CN109456927A - The recombinant bacterium and its construction method of a kind of high yield 2,4- diacetyl phloroglucin and application - Google Patents

Abstract

A recombinant bacterium with high production of 2,4-diacetylphloroglucinol and a construction method and application thereof belong to the technical field of genetic engineering. By integrating the gene marA encoding multiple resistance factors and the acetyl-CoA carboxylase gene ACC in the engineering strain produced by 2,4-diacetylphloroglucinol, the present invention improves 2,4-diacetyl-m-phenylene The yield of triphenols. The invention can be used for industrial production of 2,4-diacetylphloroglucinol.

Description

A kind of high-yield 2,4-diacetylphloroglucinol recombinant bacteria and its construction method and application

technical field

The invention belongs to the technical field of genetic engineering, and in particular relates to a recombinant bacterium with high yield of 2,4-diacetylphloroglucinol and a construction method and application thereof.

Background technique

2,4-Diacetylphloroglucinol (2,4-DAPG) is a phenolic secondary metabolite produced by a variety of Pseudomonas fluorescens. It is a spectrum antibiotic that can prevent and treat a variety of plant diseases. Its molecular structure is simple, and can be condensed into pre-monoacetylphloroglucinol MAPG by 3 molecules of acetyl-CoA and 1 molecule of malonyl-CoA, and then MAPG generates DAPG through transacetylation, which has inhibitory effect on a variety of plant pathogens. Including beet damping disease, natural decline of wheat take-all disease, tobacco black root rot, etc.

Bangear and Tomashow in 1999 analyzed the gene cluster responsible for 2,4-DAPG synthesis in the strain Pseudomnas fluorescens Q2-87. PhlD can catalyze a series of reactions in the condensation of small molecular substrate carbonyl-CoA polyketones to form long chains, cyclization of long chains, and acetylation of benzene rings. While phlACB catalyzes the acetylation of phloroglucinol to form MAPG (2-acetylphloroglucinol) and 2,4-DAPG. The phlE gene is highly homologous to the norA gene of Staphylococcus aureus and contains a transmembrane domain, which may encode a membrane permease, which is related to the transmembrane transport of harmful substances produced by the metabolism of 2,4-DAPG; PhlF protein can Repressing the expression of PhlACB and overexpressing PhlF can reduce the level of 2,4-DAPG in Pseudomonas fluorescens. At present, the synthesis methods of 2,4-DAPG include chemical synthesis and biosynthesis, and the biological method is mainly synthesized by Pseudomonas fluorescens. Chemical synthesis has harsh conditions, high energy consumption and serious pollution, while biological synthesis has received more and more attention due to its advantages of less pollution and renewable raw materials. The research on biosynthesis of 2,4-DAPG has important scientific and applied value. However, the yield of synthesizing 2,4-DAPG in the prior art is very low, making it difficult to use in industrial production. It is necessary to further genetically modify the engineered strains in order to improve the synthesis ability of 2,4-DAPG.

SUMMARY OF THE INVENTION

In view of the low yield of 2,4-DAPG obtained by the current biosynthesis method, the present invention integrates the 2,4-DAPG synthesis-related genes phlD, phlACB, phlE and the gene marA encoding a multi-resistance factor and encoding the acetyl-CoA carboxyl The acc gene of the enzyme is obtained, and the recombinant cells can improve the synthesis ability of 2,4-diacetylphloroglucinol in Escherichia coli.

The invention provides a recombinant bacterium with high production of 2,4-diacetylphloroglucinol, the recombinant bacterium overexpresses polyketide synthase gene phlD and 2,4-diacetylphloroglucinol synthase gene phlACB , efflux protein phlE gene and gene marA encoding multi-resistance factor and acc gene encoding acetyl-CoA carboxylase, the starting strain is Escherichia coli.

Preferably, the E. coli is E. coli BL21 (DE3).

