Method for biosynthesizing dicarbon organic acid by using CO2

Abstract

The invention discloses a method for biosynthesizing dicarbon organic acid by using CO2, and belongs to the technical field of bioengineering. A path for synthesizing glyoxylic acid and glycollic acid by taking CO2 as a substrate is designed, and a first reaction module for converting CO2 and acetyl coenzyme A into pyruvic acid and a second reaction module for converting pyruvic acid into malic acid are obtained through thermodynamic evaluation and screening of pathase. The third reaction module is used for converting malic acid into acetyl coenzyme A and glyoxylic acid, the fourth reaction module is used for converting glyoxylic acid into glycollic acid, and PSP circulation is carried out on the four reaction modules. Through an experimental method of a one-pot two-step method, 2.78 g / L of glyoxylic acid can be realized after 11 hours of catalytic reaction in total; and 1.78 g / L of glycollic acid. The invention successfully realizes a cell-free biosynthesis method for synthesizing glyoxylic acid and glycollic acid by taking CO2 as a substrate.

Description

Technical Field

[0001] The present invention relates to a method for utilizing CO 2 The invention discloses a method for biosynthesizing two-carbon organic acids, namely glyoxylic acid and glycolic acid, and belongs to the field of biotechnology. Background Art

[0002] Glyoxylic acid is a key intermediate metabolite in the glyoxylate cycle. Glyoxylic acid is obtained by the cleavage of isocitrate and then enters the acetic acid cycle to produce succinic acid. Succinic acid enters the tricarboxylic acid cycle metabolic process to provide nutrients for the growth and production of microorganisms. Glyoxylic acid is an important organic chemical raw material and intermediate, widely used in the production of important chemicals such as oral penicillin, vanillin, ethyl vanillin and allantoin. Glycolic acid, also known as glycolic acid, has excellent water solubility and medium-strong acidity and is an important chemical product. Since glycolic acid has hydroxyl and carboxyl groups in its molecular structure, it has the dual properties of acid and alcohol. Its characteristics make glycolic acid widely used in the textile industry, food industry and cosmetics industry.

[0003] At present, in industry, the production methods of glyoxylic acid mainly include chemical method and electrochemical oxidation method. Chemical method includes ozone oxidation maleic acid method, dichloroacetate method and glyoxal nitric acid oxidation method. Electrochemical oxidation method includes oxalic acid electrolysis and glyoxal oxidation. However, whether it is chemical method or electrochemical method, they all have problems such as complex process, difficult separation and purification of products, and non-renewable energy. At present, the chemical production of glycolic acid includes chloroacetic acid hydrolysis method, formaldehyde cyanidation method, formaldehyde hydrogen carboxylation method and dimethyl oxalate hydrogenation hydrolysis method. However, the traditional production method of glycolic acid has problems such as low raw material utilization, harsh reaction conditions, and greater environmental pollution.

[0004] The biological method of producing glyoxylic acid and glycolic acid has the advantages of environmental protection, mild conditions, and a wide range of raw material sources. Nowadays, microbial fermentation technology has become increasingly mature, and many engineered strains, such as Escherichia coli, Saccharomyces cerevisiae, and Corynebacterium glutamicum, have been used to produce glyoxylic acid or glycolic acid from glucose. However, due to the incompatibility of chassis strains with metabolic pathways, the interference and toxic effects of complex compounds in the body, and the low sugar-acid conversion rate, the production of glyoxylic acid and glycolic acid by microorganisms is limited. 2 As a clean and sustainable carbon source, CO 2 The synthesis of valuable chemicals has become an important research strategy. Therefore, at present, the biosynthesis of glyoxylate and glycolate requires a cell-free, CO-based 2 The method for substrate. Summary of the invention

[0005] In view of the above-mentioned deficiencies of the prior art, the present invention provides a method for utilizing CO 2The method for biosynthesizing dicarbon organic acids aims to solve the technical problems that the existing methods for producing glyoxylic acid or glycolic acid are limited by microbial production of glyoxylic acid and glycolic acid due to the incompatibility of chassis strains with metabolic pathways, the interference and toxic effects of complex compounds in the body, and the low sugar-acid conversion rate.

[0006] The first technical method provided by the present invention is a multi-enzyme catalytic system for biosynthesis of glyoxylic acid and glycolic acid, wherein the catalytic system comprises pyruvate synthase PS, pyruvate carboxylase PC, malate dehydrogenase MDH, malate thiokinase MTK, acyl-CoA lyase MCL and glycolate oxidoreductase GOR.

[0007] In certain embodiments, the pyruvate synthase PS is derived from Desulfocurvibacter africanus. The pyruvate synthase has an amino acid sequence as shown in SEQ ID NO.1.

