CN109456927A - The recombinant bacterium and its construction method of a kind of high yield 2,4- diacetyl phloroglucin and application - Google Patents
The recombinant bacterium and its construction method of a kind of high yield 2,4- diacetyl phloroglucin and application Download PDFInfo
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Abstract
A kind of high yield 2, the recombinant bacterium and its construction method of 4- diacetyl phloroglucin and application, belong to gene engineering technology field.The present invention encodes the gene marA and acetyl-coA carboxylase gene ACC of the multiple resistance factor by integrating in the engineered strain that 2,4- diacetyl phloroglucin produces, and improves the yield of 2,4- diacetyl phloroglucin.The present invention can be used for industrialized production 2,4- diacetyl phloroglucin.
Description
Technical field
The invention belongs to gene engineering technology fields, and in particular to a kind of high yield 2, the recombination of 4- diacetyl phloroglucin
Bacterium and its construction method and application.
Background technique
2,4- diacetyl phloroglucin (2,4-DAPG) is the phenols secondary metabolism generated by a variety of Pseudomonas fluorescens
Product is a kind of spectrum antibiotic, can prevent and treat plurality of plant diseases.Its molecular structure is simple, can be auxiliary by 3 molecule acetyl
Monoacylphosphine phloroglucin MAPG before enzyme A and 1 molecule malonyl CoA is condensed into, then MAPG is generated by transacetylation
DAPG has inhibiting effect, including beet samping off, take-all dieback, tobacco black root for various plants pathogen
Maize ear rot etc..
Bangear and Tomashow, which has been parsed in 1999 in bacterial strain Pseudomnas fluorescens Q2-87, to be responsible for
The gene cluster of 2,4-DAPG synthesis.PhlD can catalytic small molecule substrate phosphinylidyne CoA polyketone be condensed to be formed long-chain and long-chain cyclisation,
Series reaction in phenyl ring acetylation.And phlACB is then catalyzed acetylating formation MAPG (the 2- acetyl of phloroglucin
Base phloroglucin) and 2,4-DAPG.PhlE gene and the norA genetic homology of staphylococcus aureus are higher, contain cross-film knot
Structure domain may encode a film permease, the transmembrane transport correlation of the harmful substance generated with 2,4-DAPG metabolism；PhlF albumen
The expression of PhlACB can be checked, 2, the 4-DAPG that overexpression PhlF can reduce Pseudomonas fluorescens is horizontal.2,4- at present
The synthetic method of DAPG includes chemical synthesis and biosynthesis, and wherein bioanalysis is mainly synthesized by Pseudomonas fluorescens.Chemistry closes
At condition it is harsh, energy consumption is big, seriously polluted, and bioanalysis synthesis due to pollute the advantages such as small, raw material is renewable increasingly by
To attention.The research that bioanalysis synthesizes 2,4-DAPG has important science and application value.But 2 are synthesized in the prior art,
The yield of 4-DAPG is very low, it is difficult to be used for industrial production.It needs further to carry out genetic modification to engineered strain, to improve 2,
The synthesis capability of 4-DAPG.
Summary of the invention
For the problem that 2, the 4-DAPG low output that current biological synthesis method obtains, the present invention passes through integration 2,4-DAPG
Synthesis related gene phlD, phlACB, phlE and the gene marA and fgs encoder acetyl coenzyme A for encoding the multiple resistance factor
The acc gene of carboxylase, obtains recombinant cell, and the recombinant cell can be improved 2,4- diacetyl phloroglucin in large intestine bar
Synthesis capability in bacterium.
The present invention provides a kind of high yield 2, the recombinant bacterium of 4- diacetyl phloroglucin, the recombinant bacterium is overexpressed polyketone
Synthase gene phlD, 2,4- diacetyl phloroglucin synthase gene phlACB, efflux protein phlE gene and coding
The gene marA of the multiple resistance factor and the acc gene of encoding acetyl CoA carboxylase enzyme, starting strain is Escherichia coli.
Preferably, the Escherichia coli are E.coli BL21 (DE3).
It further limits, described gene phlD, phlACB, phlE derive from Pseudomonas fluorescens
(Pseudomonas protegens CHA0, DSM 19095T), or to have from other organisms and these genes
The nucleic acid sequence of same or similar function；The marA gene source is in Escherichia coli, Genebank ID:6060688, or
For the nucleic acid sequence for marA homology being more than 70%；Or the marA gene source is in other organisms and marA gene
Homology is lower than 70%, but has the nucleic acid sequence of same or similar function with marA；The acetyl CoA carboxylase gene acc
From Escherichia coli, wherein the Genebank ID of the Genebank ID:6062185 of subunit accA, subunit accB:
The Genebank ID:6059083 of the Genebank ID:6058863 of 6058890, subunit accC, subunit accD, or for and
Acc genetic homology is more than 70% nucleic acid sequence；Or the acetyl CoA carboxylase acc gene source is in other organisms,
It is lower than 70% with acc genetic homology, but there is the nucleotide sequence of same or similar function with acc.
