Method for synthesizing malonyl coa and use thereof

Abstract

The application belongs to the technical field of biology, and particularly discloses a method for synthesizing malonyl-CoA and application thereof. The method for synthesizing malonyl-CoA comprises the following steps: S1, converting an exogenous carbon source substrate into 3-hydroxypropionic acid; and S2, converting the 3-hydroxypropionic acid obtained in S1 into malonyl-CoA. The application discloses a method for synthesizing malonyl-CoA and application thereof, and the method realizes efficient, stable and controllable production of malonyl-CoA and derivatives thereof, completely releases natural feedback inhibition, and realizes stable and efficient synthesis.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for synthesizing malonyl-CoA and its application. Background Technology

[0002] Malonyl-CoA is a core precursor for the synthesis of fatty acids, polyketides, and many high-value natural products. Its traditional biosynthesis relies on the endogenous PDH-ACC pathway composed of pyruvate dehydrogenase (PDH) and acetyl-CoA carboxylase (ACC). This pathway has the following fundamental defects: (1) about one-third of the carbon is released as CO2, resulting in carbon loss; (2) the synthesis of one molecule of malonyl-CoA requires a net consumption of one molecule of ATP; (3) PDH and ACC are both key nodes in the cell's central metabolism. Overexpression or perturbation of them can easily cause cellular stress such as growth inhibition, redox imbalance, and metabolic toxicity, which seriously restricts the efficient synthesis of the target product.

[0003] To circumvent the catalytic bottleneck of the PDH-ACC pathway, existing research has developed several non-carboxylation pathways, such as the C3→C3→C3 route via "pyruvate → 3-oxopropionic acid → malonyl-CoA". However, these pathways still mainly rely on glycolytic precursors, which not only easily interfere with the cellular endogenous metabolic network, but also make it difficult to utilize non-sugar carbon sources such as CO2, methanol, and plastic hydrolysates, thus limiting their application potential in carbon recycling and high-value conversion of waste. Summary of the Invention

[0004] The present invention aims to provide a method for synthesizing malonyl-CoA and its application. This method enables efficient, stable and controllable production of malonyl-CoA and its derivatives, completely eliminates natural feedback inhibition, and achieves stable and efficient synthesis.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: A method for synthesizing malonyl-CoA includes the following steps: S1. Convert exogenous carbon source substrates into 3-hydroxypropionic acid (3-HP). S2. Convert the 3-hydroxypropionic acid obtained in S1 into malonyl-CoA.

[0006] Preferably, in S1, the exogenous carbon source substrate is a C1-C6 compound selected from at least one of CO2, formic acid, methanol, formaldehyde, ethanol, acetic acid, ethylene glycol, glycerol, 1,3-propanediol, xylose, glucose, PET hydrolysate, cellulose hydrolysate, hemicellulose hydrolysate, or chitin hydrolysate.

[0007] Preferably, in S2, the conversion of 3-hydroxypropionic acid obtained in S1 to malonyl-CoA is achieved through any of the following enzymatic catalytic pathways: Pathway 1: Under the catalysis of 3-hydroxypropionic acid dehydrogenase, 3-hydroxypropionic acid is oxidized to 3-oxopropionic acid; under the catalysis of oxidoreductase, 3-oxopropionic acid is converted to malonyl-CoA.

[0008] Pathway 2: Under the catalysis of malonyl-CoA reductase, 3-hydroxypropionic acid is converted into malonyl-CoA.

[0009] Preferably, the 3-hydroxypropionic acid dehydrogenase is selected from Bacillus masiliae (Ureaplasma masiliae). Ureibacillus massiliensis PydE from Escherichia coli ( E. coli YdfG from *Codonopsis lanceolata* ( Metallosphaera cuprina AEB94401, from *Flexobacterium orangeense* ( Chloroflexus aurantiacus CaMCR-N from Tokyo Institute of Technology and sulfur-reducing cocci ( Sulfurisphaera tokodaii One or more of FadB; The oxidoreductase is derived from *Flexobacterium orange* (… Chloroflexus aurantiacus CaMCR-C, from sulfur-reducing cocci (from Tokyo Institute of Technology) Sulfurisphaera tokodaii StMCR, from *Streptococcus lucida* ( Metallosphaera sedula Msed_0709, from *Corynebacterium neutrophils* ( Pyrobaculum neutrophilum PnSCR and from Clostridium coccidioides ( Clostridium kluyveri SucD; The malonyl-CoA reductase is selected from *Sludge Reservoir Bacterium* (Sludge Reservoir Bacterium sludge). Piscinibacter caeni PcMCR from Rhodococcus carinii ( Roseiflexus castenholzii RcMCR from Acidobacterium thermophilum ( Chloracidobacterium thermophilum CtMCR from an unnamed green sulfur bacterium ( Candidatus thermochlorobacteriaceae CtbMCR from γ-proteobacteria ( Gamma proteobacterium GpMCR from ) and from orange green flexural bacteria ( Chloroflexus aurantiacus One or more of CaMCRs.

[0010] Preferably, the amino acid sequence of PydE is shown in SEQ ID NO.1; the amino acid sequence of YdfG is shown in SEQ ID NO.2; the amino acid sequence of AEB94401 is shown in SEQ ID NO.3; the amino acid sequence of CaMCR-N is shown in SEQ ID NO.4; and the amino acid sequence of FadB is shown in SEQ ID NO.5.

[0011] Preferably, the nucleotide sequence of CaMCR-C is shown in SEQ ID NO.6; the amino acid sequence of StMCR is shown in SEQ ID NO.7; the amino acid sequence of Msed_0709 is shown in SEQ ID NO.8; the amino acid sequence of PnSCR is shown in SEQ ID NO.9; and the amino acid sequence of SucD is shown in SEQ ID NO.10.

[0012] Preferably, the amino acid sequence of the PcMCR is shown in SEQ ID NO.11; the amino acid sequence of the RcMCR is shown in SEQ ID NO.12; the amino acid sequence of the CtMCR is shown in SEQ ID NO.13; the amino acid sequence of the CtbMCR is shown in SEQ ID NO.14; the amino acid sequence of the GpMCR is shown in SEQ ID NO.15; and the amino acid sequence of the CaMCR is shown in SEQ ID NO.16.

