CN113817782B - Full biosynthesis method of pimelic acid - Google Patents

Abstract

The invention discloses a full biosynthesis method of pimelic acid, and belongs to the field of bioengineering. The invention takes colibacillus BL21 (DE 3) with knock-out lactate dehydrogenase gene (ldhA), acetyl-CoA acetyl transferase gene (atoB) and succinyl-CoA synthetase alpha subunit gene (sucD) as a host, and the genes of beta-ketothiolase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxy adipoyl dehydrogenase, 5-carboxyl-2-pentenoyl-CoA reductase and adipoyl-CoA synthetase from thermobifida fusca are expressed in a modular manner, and the yield of pimelic acid can reach 252.60 mg.L through the fermentation optimization of inorganic salt added in a culture medium ^‑1 Provides a new route for synthesizing pimelic acid in an escherichia coli host and provides a new idea for producing pimelic acid by industrial fermentation.

Description

Full biosynthesis method of pimelic acid

Technical Field

The invention relates to a full biosynthesis method of pimelic acid, belonging to the field of bioengineering.

Background

Pimelic acid (Heptanedioic acid), also known as syzygic acid, 1, 5-pentanedicarboxylic acid, is a seven-carbon dicarboxylic acid, widely used for synthesizing lubricating oil, plasticizers, copolymers, surfactants and the like, and is an important chemical raw material and intermediate in the chemical industry.

Currently, the industrial production of pimelic acid mainly depends on chemical methods. Salicylic acid was cleaved by isoamyl alcohol reduction and sodium to crystallize pimelic acid in 43-50% yield. The laboratory preparation method of pimelic acid also comprises the step of synthesizing pimelic acid by using crown ether as a phase transfer catalyst to catalyze 1, 5-pentanediol, phosphorus tribromide and potassium cyanide. The methods have the advantages of multiple processes and long reaction time, and are not beneficial to large-scale popularization.

In order to solve the above problems, people focus their eyes on the way to biosynthesis of pimelic acid. There are two main pathways reported to date for the biosynthesis of pimelic acid: one is the in vivo BioC-BioH pathway, which is the enzyme that can methylate malonyl-Acyl Carrier Protein (ACP) to malonyl methyl ester, reducing the molecular polarity, allowing fatty acid synthase to extend the latter to produce the pimelic acid derivative pimelic acid-ACP methyl ester; bioH can hydrolyze the terminal methyl groups of pimeloyl ACP methyl ester, restore the polar carboxyl groups, prevent fatty acid synthase from further extension, and further produce pimelic acid. The other is the BioI-BioW pathway of Bacillus subtilis, and studies have pointed out that in the biotin operon of Bacillus subtilisbioI-orf2-orf3The gene sequence is closely related to the synthesis of pimelic acid, and the strong maltose promoter P _glv Is connected tobioI-orf2-orf3Upstream of the cistron, the pimelic acid is integrated on a bacillus subtilis chromosome, and finally the yield of pimelic acid can be effectively increased. Both of the above-described pathways produce trace amounts of pimelic acid, but the mechanism of its synthesis is not elucidated further.

In nature, there is a natural dicarboxylic acid synthesis/catabolism pathway in which the 3-oxoadipoyl-CoA pathway of Thermobifida fusca can efficiently utilize adipic acid, and in E.coli, the pathway can be reconstituted, and acetyl-C can be utilizedThe oA and succinyl-CoA are used as substrates to enable the yield of adipic acid to reach 68. g.L ^-1 (highest value reported at present). Similarly, the pathway can also use acetyl-CoA and malonyl-CoA to synthesize glutaric acid, and the yield reaches 4. g.L ^-1 . The reason for this is that beta-ketothiolase (tfu\u)0875) The substrate spectrum is wider, and can catalyze a plurality of acyl-CoA to generate different dicarboxylic acids. Therefore, by utilizing the extremely strong plasticity of the metabolic network and combining the natural metabolic pathway and the synthetic pathway, the novel route from the cheap and renewable raw materials (glucose and the like) to the biosynthesis of pimelic acid can be designed based on the adipic acid reverse degradation pathway, and a novel idea is provided for the synthesis of pimelic acid in microorganisms.