It further limits, the nucleotide sequence of the phlD gene is as shown in SEQ ID No.1；PhlACB gene
Nucleotide sequence is as shown in SEQ ID No.2；The nucleotide sequence of phlE gene is as shown in SEQ ID No.3；MarA gene
Nucleotide sequence is as shown in SEQ ID No.4.
The present invention also provides above-mentioned high yields 2, the construction method of the recombinant bacterium of 4- diacetyl phloroglucin, and step is such as
Under:
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 obtain recombinant bacterium,
The host strain is Escherichia coli.
It further limits, 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 is obtained by PCR amplification, forward direction used is drawn
Object sequence phlD-F, nucleotide sequence is as shown in SEQ ID No.5, reverse primer phlD-R, nucleotide sequence such as SEQ ID
Shown in No.6；
Using P.protegens CHA0 genomic DNA as template, phlACB gene, forward direction used are obtained by PCR amplification
Primer sequence phlACB-F, nucleotide sequence is as shown in SEQ ID No.7, reverse primer phlACB-R, nucleotide sequence such as SEQ
Shown in ID No.8；
Using P.protegens CHA0 genomic DNA as template, phlE gene is obtained by PCR amplification, forward direction used is drawn
Object sequence phlE-F, nucleotide sequence is as shown in SEQ ID No.9, reverse primer phlE-R, nucleotide sequence such as SEQ ID
Shown in No.10；
Using E.coli genomic DNA as template, marA gene, forward primer sequence marA- used are obtained by PCR amplification
F, 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 (+) for carrier, by above-mentioned phlD gene, phlACB gene, phlE base in such a way that digestion connects
Cause and multiple resistance activity factor marA gene, are building up on pET28a (+) carrier, obtain recombinant plasmid pET28a-
phlDACBE/marA。
The present invention also provides the recombinant bacteriums to improve the application in 2,4- diacetyl phloroglucin yield.
It further limits, after the fermented culture medium culture of the recombinant bacterium, then through IPTG inducing expression, obtains 2,4-
Diacetyl phloroglucin.
It further limits, recombinant bacterium is inoculated into M9 fluid nutrient medium, culture to OD600When reaching 0.6-0.8,
The IPTG inducing expression of final concentration of 0.25mM is added.
It further limits, after the addition 0.25mM IPTG, recombinant bacterium is continued to cultivate in 30 DEG C, 180rpm
12h is to fermentation ends.
Beneficial effect
Phloroglucin is the precursor substance of biosynthesis 2,4-DAPG.Multiple resistance activity factor marA gene can be enhanced
Phloroglucin yield can be improved to the tolerance of 2,4-DAPG and phloroglucin, acc gene in Escherichia coli, and the present invention will recombinate matter
Grain pET28a-phlDACBE/marA and pA-accADBC imports e. coli bl21 (DE3), by crossing table in Escherichia coli
2,4-DAPG synthesis is improved up to marA gene and acc gene, the results showed that, in shaking flask level, containing there are two types of recombinant plasmids
On the basis of pET28a-phlDACBE/marA and pA-accADBC, the yield ratio of phloroglucin contains plasmid pET28a-
The control strain of phlDACBE increases.
Therefore, recombinant bacterium of the present invention can be used for the fermenting and producing of 2,4-DAPG, application value with higher.
Definition and abbreviation:
Following abbreviation or abbreviation is used in the present invention:
2,4- diacetyl phloroglucin: 2,4-DAPG
Isopropylthiogalactoside: IPTG
Multiple resistance activating factor gene: marA
Acetyl CoA carboxylase gene: acc
Escherichia coli (Escherichia coli): E.coli
Pseudomonas fluorescens (Pseudomonas protegens): P.Protegens
Polyketide synthases gene: phlD
2,4- diacetyl phloroglucin synthase gene: phlACB
Efflux protein gene: phlE
" heat-shock transformed " or " thermal transition " refers to one kind of rotaring dyeing technology in molecular biology, for alien gene to be integrated into
In host gene and stablize expression, using after by heat shock, there is crack in cell membrane, by alien gene import host gene or
Foreign particles are imported into host's protoplast, and heat-shock transformed or thermal transition etc..
Digestion connection: referring to and carry out digestion to target gene fragment by restriction enzyme, then again will be through same limit
One or more genetic fragments after property endonuclease digestion processed, are attached by ligase.
Detailed description of the invention
Fig. 1 phlD, phlACB, phlE coexpression vector schematic diagram.
Fig. 2 phlD, phlACB, phlE, marA coexpression vector schematic diagram.
Fig. 3 accA, accD, accB, accC coexpression vector schematic diagram.
