CN113122486A - Method for total biosynthesis of malonic acid - Google Patents

Abstract

The invention discloses a full biosynthesis method of malonic acid, and belongs to the technical field of bioengineering. The invention uses Escherichia coli BL21(DE3) as a host, and modular over-expresses phosphoenolpyruvate carboxylase gene (ppc), succinate dehydrogenase gene (sdhC), succinate semialdehyde dehydrogenase gene (yne1), aspartate enzyme gene (aspa), heterologous gene aspartate-alpha-dehydrogenase gene (panD) from Corynebacterium glutamicum and heterologous gene beta-alanine pyruvate transaminase gene (pa0123) from Pseudomonas aeruginosa, so that a complete biosynthesis pathway of malonic acid is constructed, and engineering bacteria can successfully accumulate malonic acid. The biological manufacture of the malonic acid and the precursor thereof has the advantages of less pollution, high product quality and the like, and has great development prospect.

Description

Method for total biosynthesis of malonic acid

Technical Field

The invention relates to a method for total biosynthesis of malonic acid, and belongs to the field of bioengineering.

Background

Malonic acid (malonic acid, malonate; Propane diacid) is also known as carotic acid, malic acid, or betainic acid. The molecular structure has two functional groups of active methylene and carboxyl, so that the compound can participate in various chemical reactions and is an important organic synthesis intermediate. The high-temperature-resistant flame-retardant epoxy resin is mainly applied to aspects of perfumes, adhesives, resin additives, medical intermediates, electroplating polishing agents, explosion control agents, thermal welding fluxing additives and the like.

At present, the method for hydrolyzing cyanoacetic acid and malonate is commonly used in industry to prepare malonic acid. However, the hydrolytic cyanoacetic acid method uses NaCN, and cyanide ions are extremely toxic, have great harm to the environment and have complex reaction process. The hydrolysis of malonate mainly uses dimethyl (ethyl) malonate to directly hydrolyze to prepare malonic acid, wherein the malonate is also neutralized by chloroacetic acid, cyanided and the like to prepare cyanoacetic acid, and then esterified to produce the malonate, which has the disadvantages of large environmental hazard and complex reaction process. And the malonic acid obtained by the two methods needs to be subjected to complicated and tedious purification procedures, so that the industrial production is limited.

In order to solve the above problems, attention has been focused on the way to biosynthesize malonic acid. However, due to the lack of knowledge of suitable enzymes and metabolic pathways, the research on the biological production of malonic acid has been slow. Malonic semialdehyde is an important precursor of malonic acid, but this conversion is difficult due to the absence of malonic semialdehyde dehydrogenase, and thus the progress of malonic acid biological production is slow.

Disclosure of Invention

In order to solve the problems, the invention firstly provides a recombinant escherichia coli for producing malonic acid, which takes escherichia coli BL21(DE3) as a host, and expresses phosphoenolpyruvate carboxylase gene (ppc), succinate dehydrogenase gene (sdhC), succinate semialdehyde dehydrogenase gene (yne1), aspartate enzyme gene (aspA), heterologous gene aspartate-alpha-dehydrogenase gene (panD) from corynebacterium glutamicum and heterologous gene beta-alanine pyruvate transaminase gene (pa0123) from pseudomonas aeruginosa in a modularized manner.

In one embodiment of the invention, the partial over-expression is fusion expression of genes yne1 and pa0123, fusion expression of genes aspa and panD, and fusion expression of genes sdhC and ppc.

In one embodiment, the nucleotide sequence of the ppc is shown as SEQ ID No.1, and the nucleotide sequence of the sdhC is shown as SEQ ID No. 2; the nucleotide sequence of the aspA is shown as SEQ ID NO.3, and the nucleotide sequence of the panD is shown as SEQ ID NO. 4; the nucleotide sequence of pa0123 is shown as SEQ ID NO.5, and the nucleotide sequence of yne1 is shown as SEQ ID NO. 6.