Disclosure of Invention

In view of the above problems, the present invention provides a recombinant E.coli producing pimelic acid, in which the split module overexpresses beta-ketothiolase (Tfu/u) from Thermobifida fusca0875) 3-hydroxyacyl-CoA dehydrogenase (Tfuu2399) 3-hydroxy adipyl dehydrogenase (Tfu/u)0067) 5-carboxy-2-pentenoyl-CoA reductase (Tfuu1647) And adipoyl CoA synthetase gene (Tfu_)2576，Tfu_2577) And knock out the geneldhA、atoB、sucDRealizes the efficient conversion of glutaric acid into pimelic acid, and remarkably improves the yield of pimelic acid.

The invention provides a method for producing pimelic acid by whole cells, which utilizes genetically engineered bacteria to ferment and produce pimelic acid; the genetically engineered bacterium knocks out lactate dehydrogenase gene, acetyl-CoA acetyltransferase gene and succinyl-CoA synthetase alpha subunit gene, and modularly overexpresses and encodes beta-ketothiolase (Tfu/u) from Thermobifida fusca0875) 3-hydroxyacyl-CoA dehydrogenase (Tfuu2399) 3-hydroxy adipyl dehydrogenase (Tfu/u)0067) 5-carboxy-2-pentenoyl-CoA reductase (Tfuu1647) And adipoyl CoA synthetase gene (Tfu_)2576，Tfu_2577I.e. Tfuu2576-7）。

In one embodiment, the β -ketothiolase (tfu\u)0875) NC of (2)BI accession number MH157180; the 3-hydroxyacyl-CoA dehydrogenase (Tfuu2399) Is of NCBI accession No. MH157181; the 3-hydroxyadipoyl dehydrogenase (Tfu/u)0067) Is of NCBI accession No. MH157182; the 5-carboxy-2-pentenoyl-CoA reductase (Tfuu1647) Is of NCBI accession No. MH157183; the adipoyl CoA synthetase gene (Tfu_)2576，Tfu_2577I.e. Tfuu2576-7) Is of NCBI accession No. MH157194.

In one embodiment, the beta-ketothiolase and 3-hydroxyacyl-CoA dehydrogenase are expressed using plasmid pRSFDuet-1.

In one embodiment, the 3-hydroxy adipyl dehydrogenase and 5-carboxy-2-pentenoyl-CoA reductase are expressed using plasmid pTrc99 a.

In one embodiment, the adipoyl-coa synthetase is expressed using plasmid pcdfdur-1.

In one embodiment, E.coli is used as a host.

In one embodiment, E.coli BL21 (DE 3) is used as the host.

In one embodiment, the genetically engineered bacterium is cultured at 35-37 ℃ for 12-16 hours to obtain a bacterial liquid, the bacterial liquid is inoculated to a fermentation medium according to the volume ratio of 1-5%, and the bacterial liquid is cultured at 35-37 ℃ to OD ₆₀₀ And adding IPTG with a final concentration of 1-5 mM when the concentration is 0.6-0.8, cooling to 25-30 ℃ and performing induction culture for 72 hours.

In one embodiment, the fermentation medium is an SOB medium comprising 3-5 g.L ^-1 Yeast powder 15-20 g.L ^-1 Tryptone, 1-5 g.L ^-1 NaCl，2~3 g·L ^-1 MgCl ₂ ·6H ₂ O，0.1~0.2 g·L ^{- 1} KCl，1~4 g·L ^-1 Glucose, 10-50. Mu.g/mL ^-1 Kanamycin sulfate, 10-50 mug.mL ^-1 Ampicillin, 10-50 [ mu ] g/mL ^-1 Streptomycin.

In one embodiment, the SOB medium contains 6 to 7g.L ^-1 Glutaric acid and KH of 10-50 mM ₂ PO ₄ 。

Preferably, KH ₂ PO ₄ The concentration is 20 to 50mM or 30 to 50mM.

Preferably, KH ₂ PO ₄ The concentration was 20mM, 30mM or 50mM.