Fig. 4 is to recombinate Escherichia coli fermentation product 2, the high performance liquid chromatography detection of 4-DAPG, and in figure, A is standard items 2,
4- diacetyl phloroglucin high performance liquid chromatography detection；B is that experimental group detects tunning high performance liquid chromatography detection, horizontal seat
It is designated as the time, unit is minute, and ordinate is response, and unit is AU.
Specific embodiment
The present invention will be further described combined with specific embodiments below, but the present invention should not be limited by the examples.
Material therefor, reagent, instrument and method in following embodiment are the routine in this field without specified otherwise
Material, reagent, instrument and method can be obtained by commercial channel.
Enzymatic reagent used is purchased from Thermo company, extracts reagent used in kit used in plasmid and recycling DNA fragmentation
Box is purchased from U.S. Omega company, and corresponding operation step is carried out according to product description；All culture mediums are equal unless otherwise instructed
It is prepared with deionized water.
Plasmid pACYCDuet-1 is bought from Novagen, article No. 71147-3.
Plasmid pET-28a (+) is bought from Novagen, article No. 69864-3.
Recombinant plasmid pA-accADBC be Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences according to the prior art from
Row building, as shown in figure 3, specific building process is shown in published thesis (Cao Y, Jiang X, Zhang R, Xian
M.Improved phloroglucinol production by metabolically engineered Escherichia
Coli.Applied Microbiology and Biotechnology, 2011,91:1545-1552) material and method matter
Grain content construction.
Culture medium prescription:
1) shake-flask seed culture medium
LB culture medium: yeast powder 5g/L, NaCl 10g/L, peptone 10g/L, 121 DEG C, 20min sterilizes, each constituent concentration
It is final concentration in the medium.
2) Medium of shaking flask fermentation
M9 culture medium (g/L): NH4Cl 1.0g/L, Na2HPO4·12H2O 15.2g/L, KH2PO43.0g/L, NaCl
0.5g/L, remaining is water, and 121 DEG C, 20min sterilizing, each constituent concentration is final concentration in the medium.
In practical incubation, glucose, magnesium sulfate and certain density antibiotic can be added into above-mentioned culture medium
To maintain the stability of plasmid, such as kanamycins of final concentration of 50mg/L and the chloramphenicol of 34mg/L.
The building process of the recombinant bacterium of 1. high yield 2,4- diacetyl phloroglucin of embodiment.
1) construction of recombinant plasmid:
A. recombinant plasmid I: the building of pET28a-phlDACBE
The clone of phlD gene is obtained by PCR amplification, just using P.protegens CHA0 genomic DNA as template
To primer phlD-F sequence are as follows: 5'-CATGCCATGGGCATGTCTACACTTTGCCTTCCACACGT-3', reverse primer phlD-
R sequence are as follows: 5'-ATCGCATATGTCAGGCGGTCCACTCGCCCACCGCC-3'；The clone of phlACB gene be with
P.protegens CHA0 genomic DNA is template, is obtained by PCR amplification, forward primer phlACB-F sequence are as follows:
5'-ATCGCATATGTGTTTAAGAAGGAGATATACCATGAACGTGAAAAAGATAGGTATTGT
- 3', reverse primer phlACB-R sequence are as follows: 5'-CGGGATCCTTATATATCGAGTACGAACTTATAA-3'；
PhlE gene cloning is to be obtained using P.protegens CHA0 genomic DNA as template by PCR amplification, forward primer phlE-
F sequence are as follows:
5'-CCCAAGCTTTTTAAGAAGGAGATATACCATGAACACTGGAATGCCGACCACG- 3', reverse primer
PhlE-R sequence are as follows: 5'-CCGCTCGAGCTAGTCCTTCAGGGGCAAGGGGGC-3'.
Resulting phlD gene and carrier pET28a (+) are cloned respectively after NdeI, NcoI double digestion, utilizes reclaim reagent
Purpose phlD segment and carrier pET28a (+) large fragment after digestion is separately recovered in box, then is attached, connection product conversion
E.coliDH5 α, screening positive clone obtain recombinant plasmid pET28a-phlD.
Gained phlACB gene and recombinant plasmid pET28a-phlD are cloned respectively after NdeI and EcoRI double digestion, utilization
Purpose phlACB segment and carrier pET28a-phlD large fragment after digestion is separately recovered in QIAquick Gel Extraction Kit, then is attached, connection
Product Transformed E .coli DH5 α, screening positive clone obtain recombinant plasmid pET28a-phlDACB.
Clone's gained phlE gene and recombinant plasmid pET28a-phlDACB are utilized after HindIII and XhoI double digestion
QIAquick Gel Extraction Kit recycles target fragment phlE and carrier pET28a-phlDACB large fragment after digestion, then is attached, and connects
Product Transformed E .coli DH5 α, screening positive clone obtain recombinant plasmid pET28a-phlDACBE, are denoted as recombinant plasmid I, matter
Grain map is as shown in Figure 1.