In one embodiment of the invention, the genes yne1 and pa0123 use pRSFDuet-1 as an expression vector; the genes aspa and panD take pTrc99a as an expression vector; the gene fragments sdhC and ppc use pCDFDuet-1 as an expression vector.

The invention also provides a method for constructing the recombinant escherichia coli, which comprises the following steps:

(1) connecting gene fragments yne1 and pa0123 by using the plasmid pRSFDuet-1 as a skeleton vector to obtain a recombinant plasmid pRSF-yne1-pa 0123;

(2) connecting gene fragments aspa and panD by taking the plasmid pTrc99a as a framework vector to obtain a recombinant plasmid pTrc99 a-aspa-panD;

(3) connecting gene fragments sdhC and ppc by using the plasmid pCDFDuet-1 as a skeleton vector to obtain a recombinant plasmid pCDF-sdhC-ppc;

(4) pRSF-yne1-pa0123, pTrc99a-aspa-panD and pCDF-sdhC-ppc were co-transferred into E.coli BL21(DE3) to obtain recombinant E.coli.

In one embodiment of the present invention, in step (1), the gene fragment pa0123 and the plasmid pRSFDuet-1 are cleaved with Nco I and Hind III enzymes, and then treated with T₄DNA ligase is connected to obtain a recombinant plasmid pRSF-pa 0123; both yne1 and the recombinant plasmid pRSF-pa0123 were digested simultaneously with Xho I and Nde I, and then with T₄And (3) connecting the DNA with ligase to obtain a recombinant plasmid pRSF-yne1-pa 0123.

In one embodiment of the present invention, in step (2), the gene fragment aspa and the plasmid pTrc99a are both Sal I and Xho IAfter enzymatic cleavage with T₄DNA ligase is connected to obtain a recombinant plasmid pTrc99 a-aspa; panD and the recombinant plasmid pTrc99a-aspa were digested simultaneously with Xho I and Hind III, and then with T₄The DNA ligases are connected to obtain the recombinant plasmid pTrc99 a-aspa-panD.

In one embodiment of the present invention, in step (3), the gene fragment sdhC and the plasmid pCDFDuet-1 are both digested with Nco I and EcoR I, and then treated with T₄DNA ligase is connected to obtain a recombinant plasmid pCDF-sdhC; the ppc and the recombinant plasmid pCDF-sdhC were both digested with EcoR I and Sal I, followed by T₄And (4) connecting the DNA with ligase to obtain a recombinant plasmid pCDF-sdhC-ppc.

The third purpose of the invention is to provide a method for producing malonic acid by fermenting the recombinant escherichia coli, which takes an SOB culture medium as a fermentation culture medium, and cultures the recombinant escherichia coli at 35-37 ℃ to OD₆₀₀When the concentration is 0.6-0.8, 1mM IPTG is added and the temperature is reduced to 30 ℃ for induction culture for 48 hours.

In one embodiment of the invention, the composition of the SOB medium is 2g/100ml tryptone, 0.5g/100ml yeast powder, 0.05g/100ml NaCl, 2.5mM KCl, 10mM MgCl₂0.8g/100ml glucose, 50. mu.g/ml kanamycin sulfate, 50. mu.g/ml ampicillin, 50. mu.g/ml streptomycin.

In one embodiment of the present invention, the seed solution is prepared by streaking glycerol-preserved strain on a plate, picking a single colony and inoculating into a 250ml Erlenmeyer flask containing 50ml LB liquid medium, shaking at 37 deg.C and 250rpm/min overnight. Transferring 1ml of the bacterial liquid to 50ml of LB liquid culture medium the next day, culturing at 37 ℃ and 250rpm to OD₆₀₀When reaching 0.6-0.8, the cells were inoculated into 50ml of SOB fermentation medium.

The invention also claims the application of the method in the preparation of malonic acid or a derivative product thereof.

