CN115058375A - Genetically engineered bacterium for producing methylmalonate monoacyl coenzyme A as well as preparation method and application thereof - Google Patents

Abstract

The invention discloses a genetic engineering bacterium for producing methylmalonate monoacyl-coenzyme A and a preparation method and application thereof, belonging to the technical field of genetic engineering. To provide a method for producing methylmalonate monoacyl-coenzyme A. The invention discloses a genetically engineered bacterium for producing methylmalonate monoacyl-coenzyme A, which takes escherichia coli as a starting bacterium and overexpresses acetyl coenzyme A carboxylase BCCP subunit gene accB, acetyl coenzyme A carboxylase BC subunit gene accC, propionyl coenzyme A carboxylase CT subunit gene pc and propionyl coenzyme A synthase gene pcs. The invention can reduce the production cost of the methylmalonate monoacyl-coenzyme A and promote the application of the methylmalonate monoacyl-coenzyme A in scientific research and production.

Description

A kind of genetically engineered bacteria producing methylmalonyl-CoA and preparation method thereof application

technical field

The invention belongs to the technical field of genetic engineering, and in particular relates to a genetically engineered bacteria producing methylmalonyl-CoA and a preparation method and application thereof.

Background technique

Methylmalonyl-CoA is an important intermediate metabolite formed by the combination of coenzyme A and methylmalonic acid through thioester bonds, and is involved in many biosynthesis and decomposition reactions. Methylmalonyl-CoA is also an important precursor substance for the synthesis of some drugs, participating in the synthesis of polyketides such as erythromycin, FK506, etc., in the normal life activities of organisms and the biosynthesis of secondary metabolites. ,Play an important role. Among them, in the process of biosynthesis of polyketides, adding methylmalonyl-CoA helps to increase the yield of the product, but methylmalonyl-CoA is expensive and difficult to chemically synthesize. Meet the needs of a low-carbon economy.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a method for producing methylmalonyl-CoA.

The invention provides a genetically engineered bacterium for producing methylmalonyl-CoA. The genetically engineered bacterium uses Escherichia coli as a starting bacterium and overexpresses acetyl-CoA carboxylase BCCP subunit gene accB, acetyl-CoA Carboxylase BC subunit gene accC, propionyl-CoA carboxylase CT subunit gene pcc and propionyl-CoA synthase gene pcs.

It is further defined that the acetyl-CoA carboxylase BCCP subunit gene accB is derived from Escherichia coli, and its gene sequence is shown in SEQ ID NO.1, and the acetyl-CoA carboxylase BC subunit gene accC is derived from the large intestine Bacillus, its gene sequence is shown in SEQ ID NO.2, the propionyl-CoA carboxylase CT subunit gene pcc is derived from Bacillus subtilis, and its gene sequence is shown in SEQ ID NO.3, the propionyl The coenzyme A synthase gene pcs is from Chloroflexus aurantiacus, and its gene sequence is shown in SEQ ID NO.4.

The present invention provides a preparation method of the above-mentioned genetically engineered bacteria, characterized in that, the preparation method is as follows:

Step 1: Connect the acetyl-CoA carboxylase BCCP subunit gene accB, the acetyl-CoA carboxylase BC subunit gene accC, and the propionyl-CoA carboxylase CT subunit gene pcc to the expression vector pETDuet to obtain a recombinant vector 1 ;

Step 2: connect the propionyl-CoA synthase gene pcs to the expression vector pACYCDuet to obtain the recombinant vector: 2;

Step 3: Co-transform the recombinant vector 1 and recombinant vector 2 obtained in steps 1 and 2 into Escherichia coli BL21 (DE3) to obtain genetically engineered bacteria.

The present invention provides a method for intracellular production of methylmalonyl-CoA, characterized in that the method is to ferment the above-mentioned genetically engineered bacteria in a fermentation broth containing 3-hydroxypropionic acid at a temperature of 37 ℃, fermentation time at least 5h.

Further limited, the ingredients of the fermentation broth: 14.04g/L dipotassium hydrogen phosphate trihydrate, 5.24g/L potassium dihydrogen phosphate, 1g/L sodium chloride, 1g/L ammonium chloride, 3g/L yeast powder, 4g /L sodium bicarbonate, 40mg/L biotin, 0.5g/L magnesium sulfate, 20g/L glucose and 2g/L 3-hydroxypropionic acid.

The present invention provides a method for extracellular production of methylmalonyl-CoA. The above-mentioned genetically engineered bacteria are treated to obtain a supernatant, and the supernatant is a reaction bottom of a culture solution containing 3-hydroxypropionic acid. The reaction was carried out at a temperature of 37 °C and a reaction time of at least 1 h.

Further defined, the components of the culture medium: Tris-HCl, MgCl2, DTT, KCl, NaHCO3, biotin, coenzyme A, NADPH, ATP and 3-hydroxypropionic acid.

Further limited, the method for obtaining the supernatant is as follows: inoculate the above-mentioned genetically engineered bacteria into the LB medium, cultivate at 30°C until the bacterial concentration is OD600 of 0.6-0.8, after adding IPTG, at 30°C After culturing for 6 to 8 hours, a culture solution is obtained, the culture solution is centrifuged to obtain a bacterial suspension, and the cell disrupted solution is obtained after being disrupted, and the supernatant of the cell disrupted solution is obtained after centrifugation.

Further defined, the concentration of the supernatant is 10 mg/ml.

The present invention provides the application of the above-mentioned genetically engineered bacteria in the production of methylmalonyl-CoA.

Beneficial effects: the method provided by the invention can convert 3-hydroxypropionic acid and coenzyme A into methylmalonyl coenzyme A by using enzymatic reaction in vitro, and the yield reaches 25.6 mg/L, which is methylmalonyl The in vitro preparation of coenzyme A provides a new idea.

Detailed ways

The present invention is further illustrated by the following examples. However, the present invention is not limited to the following examples.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

The enzyme reagents used were purchased from MBI Fermentas Company, the kits used for extracting plasmids and the kits used for recovering DNA fragments were purchased from OMEGA Company in the United States, and the kits for homologous recombination were purchased from Nanjing Novizan Company, and the corresponding operation steps were in accordance with the product instructions. performed; all media were prepared with deionized water unless otherwise specified.

Medium formula:

LB liquid medium: 5g/L yeast powder, 10g/L NaCl, 10g/L peptone, pH 7.

In the actual culture process, a certain concentration of antibiotics can be added to the above medium to maintain the stability of the plasmid, such as 100 mg/L of ampicillin and 50 mg/L of chloramphenicol.