It is further defined that the genes phlD, phlACB, and phlE are all derived from Pseudomonas fluorescens (Pseudomonas protegens CHA0, DSM 19095 ^T ), or are derived from other organisms, and have the same or similar functions as these genes. Nucleic acid sequences ; The marA gene is derived from Escherichia coli, Genebank ID: 6060688, or a nucleic acid sequence with more than 70% homology to marA; or the marA gene is derived from other organisms, and the homology to the marA gene is less than 70% , but a nucleic acid sequence with the same or similar function as marA; the acetyl-CoA carboxylase gene acc is derived from Escherichia coli, wherein the Genebank ID of subunit accA: 6062185, the Genebank ID of subunit accB: 6058890, the subunit accC Genebank ID: 6058863, Genebank ID of subunit accD: 6059083, or a nucleic acid sequence with more than 70% homology to the acc gene; or the acetyl-CoA carboxylase acc gene is derived from other organisms and is identical to the acc gene A nucleotide sequence that is less than 70% in origin but has the same or similar function as acc.

To be further defined, the nucleotide sequence of the phlD gene is shown in SEQ ID No.1; the nucleotide sequence of the phlACB gene is shown in SEQ ID No.2; the nucleotide sequence of the phlE gene is shown in SEQ ID No. .3; the nucleotide sequence of the marA gene is shown in SEQ ID No.4.

The present invention also provides a method for constructing the above-mentioned recombinant bacteria with high yield of 2,4-diacetylphloroglucinol, the steps are as follows:

1) Prepare a recombinant plasmid containing the genes phlD, phlACB, phlE and the multi-resistance activator marA, denoted as p-phlDACBE/marA;

2) The recombinant plasmid obtained in step 1) and the pA-accADBC recombinant plasmid are introduced into a host bacteria to obtain a recombinant bacteria, and the host bacteria is Escherichia coli.

Further limit, the construction method of the recombinant plasmid p-phlDACBE/marA described in step 1) is as follows:

Using P.protegens CHA0 genomic DNA as a template, the phlD gene was obtained by PCR amplification, the used forward primer sequence phlD-F, the nucleotide sequence was as shown in SEQ ID No.5, the reverse primer phlD-R, nucleotide The sequence is shown in SEQ IDNo.6;

Using P.protegens CHA0 genomic DNA as a template, the phlACB gene was obtained by PCR amplification, the used forward primer sequence phlACB-F, the nucleotide sequence was as shown in SEQ ID No.7, the reverse primer phlACB-R, the nucleotide sequence The sequence is shown in SEQID No.8;

Using P.protegens CHA0 genomic DNA as a template, the phlE gene was obtained by PCR amplification, the used forward primer sequence phlE-F, the nucleotide sequence was as shown in SEQ ID No.9, the reverse primer phlE-R, nucleotide The sequence is shown in SEQ IDNo.10;

Using E. coli genomic DNA as a template, the marA gene was obtained by PCR amplification. The forward primer sequence marA-F was used, and the nucleotide sequence was shown in SEQ ID No. 11. The reverse primer marA-R, the nucleotide sequence As shown in SEQ ID No.12;

Using pET28a(+) as the vector, the above-mentioned phlD gene, phlACB gene, phlE gene and multiple resistance activator marA gene were constructed into the pET28a(+) vector by enzymatic ligation to obtain the recombinant plasmid pET28a-phlDACBE/marA .

The invention also provides the application of the recombinant bacteria in improving the yield of 2,4-diacetylphloroglucinol.

It is further defined that after the recombinant bacteria are cultured in a fermentation medium, the expression is induced by IPTG to obtain 2,4-diacetylphloroglucinol.

To be further defined, the recombinant bacteria were inoculated into M9 liquid medium, cultured until the OD ₆₀₀ reached 0.6-0.8, and IPTG with a final concentration of 0.25 mM was added to induce expression.