Has the advantages that: compared with a chemical method, the Escherichia coli total biological method for synthesizing the malonic acid firstly takes glucose as a precursor, can be continuously developed, and greatly reduces the pollution degree to the environment. The method provided by the invention is a way for producing the malonic acid by fermenting the glucose, is a way with higher yield, provides environmental benefits by eliminating cyanide, chloroacetic acid and the like, can synthesize the malonic acid by utilizing the glucose by only over-expressing 2 genes, namely the heterologous gene aspartic acid-alpha-dehydrogenase gene (panD) from corynebacterium glutamicum and the heterologous gene beta-alanine pyruvate transaminase gene (pa0123) from pseudomonas aeruginosa without using other compounds as a premise, is simple, ensures that the high yield of the malonic acid of escherichia coli BL21(DE3) is possible due to mature fermentation process, and has the advantages of convenient operation and low cost, and the yield can reach 0.23g/L after 48 hours of fermentation.

Drawings

FIG. 1 is a malonic acid synthesis pathway.

FIG. 2 is a map of pRSF-yne1-pa0123 plasmid.

FIG. 3 is a map of pTrc99a-aspa-panD plasmid.

FIG. 4 is a map of the pCDF-sdhC-ppc plasmid.

FIG. 5 is a plasmid colony pcr validation map of pRSF-yne1-pa0123, 1: marker, 2-5: all the colonies are pRSF-yne1-pa0123 and pcr verifies pa 0123.

FIG. 6 is a graph of the results of fermentation of recombinant E.coli in SOB medium, IPTG 1 mM.

FIG. 7 is a liquid phase mass spectrometric detection profile of malonic acid; wherein, (A) is the liquid phase diagram of the malonic acid standard sample and the liquid phase diagram only showing the malonic acid standard sample; (B) respectively showing the mass spectrogram of a malonic acid standard sample and the mass spectrogram only showing malonic acid; (C) respectively showing a liquid phase diagram of a fermentation liquor sample and a liquid phase diagram only showing malonic acid in the fermentation liquor; (D) respectively showing the mass spectrogram of a fermentation liquor sample and the mass spectrogram of only malonic acid in the fermentation liquor; STANDARD is 1g/L of STANDARD malonic acid sample, and PA is a fermentation liquid sample.

Detailed Description

Liquid phase mass spectrum detection and result analysis of malonic acid:

pretreatment: centrifuging the fermentation sample at 12,000r/min for 2min to separate the fermentation liquid from the thallus, treating the fermentation liquid with a 0.22 μm filter membrane, and performing liquid phase mass spectrometry.

Liquid phase mass spectrum conditions: detection wavelength: 200-400n, analytical column: BEH C18(2.1 × 150mm 1.7.7 um), column temperature 45 ℃, flow rate: 0.3ml/min, sample size: 5 μ L, detector: waters Acquity PDA (200-400 nm).

The mobile phase conditions were as follows:

TABLE 1 primer sequence Listing in relation to the following examples

Example 1: constructing recombinant plasmid and obtaining recombinant Escherichia coli.

The nucleotide sequences of ppc, sdhC, aspA, panD, pa0123 and yne1 are shown in SEQ ID NO. 1-6 respectively.

Double digestion of plasmid pRSFDuet-1 with Nco I and Hind III, gel cutting to recover target gene fragment pRSF-1(3761bp), PCR amplification with primer pa0123-F/R and plasmid pUC57-pa0123 synthesized by Jinwei as template to obtain target gene fragment pa0123 (SEQ ID NO. 5), and T-digestion of both pRSF-1 and pa0123₄DNA ligase ligation, transformation JM109, colony PCR selection of positive transformants, plasmid restriction and PCR verification, verification of veri-pRSF-F/R primer, and verification of correct plasmid named pRSF-pa 0123.