Example 1. Preparation of genetically engineered strains

(1) Obtain the propionyl-CoA synthase gene pcs (gene sequence shown in SEQ ID NO. 4) derived from Chloroflexus aurantiacus, which is obtained by PCR amplification using the genome of Chloroflexus aurantiacus as a template (Primers 5'-TCATCACCACAGCCAGGGATCCatgatcgacactgcgcccctt-3' (SEQ ID NO. 5) and 5'- GCGCGCCGAGCTCGAATTCCTACCGCTCGCCGGCCGTCCACG-3' (SEQ ID NO. 6)), and then use a recovery kit to recover the target fragment to obtain the pcs gene fragment.

(2) The plasmid pACYCDuet was cut and linearized with BamHI, and after adding DpnI for demethylation, the product was recovered, and the above-mentioned pcs gene fragment was connected by the method of homologous recombination according to the steps in the homologous recombination kit. . The ligation product was transformed into E. coli DH5α, and then spread on LB solid plates supplemented with 50 mg/L chloramphenicol, and positive clones were screened by PCR. The recombinant plasmid pACYCDuet-pcs was extracted from the positive clones, and then identified by sequencing to obtain the engineering plasmid pACYCDuet-pcs.

(3) Obtain the acetyl-CoA carboxylase gene accBC (acetyl-CoA carboxylase BCCP subunit gene accB) derived from Escherichia coli, the gene sequence of which is shown in SEQ ID NO. The base gene accC, whose gene sequence is shown in SEQ ID NO.2), was obtained by PCR amplification with the genome of Escherichia coli as a template (primers: 5'-TCATCACCACAGCCAGGGATCCatggatattcgtaagattaaaaa-3' (SEQ ID NO.7) and 5 '- GCGCGCCGAGCTCGAATTCTTATTTTTCCTGAAGACCGAG-3' (SEQ ID NO. 8)), and then use a recovery kit to recover the target fragment.

(4) The plasmid pETDuet was cut and linearized by using BamHI, and after adding DpnI for demethylation, the product was recovered, and connected with the above-mentioned accBC gene fragment by the method of homologous recombination according to the steps in the homologous recombination kit . The ligation product was transformed into E. coli DH5α, and then spread on LB solid plates supplemented with 100 mg/L ampicillin, and positive clones were screened by PCR. The recombinant plasmid pETDuet-accBC was extracted from the positive clones, and then identified by sequencing to obtain the engineered plasmid pETDuet-accBC.

(5) obtaining the propionyl-CoA carboxylase CT subunit gene pcc derived from Bacillus subtilis (the gene sequence is shown in SEQ ID NO. 3), using the genome of Bacillus subtilis as a template, and obtaining by PCR amplification ( Primers: 5'-GAGATATACATATGGCAAGATCTatgaacgaacatatggatcattt-3' (SEQ ID NO. 9) and 5'-CGGCCGATATCCAATTGTTACAGCGGAATATTGCCATGT-3' (SEQ ID NO. 10)), and then the target fragment was recovered using a recovery kit.

(6) The plasmid pETDuet-accBC was cut and linearized with BglII, and after adding DpnI for demethylation, the product was recovered, and the homologous recombination method was used with the above-mentioned pcc gene fragment according to the steps in the homologous recombination kit. to connect. The ligation product was transformed into E. coli DH5α, and then spread on LB solid plates supplemented with 100 mg/L ampicillin, and positive clones were screened by PCR. The recombinant plasmid pETDuet-accBC-pcc was extracted from the positive clone, and then identified by sequencing to obtain the engineered plasmid pETDuet-accBC-pcc.

(7) The pETDuet-accBC-pcc vector and the pACYCDuet-pcs vector obtained above were introduced into E.coli BL21 (DE3) competent cells by heat shock, and coated on cells containing 100 mg/L ampicillin and 50 mg/L chloramphenicol The coated plate was placed in a 37°C constant temperature incubator, and the culture was continued until a single clone was grown to obtain the engineering strain E.coli BL21(DE3) (pETDuet-accBC-pcc, pACYCDuet-pcs).

Example 2. In vitro preparation of methylmalonyl-CoA

(1) Inoculate the genetically engineered bacteria E.coli BL21 (DE3) (pETDuet-accBC-pcc, pACYCDuet-pcs) obtained in Example 1 into LB medium, and add ampicillin and 50 mg/L ampicillin with a final concentration of 100 mg/L. L of chloramphenicol was cultured at 30°C and 220rpm until the bacterial concentration was OD600 of 0.6-0.8. After adding IPTG with a final concentration of 0.5mM, cultured at 30°C for 6-8h to obtain a culture medium.

(2) After centrifuging the obtained culture solution at 10,000 rpm for 10 min, discard the supernatant, wash three times with 20 mL of PBS solution, and then fully suspend with 5 mL of the PBS solution; 15Mpa to obtain a cell disrupted liquid; the cell disrupted liquid was centrifuged at 13000 rpm and 4° C. for 30 min to obtain a supernatant of the cell disrupted liquid, which was the protease solution.

(3) Mix the protease solution with the reaction substrate to form a reaction system containing the following components: 100 mM Tris-HCl with pH=7.5, 5 mM MgCl ₂ , 1 mM DTT, 10 mM KCl, 40 mM NaHCO ₃ , 1 mM biotin, 1 mM coenzyme A, 2 mM NADPH, 10 mM ATP, 5 mM 3-hydroxypropionic acid. The reaction system was sealed in a 2 ml centrifuge tube, placed in a shaker at 37° C. and 180 rpm to react for 1 h to obtain methylmalonyl-CoA.

(4) Detection of methylmalonyl-CoA. Methylmalonyl-CoA was characterized and quantified by HPLC-MS method. HPLC adopts Thermo-Acclaim RSLC 120C18 2.1×100mm column, the flow rate is 0.25ml/min, the column temperature is 30℃, the injection volume is 2 μl, and the gradient elution method is shown in Table 1:

Table 1

MS adopts the compact-QTOF mass spectrometer with ESI ion source. The ion mode used for detection is positive ion mode, the voltage is 4500V, the detection range is 100-1300m/z, the ion source temperature is 200℃, and the drying gas flow rate is 2.0L/min .

By extracting the ion peak map of methylmalonyl-CoA with a molecular weight of 868±0.2, the product can be qualitatively identified; by integrating, the yield of the product, methylmalonyl-CoA, was measured to be 25.6 mg/ L.

