Method for biosynthesizing four-carbon organic acid by using one-carbon compound

Abstract

The invention discloses a method for biosynthesizing four-carbon organic acid by using a one-carbon compound, and belongs to the technical field of bioengineering. According to the invention, a path for synthesizing succinic acid, fumaric acid and malic acid by taking CO2 and formaldehyde as substrates is designed, and an SFMSP path containing six modules is obtained through thermodynamic evaluation and screening of pathase. Through a one-pot experimental method, after 10 hours of catalytic reaction, 1.6 g / L succinic acid, 1.3 g / L fumaric acid and 1.5 g / L malic acid can be produced. The invention successfully realizes a cell-free biosynthesis method for synthesizing succinic acid, fumaric acid and malic acid by taking a monocarbon compound as a substrate.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for biosynthesis of four-carbon organic acids, i.e. succinic acid, fumaric acid and malic acid, using one-carbon compounds, belonging to the field of biotechnology. BACKGROUND

[0002] Succinic acid, fumaric acid and malic acid are intermediates of the tricarboxylic acid cycle (TCA), and are important organic chemical raw materials and intermediates with wide applications. Succinic acid is mainly used for synthesis of various high-value-added chemicals such as gamma-butyrolactone (GBL) in the chemical industry; is widely used as an acidulant, preservative and flavor enhancer in the food industry; and is used for synthesis of various drugs such as sedatives in the pharmaceutical field. Fumaric acid is mainly used as an acidulant, preservative and antioxidant in the food industry; is mainly used for synthesis of various drugs and intermediates or as a pharmaceutical excipient in the pharmaceutical industry; is mainly used for synthesis of polyester plastics in the plastics industry; and can be used as a plant growth regulator and soil conditioner in the agricultural field. Malic acid can be used as a food additive in the food industry; can be used for preparation of anthelmintics in the pharmaceutical industry; and can be used as a raw material for synthesis of scale removers in the chemical industry.

[0003] At present, the chemical production of succinic acid includes acetylene method, paraffin oxidation method, catalytic hydrogenation method and electrolytic reduction method, etc. The catalytic hydrogenation method is the most commonly used method for industrial production of succinic acid at present, which utilizes cis- or trans-butenedioic anhydride to undergo hydrogenation reaction under the action of a catalyst to obtain succinic acid. However, this method is heavily dependent on non-renewable fossil resources, has high energy consumption in the production process, causes serious environmental pollution, and has many by-products which are difficult to control. The main method for chemical production of fumaric acid is maleic acid isomerization, which uses maleic acid (maleic acid) as raw material to generate fumaric acid through isomerization reaction under the action of a catalyst. However, this method has high energy consumption in the production process and causes serious environmental pollution. The main methods for chemical production of malic acid include lime method and allyl alcohol method. The lime method is simple in process, but generates a large amount of waste residue and wastewater; the allyl alcohol method has good product quality, but requires the use of a noble metal catalyst, which is high in cost. Therefore, in recent years, researchers have gradually turned their attention to more sustainable and environmentally friendly biosynthesis methods.

[0004] Bio-production of fumaric acid and succinic acid has the advantages of environmental protection, mild conditions, and wide sources of raw materials. Nowadays, microbial fermentation technology has become increasingly mature, and many engineered strains, such as Escherichia coli, Saccharomyces cerevisiae, and Corynebacterium glutamicum, have been used for the production of succinic acid, fumaric acid, and malic acid from glucose. However, using multi-carbon sugars as substrates has problems such as competing with humans for food and carbon emissions. Currently, the bio-synthesis of one-carbon compounds such as CO2 is increasingly attracting attention. On the one hand, directly using one-carbon compounds can reduce the emission of greenhouse gases CO2 and methane, providing a solution to the greenhouse effect. On the other hand, resource utilization of one-carbon compounds not only can improve economic benefits, but also provides a possible method to cope with the global energy crisis. Therefore, it is necessary to establish a method for biosynthesis of succinic acid, fumaric acid, and malic acid using one-carbon compounds as substrates to solve the problems of existing technology relying on multi-carbon sources and limited net carbon emissions in the production process, and to realize the resource utilization of one-carbon compounds. SUMMARY

[0005] In view of the above problems of the prior art, the present application provides a method for biosynthesis of four-carbon organic acids using one-carbon compounds.

[0006] The present application provides a composition capable of catalyzing the generation of four-carbon organic acids from one-carbon compounds, comprising: aspartate-oxaloacetate transaminase, serine aldolase, serine deaminase, malate dehydrogenase, malate thiokinase, malonyl-CoA lyase, acetyl-CoA carboxylase, malonyl-CoA reductase / 3-hydroxypropionate dehydrogenase, propionyl-CoA synthetase, propionyl-CoA carboxylase, methylmalonyl-CoA isomerase, methylmalonyl-CoA mutase, and succinyl-CoA synthetase.