[0013] The present invention also provides a recombinant plasmid for synthesizing malonyl-CoA, comprising a gene encoding the 3-hydroxypropionate dehydrogenase and oxidoreductase or a gene encoding the malonyl-CoA reductase.

[0014] Preferably, the recombinant plasmid is pCDF- pydE-CaMCR-C (For use in Escherichia coli), wherein the amino acid sequence of PydE is shown in SEQ ID NO.1, and the nucleotide sequence of CaMCR-C is shown in SEQ ID NO.6.

[0015] Preferably, the recombinant plasmid is pki2- pydE-CaMCR-C (For use in Yersinia lipolyticis), wherein the amino acid sequence of PydE is shown in SEQ ID NO.1. CaMCR-C The amino acid sequence is shown in SEQ ID NO.17.

[0016] The present invention also provides a strain for synthesizing malonyl-CoA, the strain comprising the recombinant plasmid described above.

[0017] The present invention also provides a method for synthesizing malonyl-CoA derivatives using the recombinant plasmid, comprising the following steps: The recombinant plasmid was transferred into competent Escherichia coli cells to obtain a positive clone strain, which was then fermented to obtain a malonyl-CoA derivative.

[0018] Preferably, the malonyl-CoA derivative is a fatty acid or a polyketide compound.

[0019] Preferably, the preparation method of the polyketide (phloroglucinol) includes the following steps: mixing the recombinant plasmid pCDF- pydE-CaMCR-C The polyketide synthase expression plasmid was transformed into competent Escherichia coli cells to obtain positive clone strains, which were then fermented to obtain phloroglucinol.

[0020] The present invention also provides the application of malonyl-CoA synthesized by the method or a malonyl-CoA derivative synthesized by the method in fatty acid synthesis.

[0021] Compared with the prior art, the present invention has the following advantages and technical effects: This paper discloses a method for synthesizing malonyl-CoA and its applications. The constructed novel artificial pathway (UMCP) overcomes the multiple feedback inhibitions of the natural PDH-ACC pathway, is insensitive to key metabolites such as ATP and acetyl-CoA, and achieves stable and efficient synthesis. Furthermore, this pathway exhibits broad substrate adaptability, efficiently converting various exogenous carbon sources into malonyl-CoA. The pathway demonstrates good scalability and has been successfully coupled with downstream modules to increase fatty acid yield and synthesize polyketide compounds. It can directly utilize waste carbon sources such as CO2 electrolysis products, PET, and biomass hydrolysate to produce high-value chemicals such as phloroglucinol, providing a novel and highly promising solution for waste resource utilization and green biomanufacturing.

[0022] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0023] Figure 1 This is a process flow diagram for synthesizing malonyl-CoA according to the present invention; Figure 2 This is a graph showing the change in absorbance of NADPH at 340 nm in Example 2, where malonyl-CoA reductases from different sources catalyze the synthesis of malonyl-CoA and NADPH from 3-HP. Figure 3 The graph shows the changes in absorbance of NADPH at 340 nm in Example 2, where 3-HP dehydrogenases from different sources catalyze the synthesis of 3-oxopropionic acid and NADPH from 3-HP. Figure 4 This is a schematic diagram of the reaction for generating malonyl-CoA from different exogenous carbon substrates in Example 3; Figure 5 Example 4 describes the conversion of various waste materials into phloroglucinol and aflatoxin, wherein... Figure 5 In this context, A represents the yield of phloroglucinol. Figure 5 In this context, B represents the yield of aflatoxin; Figure 6 This is a schematic diagram of the catalytic rate in Example 5 with the addition of a natural pathway inhibitor, wherein, Figure 6 In the diagram, A represents the catalytic rate after the addition of ATP, a cellular energy source. Figure 6 In the diagram, B represents the catalytic rate after the addition of acetyl-CoA. Figure 6 C in the diagram represents the catalytic rate after the addition of palmitoyl-CoA; Figure 7 This is a schematic diagram illustrating the addition of malonyl-CoA synthesized via a natural pathway for cell growth in Example 6, wherein... Figure 7 In this context, A represents CRISPRI inhibition. accBC Schematic diagram of gene expression. Figure 7 B in the text represents inhibition. accBC This leads to cell growth arrest, but cell growth is restored after the introduction of the UMCP pathway. Figure 8 This is a diagram showing the results of Example 7 in the synthesis of caprylic acid from Yersinia lipophila; Figure 9 This is a diagram showing the results of the synthesis of phloroglucinol in Escherichia coli in Example 8. Detailed Implementation

[0024] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0025] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0026] Source of experimental materials: His-tag Protein Purification Kit: Beyotime (His-tag Protein Purification Kit), commonly known as the nickel column kit.

[0027] Construction of candidate enzyme expression plasmid: plasmid pCDF-P1- YdfG pCDF-P1- AEB94401 pCDF-P1- pydE pCDF-P1- CaMCR-C pCDF-P1- PcMCR pCDF-P1- RcMCR pCDF-P1- CtMCR pCDF-P1- CtbMCR pCDF-P1- GpMCR pCDF-P1- CaMCR pCDF-P1- CaMCR-N pCDF-P1- FadB The construction was completed by Genewiz, based on the corresponding amino acids.

[0028] The strains used in this invention are as follows: E. coli BL21(DE3) and E. coliMG1655 (DE3) was purchased from Tolo Biotech. In this invention, unless otherwise specified, all other test materials and instruments are conventional test materials in the field and can be purchased through commercial channels.

[0029] The process flow diagram for synthesizing malonyl-CoA is as follows: Figure 1 As shown.

[0030] Example 1 Purification of candidate enzymes: The purification method for CaMCR-C (nucleotide sequence as shown in SEQ ID NO. 6, amino acid sequence as shown in SEQ ID NO. 17) is as follows: (1) Take plasmid pCDF-P1- CaMCR-C 50 ng, add 50 μL E. coli In BL21(DE3) competent cells, incubate on ice for 30 min. Heat shock at 42°C for 60 s, then immediately place on ice for 2 min. Add 500 μL of SOC medium and incubate at 37°C for 1 h at 220 rpm. Centrifuge to remove the supernatant, leaving approximately 50 μL. Mix thoroughly and spread the entire volume onto LB agar plates containing spectinomycin (final concentration 50 μg / mL). After overnight incubation, positive monoclonal colonies are obtained.

