CN104388457A

CN104388457A - Gene modification method for increasing yield of phloroglucinol and application of same

Info

Publication number: CN104388457A
Application number: CN201410722779.8A
Authority: CN
Inventors: 咸漠; 赵广; 刘敏; 张汝兵
Original assignee: Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
Current assignee: Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
Priority date: 2014-12-02
Filing date: 2014-12-02
Publication date: 2015-03-04

Abstract

The invention discloses a gene modification method for increasing the yield of phloroglucinol and application of the same, and belongs to the technical field of genetic engineering. The gene modification method comprises the following steps: obtaining a mutant strain by virtue of a mode of knocking out or inserting a global regulating factor arcA gene of an original strain, converting the state of the mutant strain to a competent state, introducing recombinant plasmids containing a polyketide synthase gene phlD, a multiple resistance activating factor marA and acetyl CoA carboxylase gene ACCase, and obtaining a recombinant cell. The invention also provides a method of producing the phloroglucinol by utilizing the recombinant cell. According to the gene modification method and application disclosed by the invention, the yield of the phloroglucinol after fermentation is firstly increased by 2.06 times by adopting a mode of globally regulating carbon metabolism after post-transcriptional level, and the gene modification method and application have high industrial application value.

Description

A kind of genetic modification method and application improving Phloroglucinol output

Technical field

The present invention relates to a kind of the genetic modification method and the application that improve Phloroglucinol output, belong to gene engineering technology field.

Background technology

Phloroglucinol is a kind of important fine chemical product, and be the intermediate of synthesis flavones, osajin medicine, Phloroglucinol itself can be used as a kind of excellent smooth muscle spasmolysis medicine, is widely used in clinical.Phloroglucinol can also suppress peroxidase activity, has anti-inflammatory antioxygenation, and it can catalysis H ₂o ₂being decomposed into molecular oxygen and water, is a kind of important antioxidase.In addition, Phloroglucinol or a kind of anti-curing agent of superior performance, stablizer, fuel coupler, Tire tackifier etc., have the wide market requirement.At present, the industrialized preparing process of Phloroglucinol is mainly chemical synthesis, comprises trotyl (TNT) method, isopropyl benzene method, chlorinated benzene method and aniline process.There is various disadvantages in chemical synthesis, as raw material sources difficulty, by product is more, separating-purifying is difficult, environmental pollution is serious.Produce Phloroglucinol with fermentable, the various disadvantages of chemical method can be overcome, and the biological process method for synthesizing phloroglucinol cycle is short, safety and environmental protection.

Heterogenous expression Pseudomonas fluorescens polyketide synthases gene phlD can method for synthesizing phloroglucinol (Jihane Achkar, Mo Xian, Huimin Zhao, J.W.Frost.Biosynthesis of phloroglucinol [J] .Journal of the American ChemicalSociety, 2005,127 (15): 5332-5333.; Wenjuan Zha, Sheryl B.Rubin-Pitel, Huimin Zhao.Characterization of the substrate specificity of PhlD, a type III polyketide synthase fromPseudomonas fluorescens [J] .Journal of Biological Chemistry, 2006,281 (42): 32036-32047.).Express multiple resistance incitant marA on this basis, strengthen intestinal bacteria to the tolerance of Phloroglucinol, express the acetyl CoA carboxylase gene (ACCase) of intestinal bacteria self, in cell, the level of malonyl CoA is 3.6 times of (YujinCao of original strain, Xinglin Jiang, Rubing Zhang, Mo Xian.Improved phloroglucinol production by metabolicallyengineered Escherichia coli [J] .Appl Microbiol Biotechnol, 2011,91:1545 – 1552.).But the current output of Phloroglucinol, productive rate is still on the low side, be difficult to meet industrial demand.Need to carry out genetic modification to engineering strain further, to improving the synthesis capability of Phloroglucinol.