[0007] In one embodiment, the composition further comprises succinic acid dehydrogenase.

[0008] In one embodiment, the composition further comprises succinic acid dehydrogenase and fumarate hydratase.

[0009] In some embodiments, the nucleotide sequence encoding the aspartate- aminotransferase has the sequence as set forth in SEQ ID NO. 1; the nucleotide sequence encoding the serine aldolase has the sequence as set forth in SEQ ID NO. 2 and SEQ ID NO. 19; the nucleotide sequence encoding the serine deaminase has the sequence as set forth in SEQ ID NO. 3; the amino acid sequence of the malate enzyme has the sequence as set forth in SEQ ID NO. 4; the nucleotide sequence encoding the alpha subunit of the malate thiokinase has the sequence as set forth in SEQ ID NO. 5; the nucleotide sequence encoding the beta subunit of the malate thiokinase has the sequence as set forth in SEQ ID NO. 6; the nucleotide sequence encoding the malonyl-CoA lyase MCL has the sequence as set forth in SEQ ID NO. 7; the nucleotide sequence encoding the AccBC subunit of the acetyl-CoA carboxylase ACC has the sequence as set forth in SEQ ID NO. 8; the nucleotide sequence encoding the DtsR subunit of the acetyl-CoA carboxylase ACC has the sequence as set forth in SEQ ID NO. 9; the nucleotide sequence encoding the malonyl-CoA reductase / 3-hydroxypropionate dehydrogenase has the sequence as set forth in SEQ ID NO. 10; the nucleotide sequence encoding the propionyl-CoA synthetase PCS has the sequence as set forth in SEQ ID NO. 11; the nucleotide sequence encoding the alpha subunit of the propionyl-CoA carboxylase PCC has the sequence as set forth in SEQ ID NO. 12; the nucleotide sequence encoding the beta subunit of the propionyl-CoA carboxylase PCC has the sequence as set forth in SEQ ID NO. 13; the nucleotide sequence encoding the methylmalonyl-CoA epimerase EPI has the sequence as set forth in SEQ ID NO. 14; the nucleotide sequence encoding the methylmalonyl-CoA mutase MCM has the sequence as set forth in SEQ ID NO. 15; the nucleotide sequence encoding the succinyl-CoA synthetase SUC has the sequence as set forth in SEQ ID NO. 16.

[0010] In one embodiment, the nucleotide sequence encoding the succinate dehydrogenase SDH has the sequence as set forth in SEQ ID NO. 17; the nucleotide sequence encoding the fumarate hydratase FUM has the sequence as set forth in SEQ ID NO. 18.

[0011] In one embodiment, the one-carbon compound includes, but is not limited to, carbon dioxide, bicarbonate or carbonate.

[0012] In one embodiment, the four-carbon organic acid includes, but is not limited to, succinic acid, fumaric acid and malic acid.

[0013] The present application also provides the use of the enzyme composition in catalyzing the production of a four-carbon organic acid from a one-carbon compound.

[0014] The present invention provides a method for preparing four-carbon organic acids, using a one-carbon compound as a substrate and the enzyme composition as a catalyst; the four-carbon organic acids include, but are not limited to, succinic acid, fumaric acid and malic acid.

[0015] In one embodiment, the amounts of aspartate-glyoxylate transaminase, serine aldolase, serine deaminase, malic acid oxidase, malate thiokinase, mayl-CoA lyase, acetyl-CoA carboxylase, malonyl-CoA reductase / 3-hydroxypropionic acid dehydrogenase, propionyl-CoA synthase, propionyl-CoA carboxylase, methylmalonyl-CoA isomerase, methylmalonyl-CoA mutase, succinyl-CoA synthase, succinate dehydrogenase, and fumarate hydratase added are 3.0–7.0 μmol / L, 2.5–4.0 μmol / L, 7.0–13.0 μmol / L, 5.0–10.0 μmol / L, 12.0–18.0 μmol / L, 5.0–10.0 μmol / L, 25.0–35.0 μmol / L, and 22.0–28.0 μmol / L, respectively. μmol / L, 12.5~17.5 μmol / L, 12.5~17.5 μmol / L, 25.0~35.0 μmol / L, 4.0~8.0 μmol / L, 7.5~12.5 μmol / L, 40.0~50.0 μmol / L, 3.0~5.5 mol / L.