[0008] In certain embodiments, the pyruvate carboxylase PC is derived from Aspergillus flavus. The pyruvate carboxylase has an amino acid sequence as shown in SEQ ID NO.3.

[0009] In certain embodiments, the malate dehydrogenase (MDH) is derived from Escherichia coli. The malate dehydrogenase has an amino acid sequence as shown in SEQ ID NO.4.

[0010] In certain embodiments, the malate thiokinase MTK is derived from Methylococcus capsulatus. The malate thiokinase comprises an α subunit and a β subunit, the amino acid sequence of the malate thiokinase α subunit is shown in SEQ ID NO.5, and the amino acid sequence of the malate thiokinase β subunit is shown in SEQ ID NO.6.

[0011] In certain embodiments, the malyl-CoA lyase MCL is derived from Methylobacterium extorquens. The malyl-CoA lyase has an amino acid sequence as shown in SEQ ID NO.7.

[0012] In certain embodiments, the glycolate oxidoreductase GOR is derived from Saccharomyces cerevisiae. The glycolate oxidoreductase has an amino acid sequence as shown in SEQ ID NO.8.

[0013] In certain embodiments, the catalytic system further comprises ferredoxin:NADP + Reductase FNR. The ferredoxin: NADP+ The reductase has an amino acid sequence as shown in SEQ ID NO.2.

[0014] The present invention provides a method for synthesizing glyoxylic acid and glycolic acid, wherein the method comprises the steps of: using CO 2 As substrates, glyoxylic acid and glycolic acid are synthesized using the multi-enzyme catalytic system described in the first technical solution.

[0015] In certain embodiments, the 2 The first reaction module that converts acetyl-CoA into pyruvate is to convert pyruvate and CO 2 A second reaction module that converts malate into malate, a third reaction module that converts malate into acetyl-CoA and glyoxylate, and a fourth reaction module that converts glyoxylate into glycolate.

[0016] Furthermore, the first reaction module is composed of pyruvate synthase DaPS participating in CO 2 and acetyl-CoA. The reaction system (1 mL) of the first reaction module contains: 50 mmol / L Tris-HCl buffer (pH 8.0), 10 mmol / L magnesium chloride, 30 mmol / L sodium chloride, 100 mmol / L sodium bicarbonate, 1 mmol / L dithiothreitol, 1 mmol / L thiamine pyrophosphate, 5 mmol / L NADPH, 3 mmol / L acetyl-CoA, 12.5 U / mL FNR, 50 μg / mL Fd (ferredoxin) and 45.9 U / mL pyruvate synthase. The reaction was carried out at 50°C under anaerobic conditions for 1 h.

[0017] Furthermore, the second reaction module comprises pyruvate carboxylase AfPC participating in CO 2 and pyruvate; malate dehydrogenase EcMDH participates in the conversion of oxaloacetate. The reaction system (1 mL) of the second reaction module comprises: 50 mmol / L Tris-HCl buffer (pH 8.0), 10 mmol / L magnesium chloride, 10 mmol / L sodium pyruvate, 100 mmol / L sodium bicarbonate, 2 mmol / L NADH, 10 mmol / L potassium chloride, 5 mmol / L ATP, 169.6 U / mL malate dehydrogenase and 248.3 U / mL pyruvate carboxylase. The reaction was carried out at 30 ° C for 3 h.

[0018] Furthermore, the third reaction module is participated in the conversion of malate by malate thiokinase McMTK; and malyl-CoA lyase MeMCL is participated in the cleavage reaction of malyl-CoA. The reaction system (1 mL) of the third reaction module comprises: 50 mmol / L Tris-HCl buffer (pH 8.0), 5 mmol / L magnesium chloride, 1 mmol / L ATP, 1 mmol / L CoA, 5 mmol / L sodium malate, 13.5 U / mL malate thiokinase and 13.9 U / mL malyl-CoA lyase. The reaction is carried out at 30°C for 3 hours.

[0019] Furthermore, the fourth reaction module is involved in the oxidation of glyoxylate by glycolate oxidoreductase ScGOR. The fourth reaction module reaction system (1 mL) comprises: 50 mmol / L Tris-HCl buffer (pH 8.0), 10 mmol / L glyoxylate, 2 mmol / L NADH, 9.6 U / mL purified glycolate oxidoreductase, and the reaction is carried out at 30°C for 2 hours.