Colibacillary metabolism network comprises hundreds of metabolite, and these metabolites are by a large amount of biochemistry and regulate reacting phase mutual correlation.The different levelss such as the regulation and control of metabolic fluxes can at transcriptional level, post-transcriptional level, the movable mechanics of enzyme are realized by Various Complex mechanism.The regulation and control of transcriptional level are the most important shaping modes of intestinal bacteria.Global regulation's factor itself comprises complicated metabolism network, and present pleiotropy when playing a role, the operon that can simultaneously regulate several functions different, therefore, global regulation's factor is comparatively complicated for the impact of some specific metabolite.Arc system is the two component global regulation factors important in cell, under aerobic, anaerobic and micro-oxygen conditions, all can regulate and control the expression of multiple operon.Arc system comprises kytoplasm response factor ArcA and film in conjunction with sensitive kinase ArcB two kinds of components.ArcB can autophosphorylation and phosphate group is passed to ArcA, and the ArcA of phosphorylation can in conjunction with the promotor of several genes, regulate gene expression.ArcA can suppress the expression (S.Iuchi of TCA circulation and glyoxylate cycle genes involved, E.C.C.Lin, arcA (dye), a global regulatory gene in Escherichia coli medi-atingrepression of enzyme in aerobic pathways, Proc.Natl.Acad.Sci.U.S.A.1988, (85) 1888 – 1892.; Syed Asif Nizam, Jiangfeng Zhu, PeiYeeHo, Kazuyuki Shimizu.Effects of arcA and arcB genesknockout on the metabolism in Escherichia coli under aerobic condition [J] .BiochemicalEngineering Journal, 2009,44:240 – 250.) ArcA regulatory factor encoded by gene arcA.Research finds: the arcA gene knocking out E.coliMG1655, beta-galactosidase enzymes output increased 10%-20%, and yield of acetic acid declines 50%.On this basis, process LAN nadh oxidase (NOX), the output increased 120% of beta-galactosidase enzymes, do not detect that acetic acid generates (G.N.Vemuri, M.A.Eiteman, E.Altman.Increased Recombinant Protein Production in Escherichia coliStrainsWith Overexpressed Water-Forming NADH Oxidase and a Deleted ArcA RegulatoryProtein [J] .Biotechnology and Bioengineering.2006,538-542.).

The genes such as process LAN phlD, ACCase can improve Phloroglucinol output, but the output of current Phloroglucinol and productive rate still cannot meet industrial demand.ArcA is regulatory factor of overall importance important in intestinal bacteria, and this regulatory factor temporarily without report, improves the genetic modification method of Phloroglucinol output also without report by regulation and control of carbon metabolism of overall importance on the impact of Phloroglucinol fermentative production.

Summary of the invention

For solving the problem, the invention provides a kind of genetic modification method improving Phloroglucinol output, the technical scheme taked is as follows:

One object of the present invention is to provide a kind of genetic modification method improving Phloroglucinol output, the method is the mutant strain that mode by knocking out or inserting obtains starting strain global regulation factor arcA gene inactivation, import the recombinant plasmid containing polyketide synthases gene phlD, multiple resistance incitant marA, acetyl CoA carboxylase Gene A CCase after again mutant strain being transferred to competence, obtain reconstitution cell.

The step of described method is as follows:

1) mode passing through to insert inactivation or knock out inactivation makes the global regulation factor gene arcA inactivation of starting strain, obtains mutant strain;

2) preparation process 2) competent cell of gained mutant strain;

3) recombinant plasmid of preparation containing polyketide synthases gene phlD, multiple resistance incitant marA, acetyl CoA carboxylase Gene A CCase;

4) by step 3) recombinant plasmid of gained imports to step 2) in the competent cell of gained, obtain reconstitution cell.

Step 1) described starting strain, be intestinal bacteria, wherein preferred E.coliBL21 (DE3).

Step 3) described polyketide synthases gene phlD, derive from Pseudomonas fluorescens; Described multiple resistance incitant marA, derives from intestinal bacteria; Described acetyl CoA carboxylase Gene A CCase, derives from intestinal bacteria.

Preferably, described polyketide synthases gene phlD, GeneBank ID are 11830552; Described multiple resistance incitant marA, GeneBank ID are 6060688; Described acetyl CoA carboxylase Gene A CCase, wherein, the GeneBank ID of subunit accA is 6062185, and the GeneBank ID of subunit accB is 6058890, and the GeneBank ID of subunit accC is 6058863, and the GeneBank ID of subunit accD is 6059083.