[0016] In one embodiment, the reaction system for catalytic preparation of succinic acid comprises: 100 mmol / L Tris-HCl buffer (pH 8.0), 50 μmol / L pyridoxal phosphate, 5 mmol / L magnesium chloride, 20 mmol / L formaldehyde, 20 mmol / L glyoxylic acid, 10 mmol / L aspartic acid, 100 mmol / L sodium bicarbonate, 5 mmol / L NADH, 5 mmol / L ATP, 9 mmol / L LCoA, 7.5 μmol / L malic acid oxidase, 5.0 μmol / L aspartate-glyoxylate transaminase, 3.2 μmol / L serine aldolase, 10.0 μmol / L serine deaminase, 15.0 μmol / L malate thiokinase, and 7.5 μmol / L mayl-CoA lyase, and the reaction is carried out at 30 °C for at least 1 h. In one embodiment, the reaction system for catalytic synthesis of fumaric acid comprises: 100 mmol / L Tris-HCl buffer (pH 8.0), 5 mmol / L magnesium chloride, 100 mmol / L sodium bicarbonate, 5 mmol / L acetyl-CoA, 15 mmol / L ATP, 6 mmol / L NADPH, 15 mmol / L FAD, 30.0 μmol / L acetyl-CoA carboxylase, 25.5 μmol / L malonyl-CoA reductase / 3-hydroxypropionic acid dehydrogenase, 15.0 μmol / L propionyl-CoA synthase, 15.0 μmol / L propionyl-CoA carboxylase, 30.0 μmol / L methylmalonyl-CoA isomerase, 6.0 μmol / L methylmalonyl-CoA mutase, 10.0 μmol / L succinylated CoA synthase, 45.0 μmol / L succinate dehydrogenase, and 4.6 μmol / L... Fumarate hydratase, reacted at 30°C for 2 h.

[0017] In one embodiment, the reaction system also contains Mg. 2+ NADH, ATP, NADPH, pyridoxal phosphate, glyoxylic acid, formaldehyde, and carbon dioxide.

[0018] In one embodiment, succinate dehydrogenase and FAD are added to the succinate synthesis reaction, and the reaction is carried out at 30°C for at least 2 hours to synthesize fumaric acid.

[0019] In one embodiment, succinate dehydrogenase, fumarate hydratase, and FAD are added to the succinate synthesis reaction, and the reaction is carried out at 30°C for at least 10 h to synthesize malic acid.

[0020] In some embodiments, the reaction system for synthesizing fumarate is based on the succinate reaction system, and also contains 45.0 μmol / L succinate dehydrogenase and 15 mmol / L FAD.

[0021] In some embodiments, the reaction system for synthesizing malic acid is based on the succinic acid reaction system, and also includes the addition of 15 mmol / L FAD.

[0022] Beneficial effects: (1) The present invention screens a composition of enzymes that can synthesize fumaric acid, succinic acid or malic acid in one step, which can be used as a catalyst to achieve the catalytic preparation of four-carbon organic acids using one-carbon compounds as substrates.

[0023] (2) This invention designs a metabolic pathway (SFMSP pathway) for the biosynthesis of fumaric acid and succinic acid using one-carbon compounds as substrates. Through expression and purification of the pathway enzymes, the SFMSP pathway was successfully constructed in vitro using a one-pot method, achieving cell-free synthesis of fumaric acid and succinic acid. A reaction time of 10 h yields succinic acid up to 1.6 g / L, fumaric acid up to 1.3 g / L, or malic acid up to 1.5 g / L. This method successfully achieves de novo synthesis of fumaric acid and succinic acid using CO2 and formaldehyde as substrates. The method provided by this invention demonstrates great potential in terms of environmental protection, raw material diversification, sustainable development, and economic benefits. Attached Figure Description

[0024] Figure 1 Design of the SFMSP path.

[0025] Figure 2 For the screening and identification of SFMSP pathway enzymes; A, the results of SDS-PAGE electrophoresis of purified pathway enzymes; B, the evaluation of the catalytic activity of SFMSP pathway enzymes.

[0026] Figure 3 Modular verification of the SFMSP path.

[0027] Figure 4 For the in vitro construction and validation of the SFMSP pathway; where A, a schematic diagram of the one-pot validation of the SFMSP pathway; B, the yields of succinic acid, fumaric acid and malic acid after 10 h of reaction. Detailed Implementation

[0028] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0029] Test method: For the detection of glycine aldolase activity catalyzing the conversion of glycine and formaldehyde to pyruvate, the change in absorbance at 414 nm was measured using the acetylacetone method. The reaction system contained 100 mmol / L Tris-HCl buffer (pH 8.0), 50 μmol / L pyridoxal phosphate (PLP), 20 mM glycine, 2 mM formaldehyde, and 0.5 μmol / L glycine aldolase. For all components added to the buffer, the absorbance at 414 nm was monitored simultaneously with the reaction at 30°C. The absorbance values ​​were used to calculate the formaldehyde concentration using a standard curve. The amount of enzyme required to consume 1 μM formaldehyde in 1 min at 30°C was defined as 1 U.