Xho I and Nde I are used for enzyme digestion of plasmid pRSF-pa0123, gel cutting is carried out, a target gene fragment pRSF-2(5101bp) is recovered, the same enzyme digestion is carried out, large intestine genome is used as a template, a primer yne1-F/R is used, a yne1 fragment (shown as SEQ ID NO. 6) obtained through PCR amplification is used, and then two target fragments of pRSF-2 and yne1 are used as T₄DNA ligase ligation, JM109 transformation, colony PCR selection of positive transformants, plasmid restriction and PCR verification, verification of veri-pRSF-F/R primer, and verification of correct plasmid designated pRSF-yne1-pa0123 (plasmid map is shown as figure in the specification)Shown at 2).

The plasmid pTrc99A was digested with Sal I and Xho I, the desired vector DNA fragment pTrc99A-1(4170bp) was recovered by cutting the gel, the genome of the large intestine was used as a template, the resulting aspa fragment (shown in SEQ ID NO. 3) was PCR-amplified with primer aspa-F/R by the same enzyme digestion, and then the two desired fragments pTrc99A-1 and aspa were subjected to T-cleavage with T₄DNA ligase ligation, conversion JM109, colony PCR selection of positive transformants, plasmid digestion and PCR verification, verification of the primer veri-pTrc-F/R, and the verification of the correct plasmid named as pTrc99 a-aspa.

The plasmid pTrc99a-aspa was digested with Xho I and Hind III, the vector DNA fragment pTrc99A-2(5622bp) was recovered by gel cutting, the plasmid pUC57-panD synthesized by Kingson was digested with the same enzymes as the template, the target gene fragment panD (shown in SEQ ID NO. 4) was obtained by PCR amplification using the primer panD-F/R, and then the two target fragments pTrc99A-2 and panD were digested with T₄DNA ligase ligation, JM109 transformation, colony PCR to pick up positive transformants, plasmid digestion and PCR verification are extracted, the primers are verified to be veri-pTrc-F/R, and the plasmid after verification is named as pTrc99a-aspa-panD (a plasmid map is shown in figure 3).

The plasmid pCDFDuet-1 is digested by Nco I and EcoR I, the DNA fragment pCDF-1(3744bp) of the target vector is recovered by cutting the gel, the genome of the large intestine is used as a template, the ppc fragment (shown as SEQ ID NO. 1) obtained by PCR amplification is digested by the same enzyme with the primer ppc-F/R, and then the two target fragments pCDF-1 and ppc are digested by T₄DNA ligase ligation, transformation JM109, colony PCR selection of positive transformants, plasmid restriction enzyme digestion and PCR verification, verification primer veri-pCDF-F/R, and the plasmid after verification is named as pCDF-ppc.

Cutting plasmid pCDF-ppc with EcoR I and Sal I, cutting to recover target DNA fragment pCDF-2(6379bp), cutting with the same enzyme and using large intestine genome as template, PCR amplifying obtained sdhC fragment (shown as SEQ ID NO. 2) with primer sdhC-F/R, then cutting with T₄DNA ligase ligation, JM109 transformation, colony PCR selection of positive transformants, plasmid restriction and PCR verification, verification primer veri-pCDF-F/R, verification of correct plasmid named pCDF-ppc-sdhC (plasmid map as figure)Shown at 4).

pRSF-yne1-pa0123, pTrc99a-aspa-panD and pCDF-sdhC-ppc were co-transferred into E.coli BL21(DE3) competent cells to prepare recombinant E.coli.

Example 2: shake flask fermentation and result analysis of recombinant escherichia coli.

Fermentation medium: SOB culture medium with components of 20g/L tryptone, 5g/L yeast powder, 0.5g/L NaCl, 2.5mM KCl, 10mM MgCl₂4g/L glucose, 50. mu.g/ml kanamycin sulfate, 50. mu.g/ml ampicillin, 50. mu.g/ml streptomycin.

Preparing a seed solution: the glycerol-preserved strain was streaked on a plate, and a single colony was picked and inoculated into a 250ml Erlenmeyer flask containing 50ml of LB liquid medium, and shaken overnight at 37 ℃ and 250 rpm/min.