B. the building of recombinant plasmid I I:pET28a-phlDACBE/marA
Preparation swashs containing 2,4- diacetyl phloroglucin synthesis related gene phlD, phlACB, phlE and multiple resistance
The recombinant plasmid of factor marA living, is denoted as pET28a-phlDACBE/marA.
The clone of phlD gene is obtained by PCR amplification, just using P.protegens CHA0 genomic DNA as template
To primer phlD-F sequence are as follows: 5'-CATGCCATGGGCATGTCTACACTTTGCCTTCCACACGT-3', reverse primer phlD-
R sequence are as follows: 5'-ATCGCATATGTCAGGCGGTCCACTCGCCCACCGCC-3'；The clone of phlACB gene be with
P.protegens CHA0 genomic DNA is template, is obtained by PCR amplification, forward primer phlACB-F sequence are as follows:
5'-ATCGCATATGTGTTTAAGAAGGAGATATACCATGAACGTGAAAAAGATAGGTATTGT
- 3', reverse primer phlACB-R sequence are as follows: 5'-CGGGATCCTTATATATCGAGTACGAACTTATAA-3'；
PhlE gene cloning is to be obtained using P.protegens CHA0 genomic DNA as template by PCR amplification, forward primer phlE-
F sequence are as follows:
5'-CCCAAGCTTTTTAAGAAGGAGATATACCATGAACACTGGAATGCCGACCACG- 3', reverse primer
PhlE-R sequence are as follows: 5'-CCGCTCGAGCTAGTCCTTCAGGGGCAAGGGGGC-3'；MarA gene cloning is with E.coli
DH5 α genomic DNA is template, is obtained by PCR amplification, forward primer marA-F sequence are as follows:
5'-CGGAATTCATGTCCAGACGCAATACTGA-3', reverse primer marA-R sequence are as follows:
5'-ATCGGTCGACCTAGCTGTTGTAATGATTTA-3'。
Resulting phlD gene and carrier pET28a (+) are cloned respectively after NdeI, NcoI double digestion, utilizes reclaim reagent
Purpose phlD segment and carrier pET28a (+) large fragment after digestion is separately recovered in box, then is attached, connection product conversion
E.coliDH5 α, screening positive clone obtain recombinant plasmid pET28a-phlD.
Recombinant plasmid pET28a-phlD obtained by clone's gained marA gene and step 2) is double through EcoRI and SalI enzyme respectively
After digestion, purpose marA segment and carrier pET28a-phlD large fragment after digestion is separately recovered using QIAquick Gel Extraction Kit.It carries out again
Connection, connection product Transformed E .coli DH5 α, screening positive clone obtain recombinant plasmid pET28a-phlD/marA；
Clone's gained phlACB gene and recombinant plasmid pET28a-phlD/marA are respectively through NdeI and EcoRI double digestion
Afterwards, purpose phlACB segment and carrier pET28a-phlD/marA large fragment after digestion is separately recovered using QIAquick Gel Extraction Kit.Again
It is attached, connection product Transformed E .coli DH5 α, screening positive clone obtains recombinant plasmid pET28a-phlDACB/
marA。
Gained phlE gene and recombinant plasmid pET28a-phlDACB/marA are cloned after HindIII and XhoI double digestion,
Target fragment phlE and carrier pET28a-phlDACB/marA large fragment after digestion are recycled using QIAquick Gel Extraction Kit, then is carried out
Connection, connection product Transformed E .coli DH5 α, screening positive clone obtain recombinant plasmid pET28a-phlDACBE/marA, i.e.,
For recombinant plasmid II, plasmid map is as shown in Figure 2.
The nucleotide sequence for the phlD gene that the present embodiment PCR amplification obtains, as shown in SEQ ID No.1；PhlACB base
The nucleotide sequence of cause, as shown in SEQ ID No.2, the nucleotide sequence of phlE gene, as shown in SEQ ID No.3, marA
The nucleotide sequence of gene, as shown in SEQ ID No.4.
2) building of recombinant bacterial strain
The resulting recombinant plasmid of step 1) and pA-accADBC recombinant plasmid are imported into host strain and obtain recombinant bacterium, institute
Stating host strain is Escherichia coli, the method is as follows:
Wild type control strain E.coli BL21 is prepared according to the operating procedure of TAKARA competence reagent preparation box
(DE3) competence.
Control group 1: recombinant plasmid I converts plasmid pET28a-phlDACBE to host strain by heat shock method
E.coli BL21 (DE3) competent cell, obtains control strain, number Z0；
Control group 2: by recombinant plasmid I I, that is, the pET28a-phlDACBE/marA for preparing by heat shock method convert to
Host strain E.coli BL21 (DE3) competent cell, obtains recombinant bacterial strain, number Z1；
Experimental group: recombinant plasmid I I, i.e. pET28a-phlDACBE/marA and recombinant plasmid pA-accADBC are passed through into heat
Sharp method is converted to host strain E.coli BL21 (DE3) competent cell, obtains recombinant bacterial strain, number Z2.