[0030] For the assay of malicase activity, the change in NADH at 340 nm was detected by reacting with an excess of malate dehydrogenase. The reaction system consisted of (200 μL, 30℃): 100 mmol / L Tris-HCl buffer (pH 8.0), 10 mmol / L sodium pyruvate, 100 mmol / L sodium bicarbonate, 1 mmol / L NADH, 10 mmol / L potassium chloride, 10 mmol / L magnesium chloride, 5 mmol / L ATP, 10.0 μmol / L malate dehydrogenase, and 1.0 μmol / L malicase. 1 U is defined as the amount of enzyme required to consume 1 nM NADH per minute at 30℃.

[0031] For the detection of malate thiokinase activity, the glyoxylate-phenylhydrazone generated from the reaction of glyoxylate and phenylhydrazine at an absorbance of 324 nm was measured. A 200 μL reaction mixture was prepared, comprising (by final concentration): 100 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, 0.5 μmol / L malate thiokinase, and 2.0 μmol / L mayl-CoA lyase. The amount of enzyme required to generate 1 nM glyoxylate after a 1 min reaction at 30°C was defined as 1 U.

[0032] For the activity assay of malic yl-CoA lyase, the reaction system is the same as that for malate thiokinase, the difference being the enzyme concentration. Specifically, a final concentration of 2.0 μmol / L malate thiokinase and 0.5 μmol / L malic yl-CoA lyase are used. The amount of enzyme required to generate 1 nM glyoxylic acid after reacting at 30℃ for 1 min is defined as 1 U.

[0033] For the assay of acetyl-CoA carboxylase / propionyl-CoA carboxylase activity, enzyme activity was determined by measuring the change in NADH at 340 nm after coupling pyruvate kinase (from Sigma-Aldrich) and lactate dehydrogenase (from Sigma-Aldrich). The reaction system contained 50 mM Tris-HCl (pH 8.0), 100 mM KHCO3, 5 mM MgCl2, 5 mM ATP, 1 mM phosphoenolpyruvate, 1 mM NADH, 2 mM acetyl-CoA / propionyl-CoA, 4 U / mL Pk, 4.6 U / mL LDH, 0.5 μmol / L DtsR / PccB, and 0.5 μmol / L AccBC. 1 U was defined as the amount of enzyme required to generate 1 nM NADH per minute at 30°C.

[0034] The SFMSP reaction system (1 mL): Prepare a 1 mL succinate reaction system containing (by final concentration) 100 mmol / L Tris-HCl buffer (pH 8.0), 5 mmol / L magnesium chloride, 50 μmol / L pyridoxal phosphate, 15 mmol / L ATP, 9 mmol / L CoA, 15 mmol / L NADPH, 15 mmol / L NADH, 100 mmol / L sodium bicarbonate, 50 mmol / L formaldehyde, 20 mmol / L glyoxylic acid, 30 mmol / L aspartate, 5.0 μmol / L aspartate-glyoxylate transaminase, 3.2 μmol / L serine aldolase, 10.0 μmol / L serine deaminase, 7.5 μmol / L malate thiokinase, 15.0 μmol / L malate thiokinase, 7.5 μmol / L mayl-CoA lyase, 30.0 μmol / L succinate ... μmol / L acetyl-CoA carboxylase, 25.5 μmol / L malonyl-CoA reductase / 3-hydroxypropionate dehydrogenase, 15.0 μmol / L propionyl-CoA synthase, 15.0 μmol / L propionyl-CoA carboxylase, 30.0 μmol / L methylmalonyl-CoA isomerase, 6.0 μmol / L methylmalonyl-CoA mutase, and 10.0 μmol / L succinyl-CoA synthase.

[0035] Prepare a 1 mL fumarate reaction system by adding 15 mmol / L FAD and 45.0 μmol / L succinate dehydrogenase to the succinate reaction system.

[0036] A 1 mL reaction system for malic acid reaction was prepared. To the succinic acid reaction system, 15 mmol / L FAD, 45.0 μmol / L succinate dehydrogenase, and 4.6 μmol / L fumarate hydratase were added. The reaction was terminated after 10 h at 30 °C. The supernatant was collected by centrifugation and analyzed by HPLC and LC-MS to detect the synthesis of succinic acid, fumaric acid, and malic acid.

[0037] Analytical Methods: The concentrations of succinic acid, fumaric acid, and malic acid were 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), with 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 succinic acid, fumaric acid, and malic acid in the samples were calculated using standard curves for these three acids.

[0038] The formation of succinic acid, fumaric acid, and malic acid was verified using an LC-MS system (QTRAP 5500 LC-MS System). The system was equipped with a Waters HSS T3 column (1.8 μm, 2.1 × 100 mm), a flow rate of 0.3 mL / min, mobile phase A (0.1% formic acid-water solution) and mobile phase B (100% acetonitrile), and a temperature of 40 °C. The injection volume was 2 μL. The mobile phase elution gradient was eluted according to the following steps and program: 0–2 min: maintain 95% A and 5% B; 2–4 min: A decreases from 95% to 5% and B increases from 5% to 95%; 4–6 min: maintain 5% A and 95% B; 6–6.1 min: A increases from 5% to 95% and B decreases from 95% to 5%.