The invention also provides a method for constructing the genetically engineered bacterium, which comprises the following steps:

(1) The plasmid pRSFDuet-1 is used as a skeleton vector to connect gene fragment Tfuu0875、Tfu_2399Obtaining recombinant plasmid pAD-1;

(2) The plasmid pTrc99a is used as a skeleton vector to connect gene fragment Tfuu0067、Tfu_1647Obtaining recombinant plasmid pAD-4;

(3) The plasmid pCDFDuet-1 is used as a skeleton vector to connect gene fragments Tfuu/u2576、Tfu_2577Obtaining recombinant plasmid pAD-6;

(4) Transfer of plasmids pAD-1, pAD-4, pAD-6 into the knockdownldhA、atoB、sucDThe recombinant escherichia coli ABD-3 is obtained by the escherichia coli BL21 (DE 3) of the gene.

In one embodiment, step (1) treats plasmid pRSFDuet-1 and gene fragment Tfuu using EcoRI and HindIII double cleavage0875Through T ₄ DNA ligase ligation to obtain recombinant plasmid pRSF-0875The method comprises the steps of carrying out a first treatment on the surface of the Treatment of recombinant plasmid pRSF-by double cleavage with BglII and KpnI0875Gene fragment Tfuu2399Warp T ₄ DNA ligase ligation to obtain a ligated gene fragment Tfuu0875、Tfu_2399The recombinant plasmid pAD-1 of (E).

In one embodiment, step (2) treats plasmid pTrc99a and gene fragment Tfuu using both NcoI and HindIII cleavage0067、Tfu_1647Through T ₄ The DNA ligase was ligated to obtain recombinant plasmid pAD-4.

In one embodiment, step (3) treats plasmid pCDFDuet-1 and gene fragment Tfuu using both NcoI and HindIII cleavage2576、Tfu_2577Through T ₄ The DNA ligase was ligated to obtain recombinant plasmid pAD-6.

The invention provides application of the method in production of pimelic acid, pimelic acid derivatives and products containing pimelic acid.

The invention has the beneficial effects that: the invention is based on the reverse degradation path of adipic acid, and pimelic acid is synthesized by using glutaric acid through 5 reactions in escherichia coli, compared with the traditional synthesis method, the method has shorter synthesis steps and clearer reaction mechanism, and can provide guarantee for subsequent research. Simultaneously, the technology for synthesizing pimelic acid by a biological method is optimized, KH is added in the fermentation process ₂ PO ₄ The yield of pimelic acid is obviously improved, and the yield can reach 252.60 mg.L ^-1 Not only can provide a new idea for the industrial production of pimelic acid, but also has important effect on the development of derivatives thereof.

Drawings

FIG. 1 shows the pimelic acid synthesis pathway.

FIG. 2 shows BL21 (DE 3) knockout of lactate dehydrogenase gene [ ]ldhA) acetyl-CoA acetyltransferase geneatoB) Alpha subunit gene of succinyl-CoA synthetasesucD) Is a colony PCR verification pattern; NC-1: BL21 deltaldhANegative control (1742 bp), 1: BL21 deltaldhA（752 bp）；NC-2：BL21△atoBNegative control (1939 bp), 2: BL21 deltaatoB（754 bp）；NC-3：BL21△sucDNegative control (1623 bp), 3: BL21 deltasucD（753 bp）；M：2000 Marker。

FIG. 3 is a map of plasmids pAD-1, pAD-4, pAD-6.

FIG. 4 is a PCR validation map of recombinant E.coli ABD-3 colonies; 1: plasmid pAD-1 (3142 bp), 2: plasmid pAD-4 (2699 bp), 3: plasmid pAD-6 (2642 bp), M:5000 And (5) Marker.

FIG. 5 is a graph of SOB medium; pimelic acid production profile of all recombinant E.coli containing pAD-1, pAD-4, pAD-6 plasmids at 1mM IPTG; "-" indicates that the corresponding gene in the strain was knocked out.

FIG. 6 is a fragment pattern of a characteristic ion detected by pimelic acid liquid phase mass spectrometry.

FIG. 7 is a graph of pimelic acid yield of recombinant E.coli ABD-3 at different concentrations of different inorganic salts.

FIG. 8 shows recombinant E.coli ABD-3 at different KH ₂ PO ₄ Heptyl at concentrationDiacid yield profile.

FIG. 9 shows the mechanism associated with the synthesis of adipic acid and glutaric acid using the reverse degradation pathway of adipic acid.

