGAAAGGTGGAGCCTTCACTGGTGAAATCAGCGTGGAACAGCTCAAAGACCTTGGCTGCAAGTGGGTCATTCTTGGGCATTCCGAACGGAGACATGTCATCGGAGAAAAAGATGAGTTTATCGGGAAGAAAGCTGCATATGCATTGAGTGAGGGTCTTGGAGTGATAGCTTGTATTGGGGAAAAGCTAGAAGAGAGGGAAGCAGGCAAGACGTTTGATGTTTGCTTCGCGCAACTGAAGGCGTTTGCTGATGCTGTGCCTAGCTGGGACAATATAGTTGTTGCATACGAGCCTGTATGGGCAATTGGAACTGGTAAAGTTGCATCTCCTCAGCAAGCACAAGAAGTCCATGTAGCTGTCCGCGGTTGGCTAAAGAAGAATGTCTCTGAGGAAGTTGCTTCCAAAACGAGAATCATATATGGAGGTTCTGTCAATGGAGGCAACAGTGCAGAGCTTGCCAAAGAAGAAGACATTGATGGATTTCTTGTTGGTGGTGCCTCCTTGAAGGGTCCTGAGTTTGCAACCATTGTGAACTCAGTCACGTCGAAGAAAGTTGCTGCTTGA

[0107] SEQ ID NO:13 GAPDH nucleotide sequence

[0108]

[0109] SEQ ID NO: 14PGK nucleotide sequence

[0110]

[0111] SEQ ID NO: 15FDH nucleotide sequence

[0112]

[0113] SEQ ID NO:16 Amino acid sequence of enzyme No. 67

[0114] MRTSYTVTLPEEPPAAHFPALAKELRPRSPLSPSLLLSTFVGLLLNKAKNSKSAQGLAGLRNLGNTCFMNSILQCLSNTRELRDYCLQRLYMRDLGHTSSAHTALMEEFAKLIQTIWTSSPNDVVSPSEFKTQIQRYAPRFMGYNQQDAQEFLRFLLDGLHNEVNRVAARPKASPETLDHLPDEEKGRQMWRKYLERE DSRIGDLFVGQLKSSLTCTDCGYCSTVFDPFWDLSLPIAKRGYPEVTLMCMRLFTKEDILDGDEKPTCCRCRARKRCIKKFSVQRFPKILVLHLKRFSESRIRTSKLTTFVNFPLRDLDLREFASENTNHAVYNLYAVSNHSGTTMGGHYTAYCRSPVTGEWHTFNDSSVTPMSSSQVRTSDAYLLFYELASPPSRM

[0115] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An extracorporeal Calvin cycle system, characterized in that: The system includes carbonic anhydrase (CA), 2-phosphoglycolic acid enzyme (PGPase), formate dehydrogenase (FDH) and polyphosphatase (PKK2).

2. The system according to claim 1, characterized in that The system also includes ribulose-1,5-bisphosphate carboxylase / oxygenase (Rubisco), 3-phosphoglycerate kinase (PGK), 3-phosphoglyceraldehyde dehydrogenase (GAPDH), triosephosphate isomerase (TPI), 1,6-bisphosphate fructose aldolase (FBA), 1,6-bisphosphate fructose enzyme (FBPase), 1,7-bisphosphate sedoheptulase (SBPase), transketolase (TK), 5-phosphoribulose isomerase (RPE), 5-phosphoribose isomerase (RPI) and 5-phosphoribulose kinase (PRK).

3. The system according to claim 1 or 2, characterized in that The system also includes NADH and ATP to provide reducing power and power.

4. The system according to claim 2 or 3, characterized in that The ribulose-1,5-bisphosphate carboxylase / oxygenase (Rubisco) is Syn6301-Rubisco enzyme, and its nucleotide sequence is shown in SEQ ID NO:

1.

5. The system according to claim 4, characterized in that The system also includes HCO3 - and Mg 2+ To ensure the enzymatic activity of Syn6301-Rubisco enzyme.