Fermentation conditions are as follows: 2% inoculum size (1ml), inoculated in shake flask fermentation medium, to give initial OD₆₀₀Is 0.1. Culturing at 37 deg.C and 250r/min to OD₆₀₀When the concentration is 0.8-1.0 respectively, 1.0mM IPTG is added to induce the recombinant bacteria, and the culture is changed to 30 ℃ and 250 rpm/min.

And (4) analyzing results: sampling every 4H for the first 12H and every 12H for the last 60H during fermentation, centrifuging at 12,000r/min for 5min to separate the fermentation liquid from thallus, treating the fermentation liquid with 0.22 μm filter membrane for HPLC (high performance liquid chromatography, U.S. Bo Bio-Rad Bo Aminex HPX-87H organic acid column) detection with mobile phase of 5mM H₂SO₄The column temperature was 30 ℃ and the differential detector. According to the liquid phase results, no malonic acid production was detected for the first 24 hours, from 36 hours, malonic acid production was detected, and the yield reached a maximum of 0.23g/L at 48 hours.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> university of south of the Yangtze river

<120> method for total biosynthesis of malonic acid

<160> 6

<170> PatentIn version 3.3

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atgaacgaac aatattccgc attgcgtagt aatgtcagta tgctcggcaa agtgctggga 60

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ttgtcgaaat cttcacgcgc tggcaatgat gctaaccgcc aggagttgct caccacctta 180

caaaatttgt cgaacgacga gctgctgccc gttgcgcgtg cgtttagtca gttcctgaac 240

ctggccaaca ccgccgagca ataccacagc atttcgccga aaggcgaagc tgccagcaac 300

ccggaagtga tcgcccgcac cctgcgtaaa ctgaaaaacc agccggaact gagcgaagac 360

accatcaaaa aagcagtgga atcgctgtcg ctggaactgg tcctcacggc tcacccaacc 420

gaaattaccc gtcgtacact gatccacaaa atggtggaag tgaacgcctg tttaaaacag 480

ctcgataaca aagatatcgc tgactacgaa cacaaccagc tgatgcgtcg cctgcgccag 540

ttgatcgccc agtcatggca taccgatgaa atccgtaagc tgcgtccaag cccggtagat 600

gaagccaaat ggggctttgc cgtagtggaa aacagcctgt ggcaaggcgt accaaattac 660

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aaagatattc aggtgctggt ttctgaactg tcgatggttg aagcgacccc tgaactgctg 900

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cgtcataccg aagcgctggg cgagctgacc cgctacctcg gtatcggcga ctacgaaagc 1260

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attgccgaag caccgcaagg ctccattgcc gcctacgtga tctcgatggc gaaaacgccg 1440

tccgacgtac tggctgtcca cctgctgctg aaagaagcgg gtatcgggtt tgcgatgccg 1500

gttgctccgc tgtttgaaac cctcgatgat ctgaacaacg ccaacgatgt catgacccag 1560

ctgctcaata ttgactggta tcgtggcctg attcagggca aacagatggt gatgattggc 1620

tattccgact cagcaaaaga tgcgggagtg atggcagctt cctgggcgca atatcaggca 1680

caggatgcat taatcaaaac ctgcgaaaaa gcgggtattg agctgacgtt gttccacggt 1740

cgcggcggtt ccattggtcg cggcggcgca cctgctcatg cggcgctgct gtcacaaccg 1800

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tatggtctgc cagaaatcac cgtcagcagc ctgtcgcttt ataccggggc gattctggaa 1920

gccaacctgc tgccaccgcc ggagccgaaa gagagctggc gtcgcattat ggatgaactg 1980

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tacttccgct ccgctacgcc ggaacaagaa ctgggcaaac tgccgttggg ttcacgtccg 2100

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ggcgcaggta cttccgtaaa catgaacacc aacgaagtgc tggccaatat cggtctggaa 360