Detailed Description

TABLE 1 primer sequence listing according to the following examples

Remarks: the "" "tag sequence is a homologous sequence

Example 1: lactate dehydrogenase Gene [. About.ldhA) acetyl-CoA acetyltransferase geneatoB) Alpha subunit gene of succinyl-CoA synthetasesucD) Is knocked out of (2)

ldhAThe gene number of (a) is ECD_01352,atoBthe gene number of (a) is ECD_02151,sucDis ECD_00688.

Design primer according to nucleotide sequence of target gene published on KEGG website by CRISPR/Cas9 technology, knockoutldhAThe pTarget plasmid required for gene knockout is provided by the Suzhou Honda design.

Obtaining upstream and downstream homology arm fragments: the DNA fragments of 515 bp were amplified by PCR reaction using two pairs of primers, ldhADP 500-F, ldhAUP-R and ldhADOWN500-F, ldhADOWN-R, and the E.coli BL21 (DE 3) genome as templatesldhA515 bp upstream and downstream of the gene are homologous sequences, gel recovery and purification are performed on the amplified DNA fragments, and the purified upstream and downstream fragments are ligated into a homology arm fragment of 1000 bp in size by fusion PCR and gel recovery and purification are performed.

ldhAKnock-out of the gene: coli BL21 (DE 3) was used as the strain to be knocked out to prepare electrotransformation competence. Firstly, the pCas plasmid is introduced into competence, the transformant is selected for PCR verification, positive transformant is inoculated into LB culture medium to continuously prepare electrotransformation competence cell (the final concentration of 20mM of arabinose induction protein is added into LB culture medium for expression). The pTarget plasmid and the homology arm fragment were transferred into the sense cell at a molar ratio of 1:4In this state, transformants were picked for PCR verification, and single colonies of 752 bp size, single bands, were selected as correct knocked-out strains (control 1742 bp) using yzldhA-F and yzldhA-R as primers。

Knockout of the strain pTarget plasmid and elimination of pCas plasmid: inoculating the strain which is knocked out successfully into an LB culture medium for culture at 30 ℃, simultaneously adding IPTG for induction, then inoculating turbid bacterial liquid into the LB culture medium containing spectinomycin for culture, and if the strain does not grow in the spectinomycin, indicating that the pTarget plasmid is eliminated; the strain from which the pTarget plasmid had been eliminated was inoculated into LB medium for cultivation at 37℃and then the turbid bacterial solution was inoculated into LB medium containing kanamycin sulfate for cultivation, if the strain did not grow, it was confirmed that the pCas plasmid had been eliminated, and the plasmid-eliminated strain was designated BL21△ldhA。

Other genes are knocked out by adopting the same method, and finally 7 knocked-out strains are obtained, wherein the knocked-out strains are respectively:

3 single knockout of plantldhAGenes (gene),atoBGenes (gene),sucDColi of the gene: BL21△ldhA(named A), BL21△atoB(designated as B) and BL21△sucD(designated as D);

3-strain combined double knockoutldhAGenes (gene),atoBGenes (gene),sucDColi of the gene: BL21△ldhA△atoB(named AB), BL21△ldhA△sucD(named AD) and BL21△atoB△sucD(named BD);

1 strain knockoutldhAGenes (gene),atoBGenes (gene),sucDGene E.coli BL21△ldhA△atoB△sucD(designated as ABD).

Example 2: construction of recombinant plasmid and obtaining of recombinant E.coli

Gene Tfu_0875Is of NCBI accession No. MH157180; gene Tfu_2399Is of NCBI accession No. MH157181; gene Tfu_0067Is of NCBI accession No. MH157182; gene Tfu_1647Is of NCBI accession No. MH157183; gene Tfu_2576,2577The NCBI accession number of (i.e., tfu_2576-7) is MH157194.

EcoRI and HindIIIPlasmid pRSFDuet-1 was digested with two enzymes, and the purified vector fragment (3798 bp) was recovered and digested with the same procedures as those for plasmid pUC 57-Tfuu0875Gel recovery and purification to obtain target gene fragment Tfuu0875(shown as SEQ ID NO. 1), then connecting the two fragments by using T4DNA ligase, transforming JM109 competent cells, picking the transformant to perform colony PCR verification, extracting plasmid from positive transformant to perform enzyme digestion verification, and naming the plasmid after verification as pRSF-0875。

Recombinant plasmid pRSF-0875The purified vector fragment (4936 bp) was recovered by gel and the plasmid pUC 57-Tfuu was digested with the same enzymes2399And (shown as SEQ ID NO. 2), recovering target gene fragments by gel, connecting the two fragments by using T4DNA ligase, transforming JM109 competent cells, picking transformants, performing colony PCR verification, extracting plasmid from positive transformants, performing enzyme digestion verification, and obtaining the plasmid named pAD-1 after verification.