[0020] In certain embodiments, the method is pyruvate synthase, ferredoxin:NADP + Reductase in iron-containing redox proteins, Mg 2+ , NADPH, thiamine pyrophosphate, dithiothreitol, sodium chloride, acetyl coenzyme A and carbon dioxide in a reaction system, reacting at 50°C for at least 1 hour, which is the first step of the reaction;

[0021] On the basis of the first step reaction, pyruvate carboxylase, malate dehydrogenase, malate thiokinase, malyl-CoA lyase, glycolate oxidoreductase, ATP, NADH, sodium bicarbonate, dithiothreitol, thiamine pyrophosphate and potassium chloride are added, and the reaction is carried out at 30°C for at least 10 hours to complete the second step reaction.

[0022] In certain embodiments, pyruvate synthase DaPS, ferredoxin: NADP + The added amounts of reductase HtFNR, pyruvate carboxylase AfPC, malate dehydrogenase EcMDH, malate thiokinase McMTK, malyl-CoA lyase MeMCL, and glycolate oxidoreductase ScGOR were 45.9 U / mL, 12.5 U / mL, 248.3 U / mL, 169.6 U / mL, 13.5 U / mL, 13.9 U / mL, and 9.6 U / mL, respectively.

[0023] In certain embodiments, the reaction system (1 mL) of the first step reaction comprises: 50 mmol / L Tris-HCl buffer (pH 8.0), 10 mmol / L magnesium chloride, 30 mmol / L sodium chloride, 50 mmol / L sodium bicarbonate, 1 mmol / L dithiothreitol, 1 mmol / L thiamine pyrophosphate, 5 mmol / L NADPH and 3 mmol / L acetyl-CoA.

[0024] In certain embodiments, the reaction system (1 mL) of the second step reaction comprises: 50 mmol / L sodium bicarbonate, 1 mmol / L dithiothreitol, 1 mmol / L thiamine pyrophosphate, 5 mmol / L NADH, 10 mmol / L potassium chloride and 5 mmol / L ATP.

[0025] The third technical solution provided by the present invention is the application of the multi-enzyme catalytic system described in the first technical solution or the method described in the second technical solution in the synthesis of glyoxylic acid and / or glycolic acid.

[0026] The present invention has the following beneficial effects:

[0027] The present invention designs a method of using CO 2 The metabolic pathway (PSP cycle) for biosynthesis of glyoxylic acid and glycolic acid from substrates was successfully constructed in vitro by expressing and purifying the pathway enzymes using a one-pot two-step method, achieving cell-free synthesis of glyoxylic acid and glycolic acid. The glyoxylic acid yield in the reaction system reached 2.78 g / L; the glycolic acid yield reached 1.78 g / L. This method successfully achieved the goal of CO 2 The method provided by the invention shows great potential in environmental protection, raw material diversification, sustainable development and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Design and thermodynamic evaluation of the PSP cycle; A, the overall structure of the PSP cycle; B, the thermodynamic evaluation results of the PSP cycle.

[0029] Figure 2 For the screening and identification of enzymes in the PSP cycle pathway; A, catalytic activity evaluation of PS; B, catalytic activity evaluation of MTK; C, catalytic activity evaluation of GOR; D, sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis results after purification of the pathway enzyme.

[0030] Figure 3 This is a modular verification of the PSP cycle; A, a schematic diagram of the four modules of the PSP cycle; B, the conversion rates of the four modules.

[0031] Figure 4In vitro construction and verification of the PSP cycle; A, schematic diagram of the one-pot two-step method to verify the PSP cycle; B, yields of glyoxylic acid and glycolic acid after 10 h of reaction. DETAILED DESCRIPTION

[0032] The following is a description of the preferred embodiments of the present invention. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention. Test method:

[0033] For the detection of pyruvate synthase activity that catalyzes acetyl-CoA to pyruvate, the change in NADH at 340 nm was detected by co-reaction with excess lactate dehydrogenase. The reaction system includes (200 μL, 50°C): 50 mmol / L Tris-HCl buffer (pH 8.0), 10 mmol / L magnesium chloride, 30 mmol / L sodium chloride, 100 mmol / L sodium bicarbonate, 2 mmol / L dithiothreitol, 1 mmol / L thiamine pyrophosphate, 2 mmol / L NADPH, 2 mmol / L NADH, 3 mmol / L acetyl-CoA, 10 μg FNR, 15 U / mL LDH (commercial enzyme, derived from Staphylococcus sp), 50 μg / mL Fd (ferredoxin) and 30 μg purified pyruvate synthase. All components added to the buffer were monitored at 50°C during the reaction at 340 nm, and the absorbance value was taken into the standard curve to calculate the concentration of NADH. The amount of enzyme required to consume 1 nM NAD(P)H in a 1 min reaction at 50°C was defined as 1 U.