The concrete steps of described method are as follows:

1) with e. coli bl21 (DE3) for starting strain, knock out the sequence of 300bp on starting strain global regulation factor gene arcA, obtain mutant strain;

Described arcA gene knock out sequence as shown in SEQ ID NO.1;

2) preparation process 2) competent cell of gained mutant strain;

3) polyketide synthases gene phlD and multiple resistance incitant marA is connected on carrier pET30a, obtains recombinant plasmid pET-phlD-marA; Acetyl CoA carboxylase Gene A CCase is connected on carrier pACYC, obtains recombinant plasmid pACYC-accADBC;

Described polyketide synthases gene phlD, derives from Pseudomonas fluorescens, Genebank ID:11830552; Described multiple resistance incitant marA, derives from intestinal bacteria, Genebank ID:6060688; Described acetyl CoA carboxylase Gene A CCase, derives from intestinal bacteria, wherein, the Genebank ID:6062185 of subunit accA, the Genebank ID:6059083 of the Genebank ID:6058863 of the GenebankID:6058890 of subunit accB, subunit accC, subunit accD;

4) by step 3) recombinant plasmid pET-phlD-marA and pACYC-accADBC of gained import to step 2) obtain reconstitution cell in the competent cell of gained.

Described method is for obtaining the genetic engineering bacterium of high yield Phloroglucinol and producing Phloroglucinol.

The step of described application is as follows:

Described arcA gene knock out sequence as shown in SEQ ID NO.1;

2) preparation process 2) competent cell of gained mutant strain;

4) by step 3) recombinant plasmid of gained imports to step 2) in the competent cell of gained, obtain reconstitution cell;

5) by shake-flask culture or fermentor cultivation step 4) gained reconstitution cell production Phloroglucinol.

Described applying step 5) described cultivation, the inoculum size of reconstitution cell seed liquor is the 1%-5% of culture volume, and culture temperature is 37 DEG C, and stirring velocity is 400-800rpm, pH6.0-8.0, is cultured to OD under the condition of dissolved oxygen more than 18% ₆₀₀for 8-12, then add inductor IPTG to final concentration 100 μMs, terminate after utilizing the glucose liquid storage of massfraction 50-80% to continue fed-batch fermentation 12-24h.

The beneficial effect that the present invention obtains is as follows:

1. the invention provides a kind of the genetic modification method and the application that improve Phloroglucinol output, the method realizes in the mode of transcriptional level by regulation and control of carbon metabolism of overall importance first, improves Phloroglucinol output, has higher industrial application value.

2. the invention provides a kind of engineering strain improving Phloroglucinol output, for starting strain with bacillus coli Escherichia coliBL21 (DE3), with suicide plasmid pRE112 for medium, knock out a kind of global regulation factor arcA gene by homologous recombination technique, obtain engineering strain E.coliBL21 (DE3) △ arcA.Recombinant plasmid pET-phlD-mar and pACYC-accADBC is imported this project bacterial strain, for the fermentative production of Phloroglucinol.

3. genetic modification method provided by the invention, at shaking flask and fermentation tank level, on the basis containing two kinds of recombinant plasmid pET-phlD-mar and pACYC-accADBC, knock out arcA gene, yield increased bacterial strain Escherichia coliBL21 (DE3) of Phloroglucinol improves about 3 times.

Definition and abbreviation

Use following abbreviation or abbreviation in this article:

Phloroglucinol (Phloroglucinol): PG

Isopropylthiogalactoside: IPTG

Polyketide synthases gene: phlD

Multiple resistance incitant: marA

Acetyl CoA carboxylase gene: ACCase

Bacillus coli (Escherichia coli): E.coli

" gene knockout " refers to by certain approach, by specific gene all or part of deletion from genome, thus makes specific gene afunction.

" genotype " refers to the general name that a certain biont full gene combines, and is interior that group gene that comprise, distinctive of cell of this biology.

After specific gene is subject to various signals-modulating in " overexpression " or " process LAN " phalangeal cell, in organism, exceedes previous level express, can realize by strengthening endogenous expression or introducing foreign gene.

" bridging PCR " is over-lap PCR again, refers to adopt the primer with spacer end, makes PCR primer form overlapping chain, thus by the extension of overlapping chain in amplified reaction subsequently, is got up by the amplified fragments lap splice of different sources.

Accompanying drawing explanation

Fig. 1 is that mutant strain ZG-1949 builds schematic diagram.

Embodiment

The present invention is illustrated in detail below by example.But the present invention is not limited to following examples.

If experimental technique involved in following embodiment, without specified otherwise, is routine techniques.

If the material used in following embodiment, reagent etc., without specified otherwise, all can obtain from commercial channels.