Example 3. Method for intracellular production of methylmalonyl-CoA

The genetically engineered bacteria E.coli BL21 (DE3) (pETDuet-accBC-pcc, pACYCDuet-pcs) obtained in Example 1 were inoculated into the culture medium (14.04g/L dipotassium hydrogen phosphate trihydrate, 5.24g/L dihydrogen phosphate Potassium, 1g/L Sodium Chloride, 1g/L Ammonium Chloride, 3g/L Yeast Powder, 4g/L Sodium Bicarbonate, 40mg/L Biotin, 0.5g/L Magnesium Sulfate, 20g/L Glucose and 2g/L 3-hydroxypropionic acid), placed at 37 °C for 5 h to obtain methylmalonyl-CoA. The yield of methylmalonyl-CoA was 14.4 mg/L.

The above descriptions are only the embodiments of the present invention, and are not intended to limit the scope of the patent of the present invention. Any equivalent structure or equivalent process transformation made by the description of the present invention, or directly or indirectly applied in other related technical fields, are the same as The principles are included in the scope of patent protection of the present invention.

SEQ ID NO. 1:

atggatattcgtaagattaaaaaactgatcgagctggttgaagaatcaggcatctccgaactggaaatttctgaaggcgaagagtcagta cgcattagccgtgcagctcctgccgcaagtttccctgtgatgcaacaagcttacgctgcaccaatgatgcagcagccagctcaatctaacgcag ccgctccggcgaccgttccttccatggaagcgccagcagcagcggaaatcagtggtcacatcgtacgttccccgatggttggtactttctaccg caccccaagcccggacgcaaaagcgttcatcgaagtgggtcagaaagtcaacgtgggcgataccctgtgcatcgttgaagccatgaaaatgatgaaccagatcgaagcggacaaatccggtaccgtgaaagcaattctggtcgaaagtggacaaccggtagaatttgacgagccgctggtcgtc atcgagtaa

SEQ ID NO. 2:

atgctggataaaattgttattgccaaccgcggcgagattgcattgcgtattcttcgtgcctgtaaagaactgggcatcaagactgtcgctgt gcactccagcgcggatcgcgatctaaaacacgtattactggcagatgaaacggtctgtattggccctgctccgtcagtaaaaagttatctgaaca tcccggcaatcatcagcgccgctgaaatcaccggcgcagtagcaatccatccgggttacggcttcctctccgagaacgccaactttgccgagc aggttgaacgctccggctttatcttcattggcccgaaagcagaaaccattcgcctgatgggcgacaaagtatccgcaatcgcggcgatgaaaa aagcgggcgtcccttgcgtaccgggttctgacggcccgctgggcgacgatatggataaaaaccgtgccattgctaaacgcattggttatccgg tgattatcaaagcctccggcggcggcggcggtcgcggtatgcgcgtagtgcgcggcgacgctgaactggcacaatccatctccatgacccgt gcggaagcgaaagctgctttcagcaacgatatggtttacatggagaaatacctggaaaatcctcgccacgtcgagattcaggtactggctgac ggtcagggcaacgctatctatctggcggaacgtgactgctccatgcaacgccgccaccagaaagtggtcgaagaagcgccagcaccgggc attaccccggaactgcgtcgctacatcggcgaacgttgcgctaaagcgtgtgttgatatcggctatcgcggtgcaggtactttcgagttcctgttc gaaaacggcgagttctatttcatcgaaatgaacacccgtattcaggtagaacacccggttacagaaatgatcaccggcgttgacctgatcaaag aacagctgcgtatcgctgccggtcaaccgctgtcgatcaagcaagaagaagttcacgtt cgcggccatgcggtggaatgtcgtatcaacgcc gaagatccgaacaccttcctgccaagtccgggcaaaatcacccgtttccacgcacctggcggttttggcgtacgttgggagtctcatatctacg cgggctacaccgtaccgccgtactatgactcaatgatcggtaagctgatttgctacggtgaaaaccgtgacgtggcgattgcccgcatgaaga atgcgctgcaggagctgatcatcgacggtatcaaaaccaacgttgatctgcagatccgcatcatgaatgacgagaacttccagcatggtggca ctaacatccactatctggagaaaaaactcggtcttcaggaaaaataa

SEQ ID NO. 3:

atgaacgaacatatggatcatttctacaccaaacgtaaacaggcagaagaaggtggcggccgtgaaaaactggcacagcagcgtcag aaaggtaaactgaccgcccgcgaacgcattatttttctgctggatcaggatagttttattgaactgcatccgtttatggaaagccaggtgctgacc cgtgaacagcgtatgctgggtgacggtgttgttaccggttatggtaccattgatggtcgcagcgtttatgtttttgcacaggattttaccgtgtatgg tggtgccctgggtgaaacccatgcccgcaaaatttgcgcactgatggatctggccgccaaaaataaggcaccgattattggtctgaatgatagc ggtggtgcccgtattcaggaaggtgttctgagcctggatggttatggccatattttctatcgtaatgttctgtatagcggtgttattccgcagattag cgttattctgggtccgtgtgcaggcggtgccgtttatagcccggccctgaccgattttatttttatggccgaacagaccggtcgtatgtttattaccg gcccgaaagttattgaaaaagttaccggtgaacaggtggatgccgaaagtctgggcggcgcaggtattcataatgcagtgagtggtaatgcac attttagcggccataccgaaaaagaagttctgaccggtgtgcgtaaactgctgagttatctgccgctgaatggccgtaccaccgaaccgaaacc ggaaaaagaagcaagccgtccgctgctgaatcgtctggttccggccgataccaccaaaccgtatgatgtgcgtaaagttattcgcgaactggc cgatccgcagagctttttcgaaattcagccgtttttcgcaaaaaatattgttattggtttcgcacgtctgggtgaaaaagccattggtattgtggcaa gtcagccgaaacatctggcaggtagtctgaccattgatgcc gcagataaagcagcacgttttattcgtttttgtgatgcatttgatatcccgctgctgaccgtggaagatgtgccgggttttctgccgggcattcagcaggaacataatggtattattcgccatggcgcaaaactgctgtttgcctatgcag aagcaaccgtgccgaaagttaccctgattattcgcaaagcctatggtggcgcatatgtggccatgaatagcaaagccattggcgccgatctggt ttttgcctggccgaatgccgaaattgcagttatgggtccggaaggtgcagcaagcattctgtatgaaaaagaaattaaggccagcgccgatccg caaaaaaccaaacgtgaaaaaaccgcagaatataaaaagcagaatgcaggcccgtataaagcagccgcatgcggcatggtggatgatattat tctgccggaagaaagtcgcggtcgtctgattcaggcatttcatatgctgacccataaaaccgaagaacgtccgaaaaagaaacatggcaatatt ccgctgtaa