[0039] The molar conversion rate is calculated as follows: for the first reaction module, the molar conversion rate is calculated by dividing the number of moles of acetyl-CoA product by the number of moles of formaldehyde substrate; for the second reaction module, the molar conversion rate is calculated by dividing the number of moles of malic acid product by the number of moles of acetyl-CoA substrate.

[0040] Raw materials used in the examples: 1. Luria-Bertani (LB) medium: tryptone 10 g / L (Oxoid), yeast extract 5 g / L (Oxoid), NaCl 10 g / L (China National Pharmaceutical Group). LB medium is used for strain activation. When the cells contain recombinant plasmids, the final concentration of the corresponding antibiotic added to the medium is: kanamycin 50 mg / L.

[0041] 2. Terrific broth (TB) medium: Glycerol 4 g / L (China National Pharmaceutical Group), yeast extract 24 g / L (Angel Pharmaceutical), tryptone 12 g / L (China National Pharmaceutical Group), potassium dihydrogen phosphate 2.31 g / L (China National Pharmaceutical Group), dipotassium hydrogen phosphate trihydrate 16.42 g / L (China National Pharmaceutical Group). TB medium is used for the 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 Biotech) and antibiotics (Sangon Biotech) added to the medium are: IPTG 100 mg / L and kanamycin 50 mg / L, respectively.

[0042] 3. Both pET28a plasmid and Escherichia coli BL21(DE3) are commercially available plasmids and strains.

[0043] 4. Tris, hydrochloric acid, magnesium chloride, sodium chloride, and sodium bicarbonate are sourced from China National Pharmaceutical Group; pyridoxal phosphate, sodium pyruvate, formaldehyde, sodium malate, sodium fumarate, sodium succinate, sodium malate, NADPH, NADH, and ATP are sourced from Aladdin; CoA, acetyl-CoA, and propionyl-CoA are sourced from Maclean's.

[0044] Example 1: Design and evaluation of SFMSP pathway for the biosynthesis of succinic acid, fumaric acid and malic acid To achieve the biosynthesis of succinic acid, fumaric acid, and malic acid using CO2 as a substrate, the following CO2 fixation pathway (SFMSP pathway) with two metabolic modules was designed: (1) a first reaction module that converts CO2 and formaldehyde into acetyl-CoA; and (2) a second reaction module that converts CO2 and acetyl-CoA into succinic acid, fumaric acid, and malic acid (Figure 1). The SFMSP pathway includes a total of 15 enzymes, namely aspartate-glyoxylate transaminase, serine aldolase, serine deaminase, malate thiokinase, malate-CoA lyase, acetyl-CoA carboxylase, malonyl-CoA reductase / 3-hydroxypropionic acid dehydrogenase, propionyl-CoA synthase, propionyl-CoA carboxylase, methylmalonyl-CoA isomerase, methylmalonyl-CoA mutase, succinyl-CoA synthase, succinate dehydrogenase, and fumarate hydratase.

[0045] Example 2: Path enzyme screening of the SFMSP pathway (a) Enzyme screening in the first reaction module: selecting enzymes from... Paracoccus denitrificans Aspartate-glyoxylate transaminase (PdAGT, Genbank accession number: ABL71987.1, the nucleotide sequence of the gene is shown in SEQ ID NO.1), from Pseudomonas aeruginosa , Escherichia coli The nucleotide sequences of the serine aldolases (PaSAL, EcSAL, and PaLAT) encoding genes are shown in SEQ ID NO.2, and the nucleotide sequences of the EcLTA encoding gene are shown in SEQ ID NO.19. Selected from... Cupriavidus necator The serine deaminase (CnSDA, Genbank accession number: CAJ94679.1, nucleotide sequence of the gene is shown in SEQ ID NO.3). Screening was conducted for serine deaminases derived from... Clostridium acetobutylicum The malicase (CcME, Genbank accession number: AAK79563.1, nucleotide sequence as shown in SEQ ID NO.4). Selected from Methylobacterium extorquensThe malate thiokinase (MeMTK, GenBank accession number: ACS39574.1) contains α and β subunits. The nucleotide sequences of the genes encoding the MeMTK α and β subunits are shown in SEQ ID NO. 5 and SEQ ID NO. 6, respectively. The MeMTK gene was inserted sequentially, α and β subunits, between the BmH I and Xho I restriction sites of plasmid pET28a. [The text then abruptly shifts to a different topic:] Selected from... Methylobacterium extorquens The malic acyl-CoA lyase (MeMCL, Genbank accession number: ACS39577.1), with its nucleotide sequence shown in SEQ ID NO.7, was used. The gene encoding MeMCL was inserted between the BmH I and Xho I restriction sites of plasmid pET28a. Recombinant bacteria expressing the other enzymes mentioned above were constructed using the same strategy; that is, gene sequences encoding the enzymes were synthesized and inserted between the BmH I and Xho I restriction sites of plasmid pET28a to construct recombinant plasmids.