To be further defined, after the addition of 0.25mM IPTG, the recombinant bacteria were further cultured at 30°C and 180rpm for 12h until the fermentation was completed.

beneficial effect

Phloroglucinol is a precursor for the biosynthesis of 2,4-DAPG. The multi-resistance activator marA gene can enhance the tolerance of Escherichia coli to 2,4-DAPG and phloroglucinol, and the acc gene can increase the production of phloroglucinol. In the present invention, the recombinant plasmids pET28a-phlDACBE/marA and pA- accADBC was introduced into Escherichia coli BL21(DE3), and the synthesis of 2,4-DAPG was enhanced by overexpressing the marA gene and acc gene in Escherichia coli. On the basis of pA-accADBC, the production of phloroglucinol was improved compared to the control strain containing the plasmid pET28a-phlDACBE.

Therefore, the recombinant bacteria of the present invention can be used for the fermentation production of 2,4-DAPG, and have high application value.

Definitions and Abbreviations:

The following abbreviations or abbreviations are used in the present invention:

2,4-Diacetylphloroglucinol: 2,4-DAPG

Isopropylthiogalactoside: IPTG

Multi-resistance activator gene: marA

Acetyl-CoA carboxylase gene: acc

Escherichia coli: E.coli

Pseudomonas protegens: P. Protegens

Polyketide synthase gene: phlD

2,4-Diacetylphloroglucinol synthase gene: phlACB

efflux protein gene: phlE

"Heat shock transformation" or "heat transformation" refers to a kind of transfection technology in molecular biology, which is used to integrate foreign genes into host genes and express them stably. Introducing host genes or introducing foreign plasmids into host protoplasts, and heat shock transformation or thermal transformation.

Enzymatic ligation: refers to the restriction of the target gene fragment by restriction endonuclease, and then one or more gene fragments that have been digested by the same restriction endonuclease are connected by ligase.

Description of drawings

Fig. 1 Schematic diagram of the co-expression vector of phlD, phlACB and phlE.

Figure 2 Schematic diagram of the co-expression vectors of phlD, phlACB, phlE and marA.

Figure 3 Schematic diagram of the co-expression vectors of accA, accD, accB, and accC.

Figure 4 shows the high-performance liquid chromatography detection of recombinant Escherichia coli fermentation product 2,4-DAPG, in the figure, A is the standard product 2,4-diacetylphloroglucinol high-performance liquid chromatography detection; B is the experimental group detection fermentation The product is detected by high performance liquid chromatography, the abscissa is time, the unit is minutes, and the ordinate is the response value, and the unit is AU.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited by the embodiments.

The materials, reagents, instruments and methods used in the following examples, unless otherwise specified, are conventional materials, reagents, instruments and methods in the art, and can be obtained through commercial channels.

The enzyme reagents used were purchased from Thermo Company, and the kits used for plasmid extraction and DNA fragment recovery were purchased from Omega Company in the United States. The corresponding operation steps were carried out according to the product instructions; all media were prepared with deionized water unless otherwise specified. .

Plasmid pACYCDuet-1 was purchased from Novagen, cat. no. 71147-3.

Plasmid pET-28a(+) was purchased from Novagen, cat. no. 69864-3.

The recombinant plasmid pA-accADBC was constructed by Qingdao Institute of Bioenergy and Process, Chinese Academy of Sciences according to the existing technology, as shown in Figure 3. For the specific construction process, see published papers (Cao Y, Jiang X, Zhang R, Xian M. Improved phloroglucinol production by metabolically engineered Escherichiacoli. Applied Microbiology and Biotechnology, 2011, 91: 1545-1552) Plasmid Construction Contents of Materials and Methods.

Medium formula:

1) Shake flask seed medium

LB medium: yeast powder 5g/L, NaCl 10g/L, peptone 10g/L, sterilized at 121°C for 20min, the concentration of each component is the final concentration in the medium.

2) Shake flask fermentation medium

M9 medium (g/L): NH ₄ Cl 1.0g/L, Na ₂ HPO ₄ 12H ₂ O 15.2g/L, KH ₂ PO ₄ 3.0g/L, NaCl 0.5g/L, the rest are water, 121 ℃, 20min sterilization, the concentration of each component is the final concentration in the medium.

In the actual culture process, glucose, magnesium sulfate and a certain concentration of antibiotics can be added to the above medium to maintain the stability of the plasmid, such as kanamycin with a final concentration of 50 mg/L and chloramphenicol with a final concentration of 34 mg/L.