[0034] For the activity detection of malate thiokinase and malyl-CoA lyase, glyoxylate-phenylhydrazone generated by the reaction of glyoxylate with phenylhydrazine was detected by measuring the absorbance at 324 nm. The reaction system included (200 μL, 30°C): 50 mmol / L Tris-HCl buffer (pH 8.0), 5 mmol / L magnesium chloride, 1 mmol / L ATP, 1 mmol / L CoA, 5 mmol / L sodium malate, 10 mmol / L phenylhydrazine and appropriate amounts of malate thiokinase and malyl-CoA lyase. 8.5 μg malate thiokinase and 7.3 μg malyl-CoA lyase were used for malate thiokinase detection. 8.5 μg malate thiokinase and 10.9 μg malyl-CoA lyase were used for malyl-CoA lyase detection. The amount of enzyme required to generate 1 nM glyoxylate at 30°C for 1 min was defined as 1 U.

[0035] For the activity detection of malate dehydrogenase, the enzyme activity was determined by detecting the change in NADH at 340 nm. The reaction system included (200 μL, 30°C): 50 mmol / L Tris-HCl buffer (pH 8.0), 4 mmol / L oxaloacetate, 1 mmol / L NADH, and 0.4 μg purified malate dehydrogenase. 1 U is defined as the amount of enzyme required to consume 1 nM NADH per minute at 30°C.

[0036] For the activity determination of pyruvate carboxylase, the change in NADH at 340nm was detected by co-reaction with excess malate dehydrogenase. The reaction system includes (200μL, 30℃): 50mmol / L Tris-HCl buffer (pH 8.0), 10mmol / L sodium pyruvate, 100mmol / L sodium bicarbonate, 1mmol / L NADH, 10mmol / L potassium chloride, 10mmol / L magnesium chloride, 5mmol / L ATP, 110μg malate dehydrogenase; 270μg pyruvate carboxylase. 1U is defined as the amount of enzyme required to consume 1nM NADH per minute at 30℃.

[0037] For the activity detection of glycolate oxidoreductase, the enzyme activity was determined by detecting the change in NADH at 340 nm. The reaction system included (200 μL, 30°C): 50 mmol / L Tris-HCl buffer (pH 8.0), 10 mmol / L glyoxylic acid, 1 mmol / L NADH, and 0.5 μg purified glycolate oxidoreductase. 1 U is defined as the amount of enzyme required to consume 1 nM NADH per minute at 30°C.

[0038] Analytical method: The concentration of glyoxylic acid and glycolic acid was determined using an HPLC system (Dionex UltiMate 3000 Series, Thermo Scientific, USA). The system was equipped with an Aminex HPX-87H column (7.8×300 mm, Bio-Rad Laboratories, USA), a column temperature of 52°C, a flow rate of 0.6 mL / min, a UV detector wavelength of 210 nm, a mobile phase of 5 mmol / L sulfuric acid solution, and an injection volume of 10 μL. The concentrations of glyoxylic acid and glycolic acid in the sample were calculated using the glyoxylic acid and glycolic acid concentration standard curves.

[0039] The generation of glyoxylic acid and glycolic acid was verified by LC-MS system (QTRAP 5500LC-MS System). The system was equipped with Waters HSS T3 column (1.8μm, 2.1×100mm), flow rate was 0.3mL / min, mobile phase A (0.1% formic acid-water solution) and mobile phase B (100% acetonitrile), temperature was 40℃. The injection volume was 2μL. The mobile phase elution gradient was eluted according to the following steps and procedures: 0-2min: maintain 95% A and 5% B; 2-4min: A decreased from 95% to 5% and B increased from 5% to 95%; 4-6min: maintain 5% A and 95% B; 6-6.1min: A increased from 5% to 95% and B decreased from 95% to 5%.

[0040] The raw materials used in the examples are:

[0041] 1. Luria-Bertani (LB) medium: tryptone 10g / L (Oxoid), yeast extract 5g / L (Oxoid), NaCl 10g / L (China Pharmaceutical Group). LB medium is used for strain activation. When the cells contain recombinant plasmids, the final concentration of the corresponding antibiotics added to the medium is: kanamycin 50mg / L.

[0042] 2. Terrific broth (TB) medium: glycerol 4g / L (China Pharmaceutical Group), yeast extract 24g / L (Angel), tryptone 12g / L (China Pharmaceutical Group), potassium dihydrogen phosphate 2.31g / L (China Pharmaceutical Group), potassium dihydrogen phosphate trihydrate 16.42g / L (China Pharmaceutical Group). TB medium is used for soluble expression and in vitro purification of pathway enzymes. When the cells contain recombinant plasmids, the final concentrations of isopropyl-β-D-thiogalactoside (IPTG) (Sangon Biotechnology) and antibiotics (Sangon Biotechnology) added to the medium are: IPTG 100mg / L, kanamycin 50mg / L.