[0046] The recombinant plasmids constructed above were transformed into competent Escherichia coli BL21(DE3) cells. The transformants were screened on solid plates containing kanamycin-resistant LB medium and their genes were sequenced to obtain the correct strains containing the target expression vector. The strains were then stored in glycerol for later use.

[0047] (II) Enzyme screening in the second reaction module: selecting enzymes from... Corynebacterium glutamicum Acetyl-CoA carboxylase (CgACC) contains α and β subunits. The nucleotide sequences of the genes encoding the CgACC α and β subunits are shown in SEQ ID NO. 8 and SEQ ID NO. 9, respectively. [The text then abruptly shifts to a seemingly unrelated topic:] Selected from... Chloroflexus aurantiacus Malonyl-CoA reductase / 3-hydroxypropionate dehydrogenase CaMCR (Genbank accession number: NWF81339.1, nucleotide sequence as shown in SEQ ID NO.10). Selected from Chloroflexus aurantiacus The propionyl-CoA synthase CaPCS (Genbank accession number: MBO9316738.1, nucleotide sequence as shown in SEQ ID NO.11). Selected from... Methylobacterium extorquens The propionyl-CoA carboxylase (MePCC) contains α and β subunits. The nucleotide sequences of the genes encoding the MePCC α and β subunits are shown in SEQ ID NO.12 and SEQ ID NO.13, respectively. [The text then abruptly shifts to a seemingly unrelated topic:] Selected from... Rhodobacter Sphaeroides Methylmalonyl-CoA isomerase (RsEPI, nucleotide sequence as shown in SEQ ID NO. 14). Selected from Rhodobacter SphaeroidesMethylmalonyl-CoA mutase (RsMCM, nucleotide sequence as shown in SEQ ID NO.15). Selected from Escherichia coli The succinyl-CoA synthase (EcSUC), the nucleotide sequence encoding EcSUC is shown in SEQ ID NO.16. Selected from... Corynebacterium glutamicum The succinate dehydrogenase (CgSDH) is encoded by the nucleotide sequence shown in SEQ ID NO.17. The enzyme selected from... Escherichia coli Fumarate hydratase (EcFUM, Genbank accession number: VCV69885.1, nucleotide sequence as shown in SEQ ID NO.18) was synthesized. The encoding genes of the above enzymes were synthesized separately, and the gene fragments were inserted between the BmH I and Xho I restriction sites of plasmid pET28a to construct recombinant plasmids expressing each enzyme.

[0048] The recombinant plasmids were transformed into competent Escherichia coli BL21(DE3) cells. Transformants were screened on solid plates containing kanamycin and their genes were sequenced to obtain the correct strains containing the target expression vector. The strains were then stored in glycerol for later use.

[0049] Example 3: Preparation of SFMSP pathway enzymes The recombinant bacteria expressing each enzyme constructed in Example 2 were cultured according to the following steps: 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 was 0.6–0.8, IPTG was added to a final concentration of 100 mg / L. After induction at 16°C for 16 hours, the bacterial cells were collected, the cells were lysed, and the supernatant containing the enzyme protein was collected. After purification, the specific enzyme activity of each enzyme was measured. Specific enzyme activity is defined as the amount of enzyme required to produce 1 nM of product per minute at 30°C, which is 1 U.

[0050] The pure enzyme was tested in vitro using the following steps: The pure enzyme was added to the corresponding reaction system to initiate the reaction. After 1 minute of reaction, formic acid was added to a final concentration of 3% to terminate the reaction. A reaction sample was taken to test the amount of substrate consumed or product generated, and the enzyme activity was calculated.

[0051] like Figure 2 As shown in B, it comes from Pseudomonas aeruginosa The catalytic efficiency of the serine aldolase PaSAL is higher than that of the enzyme derived from... Escherichia coli The EcSAL enzyme activity was increased by 1.2 times.