Example 1. Construction process of recombinant bacteria with high production of 2,4-diacetylphloroglucinol.

1) Construction of recombinant plasmid:

A. Construction of recombinant plasmid I: pET28a-phlDACBE

The phlD gene was cloned using P. protegens CHA0 genomic DNA as a template and obtained by PCR amplification. The sequence of the forward primer phlD-F was: 5'-CATGCCATGGGCATGTCTACACTTTGCCTTCCACACGT-3', and the sequence of the reverse primer phlD-R was: 5'- ATCGCATATGTCAGGCGGTCCACTCGCCCACCGCC-3'; phlACB gene was cloned using P. protegens CHA0 genomic DNA as a template and obtained by PCR amplification. The sequence of the forward primer phlACB-F is:

5'-ATCGCATATGTGTTTAAGAAGGAGATATACCATGAACGTGAAAAAGATAGGTATTGT

-3', the reverse primer phlACB-R sequence is: 5'-CGGGATCCTTATATATCGAGTACGAACTTATAA-3'; the phlE gene clone is obtained by PCR amplification using P.protegens CHA0 genomic DNA as the template, and the forward primer phlE-F sequence is:

5'-CCCAAGCTTTTTAAGAAGGAGATATACCATGAACACTGGAATGCCGACCACG-3', the reverse primer phlE-R sequence is: 5'-CCGCTCGAGCTAGTCCTTCAGGGGCAAGGGGGC-3'.

The cloned phlD gene and the vector pET28a(+) were digested by NdeI and NcoI respectively, and the target phlD fragment and the vector pET28a(+) large fragment were recovered by the recovery kit respectively, and then connected, and the ligation product was transformed into E. .coliDH5α, screened positive clones, and obtained recombinant plasmid pET28a-phlD.

The cloned phlACB gene and the recombinant plasmid pET28a-phlD were digested with NdeI and EcoRI, respectively, and then the target phlACB fragment and the large fragment of the vector pET28a-phlD were recovered using a recovery kit, respectively, and then ligated, and the ligated product was transformed into E.coli DH5α, screened positive clones, and obtained recombinant plasmid pET28a-phlDACB.

The cloned phlE gene and the recombinant plasmid pET28a-phlDACB were double digested by HindIII and XhoI, and the digested target fragment phlE and the vector pET28a-phlDACB large fragment were recovered using a recovery kit, and then ligated, and the ligated product was transformed into E.coli DH5α , screened positive clones, and obtained the recombinant plasmid pET28a-phlDACBE, denoted as recombinant plasmid I, and the plasmid map is shown in Figure 1.

B. Construction of recombinant plasmid II: pET28a-phlDACBE/marA

A recombinant plasmid containing 2,4-diacetylphloroglucinol synthesis-related genes phlD, phlACB, phlE and multi-resistance activator marA was prepared, which was designated as pET28a-phlDACBE/marA.

The phlD gene was cloned using P. protegens CHA0 genomic DNA as a template and obtained by PCR amplification. The sequence of the forward primer phlD-F was: 5'-CATGCCATGGGCATGTCTACACTTTGCCTTCCACACGT-3', and the sequence of the reverse primer phlD-R was: 5'- ATCGCATATGTCAGGCGGTCCACTCGCCCACCGCC-3'; phlACB gene was cloned using P. protegens CHA0 genomic DNA as a template and obtained by PCR amplification. The sequence of the forward primer phlACB-F is:

5'-ATCGCATATGTGTTTAAGAAGGAGATATACCATGAACGTGAAAAAGATAGGTATTGT

-3', the reverse primer phlACB-R sequence is: 5'-CGGGATCCTTATATATCGAGTACGAACTTATAA-3'; the phlE gene clone is obtained by PCR amplification using P.protegens CHA0 genomic DNA as the template, and the forward primer phlE-F sequence is:

5'-CCCAAGCTTTTTAAGAAGGAGATATACCATGAACACTGGAATGCCGACCACG-3', the reverse primer phlE-R sequence is: 5'-CCGCTCGAGCTAGTCCTTCAGGGGCAAGGGGGC-3'; the marA gene clone is obtained by PCR amplification with E.coliDH5α genomic DNA as the template, and the forward primer marA-F The sequence is:

5'-CGGAATTCATGTCCAGACGACGCAATACTGA-3', the reverse primer marA-R sequence is:

5'-ATCGGTCGACCTAGCTGTTGTAATGATTTA-3'.