The shake flask fermentation of 2. recombinant bacterial strain of embodiment is tested, and the raising 2 that recombinant bacterium of the present invention has, 4- diethyl are investigated
The effect of acyl phloroglucinol yield.
The present embodiment carries out three groups of experiments altogether, to illustrate importance of the invention.
Control group: bacterial strain Z0.
Experimental group I: recombinant bacterial strain Z1.
Experimental group II: recombinant bacterial strain Z2.
1) by after activation control strain and recombinant bacterial strain be seeded in 5mL LB liquid medium respectively, and be added 50 μ
G/mL kanamycins and 50 μ g/mL chloramphenicol, 37 DEG C of growth 8-12h.The M9 liquid containing 50mL is inoculated into the ratio of 1:100
The kanamycins of addition 50mg/L and the chloramphenicol of 34mg/L are selected in the 250mL shaking flask of culture medium, in shaking flask according to plasmid, is added
Enter 20g/L glucose and the MgSO of 2mM4·7H2O.37 DEG C, shaken cultivation under the conditions of 180rpm.OD600When reaching 0.6-0.8, add
Enter the IPTG inducing expression of 0.25mM, induction be placed on 30 DEG C, 180rpm continue to cultivate 12h to fermentation ends, each concentration
It is final concentration in the medium.
2) fermentation liquid is taken, 4 DEG C, 8000rpm is centrifuged 10min, takes 20ml supernatant, is acidified to pH=2 with 1mol/L HCl
After be extracted with ethyl acetate, 1ml anhydrous methanol dissolution, the extract of high performance liquid chromatography detection fermentation liquid.
According to the present embodiment operating procedure, shaking flask is horizontal, and 2, the 4-DAPG yield of control strain Z0 is 5.7mg/L, contains matter
2, the 4-DAPG yield of grain II, i.e. the recombinant bacterial strain Z1 of pET28a-phlDACBE/marA are 14.9mg/L；Containing there are two types of recombinations
The yield of the 2,4-DAPG of the recombinant bacterial strain Z2 of plasmid pET28a-phlDACBE/marA and pA-accADBC are 18.6mg/L.I.e.
The recombinant bacterium of the gene marA and acetyl-coA carboxylase gene ACC of the present invention for incorporating the coding multiple resistance factor
Z2,2,4-DAPG yield increased bacterial strain improve 3.3 times.
It should be appreciated by those skilled in the art that above-mentioned each step is carried out according to the molecule clone technology of standard；It is above-mentioned
10 kinds of genes of expression are cloned into Escherichia coli jointly, and each step is carried out according to the molecule clone technology of standard.
Although the present invention has been disclosed in the preferred embodiment as above, it is not intended to limit the invention, any to be familiar with this
The people of technology is not departing from spirit and scope of the invention, can do various change and modification, therefore, guarantor of the invention
Shield range should subject to the definition of the claims.
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<110>Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences
<120>a kind of recombinant bacterium of high yield 2,4- diacetyl phloroglucin and its construction method and application
<130>
<160> 12
<170> PatentIn version 3.5
<210> 1
<211> 1050
<212> DNA
<213>phlD gene
<400> 1
atgtctacac tttgccttcc acacgtcatg tttccgcaac acaagatcac ccagcaacag 60
atggtcgatc acctggaaaa cctgcacgcc gaccatccac gcatggccct ggccaagcgc 120
atgatcgcca acaccgaagt caacgagcgc cacctggtgt tgccgatcga cgaactggca 180
gtgcacaccg gcttcaccca ccgcagcatc gtctacgagc gtgaagcccg gcagatgtcg 240
tcggccgcgg cgcgccaggc catcgagaat gccgggctgc agatcagcga cattcgcatg 300
gtgatcgtca cttcctgcac cggcttcatg atgccgtcgc tgaccgcgca cctgatcaac 360
gacctggccc tgccaacctc caccgtgcag ttgccgatcg cccagctggg ctgcgtggcc 420