[0052] Based on the above tests, the following enzymes were tested: aspartate-glyoxylate transaminase (PdAGT), serine aldolase (PaSAL), serine deaminase (CnSDA), malate enzyme (CcME), malate thiokinase (MeMTK), mayl-CoA lyase (MeMCL), acetyl-CoA carboxylase (CgACC), malonyl-CoA reductase / 3-hydroxypropionic acid dehydrogenase (CaMCR), propionyl-CoA synthase (CaPCS), propionyl-CoA carboxylase (MePCC), and methylmalonyl-CoA isomerase (RsEPI). Methylmalonyl-CoA mutase RsMCM, succinyl-CoA synthase EcSUC, succinate dehydrogenase CgSDH, and fumarate hydratase EcFUM were used to construct the SFMSP pathway. The specific enzyme activities of PdAGT, PaSAL, CnSDA, CcME, MeMTK, MeMCL, CgACC, CaMCR, CaPCS, MePCC, RsEPI, RsMCM, EcSUC, CgSDH, and EcFUM were 5.8 U / mg, 5.5 U / mg, 10.0 U / mg, 1.1 U / mg, 0.6 U / mg, 14.9 U / mg, 16.9 U / mg, 1.3 U / mg, 0.8 U / mg, 5.4 U / mg, 130.6 U / mg, 6.9 U / mg, 2.8 U / mg, 40.7 U / mg, and 430.5 U / mg, respectively.

[0053] Example 4: Modular Verification of SFMSP Path Based on the screening results of the pathway enzymes in Example 2, the six modules of the SFMSP pathway were modularly validated in vitro, such as... Figure 3 As shown. The enzymes required for the reaction were prepared according to the method in Example 3.

[0054] The first reaction module converts CO2 and formaldehyde into acetyl-CoA. The reaction system contains (by final concentration): 100 mmol / L Tris-HCl buffer (pH 8.0), 50 μmol / L pyridoxal phosphate, 5 mmol / L magnesium chloride, 20 mmol / L formaldehyde, 20 mmol / L glyoxylic acid, 10 mmol / L aspartic acid, 100 mmol / L sodium bicarbonate, 5 mmol / L NADH, 5 mmol / L ATP, 9 mmol / L CoA, 7.5 μmol / L malate enzyme, 5.0 μmol / L aspartate-glyoxylate transaminase, 3.2 μmol / L serine aldolase, 10.0 μmol / L serine deaminase, 15.0 μmol / L malate thiokinase, and 7.5 μmol / L mayl-CoA lyase. The reaction is carried out at 30 °C for 1 h; the supernatant is collected by centrifugation, and the product acetyl-CoA is detected.

[0055] In the second reaction module, acetyl-CoA and CO2 are converted into malic acid. The reaction system contained (at final concentrations): 100 mmol / L Tris-HCl buffer (pH 8.0), 5 mmol / L magnesium chloride, 100 mmol / L sodium bicarbonate, 5 mmol / L acetyl-CoA, 15 mmol / L ATP, 6 mmol / L NADPH, 15 mmol / L FAD, 30.0 μmol / L acetyl-CoA carboxylase, 25.5 μmol / L malonyl-CoA reductase / 3-hydroxypropionate dehydrogenase, 15.0 μmol / L propionyl-CoA synthase, 15.0 μmol / L propionyl-CoA carboxylase, 30.0 μmol / L methylmalonyl-CoA isomerase, 6.0 μmol / L methylmalonyl-CoA mutase, 10.0 μmol / L succinylated CoA synthase, 45.0 μmol / L succinate dehydrogenase, 4.6 The reaction was carried out with μmol / L fumarate hydratase at 30°C for 2 h. The supernatant was collected by centrifugation, and the malic acid content of the product was determined.

[0056] The results are as follows Figure 3 As shown, module one generates 3.1 mmol / L of acetyl-CoA, with a conversion rate of 15.4%; module two generates 0.29 mmol / L of malic acid, with a conversion rate of 5.8%.

[0057] Example 5: In vitro construction and validation of the SFMSP pathway Based on the modular verification of the SFMSP path in Example 4, the two modules are assembled to verify the feasibility of the SFMSP path, such as... Figure 4 As shown in A. The SFMSP pathway was performed using a one-pot method. After the reaction was completed, the supernatant was collected by centrifugation and analyzed by HPLC and LC-MS to detect the synthesis of succinic acid, fumaric acid, and malic acid.

[0058] Prepare a 1 mL reaction system for the succinate reaction, comprising (by final concentration) 100 mmol / L Tris-HCl buffer (pH 8.0), 5 mmol / L magnesium chloride, 50 μmol / L pyridoxal phosphate, 15 mmol / L ATP, 9 mmol / L CoA, 15 mmol / L NADPH, 15 mmol / L NADH, 100 mmol / L sodium bicarbonate, 50 mmol / L formaldehyde, 20 mmol / L glyoxylic acid, 30 mmol / L aspartic acid, 5.0 μmol / L aspartate-glyoxylate transaminase, 3.2 μmol / L serine aldolase, 10.0 μmol / L serine deaminase, 7.5 μmol / L malate thiokinase, 15.0 μmol / L malate thiokinase, 7.5 μmol / L mayl-CoA lyase, 30.0 μmol / L acetyl-CoA carboxylase, and 25.5 μmol / L succinate. μmol / L malonyl-CoA reductase / 3-hydroxypropionate dehydrogenase, 15.0 μmol / L propionyl-CoA synthase, 15.0 μmol / L propionyl-CoA carboxylase, 30.0 μmol / L methylmalonyl-CoA isomerase, 6.0 μmol / L methylmalonyl-CoA mutase, and 10.0 μmol / L succinyl-CoA synthase; reacted at 30℃ for 10 h, the results are as follows. Figure 4 As shown in B, 1.6 g / L of succinic acid can be produced by reacting via the SFMSP pathway for 10 h.