The cloned phlD gene and the vector pET28a(+) were digested by NdeI and NcoI respectively, and the target phlD fragment and the vector pET28a(+) large fragment were recovered by the recovery kit respectively, and then connected, and the ligation product was transformed into E. .coliDH5α, screened positive clones, and obtained recombinant plasmid pET28a-phlD.

The cloned marA gene and the recombinant plasmid pET28a-phlD obtained in step 2) were double digested with EcoRI and SalI enzymes, respectively, and then the target marA fragment and the large vector pET28a-phlD fragment were recovered using a recovery kit. The ligation was performed again, and the ligation product was transformed into E.coli DH5α, and positive clones were screened to obtain the recombinant plasmid pET28a-phlD/marA;

The cloned phlACB gene and the recombinant plasmid pET28a-phlD/marA were double digested with NdeI and EcoRI, respectively, and the digested phlACB fragment and the vector pET28a-phlD/marA large fragment were recovered using a recovery kit. The ligation was performed again, the ligation product was transformed into E.coli DH5α, and positive clones were screened to obtain the recombinant plasmid pET28a-phlDACB/marA.

The cloned phlE gene and the recombinant plasmid pET28a-phlDACB/marA were double digested by HindIII and XhoI, and the digested target fragment phlE and the vector pET28a-phlDACB/marA large fragment were recovered using a recovery kit, and then ligated, and the ligated product was transformed. E.coli DH5α, screen positive clones, and obtain recombinant plasmid pET28a-phlDACBE/marA, which is recombinant plasmid II, and the plasmid map is shown in FIG. 2 .

The nucleotide sequence of the phlD gene obtained by PCR amplification in this example is shown in SEQ ID No. 1; the nucleotide sequence of the phlACB gene is shown in SEQ ID No. 2, and the nucleotide sequence of the phlE gene, As shown in SEQ ID No.3, the nucleotide sequence of the marA gene is shown in SEQ ID No.4.

2) Construction of recombinant strains

The recombinant plasmid obtained in step 1) and the pA-accADBC recombinant plasmid are introduced into the host bacteria to obtain recombinant bacteria, and the host bacteria is Escherichia coli, and the method is as follows:

The wild-type control strain E. coli BL21(DE3) was prepared according to the operation steps of the TAKARA competent preparation kit.

Control group 1: The recombinant plasmid I and plasmid pET28a-phlDACBE were transformed into competent cells of host strain E.coli BL21 (DE3) by heat shock method to obtain a control strain, numbered Z0;

Control group 2: The recombinant plasmid II, that is, the prepared pET28a-phlDACBE/marA, was transformed into competent cells of the host strain E.coli BL21 (DE3) by heat shock method to obtain a recombinant strain, numbered Z1;

Experimental group: The recombinant plasmid II, namely pET28a-phlDACBE/marA and recombinant plasmid pA-accADBC were transformed into competent cells of host strain E.coli BL21(DE3) by heat shock method to obtain a recombinant strain, numbered Z2.

Example 2. Shake flask fermentation test of the recombinant strain to investigate the effect of the recombinant strain of the present invention on increasing the production of 2,4-diacetylphloroglucinol.

In this embodiment, three groups of experiments are carried out to illustrate the importance of the present invention.

Control group: strain Z0.

Experimental group I: recombinant strain Z1.

Experimental group II: recombinant strain Z2.