ggtgccgcgg ccatcaaccg cgccaacgac ttcgcccggc tcgatgcccg caaccacgta 480
ctgatcgtgt ccctggagtt ctcctcgctg tgctaccagc cggacgacac caagctgcac 540
gccttcatct ccgcggcgct gttcggcgat gcggtatccg cctgcgtgct gcgcgccgat 600
gaccaggccg gcggcttcaa gatcaagaag accgagtcgt acttcctgcc caagagcgag 660
cactacatca agtacgacgt gaaggacacc ggctttcact tcaccctcga caaggcggtg 720
atgaactcca tcaaggacgt ggcaccggtc atggagcggc tcaactacga gagtttcgaa 780
cagaactgtg cgcacaacga cttcttcatc ttccacaccg gtggtcgcaa gatcctcgac 840
gagctggtga tgcacctgga cctggcatcc aaccgggtct cgcaatcgcg cagcagcctg 900
tcggaagccg gcaacattgc cagcgtggtg gtgttcgacg tactcaagcg gcagttcgat 960
tccaacctca atcgcggcga catcggcctg ctggcggcct tcggcccggg gttcaccgcg 1020
gaaatggcgg tgggcgagtg gaccgcctga 1050
<210> 2
<211> 2768
<212> DNA
<213>phlACB gene
<400> 2
atgaacgtga aaaagatagg tattgtcagc tatggcgcgg gtattccggt atgccgcctg 60
aaagtccagg aagtgatcaa cgtctggaaa aacaccgacc tcaagctggt ggaggaaaac 120
ctcggcgtca cggaaagagc cgtgctgcaa ccggatgaag atgtcatcac cctcggggtg 180
ctggcggcgc aacgggccct ggataaagtc cccggtcatc agatcgaagc cctgtacctg 240
ggcacctgca ccaatcccta tgactcacgg gcctcggctt cgatcatcct ggaaatgctc 300
ggcagcggct acgacgccta ttgcgccgac gtgcagttcg ccggcaagtc cggaacttct 360
gccctgcaga tctgccaggc gctggtggct tccggcatga cgggcagcgc cctggccatc 420
ggcgccgaca ccatcaaccg caacaccgcg ccgggcgacc tgaccgaatc ctatgccggg 480
gccggagccg ctgcgctgct gatcggcagc caggatgtca tcgccgaatt cgacgccagc 540
ttctcctgcg ccgccgatgt cgccgacaac atccgccccc agggcgatcg ctacattcgt 600
tcgggcatgg gcctgggctc ggacaagaac agcatcggcc tggaagacca gacccgccgc 660
gccgccgaag gcctgatggc caagctgcac accagccccg ccgactatga ctatgtggtg 720
ttccagcaaa acctggtgtc cacgccctac tccctggcca agcacctggg cttcaacccc 780
aagcaggtgg aaccgggcat ctatgccggc aacgttggcg atgccggatc ggccagcccg 840
ctgctcggcc tgatcaacgt actggaccag gcacgcccgg ggcagaaaat tcttttggtg 900
tcctacggtt tcggcgccgg cagcgacgcc atcgccctga ccgtcaccga cgccatcgag 960
cagtaccaga agcacaacaa gcctctgcgc gaactgctgg aatccaagat ctacgtcgac 1020
tacggcacgt ccatcaagta cgagttcaag tacctgcggg ctgactacgc cctgaccgcc 1080
tatctctgat tacgcctgtc tacgcaagga gcatgcacat gtgcgcacgt cgtgttgcaa 1140
tcgtttcggc cgcctatacg ccgaaacccg gaagttcacg agttcggcag acgttcaagg 1200
aaatgatcgt cgagtccgcc tacaaggcac tcaaggacgc caagatgcac ccccgggaaa 1260
tccaggccgt ggcctatggt tatcacggtg aaggcatttc cgaatatggc ggcctgggcc 1320
cgaccatttc cgacgccctg ggcatcagcc cggcgccgac cttcatgagc accgccaact 1380
gcaccagcag ctcggtgtcg ttccagatgg gccaccagat ggtggcgtcc ggggagtacg 1440
acatcgtcct gtgcggcggc ttcgagaaga tgaccgatca cttcaactac gccgaataca 1500
tcggctccag cactgaatgt gaatacgatt atttcctcgg catttcccac accgatgcct 1560
tcgccctggc caccgccgag tacttccaga agttcggcta tgccggtcgc gaggccgacg 1620
tactggccac cttcggccgg cagatgcgca tctacgcaca gaacaccccc accgccaccc 1680
gttatggcca gccgattcca tccctggaag tgttgaagaa cagcgaagcc tgcggctcga 1740
tgctggcctg gggcgaagcc agtggctgcg cgatcctggt ggccgaacac ctggcccaca 1800
agtacaccga taaaccggtg ttcgtacgcg gttgcgccta taccggggtc tcccactatt 1860
tcggtacgcg ctttcacaac ccgaccctgc atcatccggg cctgcccaag gatgtgggca 1920
tggcggtttc ggccaactcc atcgcctgtg cggagatcgc ctacaagaaa gccgggatca 1980
ccgccaagga catcgacgtg gcccaggtct acgacctgct cggcgcaggg ctgatccaga 2040
tggaatccat gggcatctgt ggcaagggcc aggccggcga cttcgtgctc gaaggcggca 2100
ttgccctgga tggccagctg ccgctcaata ccgacggcgg caacatcggc cgcggccatg 2160
cctccggctg cgacggcatc ctgcacatca ccgagctgtt ccggcagctg aggggtgaat 2220
ccgacaacca ggtcaagggc gcgcgcatcg gcgtgtcgca gaaccttggc ggttacgcgg 2280
cgcacaactc agtgattgtc ctctcgaacg actaaggagc ccggaccatg tccatgtacc 2340
cagaacagat ccacagaatg accaccgcca gcatgcttcg cgaatggcgc gagcatggcg 2400
gcaagtaccg cctcgaaggc agccagtgcg aagaatgcaa cgaaatcttc ttcccccggc 2460