SEQ ID NO. 4:

atgatcgacactgcgccccttgccccaccacgggcgccccgctctaatccgattcgggatcgagttgattgggaagctcagcgcgctg ctgcgctggcagatcccggtgcctttcatggcgcgattgcccggacagttatccactggtacgacccacaacaccattgctggattcgcttcaa cgagtctagtcagcgttgggaagggctggatgccgctaccggtgcccctgtaacggtagactatcccgccgattatcagccctggcaacagg cgtttgatgatagtgaagcgccgttttaccgctggtttagtggtgggttgacaaatgcctgctttaatgaagtagaccggcatgtcacgatgggctatggcgacgaggtggcctactactttgaaggtgaccgctgggataactcgctcaacaatggtcgtggtggtccggttgtccaggagacaatca cgcggcggcgcctgttggtggaggtggtgaaggctgcgcaggtgttgcgtgatctgggcctgaagaagggtgatcggattgctctgaatatg ccgaatattatgccgcagatttattatacggaagcggcaaaacgactgggtattctgtacacgccggtcttcggtggcttctcggacaagactctt tccgaccgtattcacaatgccggtgcacgagtggtgattacctctgatggtgcgtaccgcaacgcgcaggtggtgccctacaaagaagcgtat accgatcaggcgctcgataagtatattccggttgagacggcgcaggcgattgttgcgcagaccctggccaccttgcccctgactgagtcgcag cgccagacgatcatcaccgaagtggaggccgcactggccggtgagattacggttgagcgctcggacgtgatgcgtggggttggttctgccct cgcaaagctccgcgatcttgatgcaagcgtgcaggcaaaggtgcgtacagtactggcgcagg cgctggtcgagtcgccgccgcgggttgaa gctgtggtggttgtgcgtcataccggtcaggagattttgtggaacgaggggcgagatcgctggagtcacgacttgctggatgctgcgctggcg aagattctggccaatgcgcgtgctgccggctttgatgtgcacagtgagaatgatctgctcaatctccccgatgaccagcttatccgtgcgctcta cgccagtattccctgtgaaccggttgatgctgaatatccgatgtttatcatttacacatcgggtagcaccggtaagcccaagggtgtgatccacgt tcacggcggttatgtcgccggtgtggtgcacaccttgcgggtcagttttgacgccgagccgggtgatacgatatatgtgatcgccgatccgggc tggatcaccggtcagagctatatgctcacagccacaatggccggtcggctgaccggggtgattgccgagggatcaccgctcttcccctcagc cgggcgttatgccagcatcatcgagcgctatggggtgcagatctttaaggcgggtgtgaccttcctcaagacagtgatgtccaatccgcagaat gttgaagatgtgcgactctatgatatgcactcgctgcgggttgcaaccttctgcgccgagccggtcagtccggcggtgcagcagtttggtatgc agatcatgaccccgcagtatatcaattcgtactgggcgaccgagcacggtggaattgtctggacgcatttctacggtaatcaggacttcccgctt cgtcccgatgcccatacctatcccttgccctgggtgatgggtgatgtctgggtggccgaaactgatgagagcgggacgacgcgctatcgggtc gctgatttcgatgagaagggcgagattgtgattaccgccccgtatccctacctgacccgcacactctggggtgatgtgcccggtttcgaggcgt acctgcgcggtgagattccg ctgcgggcctggaagggtgatgccgagcgtttcgtcaagacctactggcgacgtgggccaaacggtgaatg gggctatatccagggtgattttgccatcaagtaccccgatggtagcttcacgctccacggacgctctgacgatgtgatcaatgtgtcgggccac cgtatgggcaccgaggagattgagggtgccattttgcgtgaccgccagatcacgcccgactcgcccgtcggtaattgtattgtggtcggtgcg ccgcaccgtgagaagggtctgaccccggttgccttcattcaacctgcgcctggccgtcatctgaccggcgccgaccggcgccgtctcgatga gctggtgcgtaccgagaagggggcggtcagtgtcccagaggattacatcgaggtcagtgcctttcccgaaacccgcagcgggaagtatatg cggcgctttttgcgcaatatgatgctcgatgaaccactgggtgatacgacgacgttgcgcaatcctgaagtgctcgaagagattgcagccaaga tcgctgagtggaaacgccgtcagcgtatggccgaagagcagcagatcatcgaacgctatcgctacttccggatcgagtatcacccaccaacg gccagtgcgggtaaactcgcggtagtgacggtgacaaatccgccggtgaacgcactgaatgagcgtgcgctcgatgagttgaacacaattgt tgaccacctggcccgtcgtcaggatgttgccgcaattgtcttcaccggacagggcgccaggagttttgtcgccggcgctgatattcgccagttg ctcgaagagattcatacggttgaagaggcaatggccctgccgaataacgcccatcttgctttccgcaagattgagcgtatgaataagccgtgtat cgcggcgatcaacggtgtggcgctcggtggtggtctggaattcgccatggcctgccattaccgggttgccgatgtctatgcc gaattcggtcag ccagagattaatctgcgcttgctacctggttatggtggcacgcagcgcttgccgcgcctgttgtacaagcgcaacaacggcaccggtctgctcc gagcgctggagatgattctgggtgggcgtagcgtaccggctgatgaggcgctggagctgggtctgatcgatgccattgctaccggcgatcag gactcactgtcgctggcatgcgcgttagcccgtgccgcaatcggcgccgatggtcagttgatcgagtcggctgcggtgacccaggctttccgc catcgccacgagcagcttgacgagtggcgcaaaccagacccgcgctttgccgatgacgaactgcgctcgattatcgcccatccacgtatcga gcggattatccggcaggcccataccgttgggcgcgatgcggcagtgcatcgggcactggatgcaatccgctatggcattatccacggcttcg aggccggtctggagcacgaggcgaagctctttgccgaggcagtggttgacccgaacggtggcaagcgtggtattcgcgagttcctcgaccg ccagagtgcgccgttgccaacccgccgaccattgattacacctgaacaggagcaactcttgcgcgatcagaaagaactgttgccggttggttc acccttcttccccggtgttgaccggattccgaagtggcagtacgcgcaggcggttattcgtgatccggacaccggtgcggcggctcacggcg atcccatcgtggctgaaaagcagattattgtgccggtggaacgcccccgcgccaatcaggcgctgatctatgttctggcctcggaggtgaactt caacgatatctgggcgattaccggtattccggtgtcacggtttgatgagcacgaccgcgactggcacgttaccggttcaggtggcatcggcctg atcgttgcgctgggtgaagaggcgcgacgcgaaggccggctgaaggtggg tgatctggtggcgatctactccgggcagtcggatctgctctc accgctgatgggccttgatccgatggccgccgatttcgtcatccaggggaacgacacgccagatggatcgcatcagcaatttatgctggccca ggccccgcagtgtctgcccatcccaaccgatatgtctatcgaggcagccggcagctacatcctcaatctcggtacgatctatcgcgccctcttta cgacgttgcaaatcaaggccggacgcaccatctttatcgagggtgcggcgaccggtaccggtctggacgcagcgcgctcggcggcccgga atggtctgcgcgtaattggaatggtcagttcgtcgtcacgtgcgtctacgctgctggctgcgggtgcccacggtgcgattaaccgtaaagaccc ggaggttgccgattgtttcacgcgcgtgcccgaagatccatcagcctgggcagcctgggaagccgccggtcagccgttgctggcgatgttcc gggcgcagaacgacgggcgactggccgattatgtggtctcgcacgcgggcgagacggccttcccgcgcagtttccagcttctcggcgagcc acgcgatggtcacattccgacgctcacattctacggtgccaccagtggctaccacttcaccttcctgggtaagccagggtcagcttcgccgacc gagatgctgcggcgggccaatctccgcgccggtgaggcggtgttgatctactacggggttgggagcgatgacctggtagataccggcggtct ggaggctatcgaggcggcgcggcaaatgggagcgcggatcgtcgtcgttaccgtcagcgatgcgcaacgcgagtttgtcctctcgttgggct tcggggctgccctacgtggtgtcgtcagcctggcggaactcaaacggcgcttcggcgatgagtttgagtggccgcgcacgatgccgccgttg ccgaacgcccgccaggacccgc agggtctgaaagaggctgtccgccgcttcaacgatctggtcttcaagccgctaggaagcgcggtcggtg tcttcttgcggagtgccgacaatccgcgtggctaccccgatctgatcatcgagcgggctgcccacgatgcactggcggtgagcgcgatgctgatcaagcccttcaccggacggattgtctacttcgaggacattggtgggcggcgttactccttcttcgcaccgcaaatctgggtgcgccagcgcc gcatctacatgccgacggcacagatctttggtacgcacctctcaaatgcgtatgaaattctgcgtctgaatgatgagatcagcgccggtctgctg acgattaccgagccggcagtggtgccgtgggatgaactacccgaagcacatcaggcgatgtgggaaaatcgccacacggcggccacttatg tggtgaatcatgccttaccacgtctcggcctaaagaacagggacgagctgtacgaggcgtggacggccggcgagcggtag SEQ ID NO.5