[0043] 3. pET28a plasmid and Escherichia coli BL21 (DE3) are both commercial plasmids and strains.

[0044] 4. Tris, hydrochloric acid, magnesium chloride, sodium chloride, sodium bicarbonate, and potassium chloride were sourced from China National Pharmaceutical Group Corporation; dithiothreitol, thiamine pyrophosphate, NADPH, sodium pyruvate, sodium malate, glyoxylic acid, glycolic acid, NADH, and ATP were sourced from Aladdin; CoA and acetyl-CoA were sourced from Maclean.

[0045] Example 1: Design and evaluation of a PSP cycle for biosynthesis of glyoxylate and glycolate

[0046] In order to achieve CO 2For the biosynthesis of glyoxylate and glycolate as substrates, a CO2-derived 4-module metabolic system was designed as follows: 2 Fixed pathway (PSP cycle, Figure 1 A): (1) Put CO 2 The PSP cycle consists of seven enzymes: pyruvate synthase (PS), ferredox protein: NADP + Reductase FNR, pyruvate carboxylase PC, malate dehydrogenase MDH, malate thiokinase MTK, malyl-CoA lyase MCL, glycolate oxidoreductase GOR.

[0047] On this basis, the PSP cycle was thermodynamically analyzed (eQuilibrator website, https: / / equilibrator.weizmann.ac.il / ). The thermodynamic analysis of the PSP cycle was evaluated under the conditions of pH 8.0, pMg 3.0, ionic strength 0.25, and physiological concentration of 1 mmol / L. Figure 1 As shown in B, analysis of thermodynamic data revealed that there is a bottleneck reaction in the PSP cycle: pyruvate synthase catalyzes the reaction of CO 2 and the reaction of acetyl-CoA to pyruvate. Overall, the MDF (Max-min Driving Force) of the PSP cycle is 12.28 kJ / mol, indicating that this pathway is thermodynamically favorable.

[0048] Example 2: Screening of enzyme pathways in the PSP cycle

[0049] Based on Example 1, the pathway enzymes in the PSP cycle were screened and evaluated.

[0050] In the first reaction module, pyruvate synthase (DaPS, CtPS, SyPS) from Desulfocurvibacter africanus, Clostridium thermocellum, Synechocystis sp. PCC 6803 were screened. The nucleotide sequence of the gene encoding DaPS is shown in SEQ ID NO.9, and the gene encoding DaPS is inserted between the BmHⅠ and XhoⅠ restriction sites of plasmid pET28a. CtPS includes α subunit, δ subunit, β subunit and γ subunit, and the nucleotide sequences of the genes encoding CtPS α subunit, δ subunit, β subunit and γ subunit are shown in SEQ ID NO.17-20, respectively. The gene encoding CtPS is inserted into the order of α subunit, δ subunit, β subunit and γ subunit between the BmHⅠ and XhoⅠ restriction sites of plasmid pET28a. The nucleotide sequence of the gene encoding SyPS is shown in SEQ ID NO. 21. The gene encoding SyPS is inserted between the BmHI and XhoⅠ restriction sites of plasmid pET28a. + Reductase (HtFNR), the nucleotide sequence of which is shown in SEQ ID NO.10, the gene encoding HtFNR is inserted between the BmHI and XhoⅠ restriction sites of plasmid pET28a. The recombinant plasmids constructed above are transformed into competent Escherichia coli BL21 (DE3). Finally, the correct strains containing the target expression vector are obtained by screening with LB medium solid plates containing kanamycin resistance and gene sequencing, and are stored in glycerol for later use.

[0051] In the second reaction module, pyruvate carboxylase (AfPC) from Aspergillus flavus and malate dehydrogenase (EcMDH) from Escherichia coli were screened, and the nucleotide sequences are shown in SEQ ID NO.11 and SEQ ID NO.12, respectively. The above genes were inserted between the BmHⅠ and XhoⅠ restriction sites of plasmid pET28a, and then transformed into Escherichia coli BL21 (DE3) competent state. Finally, the correct strains containing the target expression vector were obtained by solid plate screening of LB medium containing kanamycin resistance and gene sequencing, and they were preserved with glycerol for later use.