[0031] (2) Take 5 positive monoclonal colonies and inoculate them into 5 mL of liquid LB seed medium containing spectinomycin (final concentration 50 μg / mL). Incubate at 220 rpm and 37℃ for 8 h until OD550 = 2.0. Inoculate the seed culture into 50 mL of liquid LB medium (containing spectinomycin at a final concentration of 50 μg / mL) with an initial OD550 of 0.1. Incubate at 220 rpm and 37℃ until OD550 = 0.5~0.8. Add the inducer isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 0.1 mM. Continue incubation at 220 rpm and 37℃ for 4~5 h.

[0032] (3) Collect the induced bacterial cells by centrifugation at 6000g for 10 min. Next, purify the protein using a His-tagged protein purification kit. Add lysis buffer at a ratio of 4 mL of non-denaturing lysis buffer per gram of bacterial precipitate wet weight and resuspend the bacterial cells thoroughly. Add lysozyme to a final concentration of 1 mg / mL and mix well. Place on ice for 30 min. Sonicate the bacteria under ice-water cooling: sonication power 150-250W, sonication for 2 seconds each time, with a 2-second interval, for a total of 5-10 min (until the bacterial solution is translucent); centrifuge at 10000g for 20-30 min at 4℃, collect the supernatant to obtain the bacterial lysis buffer, and place on ice.

[0033] (4) Protein purification is performed using a nickel affinity column. Specifically, take 1 mL of a well-mixed 50% pre-packed column, centrifuge at 4°C (1000g × 10s), discard the stock solution, add 0.5 mL of non-denaturing lysis buffer to the gel, mix well to equilibrate the gel, centrifuge at 4°C (1000g × 10s), discard the liquid, and repeat the equilibration process 1-2 times, discarding the liquid each time. Add approximately 4 mL of the bacterial lysis buffer prepared in step 3, and gently shake at 4°C for 60 min. Pack the mixture of lysis buffer and BeyoGold™ His-tag Purification Resin into the nickel affinity chromatography column. Open the cap at the bottom of the purification column and allow the liquid inside the column to flow out under gravity. Wash the column 5 times, adding 0.5-1 mL of non-denaturing wash buffer each time, and elute the target protein 6-10 times, using 0.5 mL of non-denaturing elution buffer each time. Collect the eluent from each wash into different centrifuge tubes. The collected eluent is the purified His-tag protein sample.

[0034] (5) Add the above elution buffer to a 2 mL ultrafiltration tube (Sartorius), place the ultrafiltration tube in a centrifuge, centrifuge at 4°C and 13000 rpm until all the elution buffer is concentrated to 50 μL, then add 500 μL of TEG buffer (50 mM Tris-HCl, 0.5 mM EDTA, 50 mM NaCl, 5% glycerol, pH 7.9), centrifuge at 4°C and 13000 rpm until the solution is concentrated to 50 μL, then add another 500 μL of TEG buffer (50 mM Tris-HCl, 0.5 mM EDTA, 50 mM NaCl, 5% glycerol, pH 7.9), centrifuge at 4°C and 13000 rpm until about 50-100 μL of protein solution remains in the ultrafiltration tube, mix with a pipette and transfer to a pre-cooled EP tube.

[0035] (6) The concentration of purified CaMCR-C protein was determined using a BCA protein concentration assay kit (purchased from Beyotime Biotechnology Co., Ltd.). Based on the sample quantity, prepare an appropriate amount of BCA working solution by adding 1 volume of BCA reagent B (50:1) to 50 volumes of BCA reagent A and mixing thoroughly. Take 20 μL of 25 mg / mL protein standard and add 980 μL of diluent to prepare a 0.5 mg / mL protein standard. Dilute the 0.5 mg / mL protein standard solution to 0.05 (10→90), 0.1 (20→80), 0.2 (40→60), 0.3 (60→40), and 0.4 (80→20) mg / mL, respectively. Add 20 μL of the 0-0.5 mg / mL standard to each well of a 96-well plate (three replicates). After preliminary determination using a Nano-300, dilute the sample by a certain factor and add 20 μL to the sample wells of the 96-well plate. Add 200 μL of BCA working solution to each well and incubate at 37°C for 30 min. Measure the absorbance at 562 nm using a microplate reader. Construct a standard curve based on the absorbance and concentration of the standard protein solution at 562 nm, and calculate the concentration of the purified protein based on the standard curve.

[0036] 3-Hydroxypropionate dehydrogenase: PydE (SEQ ID NO.1), YdfG (SEQ ID NO.2), AEB94401 (SEQ ID NO.3), CaMCR-N (SEQ ID NO.4), FadB (SEQ ID NO.5); Oxidoreductase: StMCR (SEQ ID NO.7), Msed_0709 (SEQ ID NO.8), PnSCR (SEQ ID NO.9) and SucD (SEQ ID NO.10); Malonyl-CoA reductase: PcMCR (SEQ ID NO.11), RcMCR (SEQ ID NO.12), CtMCR (SEQ ID NO.13), CtbMCR (SEQ ID NO.14), GpMCR (SEQ ID NO.15), CaMCR (SEQ ID NO.16); Purification method is the same as that for CaMCR-C.

[0037] Example 2 In vitro reaction (artificial synthesis of malonyl-CoA): Malonyl-CoA reductase activity assay using 3-HP as a substrate: The in vitro reaction was carried out in a total reaction volume of 200 µL containing 100 mM Tris buffer (pH 9.5), 2 mM MgCl2, 3 mM dithiothreitol (DTT), 36 mM 3-HP, and 2.5 mM NADP. +2.5 mM coenzyme A and 1 µM purified malonyl-CoA reductase were added. The reaction mixture was incubated at 37 °C, and the increase in 340 nm (where the generated NADPH has an absorption peak) was monitored. The results are as follows: Figure 2 As shown.

[0038] Depend on Figure 2 It can be seen that only CaMCR exhibited significant catalytic activity, with a catalytic rate of 0.2 µmol / min. -1 mg -1 .

[0039] CaMCR is split into two parts: CaMCR-N is responsible for catalyzing the reaction of 3-HP to 3-oxopropionic acid, and CaMCR-C is responsible for catalyzing the reaction of 3-oxopropionic acid to malonyl-CoA.