TCATCACCACAGCCAGGGATCCatgatcgacactgcgcccctt

SEQ ID NO.6

GCGCGCCGAGCTCGAATTCCTACCGCTCGCCGGCCGTCCACG

SEQ ID NO.7

TCATCACCACAGCCAGGGATCCatggatattcgtaagattaaaaa

SEQ ID NO. 8

GCGCGCCGAGCTCGAATTCTTATTTTTCCTGAAGACCGAG

SEQ ID NO.9

GAGATATACATATGGCAAGATCTatgaacgaacatatggatcattt

SEQ ID NO. 10

CGGCCGATATCCAATTGTTACAGCGGAATATTGCCATGT

SEQUENCE LISTING

<110> Shandong University of Technology

<120> A genetically engineered bacterium producing methylmalonyl-CoA and its preparation method and application

<160> 10

<170> PatentIn version 3.5

<210> 1

<211> 471

<212> DNA

<213> Synthetic

<400> 1

atggatattc gtaagattaa aaaactgatc gagctggttg aagaatcagg catctccgaa 60

ctggaaattt ctgaaggcga agagtcagta cgcattagcc gtgcagctcc tgccgcaagt 120

ttccctgtga tgcaacaagc ttacgctgca ccaatgatgc agcagccagc tcaatctaac 180

gcagccgctc cggcgaccgt tccttccatg gaagcgccag cagcagcgga aatcagtggt 240

cacatcgtac gttccccgat ggttggtact ttctaccgca ccccaagccc ggacgcaaaa 300

gcgttcatcg aagtgggtca gaaagtcaac gtgggcgata ccctgtgcat cgttgaagcc 360

atgaaaatga tgaaccagat cgaagcggac aaatccggta ccgtgaaagc aattctggtc 420

gaaagtggac aaccggtaga atttgacgag ccgctggtcg tcatcgagta a 471

<210> 2

<211> 1350

<212> DNA

<213> Synthetic

<400> 2

atgctggata aaattgttat tgccaaccgc ggcgagattg cattgcgtat tcttcgtgcc 60

tgtaaagaac tgggcatcaa gactgtcgct gtgcactcca gcgcggatcg cgatctaaaa 120

cacgtattac tggcagatga aacggtctgt attggccctg ctccgtcagt aaaaagttat 180

ctgaacatcc cggcaatcat cagcgccgct gaaatcaccg gcgcagtagc aatccatccg 240

ggttacggct tcctctccga gaacgccaac tttgccgagc aggttgaacg ctccggcttt 300

atcttcattg gcccgaaagc agaaaccatt cgcctgatgg gcgacaaagt atccgcaatc 360

gcggcgatga aaaaagcggg cgtcccttgc gtaccgggtt ctgacggccc gctgggcgac 420

gatatggata aaaaccgtgc cattgctaaa cgcattggtt atccggtgat tatcaaagcc 480

tccggcggcg gcggcggtcg cggtatgcgc gtagtgcgcg gcgacgctga actggcacaa 540

tccatctcca tgacccgtgc ggaagcgaaa gctgctttca gcaacgatat ggtttacatg 600

gagaaatacc tggaaaatcc tcgccacgtc gagattcagg tactggctga cggtcagggc 660

aacgctatct atctggcgga acgtgactgc tccatgcaac gccgccacca gaaagtggtc 720

gaagaagcgc cagcaccggg cattaccccg gaactgcgtc gctacatcgg cgaacgttgc 780

gctaaagcgt gtgttgatat cggctatcgc ggtgcaggta ctttcgagtt cctgttcgaa 840

aacggcgagt tctatttcat cgaaatgaac acccgtattc aggtagaaca cccggttaca 900

gaaatgatca ccggcgttga cctgatcaaa gaacagctgc gtatcgctgc cggtcaaccg 960

ctgtcgatca agcaagaaga agttcacgtt cgcggccatg cggtggaatg tcgtatcaac 1020

gccgaagatc cgaacacctt cctgccaagt ccgggcaaaa tcacccgttt ccacgcacct 1080

ggcggttttg gcgtacgttg ggagtctcat atctacgcgg gctacaccgt accgccgtac 1140

tatgactcaa tgatcggtaa gctgatttgc tacggtgaaa accgtgacgt ggcgattgcc 1200

cgcatgaaga atgcgctgca ggagctgatc atcgacggta tcaaaaccaa cgttgatctg 1260

cagatccgca tcatgaatga cgagaacttc cagcatggtg gcactaacat ccactatctg 1320

gagaaaaaac tcggtcttca ggaaaaataa 1350

<210> 3

<211> 1524

<212> DNA

<213> Synthetic

<400> 3

atgaacgaac atatggatca tttctacacc aaacgtaaac aggcagaaga aggtggcggc 60

cgtgaaaaac tggcacagca gcgtcagaaa ggtaaactga ccgcccgcga acgcattatt 120

tttctgctgg atcaggatag ttttattgaa ctgcatccgt ttatggaaag ccaggtgctg 180

acccgtgaac agcgtatgct gggtgacggt gttgttaccg gttatggtac cattgatggt 240

cgcagcgttt atgtttttgc acaggatttt accgtgtatg gtggtgccct gggtgaaacc 300

catgcccgca aaatttgcgc actgatggat ctggccgcca aaaataaggc accgattatt 360

ggtctgaatg atagcggtgg tgcccgtatt caggaaggtg ttctgagcct ggatggttat 420

ggccatattt tctatcgtaa tgttctgtat agcggtgtta ttccgcagat tagcgttatt 480

ctgggtccgt gtgcaggcgg tgccgtttat agcccggccc tgaccgattt tatttttatg 540

gccgaacaga ccggtcgtat gtttattacc ggcccgaaag ttattgaaaa agttaccggt 600

gaacaggtgg atgccgaaag tctgggcggc gcaggtattc ataatgcagt gagtggtaat 660

gcacatttta gcggccatac cgaaaaagaa gttctgaccg gtgtgcgtaa actgctgagt 720

tatctgccgc tgaatggccg taccaccgaa ccgaaaccgg aaaaagaagc aagccgtccg 780

ctgctgaatc gtctggttcc ggccgatacc accaaaccgt atgatgtgcg taaagttatt 840