[0052] In the third reaction module, malate thiokinase (PaMTK, McMTK, RlMTK) from Paracoccus aminovorans, Methylococcuscapsulatus, and Roseobacter litoralis was screened, and PaMTK, McMTK, and RlMTK all contained α subunits and β subunits. The nucleotide sequences of the genes encoding PaMTKα subunit, PaMTKβ subunit, McMTKα subunit, McMTKβ subunit, RlMTKα subunit, and RlMTKβ subunit are SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24, and SEQ ID NO.25, respectively. The gene encoding PaMTK was inserted into the BmHI and XhoⅠ restriction sites of plasmid pET28a in the order of α subunit and β subunit, the gene encoding McMTK was inserted into the BmHI and XhoⅠ restriction sites of plasmid pET28a in the order of α subunit and β subunit, and the gene encoding RlMTK was inserted into the BmHI and XhoⅠ restriction sites of plasmid pET28a in the order of α subunit and β subunit. The malyl-CoA lyase MeMCL from Methylobacterium extorquens, the nucleotide sequence of which is shown in SEQ ID NO.15, was inserted into the BmHI and XhoⅠ restriction sites of plasmid pET28a. The recombinant plasmids constructed above were transformed into competent Escherichia coli BL21 (DE3) respectively. Finally, the correct strains containing the target expression vector were obtained through LB medium solid plate screening containing kanamycin resistance and gene sequencing, and were preserved in glycerol for later use.

[0053] In the fourth reaction module, glycolate oxidoreductase (AaGOR, ScGOR, NcGOR) from Acetobacter aceti, Saccharomycescerevisiae, and Neurospora crassa were screened, and the nucleotide sequences were shown in SEQ ID NO.26, SEQ ID NO.16, and SEQ ID NO.27, respectively. The above genes were inserted between the BmHI and XhoⅠ restriction sites of plasmid pET28a, and then transformed into Escherichia coli BL21 (DE3) competent state. Finally, the correct strains containing the target expression vector were obtained by solid plate screening of LB medium containing kanamycin resistance and gene sequencing, and they were preserved with glycerol for later use.

[0054] The recombinant bacteria were cultured as follows, and the cells were collected and the specific enzyme activity was determined. The recombinant bacteria were inoculated into LB medium for activation, and then the activated bacteria were transferred to TB medium and cultured at 37°C until the cells grew to OD 600 When the pH value was 0.6 to 0.8, IPTG was added at a final concentration of 100 mg / L. After induction at 16°C for 16 hours, the collected bacteria were purified and the specific enzyme activity was determined.

[0055] In vitro testing of pure enzymes, e.g. Figure 2 As shown, pyruvate synthase from Desulfocurvibacter africanus, malate thiokinase from Methylococcus capsulatus, and glycolate oxidoreductase from Saccharomyces cerevisiae had the best catalytic effect.

[0056] Based on the above tests, pyruvate synthase DaPS, ferredoxin: NADP + Reductase HtFNR, pyruvate carboxylase AfPC, malate dehydrogenase EcMDH, malate thiokinase McMTK, malyl-CoA lyase MeMCL, glycolate oxidoreductase ScGOR were used to construct the PSP cycle. The specific enzyme activities of DaPS, AfPC, EcMDH, McMTK, MeMCL, and ScGOR were 13 U / mg, 230 U / mg, 1555.55 U / mg, 92.05 U / mg, 80 U / mg, and 480.28 U / mg, respectively.

[0057] Example 3: Modular Verification of PSP Cycles

[0058] Based on the screening results of the pathway enzymes in Example 2, the four modules of the PSP cycle were modularly verified in vitro, such as Figure 3 As shown in A.

[0059] In the first reaction module, pyruvate synthase catalyzes the reaction of acetyl-CoA with CO 2 The reaction module was reacted at 50°C under anaerobic conditions for 1 hour. The reaction system (1 mL) contained: 50 mmol / L Tris-HCl buffer (pH 8.0), 10 mmol / L magnesium chloride, 30 mmol / L sodium chloride, 100 mmol / L sodium bicarbonate, 1 mmol / L dithiothreitol, 1 mmol / L thiamine pyrophosphate, 5 mmol / L NADPH, 3 mmol / L acetyl-CoA, 12.5 U / mL FNR, 45.7 U / mL Fd and 45.9 U / mL purified pyruvate synthase. The reaction was terminated after 1 hour, the supernatant was centrifuged and the product pyruvate was detected.

[0060] In the second reaction module, pyruvate carboxylase catalyzes the reaction of pyruvate and CO 2 The reaction of oxaloacetate to malic acid was catalyzed by malate dehydrogenase. The reaction system (1 mL) contained: 50 mmol / L Tris-HCl buffer (pH 8.0), 10 mmol / L magnesium chloride, 10 mmol / L sodium pyruvate, 100 mmol / L sodium bicarbonate, 2 mmol / L NADH, 10 mmol / L potassium chloride, 5 mmol / L ATP, 169.6 U / mL malate dehydrogenase and 248.3 U / mL pyruvate carboxylase. The reaction was terminated at 30 ° C for 3 h, and the supernatant was taken to detect the product malic acid.