[0040] The in vitro reaction was carried out in a total reaction volume of 200 µL, containing 100 mM Tris buffer (pH 7.8), 2 mM MgCl2, 3 mM DTT, 36 mM 3-HP, and 2.5 mM NADP. + 2.5 mM coenzyme A and 1 µM purified 3-HP dehydrogenase were added. The reaction mixture was incubated at 37 °C, and the increase in 340 nm (where the generated NADPH has an absorption peak) was monitored. The results are as follows: Figure 3 As shown.

[0041] Depend on Figure 3 It was found that four enzymes exhibited activity, namely PydE (1.26 µmol min). -1 mg -1 YdfG (0.624µmol min) -1 mg -1 ), CaMCR-N (0.229µmol min) -1 mg -1 ) and AEB94401 (0.02µmol min) -1 mg -1 ).

[0042] Example 3 Systematic validation of the in vitro transformation feasibility of 20 C1-C6 substrates (e.g.) Figure 4 As shown in the figure, all 20 recombinant proteins from the C1-C6 substrate in vitro pathway were constructed using pCDF as a vector and named pCDF-XX after the gene. The strain construction and protein purification methods were the same as above.

[0043] Recombinant proteins from the C1-C6 substrate extracorporeal pathway: The recombinant protein constructed in this invention: pCDF-P1- pydE pCDF-P1- CaMCR-C pCDF-P1- AEB94401 pCDF-P1- YdfG pCDF-P1- FadB pCDF-P1- CaMCR-N pCDF- DhaB pCDF- DhaS pCDF- DhaT pCDF- PduP pCDF- ACX4 pCDF- KatE pCDF- AcuK pCDF- AdhE pCDF- PddABC pCDF- ACS6 pCDF- PCT pCDF- Lcd pCDF- GldA pCDF- FLS pCDF- Mdh pCDF- LmACDH2 pCDF- DERA pCDF- Kdc pCDF- MaDH pCDF- XylA pCDF -XylB pCDF -DAS pCDF- TpiA pCDF- YidA pCDF- Glk pCDF- Pgi pCDF- PfkA pCDF- FbaA .

[0044] pCDF-duet (Invitrogen), pMCR-C (C Liu et al., 2016), pEcCas9 (Li QI et al., 2021), pEcgRNA (Li QI et al., 2021), pQL1 (Q Liu et al., 2015), pCum (Hou J et al., 2020), pCF-tesA (L Fang, et al., 2020) al., 2020), pACYC- dCas9 、Sg-accBC、pTrc- TE10 (ZTan, et al, 2016) is derived from existing technology.

[0045] A method for synthesizing malonyl-CoA from formic acid includes the following steps: S1. Preparation of the reaction system: On ice, add the following components sequentially to a sterile centrifuge tube to prepare a total reaction solution of 200 µL: Buffer components: 50mM HEPES buffer (pH 9.5), 2mM MgCl2, 3mM DTT.

[0046] Substrate and cofactors: Formic acid, final concentration 5 mM; ATP, final concentration 5 mM; Coenzyme B 12 Final concentration 15µM; KCl, final concentration 10mM; NAD + Final concentration 5mM; NADP + Final concentration 5mM; Coenzyme A, final concentration 5mM; NADH, final concentration 0.125mM; Thiamine pyrophosphate (TPP), final concentration 2.5mM; Flavin adenine dinucleotide (FAD), final concentration 5mM.

[0047] Enzyme mixture: Add purified recombinant enzymes at a final concentration of 1 µM each, including ACS6, LmACDH2, FLS, GldA, DhaB, DhaS, PydE, and CaMCR-C.

[0048] S2. Gently mix the reaction solution prepared in S1, set the microplate reader to 37℃, place it in the microplate reader for monitoring and reaction for 1 hour, collect the reaction solution, add an equal volume of methanol, centrifuge at 12000 rpm for 10 minutes to obtain the supernatant containing malonyl-CoA.

[0049] A method for synthesizing malonyl-CoA from propionaldehyde includes the following steps: S1. Preparation of reaction system: Add the following components sequentially to a sterile centrifuge tube to prepare a total reaction solution of 200µL: Buffer components: 50mM HEPES buffer (pH 9.5), 2mM MgCl2, 3mM DTT.

[0050] Substrate and cofactors: propionaldehyde, final concentration 5 mM; NAD + Final concentration 5mM; NADP + Final concentration 5mM; Coenzyme A, final concentration 5mM; NADH, final concentration 0.125mM; NADPH, final concentration 0.125mM.

[0051] Enzyme mixture: Add purified recombinant enzymes at a final concentration of 1 µM each, including PduP, Acx4, KatE, AcuK, PydE, and CaMCR-C.

[0052] S2. Gently mix the reaction solution prepared in S1, set the microplate reader to 37℃, place it in the microplate reader for monitoring and reaction for 1 hour, collect the reaction solution, add an equal volume of methanol, centrifuge at 12000 rpm for 10 minutes to obtain the supernatant containing malonyl-CoA.

[0053] Systematic validation of the in vitro transformation feasibility of 20 C1-C6 substrates (e.g.) Figure 4 As shown in Table 1, the enzymes used to generate malonyl-CoA from different exogenous carbon substrates are shown in Table 1.

[0054] Table 1. Enzymes used to generate malonyl-CoA from different exogenous carbon substrates.

[0055] HPLC-MS was used to detect malonyl-CoA products. The generation of NADH and NADPH was monitored by absorbance at 340 nm, and the enzyme reaction rate corresponding to the increase in absorbance per unit time was calculated. The results are shown in Table 2.

[0056] Table 2. Rates of malonyl-CoA formation from different substrates

[0057] Table 2 shows that all 20 C1-C6 substrates were successfully synthesized in vitro using the UMCP platform with 100% carbon yield (no CO2 release) and zero net ATP consumption (the ATP consumption of some substrate front-end adaptation modules can be replaced by ATP-independent acyl-CoA transferases). Dihydroxyacetone showed the highest conversion rate (31.5 μM min). -1 Formic acid (108.0 μM min) -1 ), Acetic acid (28.8 μM min) -1 ), propionaldehyde (206.67 μM min) -1 It exhibited high conversion activity; among C1 substrates, the conversion rate of formic acid was significantly higher than that of methanol (1.0 μM min). -1 ) and formaldehyde (0.7μM min) -1 In C4-C6 substrates, glucose (13.7 μM min) -1 xylose (5.8 μM min) -1 The conversion efficiency of ) is better than that of oxaloacetic acid (3.1 μM min). -1 ) and malic acid (1.6 μM min) -1 ).