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tgcgtaccgg ctgaagacct gatcgaagcg acctctgact gcggcgctta tgttatggtt 840

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gagggctcct ggctgaccga cgacaagggc cgtaaagtat acgatagcct gtcaggatta 180

tggacctgcg gtgcggggca tagccgcaag gaaattcagg aagcggttgc tcgtcaactg 240

gggactttgg actattcgcc aggattccaa tatggacatc cattgtcttt ccagttggcc 300

gagaagattg ctgggttatt acctggggaa ttaaaccatg tcttttttac gggatcaggg 360

tcggagtgcg cagacacttc gattaagatg gcccgcgcct actggcgctt aaagggacaa 420

ccccagaaga ctaagctgat tggacgcgca cgcggttacc acggcgtgaa tgtcgcgggc 480

acaagccttg gagggatcgg ggggaaccgc aagatgttcg gacagctgat ggatgtggac 540

catcttcccc atacccttca gccaggtatg gcattcactc gcgggatggc gcagacagga 600

ggcgttgaac tggcgaatga gttattaaag ttaattgaat tgcacgatgc gtctaacatc 660

gcagcggtta ttgtcgagcc catgtccggt tccgcaggag ttttagtgcc acccgtgggc 720

tatctgcagc gtttgcgcga aatctgtgac caacacaata ttctgcttat ctttgatgaa 780

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ccggacttga tgaatgttgc aaaacaggtc acgaatggtg cagtacctat gggtgctgta 900

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gaattttccc acggttatac atattcagcg cacccagtgg catgtgctgc gggattagca 1020