1) The activated control strain and recombinant strain were inoculated into 5 mL LB liquid medium, respectively, and 50 μg/mL kanamycin and 50 μg/mL chloramphenicol were added, and grown at 37°C for 8-12 h. The ratio of 1:100 was inoculated into a 250 mL shake flask containing 50 mL of M9 liquid medium, and 50 mg/L of kanamycin and 34 mg/L of chloramphenicol were added to the shake flask according to plasmid selection, and 20 g/L of glucose was added. and 2 mM MgSO ₄ ·7H ₂ O. Shake culture at 37°C and 180 rpm. When the OD ₆₀₀ reached 0.6-0.8, 0.25 mM IPTG was added to induce expression, and after induction, the cells were placed at 30° C. and 180 rpm to continue culturing for 12 h until the end of the fermentation. The above concentrations were the final concentrations in the medium.

2) Take the fermentation broth, centrifuge at 8000 rpm for 10 min at 4°C, take 20 ml of the supernatant, acidify with 1mol/L HCl to pH=2, extract with ethyl acetate, dissolve in 1 ml of anhydrous methanol, and detect the fermentation broth by high performance liquid chromatography. Extracts.

According to the operation steps of this example, at the shake flask level, the 2,4-DAPG yield of the control strain Z0 was 5.7 mg/L, and the 2,4-DAPG yield of the recombinant strain Z1 containing plasmid II, namely pET28a-phlDACBE/marA was 14.9 mg/L; the yield of 2,4-DAPG of recombinant strain Z2 containing two recombinant plasmids pET28a-phlDACBE/marA and pA-accADBC was 18.6 mg/L. That is, the recombinant strain Z2, 2,4-DAPG, which integrates the multi-resistance factor-encoding gene marA and the acetyl-CoA carboxylase gene ACC according to the present invention, is 3.3 times higher than the control strain.

Those skilled in the art should understand that each of the above steps is performed according to standard molecular cloning technology; the 10 genes expressed above are co-cloned into Escherichia coli, and each step is performed according to standard molecular cloning technology.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone who is familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, , the protection scope of the present invention should be defined by the claims.

Nucleotide sequence listing

<110> Qingdao Institute of Bioenergy and Processes, Chinese Academy of Sciences

<120> A kind of recombinant bacteria with high yield of 2,4-diacetylphloroglucinol and its construction method and application

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<170> PatentIn version 3.5

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aaaccgtgcc gcgcatcatg gccatggtgc gcctggacga cggcctggtg atcgcttcgg 2640