[0058] Example 4 The waste materials, including CO2 electrolysis products, PET hydrolysate, cellulose hydrolysate, chitin hydrolysate, and hemicellulose hydrolysate, are converted into phloroglucinol and aflatoxin. The method includes the following steps: Pretreatment of waste materials and preparation of hydrolysate: Preparation of CO2 electrolysis products: CO2 was converted to formate using an electrochemical reduction method. The specific steps were as follows: A three-electrode system (Bi-based material as the working electrode, Ag / AgCl as the reference electrode, and a Pt sheet as the counter electrode) was used in an H-type electrolytic cell (with a Nafion membrane separating the anode and cathode chambers). Both the cathode and anode chambers were filled with a 0.5 mol / L KHCO3 solution saturated with CO2. Constant potential electrolysis was performed at a potential of -1.5 V to -1.6 V (vs. Ag / AgCl), reducing CO2 to formate at the cathode. The electrolyte was collected, and the formate concentration was adjusted to 20 mM for use as the reaction raw material.

[0059] Preparation of PET plastic hydrolysate: Waste PET plastic fragments were placed in a reactor, and a PET enzyme (LCCICCG-L883-GsCbe) solution was added. Enzymatic hydrolysis was carried out at 50℃ and pH 7.0. After the reaction, insoluble precipitates were removed by centrifugation, and the supernatant was collected. The ethylene glycol concentration in the supernatant was adjusted to 15 mM and used as the reaction raw material.

[0060] Preparation of chitin hydrolysate: Chitin powder was placed in a reactor, and Serratia marcescens was added ( Serratia marcescens The chitinase solution was subjected to enzymatic hydrolysis at 37°C and pH 6.0. After the reaction, the precipitate was removed by centrifugation, and the supernatant was collected. The N-acetylglucosamine concentration in the supernatant was adjusted to 20 mM and used as the reaction starting material.

[0061] Preparation of cellulose hydrolysate: Corn stalks were crushed to 40 mesh. After removing some lignin using a dilute alkali method, the mixture was hydrolyzed with 72% (w / w) concentrated sulfuric acid at 30°C for 3 hours. After hydrolysis, the mixture was neutralized, decolorized, and filtered, and the filtrate was collected. The glucose concentration in the filtrate was adjusted to 20 mM and used as a reaction feedstock.

[0062] Preparation of hemicellulose hydrolysate: Corn cobs were crushed to 40 mesh. Hydrolysis was performed using 0.5% (w / w) dilute sulfuric acid at 100℃ for 3 hours. After hydrolysis, the hydrolysate was deacidified and impurities removed, and the clear liquid was collected. The xylose concentration in the clear liquid was adjusted to 20 mM and used as a reaction feedstock.

[0063] Glucose (final concentration 20mM) and xylose (final concentration 20mM) were centrifuged to remove precipitates and then used as reaction raw materials.

[0064] Using the aforementioned waste hydrolysate as substrates, malonyl-CoA and downstream products were synthesized. The reaction system followed the general in vitro reaction conditions described in Example 3 of this invention, with enzyme combinations added as shown in Table 1. Specifically, for the synthesis of phloroglucinol, an additional 5 µM of PhlD enzyme was added. For the synthesis of aflatoxin, an additional 5 µM of RppA enzyme was added.

[0065] The reaction was carried out by incubating a total reaction solution of 200 µL at 37 °C.

[0066] The content of phloroglucinol in the reaction solution was detected using HPLC 1260-QQQ 6470, and the content of aflatoxin in the reaction solution was detected using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 520 nm. The results are as follows: Figure 5 As shown.

[0067] Depend on Figure 5 It can be seen that the CO2 electrolysis products yielded 4.8 mg L. -1 Phloroglucinol, 3.9 mg / L -1 Paleoxacin ( Figure 5 (A-4 in B) represents the first fully enzymatic pathway from CO2 to drug precursor. 7.5 μg L was obtained from the PET enzymatic hydrolysate. -1 The yield of phloroglucinol and 0.9 mg L -1 Paleoxane enables the direct conversion of plastic waste into high-value pharmaceuticals. The yield of phloroglucinol from cellulose hydrolysate is 0.75 mg / L. -1 The yield of aflatoxin was 2.2 mg / L. -1 The conversion efficiency is better than that of chitin hydrolysate (phloroglucinol 6.1 μg / L). -1 Paleoxacin 0.4 mg / L -1 ) and hemicellulose hydrolysate (phloroglucinol 7.5 μg L) -1 Paleoxacin 1.0 mg / L -1 ).

[0068] Example 5 Using an in vitro reaction system similar to that in Example 2, with 3-HP as the starting substrate, the reaction was carried out in a 200 µL system containing: 100 mM Tris-HCl buffer (pH 9.5), 2 mM MgCl2, 3 mM DTT, 36 mM 3-HP, and 2.5 mM NADP. + The use of 2.5 mM coenzyme A and purified recombinant enzymes PydE and CaMCR-C at final concentrations of 1 µM each verified that the new Malonyl-CoA synthetic pathway relieved multiple feedback inhibition. Inhibitor addition experimental setup: Different combinations of potential inhibitors were added to the above basic reaction system to simulate the inhibitory conditions experienced by the natural pathway: (1) Add ATP and ADP to make the total adenosine concentration 3.75 mM, and set two extreme ADP / ATP ratios: 20 and 0.1; (2) Add acetyl-CoA and set the concentrations to 0 mM and 2 mM; (3) Add palmitoyl-CoA at concentrations of 0 µM and 24 µM; All reaction systems were incubated at 37°C. The absorbance at 340 nm was monitored in real-time, and the results are as follows: Figure 6 As shown.

[0069] Depend on Figure 6 As shown in A, the catalytic rate of the UMCP pathway did not change significantly under the conditions of ADP / ATP ratios of 20 and 0.1, indicating that the pathway is not regulated by cellular energy state (ATP level). Figure 6 As shown in B, the catalytic rate of the experimental group with added 2mM acetyl-CoA was basically the same as that of the control group without added acetyl-CoA, proving that the pathway is not affected by the feedback inhibition of the product. Figure 6 The C-value in the figure shows that even in the presence of a high concentration (24 µM) of palmitoyl-CoA, the catalytic activity of this pathway was not different from that of the control group, indicating that it is completely unaffected by such end products. These in vitro experimental results demonstrate that typical inhibitors of these natural synthetic pathways have no inhibitory effect on the artificial UNCM pathway.