Restriction enzyme used and T4DNA ligase enzyme are all purchased from MBI Fermentas company, and plasmid extraction and glue reclaim used kit purchased from American OMEGA company, and operation steps is carried out according to product description; All substratum are all prepared with deionized water if no special instructions.

Culture medium prescription:

1) seed liquor substratum

LB substratum: yeast powder 5g/L, NaCl 10g/L, peptone 10g/L, adds kantlex 50 μ g/mL during inoculation, paraxin 50 μ g/mL.

M9 substratum: NH ₄cl 1.0g/L, Na ₂hPO ₄12H ₂o 15.2g/L, KH ₂pO ₄3.0g/L, NaCl 0.5g/L, glucose 20g/L, MgSO ₄7H ₂o 0.4g/L, 1000 × trace element ((NH ₄) ₆mo ₇o ₂₄4H ₂o 3.7g/L; ZnSO ₄7H ₂o2.9g/L; H ₃bO ₃24.7g/L; CuSO ₄5H ₂o 2.5g/L; MnCl ₂4H ₂o 15.8g/L), add kantlex 50 μ g/mL during inoculation, paraxin 50 μ g/mL.

2) fermention medium

K ₂hPO ₄3H ₂o 9.8g/L, Citric acidH ₂o 2.1g/L, ferric ammonium citrate 0.3g/L, (NH ₄) ₂sO ₄3.0g/L, glucose 20g/L, MgSO ₄7H ₂o 0.4g/L, 1000 × trace element ((NH ₄) ₆mo ₇o ₂₄4H ₂o 3.7g/L; ZnSO ₄7H ₂o 2.9g/L; H ₃bO ₃24.7g/L; CuSO ₄5H ₂o 2.5g/L; MnCl ₂4H ₂o 15.8g/L), kantlex 50 μ g/mL, paraxin 50 μ g/mL.

Wherein: K ₂hPO ₄3H ₂o 9.8g/L, Citric acidH ₂o 2.1g/L, ferric ammonium citrate 0.3g/L, (NH4) ₂sO ₄pH 7.0 is adjusted to, 121 DEG C, 20min autoclaving after 3.0g/L mixing.Glucose liquid storage is 500g/L, 115 DEG C, the independent sterilizing of 20min, MgSO ₄7H ₂o liquid storage is 200g/L, 121 DEG C, the independent sterilizing of 20min, and 1000 × trace element adopts 0.22 μm of bacteriological filtration membrane filtration degerming, adds above-mentioned glucose degerming separately, MgSO respectively during switching seed liquor ₄7H ₂o, 1000 × micro-liquid storage and microbiotic.

The structure of embodiment 1 bacterial strain

With bacillus coli E.coliBL21 (DE3) for starting strain, knock out the arcA gene in genome, build engineering strain ZG1949 (building flow process as shown in Figure 1), related plasmids pET-phlD-mar and pACYC-accADBC producing Phloroglucinol is imported the Phloroglucinol fermentation carrying out shaking flask and fermentation tank level in ZG1949 and control strain E.coliBL21 (DE3) and detect.

It should be appreciated by those skilled in the art that the gene knockout experiment of above-mentioned bacillus coli E.coliBL21 (DE3), each step is all carried out according to the molecule clone technology of standard.

The structure of 1.1 homology arms

With the upstream of bacillus coli E.coliBL21 (DE3) wild strain arcA gene and downstream (containing arcA Gene Partial sequence) about 600bp base fragment for template design primer, the upstream and downstream homology arm fragment of pcr amplification arcA gene, utilizes glue to reclaim test kit and reclaims goal gene fragment.

Amplimer sequence is:

arcA-Up-5'：

5'-CGGGGTACCGCTGGCGCTTTCTGAAC-3'

arcA-Up-3'：

5'-GTGAACTGCGCGAGCAGGCTGAAAACCCAGGCAAA-3'

arcA-Down-5'：

5'-GCCTGCTCGCGCAGTTCACG-3'

arcA-Down-3'：

5'-CGAGCTCCGTTTATTAGTTGTATGATGC-3'

With the upstream and downstream homology arm fragment after recovery for template carries out bridging PCR, utilize glue to reclaim test kit and reclaim homology arm fragment Δ arcA, restriction enzyme KpnI, SacI be double digestion homology arm fragment Δ arcA and suicide plasmid pRE112 respectively, ratio 16 DEG C of enzymes that spend the night under the effect of ligase enzyme of fragment and carrier 2:1 in molar ratio connect, enzyme connects the heat-shock transformed E.coliCC118 competence of product 42 DEG C, PCR screening obtains positive colony, obtains recombinant plasmid pRE112-Δ arcA.