gcaccgtctg cggcgcttgc aactccctga gtgtgaagcc gtatcgctgc gcgcgcagtg 2520
gcaagatcga ggtcatggcc ccggccgaga acccgatcct ggccgccatg ggctatggcg 2580
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 19095T),
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.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109439606A (en) * | 2018-11-14 | 2019-03-08 | 中国科学院青岛生物能源与过程研究所 | A kind of genetic engineering bacterium improving phloroglucin yield and its construction method and application |
Citations (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101084311A (en) * | 2004-10-12 | 2007-12-05 | 密歇根州州立大学托管理事会 | Biosynthesis of phloroglucinol and preparation of 1,3-dihydroxybenzene therefrom |
| WO2012003461A2 (en) * | 2010-07-02 | 2012-01-05 | Draths Corporation | Phloroglucinol synthases and methods of making and using the same |
| WO2012006244A1 (en) * | 2010-07-06 | 2012-01-12 | Draths Corporation | Method for producing phloroglucinol and dihydrophloroglucinol |
| WO2012006245A1 (en) * | 2010-07-06 | 2012-01-12 | Draths Corporation | Phloroglucinol reductase and methods of making and using the same |
| CN102787135A (en) * | 2011-05-18 | 2012-11-21 | 中国科学院青岛生物能源与过程研究所 | Method for improving phloroglucinol synthetic capability of engineering escherichia coli |
| EP2788371A2 (en) * | 2011-12-08 | 2014-10-15 | Biocartis NV | Biomarkers and test panels useful in systemic inflammatory conditions |
| CN104357495A (en) * | 2014-11-05 | 2015-02-18 | 中国科学院青岛生物能源与过程研究所 | Method for increasing cell phloroglucinol synthesis yield and application |
| CN104371966A (en) * | 2014-11-12 | 2015-02-25 | 中国科学院青岛生物能源与过程研究所 | Gene engineering strain capable of synthesizing phloroglucinol from acetic acid and construction method and application thereof |
| CN104388371A (en) * | 2014-12-02 | 2015-03-04 | 中国科学院青岛生物能源与过程研究所 | Genetic engineering bacterium for high-yield phloroglucinol as well as construction method and application of genetic engineering bacterium |
| CN104388457A (en) * | 2014-12-02 | 2015-03-04 | 中国科学院青岛生物能源与过程研究所 | Gene modification method for increasing yield of phloroglucinol and application of same |
| CN104725208A (en) * | 2013-12-18 | 2015-06-24 | 上海塞恩泰恩生物科技有限公司 | 2,4-diacetylphloroglucinol separation and purification method |
| CN104988172A (en) * | 2015-07-30 | 2015-10-21 | 中国科学院青岛生物能源与过程研究所 | Construction method and application of high-yield phloroglucinol gene engineering bacterium |
| CN105622379A (en) * | 2016-03-15 | 2016-06-01 | 中国科学院华南植物园 | 2,4-DAPG (2,4-Diacetylphloroglucinol) analogue and preparation method and application thereof |
| CN106905157A (en) * | 2017-01-10 | 2017-06-30 | 西北农林科技大学 | 2,4 diacetyl phloroglucin ester type compounds and its application of sterilization |
| CN106929527A (en) * | 2017-04-24 | 2017-07-07 | 中国科学院青岛生物能源与过程研究所 | A kind of genetic engineering bacterium of phloroglucin synthesis capability high and construction method and application |
| CN107557326A (en) * | 2017-10-27 | 2018-01-09 | 山东大学 | One plant of sterilization fixed nitrogen Pseudomonas fluorescens and its fermentation process and application |
| CN105018511B (en) * | 2015-07-30 | 2018-08-17 | 中国科学院青岛生物能源与过程研究所 | A kind of method of external enzyme reaction method for synthesizing phloroglucinol and application |
-
2018
- 2018-11-14 CN CN201811354253.3A patent/CN109456927A/en active Pending
Patent Citations (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101084311A (en) * | 2004-10-12 | 2007-12-05 | 密歇根州州立大学托管理事会 | Biosynthesis of phloroglucinol and preparation of 1,3-dihydroxybenzene therefrom |
| WO2012003461A2 (en) * | 2010-07-02 | 2012-01-05 | Draths Corporation | Phloroglucinol synthases and methods of making and using the same |
| WO2012006244A1 (en) * | 2010-07-06 | 2012-01-12 | Draths Corporation | Method for producing phloroglucinol and dihydrophloroglucinol |