6. The system according to any one of claims 3 to 5, characterized in that The system also includes formate to regenerate NADH with the help of formate dehydrogenase (FDH).

7. The system according to any one of claims 3 to 6, characterized in that The system further includes PolyP6 to regenerate ATP with the help of polyphosphatase (PKK2), optionally the system further includes Tris-HCl, optionally the system further includes TPP, optionally the system further includes DTT.

8. The system according to any one of claims 2 to 6, characterized in that The system further comprises ribulose-1,5-bisphosphate (RuBP) to start the cycle reaction. Preferably, the pH of the system is 6-10, preferably pH 8.

9. A method for reconstructing a Calvin cycle system in vitro, characterized in that: The method comprises: a. recombinantly expressing ribulose-1,5-bisphosphate carboxylase / oxygenase (Rubisco), 3-phosphoglycerate kinase (PGK), 3-phosphoglyceraldehyde dehydrogenase (GAPDH), triosephosphate isomerase (TPI), 1,6-bisphosphate fructose aldolase (FBA), 1,6-bisphosphate fructose enzyme (FBPase), 1,7-bisphosphate sedoheptulase (SBPase), transketolase (TK), 5-phospholipidose isomerase (RPE), 5-phospholipidose isomerase (RPI), 5-phospholipidose kinase (PRK), carbonic anhydrase (CA), 2-phosphoglycolic acid enzyme (PGPase), formate dehydrogenase (FDH) and polyphosphatase (PKK2) in vitro, optionally the nucleotide sequence of the carbonic anhydrase (CA) is as shown in SEQ ID NO: 11, optionally the nucleotide sequence of the 2-phosphoglycolic acid enzyme (PGPase) is as shown in SEQ ID wherein the nucleotide sequence of the formate dehydrogenase (FDH) is as shown in SEQ ID NO:10, the nucleotide sequence of the formate dehydrogenase (FDH) is as shown in SEQ ID NO:15, the nucleotide sequence of the polyphosphatase (PKK2) is as shown in SEQ ID NO:9, the nucleotide sequence of the 3-phosphoglycerate kinase (PGK) is as shown in SEQ ID NO:14, the nucleotide sequence of the 3-phosphoglyceraldehyde dehydrogenase (GAPDH) is as shown in SEQ ID NO:13, the nucleotide sequence of the triosephosphate isomerase (TPI) is as shown in SEQ ID NO:12, the nucleotide sequence of the 1,6-bisphosphate fructose aldolase (FBA) is as shown in SEQ ID NO:2, the nucleotide sequence of the 1,6-bisphosphate fructose enzyme (FBPase) is as shown in SEQ ID NO:3, the nucleotide sequence of the 1,7-bisphosphate sedoheptulose enzyme (SBPase) is as shown in SEQ ID NO:5, the nucleotide sequence of the transketolase (TK) is as shown in SEQ ID NO: NO:4, optionally the nucleotide sequence of 5-phosphoribosyl isomerase (RPE) is shown in SEQ ID NO:7, optionally the nucleotide sequence of 5-phosphoribosyl isomerase (RPI) is shown in SEQ ID NO:6, optionally the nucleotide sequence of 5-phosphoribosyl kinase (PRK) is shown in SEQ ID NO:8; b. purifying the enzyme expressed in step a; c. Add Tris-HCl, NaHCO3, Mg(COOH)2, TPP, PolyP6, ATP, DTT and NADH to the purified enzyme in step b; d. Add ribulose-1,5-bisphosphate (RuBP) to start the cycle reaction, preferably adjusting the pH of the system to 6-10, preferably adjusting the pH to 8.

10. Use of the system according to any one of claims 1 to 8 or the system reconstructed by the method according to claim 9 in the study of Calvin cycle, for example, in the study of Calvin cycle carbon fixation efficiency and key enzymes of Calvin cycle.

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