Other plasmids were constructed in the same manner and the final fragment Tfuu0067、Tfu_1647Ligation to plasmid pTrc99a using NcoI and HindIII (as shown in SEQ ID NO. 3) gives recombinant plasmid pAD-4; fragment Tfuu2576、Tfu_2577As shown in SEQ ID No.4, the recombinant plasmid pAD-6 was obtained by ligating the plasmid pCDFDuet-1 with NcoI and HindIII.

The plasmids pAD-1, pAD-4 and pAD-6 are respectively transferred into the 7 knocked-out strains to respectively obtain recombinant escherichia coli A-3, B-3, D-3, AB-3, AD-3, BD-3 and ABD-3.

Example 3: shake flask fermentation and result analysis of recombinant escherichia coli

Shake flask fermentation system 50 mL.

Fermentation medium:

SOB culture medium with a composition of 5 g.L ^-1 Yeast powder, 20 g · ^L-1 Tryptone, 5 g.L ^-1 NaCl ，2.03 g·L ^-1 MgCl ₂ ·6H ₂ O ，0.186 g·L ^-1 KCl，4 g·L ^-1 Glucose, 6. g.L ^-1 Glutaric acid, 50. Mu.g.mL ^-1 Kanamycin sulfate, 50. Mu.g.mL ^-1 Ampicillin, 50. Mu.g.mL ^-1 Streptomycin.

Seed liquid preparation: marking the strain preserved in glycerol on a flat plate, picking single colony, inoculating into a triangular flask containing 50ml LB culture medium, and culturing at 37deg.C and 250 rpm min ^-1 Shake flask overnight. Transferring 1ml of fungus liquid into 50ml of LB liquid medium at 37deg.C and 250 rpm/min ^-1 Culturing for 12-16 hr.

Fermentation conditions: inoculating the strain into fermentation medium at 37deg.C and 250 rpm/min according to inoculum size of 2% (2 mL/100 mL) ^-1 Culturing until OD is 0.6-0.8, adding 1mM IPTG to induce recombinant bacteria, and cooling to 30deg.C for culturing.

Analysis of results: sampling every 12 h samples during fermentation, 10000 rpm min ^-1 Separating the fermentation broth from thallus after centrifuging for 2 min, treating the fermentation broth with 0.22 μm water system filter membrane for HPLC (high performance liquid chromatography, bio-Rad HPX-87H organic acid column in America), wherein mobile phase is 5mM H ₂ SO ₄ The column temperature was 35℃and the UV detector wavelength was 218 nm.

As a result, as shown in FIG. 5, the E.coli BL21 (DE 3) plasmid-free strain was used as a control strain, and under the same conditions, BL21 (DE 3) plasmid-free strain did not produce pimelic acid; all the knock-out strains containing pAD-1, pAD-4 and pAD-6 plasmids produced a certain amount of pimelic acid, with the highest pimelic acid yield of strain ABD-3 being 108.43 mg.L ^-1 。

Example 4: shake flask fermentation optimization and result analysis of recombinant escherichia coli

1. External inorganic salt type optimization

Fermentation medium:

SOB culture medium with a composition of 5 g.L ^-1 Yeast powder, 20 g.L ^-1 Tryptone, 5 g.L ^-1 NaCl，2.03 g·L ^-1 MgCl ₂ ·6H ₂ O，0.186 g·L ^-1 KCl，4 g·L ^-1 Glucose, 6. g.L ^-1 Glutaric acid, 50. Mu.g.mL ^-1 Kanamycin sulfate, 50. Mu.g.mL ^-1 Ampicillin, 50. Mu.g.mL ^-1 Streptomycin; simultaneously adding inorganic salt solutions with different concentrations into the culture mediumThe corresponding inorganic salt types and concentrations are respectively as follows: KCl 25 mM, mnCl ₂ 0.5 mM，MgSO ₄ 5 mM，KH ₂ PO ₄ 20 mM。

Seed liquid preparation: marking the strain ABD-3 deposited in glycerol on a flat plate, picking single colony, inoculating into a triangular flask containing 50ml LB culture medium, and culturing at 37deg.C and 250 rpm min ^-1 Shaking the flask overnight, transferring 1ml of bacterial liquid into 50ml of LB liquid medium in the next day, and transferring the bacterial liquid into the LB liquid medium at 37 ℃ and 250 rpm min ^-1 Culturing for 12-16 hr.