[0070] Example 6 The new synthetic pathway can replace the natural pathway for the synthesis of intracellular malonyl-CoA. CRISPR interference (CRISPRi) technology was used to inhibit the expression of the accBC gene, a key gene in the natural synthesis pathway of malonyl-CoA in Escherichia coli MG1655 (DE3).

[0071] The plasmids pACYC-dCas9 and Sg-accBC are derived from existing technologies.

[0072] Construction of strains containing pACYC-dCas9 and Sg-accBC expression vectors: 1 μL of pACYC-dCas9 and 1 μL of Sg-accBC were added to 50 μL of MG1655(DE3) competent cells, and the cells were incubated on ice for 30 min. The cells were then heat-shocked at 42℃ for 30 seconds and immediately placed on ice for 2 min. 500 μL of SOC medium (purchased from Shanghai Sangon Biotech Co., Ltd.) was added, and the cells were incubated at 37℃ for 1 hour at 220 rpm. 20 μL of the bacterial culture was plated on an LB agar plate containing chloramphenicol and ampicillin (final concentration 50 μg / mL). After overnight incubation, 5 positive single colonies were selected for colony PCR verification, yielding MG1655(DE3), pACYC-dCas9, and Sg-accB positive bacteria (strain A).

[0073] CRISPRi experiment: Seed culture: Select 3-5 single clones of strain A and inoculate them into 15 mL test tubes containing 5 mL MOPS medium (containing 20 g / L glucose and 50 mM 3-hydroxypropionic acid). The medium contains chloramphenicol and ampicillin (final concentration 50 μg / mL). Incubate at 220 rpm and 37 °C until the OD550 is about 1.5-1.8, which will be used as seed culture.

[0074] The seed culture was inoculated into MOPS medium (containing 20 g / L glucose, 0.1 mM IPTG, and chloramphenicol and ampicillin at a final concentration of 50 μg / ml) at an initial OD550 of 0.1. After inoculation, the mixture was shaken to mix, and 200 µL was transferred to a 96-well plate. OD550 was measured every 10 min using a Biotek microplate reader to determine the growth curve of the strain. The results are shown below. Figure 7 As shown.

[0075] The results showed that when the interference vectors pACYC-dCas9 and Sg-accBC were introduced into MG1655 (DE3), the strain could not grow in MOPS + 2% (w / v) glucose medium due to the nutrient deficiency of malonyl-CoA. Figure 7 (B in the middle).

[0076] The preparation method for MOPS medium is as follows: MOPS medium: 25 mL 40×M stock solution, 1 mL 0.528M MgCl2, 1 mL 0.276M K2SO4, 1 mL 1M K2HPO4, 100 mL 200 g / L glucose, distilled water to a final volume of 1 L, filtered through a 0.22 μm sterile filter membrane and ready for use.

[0077] The 40×M mother liquor was prepared as follows: Weigh 334.8g MOPS powder, 28.2g Tricine, 116.8g NaCl, 20.4g NH4Cl, and 32g KOH, add distilled water to 900mL and stir well. Adjust the pH to 7.3-7.4 with 10M HCl or 10M KOH. While stirring, add 2mL of "trace metal mother liquor" and 0.2mL of ZnCl2 solution (34g / L), 0.2mL of Na2SeO3 solution (43g / L), and 0.2mL of Na2MoO4 solution (60.5g / L), and then add distilled water to a final volume of 1L. Dispense the prepared solution into 20 50mL centrifuge tubes.

[0078] The preparation method of the "trace metal mother liquor" is as follows: Weigh 5g FeCl2·4H2O, 184mg CaCl2·2H2O, 62mg H3BO3, 40mg MnCl2·4H2O, 18mg CoCl2·6H2O, and 4mg CuCl2·2H2O, add 70mL of distilled water, add 8mL of concentrated hydrochloric acid (37% HCl), stir well, transfer to a 100mL graduated cylinder, and make up to 100mL with ddH2O.

[0079] pCDF- pydE-CaMCR-C Construction and introduction: B1. Using pCDF-P1-PydE plasmid as a template, the pCDF-P1-pydE vector backbone fragment was amplified using primers P2-MCRC-R (sequence shown in SEQ ID NO.18) and P2-MCRC-F (sequence shown in SEQ ID NO.19). This fragment contains the T7 promoter, the Ori sequence, the pydE coding sequence, and the spectinomycin resistance gene Smr, and is referred to as fragment I (sequence shown in SEQ ID NO.20). B2. Using pCDF-P1-CaMCR-C plasmid as a template, primers MCR-C-pCDF-P2-up (sequence shown in SEQ ID NO. 21) and MCR-C-pCDF-P2-down (sequence shown in SEQ ID NO. 22) were used to amplify the CaMCR-C encoding gene, which is called fragment II (sequence shown in SEQ ID NO. 23). B3. Remove template from fragments I and II using the restriction enzyme DpnI; after electrophoresis, wash with a PCR purification kit; take the template-removed fragments I and II and add a seamless cloning enzyme for reaction; B4. Take the reaction product of B3, transform it into Trans-T1 competent cells, select positive single colonies, and extract plasmids.

[0080] B5. Take 1 μL of pCDF- pydE-CaMCR-C Plasmids were added to 50 μL of MG1655 (DE3) competent cells containing the interference vectors pACYC-dCas9 and Sg-accBC, and incubated on ice for 30 min. The cells were then heat-shocked at 42℃ for 30 seconds and immediately placed on ice for 2 min. 500 μL of SOC medium (purchased from Shanghai Sangon Biotech Co., Ltd.) was added, and the cells were incubated at 37℃ for 1 hour at 220 rpm. 20 μL of the bacterial culture was plated onto LB agar plates containing chloramphenicol, ampicillin, and spectinomycin (final concentration 50 μg / ml), and cultured overnight to obtain MG1655(DE3), pACYC-dCas9, Sg-accB, and pCDF- pydE-CaMCR-C (Strain B).

[0081] Growth experiments were then conducted: Seed culture: Select 3-5 single clones (strain B) and inoculate them into 15 mL test tubes containing 5 mL of MOPS medium (containing 20 g / L glucose). The medium contains chloramphenicol, ampicillin, and spectinomycin (final concentration 50 μg / mL). Incubate at 220 rpm and 37 °C until the OD550 is about 1.5-1.8, which will be used as seed culture.