gcactggaca tcttggcgcg cgataactta gtacagcagt cagcagagtt agctccacac 1080

ttcgagaagg gattgcatgg ccttcaaggt gccaagaatg ttatcgacat tcgcaactgc 1140

ggcttagcag gcgcgatcca gatcgctccc cgtgatgggg atccgacagt tcgccccttt 1200

gaagccggga tgaaactgtg gcaacaaggg ttttacgtcc gtttcggcgg cgacactctg 1260

cagtttgggc caacatttaa tgcacgccca gaggaattgg accgtctttt tgacgctgta 1320

ggtgaggctc tgaacggaat tgcctga 1347

<210> 6

<211> 1149

<212> DNA

<213> Escherichia coli

<400> 6

aaaccaatca accaggcgcg cgctgaagtg gcgaaatcgg cgaatttgtg tgactggtat 60

gcagaacatg gtccggcaat gctgaaggcg gaacctacgc tggtggaaaa tcagcaggcg 120

gttattgagt atcgaccgtt ggggacgatt ctggcgatta tgccgtggaa ttttccgtta 180

tggcaggtga tgcgtggcgc tgttcccatc attcttgcag gtaacggcta cttacttaaa 240

catgcgccga atgtgatggg ctgtgcacag ctcattgccc aggtgtttaa agatgcgggt 300

atcccacaag gcgtatatgg ctggctgaat gccgacaacg acggtgtcag tcagatgatt 360

aaagactcgc gcattgctgc tgtcacggtg accggaagtg ttcgtgcggg agcggctatt 420

ggcgcacagg ctggagcggc actgaaaaaa tgcgtactgg aactgggcgg ttcggatccg 480

tttattgtgc ttaacgatgc cgatctggaa ctggcggtga aagcggcggt agccggacgt 540

tatcagaata ccggacaggt atgtgcagcg gcaaaacgct ttattatcga agagggaatt 600

gcttcggcat ttaccgaacg ttttgtggca gctgcggcag ccttgaaaat gggcgatccc 660

cgtgacgaag agaacgctct cggaccaatg gctcgttttg atttacgtga tgagctgcat 720

catcaggtgg agaaaaccct ggcgcagggt gcgcgtttgt tactgggcgg ggaaaagatg 780

gctggggcag gtaactacta tccgccaacg gttctggcga atgttacccc agaaatgacc 840

gcgtttcggg aagaaatgtt tggccccgtt gcggcaatca ccattgcgaa agatgcagaa 900

catgcactgg aactggctaa tgatagtgag ttcggccttt cagcgaccat ttttaccact 960

gacgaaacac aggccagaca gatggcggca cgtctggaat gcggtggggt gtttatcaat 1020

ggttattgtg ccagcgacgc gcgagtggcc tttggtggcg tgaaaaagag tggctttggt 1080

cgtgagcttt cccatttcgg cttacacgaa ttctgtaata tccagacggt gtggaaagac 1140

cggatctga 1149

Claims (10)

1. The recombinant Escherichia coli for producing the malonic acid is characterized in that Escherichia coli BL21(DE3) is used as a host, and phosphoenolpyruvate carboxylase genes, succinate dehydrogenase genes, succinate semialdehyde dehydrogenase genes and aspartate enzyme genes from the Escherichia coli, heterologous genes aspartate-alpha-dehydrogenase genes from Corynebacterium glutamicum and beta-alanine pyruvate transaminase genes from Pseudomonas aeruginosa are expressed in a modularized over-expression manner.

2. The recombinant Escherichia coli of claim 1, wherein said modular overexpression is fusion expression of a succinate semialdehyde dehydrogenase gene and a β -alanine pyruvate transaminase gene, fusion expression of an aspartate gene and an aspartate- α -dehydrogenase gene, and fusion expression of a succinate dehydrogenase gene and a phosphoenolpyruvate carboxylase gene.

3. The recombinant Escherichia coli of claim 1 or 2, wherein the nucleotide sequence of the phosphoenolpyruvate carboxylase gene is represented by SEQ ID No.1, and the nucleotide sequence of the succinate dehydrogenase gene is represented by SEQ ID No. 2; the nucleotide sequence of the aspartate gene is shown as SEQ ID NO.3, and the nucleotide sequence of the aspartate-alpha-dehydrogenase gene is shown as SEQ ID NO. 4; the nucleotide sequence of the beta-alanine pyruvate transaminase gene is shown as SEQ ID NO.5, and the nucleotide sequence of the succinic semialdehyde dehydrogenase gene is shown as SEQ ID NO. 6.

4. The recombinant E.coli of claim 3, wherein the vector for modular overexpression is selected from the group consisting of pRSFDuet-1, pTrc99a and pCDFDuet-1.

5. A method for constructing the recombinant Escherichia coli of claim 4, comprising the steps of:

(1) connecting gene fragments yne1 and pa0123 by using the plasmid pRSFDuet-1 as a skeleton vector to obtain a recombinant plasmid pRSF-yne1-pa 0123;

(2) connecting gene fragments aspa and panD by taking the plasmid pTrc99a as a framework vector to obtain a recombinant plasmid pTrc99 a-aspa-panD;

(3) connecting gene fragments sdhC and ppc by using the plasmid pCDFDuet-1 as a skeleton vector to obtain a recombinant plasmid pCDF-sdhC-ppc;

(4) pRSF-yne1-pa0123, pTrc99a-aspa-panD and pCDF-sdhC-ppc were co-transferred into E.coli BL21(DE3) to obtain recombinant E.coli.

6. A method for producing malonic acid, comprising inoculating the recombinant Escherichia coli of any one of claims 1 to 4 to a fermentation medium containing glucose, and fermenting to produce malonic acid.

7. The method of claim 6, wherein the recombinant Escherichia coli is cultured to OD at 35-37 ℃₆₀₀When the concentration is 0.6-0.8, IPTG is added for induction.

8. The method of claim 7, wherein the inducing is performed at 28-30 ℃ for 36-60 h.

9. The method according to any one of claims 6 to 8, wherein the fermentation medium is an SOB medium.

10. Use of the recombinant E.coli of any one of claims 1 to 4 or the method of any one of claims 6 to 9 for the preparation of malonic acid or a derivative thereof.

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