Fermentation conditions: inoculating the strain into fermentation medium at 37deg.C and 200 rpm/min according to 2% (2 mL/100 mL) ^-1 Culturing until OD is 0.6-0.8, adding 1mM IPTG to induce recombinant bacteria, and cooling to 30deg.C for culturing.

The results are shown in FIG. 7: the strain ABD-3 is used as a fermentation strain, inorganic salts with different types and different concentrations are added, and the yield of the pimelic acid of the strain is changed along with the change of the types of the inorganic salts. Add 5mM MgSO ₄ When the pimelic acid yield is improved; add 20mM KH ₂ PO ₄ The contribution to the synthesis of pimelic acid by the strain is greatest.

2. Externally added KH ₂ PO ₄ Concentration optimization

Fermentation medium:

SOB culture medium with a composition of 5 g.L ^-1 Yeast powder, 20 g.L ^-1 Tryptone, 5 g.L ^-1 NaCl，2.03 g·L ^-1 MgCl ₂ ·6H ₂ O，0.186 g·L ^-1 KCl，4 g·L ^-1 Glucose, 6. g.L ^-1 Glutaric acid, 50. Mu.g.mL ^-1 Kanamycin sulfate, 50. Mu.g.mL ^-1 Ampicillin, 50. Mu.g.mL ^-1 Streptomycin.

Simultaneously adding a certain amount of 1M KH to the culture medium ₂ PO ₄ Controlling KH in culture medium ₂ PO ₄ The final concentrations of the solutions were 0,5, 10, 20, 30 and 50mM, respectively.

Seed liquid preparation: marking the strain preserved in glycerol on a flat plate, picking single colony, inoculating into a triangular flask containing 50ml LB culture medium, and culturing at 37deg.C and 250 rpm min ^-1 Shake flask overnight. The next day1ml of the bacterial liquid is transferred into 50ml of LB liquid medium, the temperature is 37 ℃ and the rpm is 250 min ^-1 Culturing for 12-16 hr.

Fermentation conditions: inoculating the strain into fermentation medium at 37deg.C and 200 rpm/min according to 2% (2 mL/100 mL) ^-1 Culturing until OD is 0.6-0.8, adding 1mM IPTG to induce recombinant bacteria, and cooling to 30deg.C for culturing.

The results are shown in FIG. 8: the strain ABD-3 is used as a fermentation strain, and the pimelic acid yield is along with KH in a culture medium ₂ PO ₄ The increase in concentration increases. KH in the culture medium ₂ PO ₄ At a concentration of 50mM, pimelic acid yield was highest, 252.89 mg.L ^-1 KH in the culture medium ₂ PO ₄ At a concentration of 30mM, pimelic acid yield was highest, 252.60 mg.L ^-1 。

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and 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 Jiangnan

<120> a method for total biosynthesis of pimelic acid

<130> BAA211007A

<160> 4

<170> PatentIn version 3.3

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gctctcgcga ctctgttaga cgcggaacgc atcgatgccg aggaggcgtt ccgtgctggc 540