[0082] The seed culture was inoculated into MOPS medium (containing 20 g / L glucose, 0.1 mM IPTG, 50 mM 3-HP, and chloramphenicol, ampicillin, and spectinomycin at a final concentration of 50 μg / ml) at an initial OD550 of 0.1. After inoculation, the medium was shaken to mix thoroughly, and 200 µL was transferred to a 96-well plate. OD550 was measured every 10 min using a Biotek microplate reader to determine the growth curve of the strain.

[0083] The results show that, with the introduction of pCDF- pydE-CaMCR-C Subsequently, the growth damaged by accBC interference was greatly restored. Figure 7 (B in the figure), indicating that the artificial pathway can replace the natural intracellular pathway for the synthesis of malonyl-CoA.

[0084] Example 7 The specific experimental protocol for fatty acid synthesis is as follows: The plasmid used for octanoic acid synthesis is pTrc-TE10, which contains thioesterase TE10 derived from Anaerococcus tetradius, which can specifically hydrolyze octanoyl-ACP to form octanoic acid.

[0085] The fermentation of caprylic acid products involves three steps: Building pki2- pydE-CaMCR-C The construction method is the same as pCDF- pydE-CaMCR-CThe pki plasmid is derived from existing technology (Liu X, et al. 2022).

[0086] (1) Use SspI endonuclease to digest pki2- pydE-CaMCR-C The sample was linearized and then transformed into *Yersinia lipolytica* PO1f (Liu X, et al. 2022) to obtain strain 1. The plasmid YLEP-TE10 was then transformed into *Yersinia lipolytica* PO1f and strain 1, respectively, to obtain control strain 2 and engineered strain 3 for fatty acid synthesis.

[0087] (2) Seed culture: Select strain 3 and control strain 2 and inoculate them into YNB medium lacking biotin for 30h.

[0088] (3) Fermentation culture: Prepare 30 mL of biotin-deficient YNB fermentation medium in a 250 mL Erlenmeyer flask, inoculate the seed liquid with an initial OD600=0.1, and culture at 220 r and 30 ℃ for 72 h. OD550 is measured and samples are taken every 24 h.

[0089] The culture medium used for seed culture and fermentation was biotin-deficient YNB medium, with the following formula: glucose 20 g / L, YNB (biotin-deficient) 1.7 g / L, and Leu-Ura double-deficient amino acid mixture 8 g / L.

[0090] Detection of fatty acids: After fermentation, 1 mL of fermentation broth was mixed with 125 μL of 10% NaCl (wt / v) and 125 μL of acetic acid; then 10 μL of 5 mg / L internal standard (C7:0 / C15:0, final concentration 50 mg / L) was added, and after brief mixing, 500 μL of ethyl acetate was added. The sample was vortexed for 15 s and centrifuged at 16000×g for 10 min. 250 μL of the upper organic phase was transferred to a new glass tube, and 2.25 mL of ethanol:hydrogen chloride (30:1 v / v) was added. After incubation at 55 ℃ for 1 h, the mixture was cooled to room temperature, and 1.25 mL of ddH2O and 1.25 mL of n-hexane were added. After vortexing, the mixture was centrifuged at 2000×g for 2 min. The upper n-hexane layer was used for GC-MS analysis. Malonyl-CoA was quantified using an Agilent 5975 mass spectrometer and an Agilent 7890 gas chromatograph equipped with a gas chromatograph-flame ionization detector / mass spectrometer (GC-FID / MS). The analytical program was as follows: initial temperature of 50 °C, hold for 2 min; increase to 200 °C at 25 °C / min, hold for 1 min; then increase to 315 °C at 25 °C / min, hold for 2 min. Helium was used as the carrier gas at a flow rate of 1 mL / min. A DB-5MS column (30 m, 0.25 mm ID, 0.25 μm, Agilent) was used for separation, and gas chromatograph-mass spectrometer was used for detection.

[0091] Results: The growth of control strain 2 was restricted, with an octanoic acid production of 0, while the engineered strain (strain 3) containing the new malonyl-CoA synthetic pathway produced octanoic acid at a rate of 121 ± 13 mg / L. Figure 8 ).

[0092] Example 8 The fermentation of phloroglucinol products involves three steps: (1) The bioH gene of E. coli MG1655(DE3) was knocked out using the λ red recombination method in the existing technology to obtain strain JP01. The specific experimental protocol is as follows: 50 ng of pkD46 plasmid (containing a thermosensitive replicon and ampicillin resistance) was transformed into the target strain and incubated at 30°C. 100 μL of the plasmid was then spread onto LB+Amp plates and incubated overnight at 30°C. The obtained positive transformants were cultured, and at OD550 = 0.5, the culture medium was placed on ice for 30 min. The cells were then washed with pre-chilled water, centrifuged at 5000 rpm, and then pre-chilled 10% glycerol solution was added to prepare electrically transformed competent cells. Using pKD4 plasmid as a template, PCR amplification was performed using bioH-KO-up (sequence shown in SEQ ID NO.24) / bioH-KO-down (sequence shown in SEQ ID NO.25) primers. The amplification program was as follows: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 2 min, followed by 95℃ denaturation for 15 s, repeated 30 times. After denaturation, the cells were held at 72℃ for 5 min and cooled to 4℃. The resulting fragment contained a bioH homologous fragment, the kanamycin encoding gene, and the FRT sequence (sequence shown in SEQ ID NO.26). The obtained fragment was added to competent cells for transformation, yielding strain JP01.

[0093] (2) Take plasmids pCDF-duet and pCum- phlD Add 50 ng each of JP01 competent cells and incubate on ice for 30 min. Heat shock at 42℃ for 60 seconds, then immediately place on ice for 2 min. Add 500 μL of SOC medium (purchased from Shanghai Sangon Biotech Co., Ltd.), and incubate at 37℃ for 1 hour at 220 rpm. Centrifuge to remove the supernatant, leaving approximately 50 μL. Mix well and spread the entire amount onto LB agar plates containing spectinomycin (final concentration 50 μg / mL) and kanamycin (final concentration 50 μg / mL). After overnight culture, positive clones JP01, pCDF-duet, and pCum- are obtained. phlD This is called strain 4.