aaatcgtcga tgtgtgcgat cagcaacagc tgaaagtcgg tgcgccggtg cgcatggtga 2700

tccgcaagca tgtgcgcgaa agcaacctgg cctggcaata cgcttataag ttcgtactcg 2760

atatataa 2768

<210> 3

<211> 1266

<212> DNA

<213> phlE gene

<400> 3

atgaacactg gaatgccgac cacggctgcc aacagccgtt acgaaagatc catggtgctg 60

ttgctgtcac tgagtttcgg gctggtgggg ctggatcgct tcatcatcct gccgctgttt 120

ccggtgatca tgcacgacct gcaactggac tatcaggacc tgggctacct gtccgccgcg 180

ctggccttca cctgggggct gtccgccctg ggcatgggca gcgtgatcga gcgcctgggc 240

acccgccgcg tgctggtgac ctcgattgcc gtgctttcgc tactgtccgg tttttccgcg 300

ctggccaccg gcgtgctggg gctggtgatt ctgcgcgggc tgatgggcat ctgcgagggt 360

gccttcaccc ccaccagcat cattgtcacc aatgaagtct cgcgccccga ccggcgcggg 420

ctgaacctgg gtatccagca ggcgctgttc ccgattctcg gcctgtgcct ggggccgctg 480

atcgccgcat acctcctgca aatcactggc tcgtggcgtg cggtgttcgc catcatttcc 540

ctgccgggcc tgctgctggc cggctacctg tggaaaatct accgaccgct gccggcgccc 600

caggccgacc gcaccgcagg cacctggctg gcggccttca agagcggcaa cgtgagcctg 660

aacatcctga tcatgttctg catactcacc tgccagttcg tcctctgcgc catgctgccc 720

agctacctga ccgaccatat gcacctggaa accctgtcca tggccttcgt catctcggcc 780

atcggcgtgg gcggcttcat cggccaattg atcatccccg gcatgtccga ccgtctcggg 840

cgcaagccgg tggtgtcggt gtgcttcatg accagcagca ccctggtggg cctgctgatc 900

gtcagcccgc cccttccctg gctgctgttc ctgctgctgt tcctgctgtc gttcttcaac 960

ttcagcctga tctgcatgac agtcggccct ttgaccagcg aatcggtcac cccggccctg 1020

ctgccggcgg cgaccggaat cgtggtgggg ttcggcgaga tcctgggtgg cgggttgtca 1080

ccggccgtgg ccgggttcgc cgccagccat ttcggcctgc catcgatcct gtacgtggcc 1140

ctgagcggca gcctggccgg cttgctgctg tcattcctgc tcaaggaaca ggccctcgaa 1200

ccgcgccgtt cgcgcgtgcg cgaagcgctg cccatcctcc ccgccccctt gcccctgaag 1260

gactag 1266

<210> 4

<211> 384

<212> DNA

<213> marA gene

<400> 4

atgtccagac gcaatactga cgctattacc attcatagca ttttggactg gatcgaggac 60

aacctggaat cgccactgtc actggagaaa gtgtcagagc gttcgggtta ctccaaatgg 120

cacctgcaac ggatgtttaa aaaagaaacc ggtcattcat taggccaata catccgcagc 180

cgtaagatga cggaaatcgc gcaaaagctg aaggaaagta acgagccgat actctatctg 240

gcagaacgat atggcttcga gtcgcaacaa actctgaccc gaaccttcaa aaattacttt 300

gatgttccgc cgcataaata ccggatgacc aatatgcagg gcgaatcgcg ctttttacat 360

ccattaaatc attacaacag ctag 384

<210> 5

<211> 38

<212> DNA

<213>phlD-F

<400> 5

catgccatgg gcatgtctac actttgcctt ccacacgt 38

<210> 6

<211> 35

<212> DNA

<213>phlD-R

<400> 6

atcgcatatg tcaggcggtc cactcgccca ccgcc 35

<210> 7

<211> 57

<212> DNA

<213>phlACB-F

<400> 7

atcgcatatg tgtttaagaa ggagatatac catgaacgtg aaaaagatag gtattgt 57

<210> 8

<211> 33

<212> DNA

<213>phlACB-R

<400> 8

cggaattctt atatatcgag tacgaactta taa 33

<210> 9

<211> 52

<212> DNA

<213>phlE-F

<400> 9

cccaagcttt ttaagaagga gatataccat gaacactgga atgccgacca cg 52

<210> 10

<211> 33

<212> DNA

<213>phlE-R

<400> 10

ccgctcgagc tagtccttca ggggcaaggg ggc 33

<210> 11

<211> 47

<212> DNA

<213> marA-F

<400> 11

cggaattctt taagaaggag atataccatg tccagacgca atactga 47

<210> 12

<211> 30

<212> DNA

<213> marA-R

<400> 12

atcggtcgac ctagctgttg taatgattta 30

Claims (10)

1. a kind of high yield 2, the recombinant bacterium of 4- diacetyl phloroglucin, which is characterized in that the recombinant bacterium is overexpressed polyketone and closes It is more at enzyme gene phlD, 2,4- diacetyl phloroglucin synthase gene phlACB, efflux protein phlE gene and coding The gene marA of weight resistance factor and the acc gene of encoding acetyl CoA carboxylase enzyme, starting strain is Escherichia coli.

2. high yield 2 according to claim 1, the recombinant bacterium of 4- diacetyl phloroglucin, which is characterized in that the large intestine Bacillus is E.coli BL21 (DE3).