cgcgaactgg ccgatccgca gagctttttc gaaattcagc cgtttttcgc aaaaaatatt 900

gttattggtt tcgcacgtct gggtgaaaaa gccattggta ttgtggcaag tcagccgaaa 960

catctggcag gtagtctgac cattgatgcc gcagataaag cagcacgttt tattcgtttt 1020

tgtgatgcat ttgatatccc gctgctgacc gtggaagatg tgccgggttt tctgccgggc 1080

attcagcagg aacataatgg tattattcgc catggcgcaa aactgctgtt tgcctatgca 1140

gaagcaaccg tgccgaaagt taccctgatt attcgcaaag cctatggtgg cgcatatgtg 1200

gccatgaata gcaaagccat tggcgccgat ctggttttttg cctggccgaa tgccgaaatt 1260

gcagttatgg gtccggaagg tgcagcaagc attctgtatg aaaaagaaat taaggccagc 1320

gccgatccgc aaaaaaccaa acgtgaaaaa accgcagaat ataaaaagca gaatgcaggc 1380

ccgtataaag cagccgcatg cggcatggtg gatgatatta ttctgccgga agaaagtcgc 1440

ggtcgtctga ttcaggcatt tcatatgctg acccataaaa ccgaagaacg tccgaaaaag 1500

aaacatggca atattccgct gtaa 1524

<210> 4

<211> 5469

<212> DNA

<213> Synthetic

<400> 4

atgatcgaca ctgcgcccct tgccccacca cgggcgcccc gctctaatcc gattcgggat 60

cgagttgatt gggaagctca gcgcgctgct gcgctggcag atcccggtgc ctttcatggc 120

gcgattgccc ggacagttat ccactggtac gacccacaac accattgctg gattcgcttc 180

aacgagtcta gtcagcgttg ggaagggctg gatgccgcta ccggtgcccc tgtaacggta 240

gactatcccg ccgattatca gccctggcaa caggcgtttg atgatagtga agcgccgttt 300

taccgctggt ttagtggtgg gttgacaaat gcctgcttta atgaagtaga ccggcatgtc 360

acgatgggct atggcgacga ggtggcctac tactttgaag gtgaccgctg ggataactcg 420

ctcaacaatg gtcgtggtgg tccggttgtc caggagacaa tcacgcggcg gcgcctgttg 480

gtggaggtgg tgaaggctgc gcaggtgttg cgtgatctgg gcctgaagaa gggtgatcgg 540

attgctctga atatgccgaa tattatgccg cagatttatt atacggaagc ggcaaaacga 600

ctgggtattc tgtacacgcc ggtcttcggt ggcttctcgg acaagactct ttccgaccgt 660

attcacaatg ccggtgcacg agtggtgatt acctctgatg gtgcgtaccg caacgcgcag 720

gtggtgccct acaaagaagc gtataccgat caggcgctcg ataagtatat tccggttgag 780

acggcgcagg cgattgttgc gcagaccctg gccaccttgc ccctgactga gtcgcagcgc 840

cagacgatca tcaccgaagt ggaggccgca ctggccggtg agattacggt tgagcgctcg 900

gacgtgatgc gtggggttgg ttctgccctc gcaaagctcc gcgatcttga tgcaagcgtg 960

caggcaaagg tgcgtacagt actggcgcag gcgctggtcg agtcgccgcc gcgggttgaa 1020

gctgtggtgg ttgtgcgtca taccggtcag gagattttgt ggaacgaggg gcgagatcgc 1080

tggagtcacg acttgctgga tgctgcgctg gcgaagattc tggccaatgc gcgtgctgcc 1140

ggctttgatg tgcacagtga gaatgatctg ctcaatctcc ccgatgacca gcttatccgt 1200

gcgctctacg ccagtattcc ctgtgaaccg gttgatgctg aatatccgat gtttatcatt 1260

tacacatcgg gtagcaccgg taagcccaag ggtgtgatcc acgttcacgg cggttatgtc 1320

gccggtgtgg tgcacacctt gcgggtcagt tttgacgccg agccgggtga tacgatatat 1380

gtgatcgccg atccgggctg gatcaccggt cagagctata tgctcacagc cacaatggcc 1440

ggtcggctga ccggggtgat tgccgaggga tcaccgctct tcccctcagc cgggcgttat 1500

gccagcatca tcgagcgcta tggggtgcag atctttaagg cgggtgtgac cttcctcaag 1560

acagtgatgt ccaatccgca gaatgttgaa gatgtgcgac tctatgatat gcactcgctg 1620

cgggttgcaa ccttctgcgc cgagccggtc agtccggcgg tgcagcagtt tggtatgcag 1680

atcatgaccc cgcagtatat caattcgtac tgggcgaccg agcacggtgg aattgtctgg 1740

acgcatttct acggtaatca ggacttcccg cttcgtcccg atgcccatac ctatcccttg 1800

ccctgggtga tgggtgatgt ctgggtggcc gaaactgatg agagcgggac gacgcgctat 1860

cgggtcgctg atttcgatga gaagggcgag attgtgatta ccgccccgta tccctacctg 1920

acccgcacac tctggggtga tgtgcccggt ttcgaggcgt acctgcgcgg tgagattccg 1980

ctgcgggcct ggaagggtga tgccgagcgt ttcgtcaaga cctactggcg acgtgggcca 2040

aacggtgaat ggggctatat ccagggtgat tttgccatca agtaccccga tggtagcttc 2100

acgctccacg gacgctctga cgatgtgatc aatgtgtcgg gccaccgtat gggcaccgag 2160

gagattgagg gtgccatttt gcgtgaccgc cagatcacgc ccgactcgcc cgtcggtaat 2220

tgtattgtgg tcggtgcgcc gcaccgtgag aagggtctga ccccggttgc cttcattcaa 2280

cctgcgcctg gccgtcatct gaccggcgcc gaccggcgcc gtctcgatga gctggtgcgt 2340

accgagaagg gggcggtcag tgtcccagag gattacatcg aggtcagtgc ctttcccgaa 2400

acccgcagcg ggaagtatat gcggcgcttt ttgcgcaata tgatgctcga tgaaccactg 2460

ggtgatacga cgacgttgcg caatcctgaa gtgctcgaag agattgcagc caagatcgct 2520