[0094] Take plasmid pCDF- pydE-CaMCR-C and pCum- phlD Add 50 ng each of JP01 competent cells to 50 μL of the medium and incubate on ice for 30 min. Heat shock at 42°C for 60 seconds, then immediately place on ice for 2 min. Add 500 μL of SOC medium (purchased from Shanghai Sangon Biotech Co., Ltd.) and incubate at 37°C for 1 hour at 220 rpm. Centrifuge to remove the supernatant, leaving approximately 50 μL. Mix well and spread the entire amount onto LB agar plates containing spectinomycin (final concentration 50 μg / mL) and kanamycin (final concentration 50 μg / mL). After overnight culture, the positive clone JP01, pCDF- is obtained. pydE-CaMCR-C , pCum- phlD This is called strain 5.

[0095] (3) Seed culture: Select strain 4 and strain 5 and inoculate them into 50 mL shaker tubes containing 10 mL LB medium containing spectinomycin (final concentration of 50 μg / mL) and kanamycin (final concentration of 50 μg / mL), respectively. Incubate overnight at 37°C and 220 rpm to prepare seed culture.

[0096] (4) Fermentation culture: Prepare 50 mL of M9 fermentation medium (containing 50 mM 3-HP, spectinomycin (final concentration 50 μg / mL) and kanamycin (final concentration 50 μg / mL) in a 250 mL Erlenmeyer flask. Inoculate the seed culture of strains 4 and 5 with an initial OD550 of 0.1, and incubate at 220 rpm and 37 °C until the OD550 is about 0.6. Add IPTG to a final concentration of 0.1 mM and cumate to a final concentration of 0.25 mM. Continue fermentation culture at 220 rpm and 37 °C for 24 h.

[0097] The fermentation medium used was M9 medium, with the following formula: glucose 20 g / L, Na2HPO4•7H2O 12.8 g / L, NaCl 0.5 g / L, NH4Cl 1 g / L, KH2PO4 3 g / L, 2 mM MgSO4 and 0.1 mM CaCl2.

[0098] The preparation method is to take 10 mL of 200 g / L glucose, 20 mL of 5×M9, 200 μL of 1 M MgSO4 and 10 μL of 1 M CaCl2, and then add distilled water to 100 mL.

[0099] Among them, 5×M9 consists of 64 g / L Na2HPO4•7H2O, 15 g / L KH2PO4, 2.5 g / L NaCl, and 5 g / L NH4Cl.

[0100] Analytical method: After fermentation, take 2 mL of sample, centrifuge at 12000×g for 2 min, collect the supernatant, and perform HPLC analysis. A C18 column was used, with a flow rate of 0.8 mL / min, a column temperature of 30℃, a mobile phase of 40% acetonitrile, and a UV detector at a wavelength of 254 nm.

[0101] Results: Malonyl-CoA deficiency in the control strain led to cell death and no phloroglucinol production. The UMCP-engineered strain (strain 5) produced phloroglucinol at a yield of 152 ± 17 mg / L. Figure 9 ).

[0102] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for synthesizing malonyl-CoA, characterized in that, Includes the following steps: S1. Convert exogenous carbon source substrates into 3-hydroxypropionic acid; S2. Convert the 3-hydroxypropionic acid obtained in S1 into malonyl-CoA.

2. The method according to claim 1, characterized in that, In S2, the conversion of 3-hydroxypropionic acid obtained in S1 to malonyl-CoA is achieved through any of the following enzymatic catalytic pathways: Pathway 1: Under the catalysis of 3-hydroxypropionic acid dehydrogenase, 3-hydroxypropionic acid is oxidized to 3-oxopropionic acid; under the catalysis of oxidoreductase, 3-oxopropionic acid is converted to malonyl-CoA. Pathway 2: Under the catalysis of malonyl-CoA reductase, 3-hydroxypropionic acid is converted into malonyl-CoA.

3. The method according to claim 2, characterized in that, The 3-hydroxypropionate dehydrogenase is selected from one or more of PydE, YdfG, AEB94401, CaMCR-N or FadB; The oxidoreductase is selected from one or more of CaMCR-C, StMCR, Msed_0709, PnSCR or SucD; The malonyl-CoA reductase is selected from one or more of PcMCR, RcMCR, CtMCR, CtbMCR, GpMCR, or CaMCR.

4. The method according to claim 3, characterized in that, The amino acid sequence of PydE is shown in SEQ ID NO.1; the amino acid sequence of YdfG is shown in SEQ ID NO.2; the amino acid sequence of AEB94401 is shown in SEQ ID NO.3; the amino acid sequence of CaMCR-N is shown in SEQ ID NO.4; and the amino acid sequence of FadB is shown in SEQ ID NO.

5.

5. The method according to claim 3, characterized in that, The nucleotide sequence of CaMCR-C is shown in SEQ ID NO.6; the amino acid sequence of StMCR is shown in SEQ ID NO.7; the amino acid sequence of Msed_0709 is shown in SEQ ID NO.8; the amino acid sequence of PnSCR is shown in SEQ ID NO.9; and the amino acid sequence of SucD is shown in SEQ ID NO.

10.

6. The method according to claim 3, characterized in that, The amino acid sequence of the PcMCR is shown in SEQ ID NO.11; the amino acid sequence of the RcMCR is shown in SEQ ID NO.12; the amino acid sequence of the CtMCR is shown in SEQ ID NO.13; the amino acid sequence of the CtbMCR is shown in SEQ ID NO.14; the amino acid sequence of the GpMCR is shown in SEQ ID NO.15; and the amino acid sequence of the CaMCR is shown in SEQ ID NO.

16.

7. A recombinant plasmid for synthesizing malonyl-CoA, characterized in that, It includes a gene encoding the 3-hydroxypropionate dehydrogenase and oxidoreductase of claim 3 or a gene encoding the malonyl-CoA reductase of claim 3.

8. A strain for synthesizing malonyl-CoA, characterized in that, The strain includes the recombinant plasmid as described in claim 7.

9. A method for synthesizing malonyl-CoA derivatives using the recombinant plasmid according to claim 8, characterized in that, Includes the following steps: The recombinant plasmid was transferred into competent Escherichia coli cells to obtain a positive clone strain, which was then fermented to obtain a malonyl-CoA derivative.

10. The use of malonyl-CoA synthesized by the method of any one of claims 1-6 or the malonyl-CoA derivative synthesized by the method of claim 9 in the preparation of a medicament.

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