1.2 homologous recombination

Recombinant plasmid pRE112-Δ arcA is converted into E.coli χ 7213, and carry out twice homologous recombination as donor bacterium and E.coliBL21 (DE3) wild strain, arcA gene knockout part is about 300bp, verify that positive colony molecular weight is less than wild mushroom by PCR, obtain engineering strain E.coliBL21 (DE3) the Δ arcA knocking out arcA gene.

Checking primer sequence:

arcA-Up-5'：

5'-CGGGGTACCGCTGGCGCTTTCTGAAC-3'

ID-arcA-3'

5'-GCTACATATCCTTCTGTTTAC-3'

1.3 expression vector transformed host cells

The operation steps preparing test kit according to TAKARA competence prepares the competent cell of wild type control strain E.coli BL21 (DE3) and engineering strain ZG1949, two kinds of recombinant plasmid pET-phlD-mar and pACYC-accADBC is converted into the competent cell of above-mentioned preparation by heat shock method.

The fermenting experiment of embodiment 2. engineering strain

2.1 shake flask fermentation experiments

1) cultivation of primary seed solution, wild control strain E.coliBL21 (DE3) containing pET-phlD-marA and pACYC-accADBC recombinant plasmid and ZG1949 are seeded in 3mL LB liquid nutrient medium respectively, and add 50 μ g/mL kantlex and 50 μ g/mL paraxin, 37 DEG C of growth 8-12h.

2) primary seed solution is forwarded in 250mL fermentation shake flask by 1% inoculum size, containing 50mL fermention medium, 200g/L MgSO4.7H2O 100 μ L is added during switching seed liquor, 500g/L glucose 2mL, 1000 × micro-50 μ L, 50 μ g/mL kantlex, 50 μ g/mL paraxin are Double, often kind of bacterial strain sets 3 parallel controls, 37 DEG C, 180rpm cultivation.

3) cell OD600 reaches and can add 100 μMs/L IPTG between 0.6-1.0 and induce.

4) after IPTG induction, 37 DEG C, 180rpm continues to cultivate after 24h and collect bacterium liquid, centrifuging and taking supernatant, measures Phloroglucinol content.

2.2 fed-batch fermentation experiments

1) cultivation of primary seed solution, according to above-mentioned 2.1 inoculation methods

2) cultivation of secondary seed solution, primary seed solution is forwarded in 250mL triangular flask by 1% inoculum size, containing 50mL M9 substratum, 200g/L MgSO4.7H2O 100 μ L is added during switching seed liquor, 500g/L glucose 2mL, 1000 × micro-50 μ L, 50 μ g/mL kantlex, 50 μ g/mL paraxin are Double, 37 DEG C, 180rpm cultivates 8-12h.

3) secondary seed solution is forwarded in 5L fermentor tank by 1% inoculum size, ferment containing in 2-3L fermention medium, be cultured to OD600 under the condition of culture temperature 37 DEG C, stirring velocity 400-800rpm, pH 6.0-8.0 and dissolved oxygen more than 18% and be about 8, add inductor IPTG to final concentration 100uM/L, 50%-80% glucose liquid storage continues fed-batch fermentation 24 hours, centrifuging and taking supernatant, measures Phloroglucinol content.

The content detection of 2.3 Phloroglucinols

Phloroglucinol (PG) concentration determination: phenylacrolein development process

Adopt anti-Weisner detection method (Wenjuan Zha, Sheryl B.Rubin-Pitel, Huimin Zhao.Exploitinggenetic diversity by directed evolution:molecular breeding of type III polyketide synthasesimproves productivity [J] .Molecular BioSystems, 2008,4 (3): 246-248.) content of Phloroglucinol in fermented liquid is measured, its principle is the color reaction according to Phloroglucinol and phenylacrolein, and concrete steps are as follows:

1) the phenylacrolein nitrite ion (phenylacrolein is directly dissolved in the concentrated hydrochloric acid/ethanolic soln of volume ratio 1:3) of 10mg/L is prepared.

2) in 1.5mL centrifuge tube, add 1mL phenylacrolein nitrite ion, then add 5 μ L fermented liquid supernatant, put upside down mixing, room temperature places 15min.