gagtggaaac gccgtcagcg tatggccgaa gagcagcaga tcatcgaacg ctatcgctac 2580

ttccggatcg agtatcaccc accaacggcc agtgcgggta aactcgcggt agtgacggtg 2640

acaaatccgc cggtgaacgc actgaatgag cgtgcgctcg atgagttgaa cacaattgtt 2700

gaccacctgg cccgtcgtca ggatgttgcc gcaattgtct tcaccggaca gggcgccagg 2760

agttttgtcg ccggcgctga tattcgccag ttgctcgaag agattcatac ggttgaagag 2820

gcaatggccc tgccgaataa cgcccatctt gctttccgca agattgagcg tatgaataag 2880

ccgtgtatcg cggcgatcaa cggtgtggcg ctcggtggtg gtctggaatt cgccatggcc 2940

tgccattacc gggttgccga tgtctatgcc gaattcggtc agccagagat taatctgcgc 3000

ttgctacctg gttatggtgg cacgcagcgc ttgccgcgcc tgttgtacaa gcgcaacaac 3060

ggcaccggtc tgctccgagc gctggagatg attctgggtg ggcgtagcgt accggctgat 3120

gaggcgctgg agctgggtct gatcgatgcc attgctaccg gcgatcagga ctcactgtcg 3180

ctggcatgcg cgttagcccg tgccgcaatc ggcgccgatg gtcagttgat cgagtcggct 3240

gcggtgaccc aggctttccg ccatcgccac gagcagcttg acgagtggcg caaaccagac 3300

ccgcgctttg ccgatgacga actgcgctcg attatcgccc atccacgtat cgagcggatt 3360

atccggcagg cccataccgt tgggcgcgat gcggcagtgc atcgggcact ggatgcaatc 3420

cgctatggca ttatccacgg cttcgaggcc ggtctggagc acgaggcgaa gctctttgcc 3480

gaggcagtgg ttgacccgaa cggtggcaag cgtggtattc gcgagttcct cgaccgccag 3540

agtgcgccgt tgccaacccg ccgaccattg attacacctg aacaggagca actcttgcgc 3600

gatcagaaag aactgttgcc ggttggttca cccttcttcc ccggtgttga ccggattccg 3660

aagtggcagt acgcgcaggc ggttattcgt gatccggaca ccggtgcggc ggctcacggc 3720

gatcccatcg tggctgaaaa gcagattatt gtgccggtgg aacgcccccg cgccaatcag 3780

gcgctgatct atgttctggc ctcggaggtg aacttcaacg atatctgggc gattaccggt 3840

attccggtgt cacggtttga tgagcacgac cgcgactggc acgttaccgg ttcaggtggc 3900

atcggcctga tcgttgcgct gggtgaagag gcgcgacgcg aaggccggct gaaggtgggt 3960

gatctggtgg cgatctactc cgggcagtcg gatctgctct caccgctgat gggccttgat 4020

ccgatggccg ccgatttcgt catccagggg aacgacacgc cagatggatc gcatcagcaa 4080

tttatgctgg cccaggcccc gcagtgtctg cccatcccaa ccgatatgtc tatcgaggca 4140

gccggcagct acatcctcaa tctcggtacg atctatcgcg ccctctttac gacgttgcaa 4200

atcaaggccg gacgcaccat ctttatcgag ggtgcggcga ccggtaccgg tctggacgca 4260

gcgcgctcgg cggcccggaa tggtctgcgc gtaattggaa tggtcagttc gtcgtcacgt 4320

gcgtctacgc tgctggctgc gggtgcccac ggtgcgatta accgtaaaga cccggaggtt 4380

gccgattgtt tcacgcgcgt gcccgaagat ccatcagcct gggcagcctg ggaagccgcc 4440

ggtcagccgt tgctggcgat gttccgggcg cagaacgacg ggcgactggc cgattatgtg 4500

gtctcgcacg cgggcgagac ggccttcccg cgcagtttcc agcttctcgg cgagccacgc 4560

gatggtcaca ttccgacgct cacattctac ggtgccacca gtggctacca cttcaccttc 4620

ctgggtaagc cagggtcagc ttcgccgacc gagatgctgc ggcgggccaa tctccgcgcc 4680

ggtgaggcgg tgttgatcta ctacggggtt gggagcgatg acctggtaga taccggcggt 4740

ctggaggcta tcgaggcggc gcggcaaatg ggagcgcgga tcgtcgtcgt taccgtcagc 4800

gatgcgcaac gcgagtttgt cctctcgttg ggcttcgggg ctgccctacg tggtgtcgtc 4860

agcctggcgg aactcaaacg gcgcttcggc gatgagtttg agtggccgcg cacgatgccg 4920

ccgttgccga acgcccgcca ggacccgcag ggtctgaaag aggctgtccg ccgcttcaac 4980

gatctggtct tcaagccgct aggaagcgcg gtcggtgtct tcttgcggag tgccgacaat 5040

ccgcgtggct accccgatct gatcatcgag cgggctgccc acgatgcact ggcggtgagc 5100

gcgatgctga tcaagccctt caccggacgg attgtctact tcgaggacat tggtgggcgg 5160

cgttactcct tcttcgcacc gcaaatctgg gtgcgccagc gccgcatcta catgccgacg 5220

gcacagatct ttggtacgca cctctcaaat gcgtatgaaa ttctgcgtct gaatgatgag 5280

atcagcgccg gtctgctgac gattaccgag ccggcagtgg tgccgtggga tgaactaccc 5340

gaagcacatc aggcgatgtg ggaaaatcgc cacacggcgg ccacttatgt ggtgaatcat 5400

gccttaccac gtctcggcct aaagaacagg gacgagctgt acgaggcgtg gacggccggc 5460

gagcggtag 5469

<210> 5

<211> 43

<212> DNA

<213> Synthetic

<400> 5

tcatcaccac agccagggat ccatgatcga cactgcgccc ctt 43

<210> 6

<211> 42

<212> DNA

<213> Synthetic

<400> 6

gcgcgccgag ctcgaattcc taccgctcgc cggccgtcca cg 42

<210> 7

<211> 45

<212> DNA

<213> Synthetic

<400> 7

tcatcaccac agccagggat ccatggatat tcgtaagatt aaaaa 45