[0061] In the third reaction module, malate thiokinase catalyzes the reaction of malate to maloyl-CoA, and maloyl-CoA lyase catalyzes the reaction of maloyl-CoA to acetyl-CoA and glyoxylate. The reaction system (1 mL) contains: 50 mmol / L Tris-HCl buffer (pH 8.0), 5 mmol / L magnesium chloride, 1 mmol / L ATP, 1 mmol / L CoA, 5 mmol / L sodium malate, 13.5 U / mL malate thiokinase and 13.9 U / mL maloyl-CoA lyase. The reaction was terminated after 3 h at 30 ° C, and the supernatant was taken to detect the product glyoxylate.

[0062] In the fourth reaction module, glycolate oxidoreductase catalyzes the reaction of glyoxylate to glycolate. The reaction system (1 mL) contains: 50 mmol / L Tris-HCl buffer (pH 8.0), 10 mmol / L glyoxylate, 2 mmol / L NADH, and 9.6 U / mL purified glycolate oxidoreductase. The reaction was terminated after 2 h at 30 °C, and the supernatant was taken to detect the product glycolate.

[0063] The results are as follows Figure 3 As shown in B, the conversion rate of module one is 4.98%, the conversion rate of module two is 55.57%, the conversion rate of module three is 50.78%, and the conversion rate of module four is 35.64%.

[0064] Example 4: In vitro construction and verification of PSP cycle

[0065] Based on the modular verification of the PSP cycle in Example 3, four modules were assembled to verify the feasibility of the PSP cycle, as shown in FIG. Figure 4 As shown in A.

[0066] The PSP cycle was carried out in a one-pot two-step method. One-pot two-step reaction system: In the first step, only the reaction of pyruvate synthase was carried out. The reaction system (1 mL) contained: 50 mmol / L Tris-HCl buffer (pH 8.0), 10 mmol / L magnesium chloride, 30 mmol / L sodium chloride, 50 mmol / L sodium bicarbonate, 1 mmol / L dithiothreitol, 1 mmol / L thiamine pyrophosphate, 5 mmol / L NADPH, 3 mmol / L acetyl-CoA, 12.5 U / mL FNR, 45.7 U / mL Fd and 45.9 U / mL purified pyruvate synthase. After reacting at 50 °C for 1 h, the remaining components of the second step reaction were added: 50 mmol / L sodium bicarbonate, 1 mmol / L dithiothreitol, 1 mmol / L thiamine pyrophosphate, 5 mmol / L NADH, 10 mmol / L potassium chloride, 5 mmol / L ATP, 169.6U / mL malate dehydrogenase and 248.3U / mL pyruvate carboxylase, 13.5U / mL malate thiokinase and 13.9U / mL malyl-CoA lyase, 9.6U / mL purified glycolate oxidoreductase. The reaction was terminated after 10h at 30℃, and the supernatant was centrifuged to detect glyoxylate and glycolate. The results are as follows Figure 4 As shown in B, after a total of 11 h of reaction, the PSP cycle can produce 2.78 g / L of glyoxylic acid and 1.78 g / L of glycolic acid, proving that the PSP cycle can be successfully constructed in vitro and achieve the CO 2 Produces glyoxylate and glycolate as substrates.

[0067] 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 should be based on the definition of the claims.

Claims

1. A multi-enzyme catalytic system for biosynthesis of glyoxylic acid and glycolic acid, characterized in that: The catalytic system includes pyruvate synthase PS from Desulfocurvibacter africanus, pyruvate carboxylase PC from Aspergillus flavus, malate dehydrogenase MDH from Escherichia coli, malate thiokinase MTK from Methylococcus capsulatus, acyl-CoA lyase MCL from Methylobacterium extorquens and glycolate oxidoreductase GOR from Saccharomyces cerevisiae.

2. The multi-enzyme catalytic system according to claim 1, characterized in that: The catalytic system also includes ferredoxin: NADP + Reductase FNR, the FNR is derived from Hydrogenobater thermophiles.

3. A method for synthesizing glyoxylic acid and glycolic acid, characterized in that: The method uses CO2 as a substrate and utilizes the multi-enzyme catalysis system of claim 1 or 2 to synthesize glyoxylic acid and glycolic acid.