cttgtgactc gcctcgtcga ggatcctgtg gcagaagctc ttgcgatggc acatagctat 600

gcggaacggg acccaggcct ggtacgggac atgaaacgtg ccgtccgcat ggctgaaact 660

gccgatcttg ctaccgtgct tgaatttgag tcgtgggcgc aggcttcaag cgtaaactcc 720

ccgcgcttcc aggagttttt agcggagttt gcggcgcgca aaaataaaaa agaataaaag 780

aggtatatat taatgtctga ttttgatctg taccggccga ctgaagaaca tgaggctttg 840

cgtgaagcaa ttcggagtgt ggccgaagac aaaatcgcgc cgcatgctgc cgatgtagat 900

gaacaaagcc gctttcctca ggaggcatac gaagctctcc gcgcatccga ttttcatgcc 960

ccgcatgtcg cggaggagta cggcggtgtc ggagcagatg ccttggcgac ctgcatcgtc 1020

attgaagaaa tcgcccgggt gtgtgcaagc tcgtcactga tcccggccgt taataaactg 1080

ggtagtatgc ctttgatttt aagcggctca gacgaagtga aacaacgtta tttgcccgag 1140

cttgcatccg gggaagcgat gtttagttat ggattaagtg agcgcgaagc cggttcggac 1200

acggcttcaa tgcgtactcg cgcggtccgt gatggcgacg attggatcct gaacggtcag 1260

aaatcgtgga ttacaaacgc gggcatttca aaatattata cggtcatggc ggtcaccgac 1320

ccggacggcc cgcgtggccg caacattagt gcatttgttg ttcatattga tgaccctggc 1380

ttctcttttg gtgagccaga gcgtaaatta ggtatcaaag gatcaccgac ccgcgaactg 1440

atttttgaca acgtgcgtat cccaggtgat cggcttgttg gtaaggttgg cgaaggactg 1500

cgtaccgctc tgcgcacact ggaccatacc cgcgtcacca tcggtgcaca ggccgttggt 1560

attgctcagg gtgcgcttga ctacgcatta ggctatgtta aagaacgtaa acaattcggt 1620

aaagcgattg ctgattttca gggaattcag ttcatgttag ctgacatggc gatgaagctg 1680

gaagcagcac gtcagatggt ttatgtcgca gcggcaaaat ctgaacgcga tgatgccgat 1740

ctgtccttct atggcgcggc agcaaagtgc ttcgccagtg atgtcgctat ggagattact 1800

actgatgccg tgcaactgct gggtggttat ggttacaccc gggactatcc tgttgagcgc 1860

atgatgcgcg acgcgaaaat cacacaaatc tacgaaggta cgaaccaaat ccagcgcgtt 1920

gtcatggcac ggcagctgct taaaaagtaa aagctt 1956

<210> 4

<211> 2046

<212> DNA

<213> artificial sequence

<400> 4

ccatggatgg cgatctttct gaccaaagat tctaaggtgc tggtgcaggg catgaccggc 60

agtgaaggga ccaagcacac gcgccgcatg ctggctgcag gaactaatat tgttgggggt 120

gtcaacccac gcaaagctgg ccaagttgta gattttgacg ggacgcaggt gccagtattt 180

ggtagtgtcg cggaaggcat gaaagctacg ggagcggatg tgacagtcat ttttgttccc 240

ccaaaatttg cgaaagacgc ggtgattgag gccattgacg ctgaaattgg cctggcagtt 300

gttattaccg aaggaatccc ggtacacgac actgctacgt tctgggcgca tgcgtgctct 360

aaaggaaata aaacgcgtat catcgggcca aattgcccgg gtttaattac accggggcag 420

agcaacgctg gcattattcc tgcagatatt accaaacccg gccgcatcgg cctggtgagc 480

aaatcgggga cgttaactta ccagatgatg tatgaattac gtgacatcgg atttagtacc 540

tgtgtgggta ttggcgggga tccgattatc ggtaccacgc atattgatgc actcgccgca 600

tttgaagcgg atccggatac ggacgttatc gtgatgattg gagaaattgg cggcgacgcg 660

gaagagcgcg cagcggagta catcaaaaaa catgttacca aaccggtggt ggggtatatt 720

gcggggttta cagcaccgga aggtaaaacg atgggtcacg ccggagcgat cgtctccggc 780

tcttctggga cggccgccgc aaaaaaagaa gccctggagg cagtcggcgt gaaggtgggt 840

aaaactccga gtgaagctgc gaagttagtc cgttcgctgt tcatgagcga tttcgatctg 900

taccggccaa ccgaagaaca tgaagcgtta cgcgaagcta tccgctcagt ggccgaagat 960

aaaatcgcac cacatgccgc agacgtggat gaacagagcc gcttcccgca ggaagcgtac 1020

gaagcacttc gcgcatcaga tttccatgct cctcatgttg cggaagaata tggaggcgta 1080

ggcgccgacg ccctggcaac gtgtattgtg atcgaagaaa ttgcccgtgt ttgtgcatca 1140

agctccctca tccctgcggt gaataagctg ggctcaatgc ccctgattct gtcgggttct 1200