<210> 8

<211> 40

<212> DNA

<213> Synthetic

<400> 8

gcgcgccgag ctcgaattct tatttttcct gaagaccgag 40

<210> 9

<211> 46

<212> DNA

<213> Synthetic

<400> 9

gagatataca tatggcaaga tctatgaacg aacatatgga tcattt 46

<210> 10

<211> 39

<212> DNA

<213> Synthetic

<400> 10

cggccgatat ccaattgtta cagcggaata ttgccatgt 39

Claims (10)

1. The genetically engineered bacterium for producing methylmalonate monoacyl-coenzyme A is characterized in that escherichia coli is used as an initial bacterium, and an acetyl coenzyme A carboxylase BCCP subunit gene accB, an acetyl coenzyme A carboxylase BC subunit gene accC, a propionyl coenzyme A carboxylase CT subunit gene pc and a propionyl coenzyme A synthase gene pcs are overexpressed.

2. The genetically engineered bacterium of claim 1, wherein the acetyl-CoA carboxylase BCCP subunit gene accB is derived from Escherichia coli and has a gene sequence shown in SEQ ID No.1, the acetyl-CoA carboxylase BC subunit gene accC is derived from Escherichia coli and has a gene sequence shown in SEQ ID No.2, the propionyl-CoA carboxylase CT subunit gene pc is derived from Bacillus subtilis and has a gene sequence shown in SEQ ID No.3, and the propionyl-CoA synthase gene pcs is derived from Chlorotrichum aurantium and has a gene sequence shown in SEQ ID No. 4.

3. The method for producing a genetically engineered bacterium according to claim 1 or 2, characterized in that the production method is as follows:

step 1: connecting acetyl coenzyme A carboxylase BCCP subunit gene accB, acetyl coenzyme A carboxylase BC subunit gene accC and propionyl coenzyme A carboxylase CT subunit gene pc to an expression vector pETDuet to obtain a recombinant vector 1;

step 2: propionyl coenzyme A synthase gene pcs is connected to an expression vector pACYCDuet to obtain a recombinant vector 2;

and step 3: and (3) co-transferring the recombinant vector 1 and the recombinant vector 2 obtained in the steps (1) and (2) to escherichia coli BL21(DE3) to obtain the genetically engineered bacterium.

4. A method for intracellular production of methylmalonate monoacyl-coenzyme A, characterized in that the genetically engineered bacterium of claim 1 or 2 is fermented in a fermentation broth containing 3-hydroxypropionic acid at 37 ℃ for at least 5 h.

5. The method of claim 4, wherein the fermentation broth has a composition: 14.04g/L dipotassium hydrogen phosphate trihydrate, 5.24g/L potassium dihydrogen phosphate, 1g/L sodium chloride, 1g/L ammonium chloride, 3g/L yeast powder, 4g/L sodium bicarbonate, 40mg/L biotin, 0.5g/L magnesium sulfate, 20g/L glucose and 2 g/L3-hydroxypropionic acid.

6. A method for extracellular production of methylmalonate monoacyl-coenzyme A, characterized in that the genetically engineered bacterium of claim 1 or 2 is treated to obtain a supernatant, and the supernatant is reacted at 37 ℃ for at least 1h by using a culture solution containing 3-hydroxypropionic acid as a reaction substrate.

7. The method according to claim 6, wherein the composition of the culture solution is: Tris-HCl, MgCl2, DTT, KCl, NaHCO3, biotin, coenzyme A, NADPH, ATP and 3-hydroxypropionic acid.

8. The method according to claim 6, wherein the supernatant is obtained by the following method: inoculating the genetically engineered bacterium of claim 1 or 2 into an LB culture medium, culturing at 30 ℃ until the bacterial concentration is 0.6-0.8 OD600, adding IPTG, culturing at 30 ℃ for 6-8 h to obtain a culture solution, centrifuging the culture solution to obtain a bacterial suspension, crushing to obtain a cell crushing solution, and centrifuging to obtain a supernatant of the cell crushing solution.

9. The method of claim 6, wherein the supernatant is at a concentration of 10 mg/ml.

10. The use of the genetically engineered bacterium of claim 1 or 2 for the production of methylmalonate monoacyl-coenzyme A.