| WO2012006245A1 (en) * | 2010-07-06 | 2012-01-12 | Draths Corporation | Phloroglucinol reductase and methods of making and using the same |
| CN102787135A (en) * | 2011-05-18 | 2012-11-21 | 中国科学院青岛生物能源与过程研究所 | Method for improving phloroglucinol synthetic capability of engineering escherichia coli |
| EP2788371A2 (en) * | 2011-12-08 | 2014-10-15 | Biocartis NV | Biomarkers and test panels useful in systemic inflammatory conditions |
| CN104725208A (en) * | 2013-12-18 | 2015-06-24 | 上海塞恩泰恩生物科技有限公司 | 2,4-diacetylphloroglucinol separation and purification method |
| CN104357495A (en) * | 2014-11-05 | 2015-02-18 | 中国科学院青岛生物能源与过程研究所 | Method for increasing cell phloroglucinol synthesis yield and application |
| CN104371966A (en) * | 2014-11-12 | 2015-02-25 | 中国科学院青岛生物能源与过程研究所 | Gene engineering strain capable of synthesizing phloroglucinol from acetic acid and construction method and application thereof |
| CN104388371A (en) * | 2014-12-02 | 2015-03-04 | 中国科学院青岛生物能源与过程研究所 | Genetic engineering bacterium for high-yield phloroglucinol as well as construction method and application of genetic engineering bacterium |
| CN104388457A (en) * | 2014-12-02 | 2015-03-04 | 中国科学院青岛生物能源与过程研究所 | Gene modification method for increasing yield of phloroglucinol and application of same |
| CN104988172A (en) * | 2015-07-30 | 2015-10-21 | 中国科学院青岛生物能源与过程研究所 | Construction method and application of high-yield phloroglucinol gene engineering bacterium |
| CN104988172B (en) * | 2015-07-30 | 2018-05-04 | 中国科学院青岛生物能源与过程研究所 | A kind of construction method of high yield phloroglucin genetic engineering bacterium and application |
| CN105018511B (en) * | 2015-07-30 | 2018-08-17 | 中国科学院青岛生物能源与过程研究所 | A kind of method of external enzyme reaction method for synthesizing phloroglucinol and application |
| CN105622379A (en) * | 2016-03-15 | 2016-06-01 | 中国科学院华南植物园 | 2,4-DAPG (2,4-Diacetylphloroglucinol) analogue and preparation method and application thereof |
| CN106905157A (en) * | 2017-01-10 | 2017-06-30 | 西北农林科技大学 | 2,4 diacetyl phloroglucin ester type compounds and its application of sterilization |
| CN106929527A (en) * | 2017-04-24 | 2017-07-07 | 中国科学院青岛生物能源与过程研究所 | A kind of genetic engineering bacterium of phloroglucin synthesis capability high and construction method and application |
| CN107557326A (en) * | 2017-10-27 | 2018-01-09 | 山东大学 | One plant of sterilization fixed nitrogen Pseudomonas fluorescens and its fermentation process and application |
Non-Patent Citations (8)
| Title |
|---|
| JIHANE ACHKAR 等: "Biosynthesis of Phloroglucinol", 《JACS》 * |
| M. GITA BANGERA 等: "Identification and Characterization of a Gene Cluster for Synthesis of the Polyketide Antibiotic 2,4-Diacetylphloroglucinol from Pseudomonas fluorescens Q2-87", 《JOURNAL OF BACTERIOLOGY》 * |
| NCBI: "Pseudomonas protegens CHA0 genome assembly, chromosome: 1", 《GENBANK DATABASE》 * |
| NCBI: "Pseudomonas protegens CHA0, complete genome", 《GENBANK DATABASE》 * |
| XU YAN 等: "Transcriptional Regulator PhlH Modulates 2,4-Diacetylphloroglucinol Biosynthesis in Response to the Biosynthetic Intermediate and End Product", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 * |
| 周洪友 等: "抗生素2,4-二乙酰基间苯三酚合成基因在假单胞杆菌P32中的异源表达", 《中国植物病理学会2004年学术年会论文集》 * |
| 周洪友: "荧光假单胞菌2P24抗生素2，4-二乙酰基间苯三酚生物合成的基因调控研究", 《中国优秀博士学位论文全文数据库（电子期刊）农业科技辑》 * |
| 周田甜: "番茄青枯病拮抗菌J12和J2的分离鉴定及抗生素2,4-DAPG合成和调控研究", 《中国优秀博士学位论文全文数据库（电子期刊）农业科技辑》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109439606A (en) * | 2018-11-14 | 2019-03-08 | 中国科学院青岛生物能源与过程研究所 | A kind of genetic engineering bacterium improving phloroglucin yield and its construction method and application |
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