[0059] To prepare a 1 mL fumarate reaction system, add 15 mmol / L FAD and 45.0 μmol / L succinate dehydrogenase (based on the final concentration) to the succinate reaction system. React at 30℃ for 10 h. The results are as follows: Figure 4 As shown in B, 1.3 g / L of fumaric acid can be produced by reacting via the SFMSP pathway for 10 h.

[0060] A 1 mL reaction system for the malic acid reaction was prepared by adding 15 mmol / L LFAD, 45.0 μmol / L succinate dehydrogenase, and 4.6 μmol / L fumarate hydratase to the succinate reaction system. The reaction was carried out at 30℃ for 10 h, and the results are as follows. Figure 4 As shown in B, 1.5 g / L of malic acid can be produced by reacting via the SFMSP pathway for 10 h.

[0061] The above results demonstrate that the pathway can be successfully constructed in vitro, enabling the production of succinic acid, fumaric acid, and malic acid using CO2 and formaldehyde as substrates.

[0062] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A composition capable of catalyzing the formation of a four-carbon organic acid from a one-carbon compound, characterized in that, It contains: aspartate-glyoxylate transaminase, serine aldolase, serine deaminase, malic acid kinase, malate thiokinase, maloyl-CoA lyase, acetyl-CoA carboxylase, malonyl-CoA reductase / 3-hydroxypropionic acid dehydrogenase, propionyl-CoA synthase, propionyl-CoA carboxylase, methylmalonyl-CoA isomerase, methylmalonyl-CoA mutase, and succinyl-CoA synthase.

2. The composition according to claim 1, characterized in that, The composition also contains succinate dehydrogenase.

3. The composition according to claim 1, characterized in that, The composition also contains succinate dehydrogenase and fumarate hydratase.

4. The composition according to any one of claims 1 to 3, characterized in that, The one-carbon compounds include, but are not limited to, carbon dioxide, bicarbonates, or carbonates; the four-carbon organic acids include, but are not limited to, succinic acid, fumaric acid, and malic acid.

5. The use of the enzyme composition according to any one of claims 1 to 4 in catalyzing the formation of four-carbon organic acids from one-carbon compounds.

6. A method for preparing a four-carbon organic acid, characterized in that, Using a one-carbon compound as a substrate and the enzyme composition according to any one of claims 1 to 3 as a catalyst, a four-carbon organic acid is prepared; the four-carbon organic acid includes, but is not limited to, succinic acid, fumaric acid and malic acid.

7. The method according to claim 6, characterized in that, The addition amounts of aspartate-glyoxylate transaminase, serine aldolase, serine deaminase, malic acid kinase, malate thiokinase, mayl-CoA lyase, acetyl-CoA carboxylase, malonyl-CoA reductase / 3-hydroxypropionic acid dehydrogenase, propionyl-CoA synthase, propionyl-CoA carboxylase, methylmalonyl-CoA isomerase, methylmalonyl-CoA mutase, succinyl-CoA synthase, succinate dehydrogenase, and fumarate hydratase are 3.0–7.0 μmol / L, 2.5–4.0 μmol / L, 7.0–13.0 μmol / L, 5.0–10.0 μmol / L, 12.0–18.0 μmol / L, 5.0–10.0 μmol / L, 25.0–35.0 μmol / L, 22.0–28.0 μmol / L, and 12.5–17.5 μmol / L, respectively. μmol / L, 12.5~17.5 μmol / L, 25.0~35.0 μmol / L, 4.0~8.0 μmol / L, 7.5~12.5 μmol / L, 40.0~50.0 μmol / L, 3.0-5.5 mol / L.

8. The method according to claim 7, characterized in that, The reaction system also contains Mg 2+ NADH, ATP, NADPH, pyridoxal phosphate, glyoxylic acid, formaldehyde, and carbon dioxide.

9. The method according to claim 8, characterized in that, Based on the succinate synthesis reaction, succinate dehydrogenase and FAD are added, and the reaction is carried out at 30°C for at least 2 hours to synthesize fumaric acid; or Based on the succinic acid synthesis reaction, succinate dehydrogenase, fumarate hydratase and FAD are added, and the reaction is carried out at 30°C for at least 10 h to synthesize malic acid.

10. The use of the method according to any one of claims 6 to 9 in the preparation of products containing succinic acid, fumaric acid and / or malic acid.

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