3. high yield 2 according to claim 1, the recombinant bacterium of 4- diacetyl phloroglucin, which is characterized in that the gene PhlD, phlACB, phlE derive from Pseudomonas fluorescens (Pseudomonas protegens CHA0, DSM 19095^T), Or the nucleic acid sequence to have same or similar function from other organisms and these genes；The marA gene comes Derived from Escherichia coli, Genebank ID:6060688, or the nucleic acid sequence with marA homology to be more than 70%；Or institute MarA gene source is stated in other organisms and marA genetic homology lower than 70%, but there is same or similar function with marA The nucleic acid sequence of energy；The acetyl CoA carboxylase gene acc derives from Escherichia coli, wherein the Genebank of subunit accA The Genebank ID:6058863 of the Genebank ID:6058890 of ID:6062185, subunit accB, subunit accC, subunit The Genebank ID:6059083 of accD, or the nucleic acid sequence with acc genetic homology to be more than 70%；Or the second Acyl CoA carboxylase acc gene source is lower than 70% in other organisms and acc genetic homology, but with acc have it is identical or The nucleotide sequence of identity function.

4. high yield 2 according to claim 3, the recombinant bacterium of 4- diacetyl phloroglucin, which is characterized in that the phlD base The nucleotide sequence of cause is as shown in SEQ ID No.1；The nucleotide sequence of phlACB gene is as shown in SEQ ID No.2；phlE The nucleotide sequence of gene is as shown in SEQ ID No.3；The nucleotide sequence of marA gene is as shown in SEQ ID No.4.

5. high yield 2 described in claim 1-4 any one, the construction method of the recombinant bacterium of 4- diacetyl phloroglucin is special Sign is that steps are as follows:

1) recombinant plasmid containing gene phlD, phlACB, phlE and multiple resistance activity factor marA is prepared, p- is denoted as phlDACBE/marA；

2) the resulting recombinant plasmid of step 1) and pA-accADBC recombinant plasmid are imported into host strain and obtains recombinant bacterium, it is described Host strain is Escherichia coli.

6. high yield 2 according to claim 5, the construction method of the recombinant bacterium of 4- diacetyl phloroglucin, feature exist In the construction method of recombinant plasmid p-phlDACBE/marA described in step 1) is as follows:

Using P.protegens CHA0 genomic DNA as template, phlD gene, forward primer sequence used are obtained by PCR amplification PhlD-F is arranged, nucleotide sequence is as shown in SEQ ID No.5, reverse primer phlD-R, nucleotide sequence such as SEQ ID No.6 institute Show；

Using P.protegens CHA0 genomic DNA as template, phlACB gene, forward primer used are obtained by PCR amplification Sequence phlACB-F, nucleotide sequence is as shown in SEQ ID No.7, reverse primer phlACB-R, nucleotide sequence such as SEQ ID Shown in No.8；

Using P.protegens CHA0 genomic DNA as template, phlE gene, forward primer sequence used are obtained by PCR amplification PhlE-F is arranged, nucleotide sequence is as shown in SEQ ID No.9, reverse primer phlE-R, nucleotide sequence such as SEQ ID No.10 It is shown；

Using E.coli genomic DNA as template, marA gene, forward primer sequence marA-F used, core are obtained by PCR amplification Nucleotide sequence is as shown in SEQ ID No.11, reverse primer marA-R, and nucleotide sequence is as shown in SEQ ID No.12；

With pET28a (+) be carrier, digestion connect by way of by above-mentioned phlD gene, phlACB gene, phlE gene and Multiple resistance activity factor marA gene, is building up on pET28a (+) carrier, obtains recombinant plasmid pET28a-phlDACBE/ marA。

7. recombinant bacterium described in claim 1-4 any one is improving the application in 2,4- diacetyl phloroglucin yield.

8. application according to claim 7, which is characterized in that after the fermented culture medium culture of the recombinant bacterium, then pass through IPTG inducing expression obtains 2,4- diacetyl phloroglucin.

9. application according to claim 8, which is characterized in that recombinant bacterium is inoculated into M9 fluid nutrient medium, culture is extremely OD₆₀₀When reaching 0.6-0.8, the IPTG inducing expression of final concentration of 0.25mM is added.

10. application according to claim 9, which is characterized in that after the addition 0.25mM IPTG, by recombinant bacterium in 30 DEG C, 180rpm continue to cultivate 12h to fermentation ends.