4. The method according to claim 3, characterized in that It includes a first reaction module for converting CO2 and acetyl-CoA into pyruvate, a second reaction module for converting pyruvate and CO2 into malate, a third reaction module for converting malate into acetyl-CoA and glyoxylate, and a fourth reaction module for converting glyoxylate into glycolate.

5. The method according to claim 4, characterized in that The first reaction module comprises pyruvate synthase DaPS involved in the conversion of CO2 and acetyl-CoA; In the second reaction module, pyruvate carboxylase AfPC participates in the conversion of CO2 and pyruvate, and malate dehydrogenase EcMDH participates in the conversion of oxaloacetate; In the third reaction module, malate thiokinase McMTK participates in the conversion of malate, and malyl-CoA lyase MeMCL participates in the cleavage reaction of malyl-CoA; The fourth reaction module involves glycolate oxidoreductase ScGOR in the oxidation of glyoxylate.

6. The method according to claim 4 or 5, characterized in that: The reaction system of the first reaction module includes: 50mmol / L Tris-HCl buffer (pH 8.0), 10mmol / L magnesium chloride, 30mmol / L sodium chloride, 100mmol / L sodium bicarbonate, 1mmol / L dithiothreitol, 1mmol / L thiamine pyrophosphate, 5mmol / L NADPH, 3mmol / L acetyl CoA, 12.5U / mL FNR, 50μg / mL Fd and 45.9U / mL pyruvate synthase, and the reaction is carried out at 50°C under anaerobic conditions for 1h; The reaction system of the second reaction module comprises: 50mmol / L Tris-HCl buffer (pH 8.0), 10mmol / L magnesium chloride, 10mmol / L sodium pyruvate, 100mmol / L sodium bicarbonate, 2mmol / L NADH, 10mmol / L potassium chloride, 5mmol / L ATP, 169.6U / mL malate dehydrogenase and 248.3U / mL pyruvate carboxylase, reacting at 30°C for 3h; The reaction system of the third reaction module comprises: 50 mmol / L Tris-HCl buffer with a pH of 8.0, 5 mmol / L magnesium chloride, 1 mmol / L ATP, 1 mmol / L CoA, 5 mmol / L sodium malate, 13.5 U / mL malate thiokinase and 13.9 U / mL malyl-CoA lyase; the reaction is carried out at 30° C. for 3 hours; The fourth reaction module reaction system comprises: 50mmol / L Tris-HCl buffer (pH 8.0), 10mmol / L glyoxylic acid, 2mmol / L NADH, 9.6U / mL glycolate oxidoreductase, and reacts at 30°C for 2h.

7. The method according to claim 4, characterized in that The method is pyruvate synthase PS, ferredoxin: NADP + Reductase FNR in iron-containing redox proteins, Mg 2+ , NADPH, thiamine pyrophosphate, dithiothreitol, sodium chloride, acetyl coenzyme A and carbon dioxide in a reaction system, reacting at 50°C for at least 1 hour, which is the first step of the reaction; On the basis of the first step reaction, pyruvate carboxylase PS, malate dehydrogenase MDH, malate thiokinase MTK, malyl-CoA lyase MCL, glycolate oxidoreductase GOR, ATP, NADH, sodium bicarbonate, dithiothreitol, thiamine pyrophosphate and potassium chloride are added, and the reaction is carried out at 30°C for at least 10 hours to complete the second step reaction.

8. The method according to claim 7, characterized in that Pyruvate synthase PS, ferredoxin: NADP + The added amounts of reductase FNR, pyruvate carboxylase PC, malate dehydrogenase MDH, malate thiokinase MTK, malyl-CoA lyase MCL, and glycolate oxidoreductase GOR were 45.9 U / mL, 12.5 U / mL, 248.3 U / mL, 169.6 U / mL, 13.5 U / mL, 13.9 U / mL, and 9.6 U / mL, respectively.

9. The method according to claim 7 or 8, characterized in that: The reaction system of the first step reaction includes: 50mmol / L Tris-HCl buffer with a pH of 8.0, 10mmol / L magnesium chloride, 30mmol / L sodium chloride, 50mmol / L sodium bicarbonate, 1mmol / L dithiothreitol, 1mmol / L thiamine pyrophosphate, 5mmol / L NADPH, 50μg / mL Fd and 3mmol / L acetyl CoA; The reaction system of the second step reaction includes: 50mmol / L sodium bicarbonate, 1mmol / L dithiothreitol, 1mmol / L thiamine pyrophosphate, 5mmol / L NADH, 10mmol / L potassium chloride and 5mmol / L ATP.

10. Use of the multi-enzyme catalytic system according to claim 1 or 2 or the method according to any one of claims 3 to 9 in the synthesis of glyoxylic acid and / or glycolic acid.

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