gatgaagtga aacagcggta cttaccggaa ctggcctctg gtgaggcgat gtttagttat 1260

ggcctgagcg aacgtgaagc cggcagtgac actgcttcga tgcgtacgcg tgctgtacgc 1320

gacggtgacg attggattct gaacggccag aaaagttgga tcacgaacgc gggtattagc 1380

aaatactaca ccgtgatggc ggttacggat ccggatggcc cgcgcggtcg taacatctcc 1440

gcatttgttg tacacattga tgaccctgga ttttcgttcg gcgaaccgga acgtaaatta 1500

gggatcaaag gctcgccgac gcgtgaattg atcttcgaca atgttcgcat tcccggagac 1560

cgtctggtgg ggaaagtagg tgagggcctg cgtacagctc tgcgtaccct tgaccatact 1620

cgcgtgacga ttggtgcgca agccgttggg atcgcgcaag gtgcactgga ttatgcgctt 1680

gggtatgtta aagaacgcaa acagtttgga aaagcgattg ccgatttcca gggcatccaa 1740

tttatgctgg ccgacatggc catgaaactg gaggcggctc gccagatggt ctacgtggct 1800

gcagcgaaat cggagcgcga cgatgcggat ctgagcttct atggcgcggc ggccaaatgt 1860

tttgcctcag atgtggcgat ggaaattacc acggatgcag tccaactgtt aggcggttac 1920

ggctatacgc gcgactaccc cgttgaacgt atgatgcggg acgcgaaaat cacccagatt 1980

tacgaaggta ccaatcaaat ccaacgcgtg gtgatggcgc gccagctgct gaagaaataa 2040

aagctt 2046

Claims (8)

1. A method for producing pimelic acid by whole cells is characterized in that the pimelic acid is produced by fermenting genetically engineered bacteria; the genetically engineered bacterium takes escherichia coli as a host, knocks out lactate dehydrogenase genes, acetyl-CoA acetyl transferase genes and succinyl-CoA synthetase alpha subunit genes, and is obtained by modular overexpression of genes encoding beta-ketothiolase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxy adipyl dehydrogenase, 5-carboxyl-2-pentenoyl-CoA reductase and adipyl CoA synthetase from brown thermobifida;

the NCBI accession number of the beta-ketothiolase is MH157180; the NCBI accession number of the 3-hydroxyacyl-CoA dehydrogenase is MH157181; the NCBI accession number of the 3-hydroxy adipyl dehydrogenase is MH157182; the NCBI accession number of the 5-carboxyl-2-pentenoyl-CoA reductase is MH157183; the NCBI accession number of the gene of adipoyl-CoA synthetase is MH157194.

2. The method according to claim 1, characterized in that the beta-ketothiolase and 3-hydroxyacyl-coa dehydrogenase are expressed using plasmid prsfpet-1.

3. The method according to claim 2, characterized in that the 3-hydroxy adipoyl dehydrogenase and 5-carboxy-2-pentenoyl-CoA reductase are expressed using plasmid pTrc99 a.

4. The method of claim 3, wherein the adipoyl-coa synthetase is expressed using plasmid pcdfdur-1.

5. The method according to claim 4, wherein the genetically engineered bacterium is cultured at 35-37 ℃ for 12-16 hours to obtain a bacterial liquid, the bacterial liquid is inoculated to a fermentation medium according to a volume ratio of 1-5%, and the bacterial liquid is cultured at 35-37 ℃ to OD ₆₀₀ And adding IPTG with a final concentration of 1-5 mM when the concentration is 0.6-0.8, cooling to 25-30 ℃ and performing induction culture for 72 hours.

6. The method of claim 5, wherein the fermentation medium is a SOB medium.

7. The method according to claim 6, wherein the SOB medium contains 6 to 7 g.L ^-1 Glutaric acid and KH of 10-50 mM ₂ PO ₄ 。

8. Use of the method according to any one of claims 1 to 7 for the production of pimelic acid and pimelic acid containing products.