3) read OD446 value with 10mm optical path cuvette, OD446 value is stable in 2h;

4) draw Phloroglucinol typical curve, calculate Phloroglucinol content according to typical curve.

According to the present embodiment operation steps, on the basis containing two kinds of recombinant plasmid pET-phlD-mar and pACYC-accADBC, shaking flask level, the PG output of wild control strain is 0.30g/L, and the output of mutant strain ZG1949 is 0.93g/L; In fed-batch fermentation tank level, the PG output of wild control strain is 3.8g/L, and the output of mutant strain ZG1949 is 11.2g/L.Under identical fermentation condition, on the basis containing two kinds of recombinant plasmid pET-phlD-mar and pACYC-accADBC, shaking flask and fermentation tank level, mutant strain ZG1949 provided by the invention, yield increased bacterial strain Escherichia coliBL21 (DE3) of Phloroglucinol improves 1.95 times.

The fermenting experiment of embodiment 3. engineering strain

3.1 shake flask fermentation experiments

1) cultivation of primary seed solution, according to embodiment 2.1 inoculation method.

2) primary seed solution is forwarded in 250mL fermentation shake flask by 3% inoculum size, containing 50mL fermention medium, during switching seed liquor, adds 200g/L MgSO ₄.7H ₂o 100 μ L, 500g/L glucose 2mL, 1000 × micro-50 μ L, 50 μ g/mL kantlex, 50 μ g/mL paraxin are Double, and often kind of bacterial strain sets 3 parallel controls, 37 DEG C, 180rpm cultivation.

3) cell OD600 reaches and can add 100 μMs/L IPTG between 0.6-1.0 and induce.

The fed-batch fermentation of 3.2 Phloroglucinols is produced

1) cultivation of primary seed solution, according to embodiment 2.1 inoculation method

2) cultivation of secondary seed solution, is forwarded in 250mL triangular flask by primary seed solution by 3% inoculum size, containing 50mL M9 substratum, adds 200g/L MgSO during switching seed liquor ₄.7H ₂o 100 μ L, 500g/L glucose 2mL, 1000 × micro-50 μ L, 50 μ g/mL kantlex, 50 μ g/mL paraxin are Double, 37 DEG C, 180rpm cultivates 8-12h.

3) secondary seed solution is forwarded in 5L fermentor tank by 3% inoculum size, ferment containing in 2-3L fermention medium, be cultured to OD600 under the condition of culture temperature 37 DEG C, stirring velocity 400-800rpm, pH 6.0-8.0 and dissolved oxygen more than 18% and be about 10, add inductor IPTG to final concentration 100uM/L, 50%-80% glucose liquid storage continues fed-batch fermentation 16 hours, centrifuging and taking supernatant, measures Phloroglucinol content.

The content detection of 3.3 Phloroglucinols

The assay of Phloroglucinol is with embodiment 2.3

According to the present embodiment operation steps, on the basis containing two kinds of recombinant plasmid pET-phlD-mar and pACYC-accADBC, shaking flask level, the PG output of wild control strain is 0.33g/L, and the PG output of mutant strain ZG1949 is 0.84g/L; In fed-batch fermentation tank level, the PG output of wild control strain is 3.5g/L, and the PG output of mutant strain ZG1949 is 10.1g/L.Under identical fermentation condition, on the basis containing two kinds of recombinant plasmid pET-phlD-mar and pACYC-accADBC, shaking flask and fermentation tank level, engineering strain ZG1949 provided by the invention, yield increased bacterial strain Escherichia coli BL21 (DE3) of Phloroglucinol improves 1.89 times.

The fermenting experiment of embodiment 4. engineering strain

4.1 shake flask fermentation experiments

1) cultivation of primary seed solution, according to above-described embodiment 2.1 inoculation method.

2) primary seed solution is forwarded in 250mL fermentation shake flask by 5% inoculum size, containing 50mL fermention medium, during switching seed liquor, adds 200g/L MgSO ₄.7H ₂o 100 μ L, 500g/L glucose 2mL, 1000 × micro-50 μ L, 50 μ g/mL kantlex, 50 μ g/mL paraxin are Double, and often kind of bacterial strain sets 3 parallel controls, 37 DEG C, 180rpm cultivation.

3) cell OD600 reaches and can add 100 μMs/L IPTG between 0.6-1.0 and induce.

The fed-batch fermentation of 4.2 Phloroglucinols is produced

2) cultivation of secondary seed solution, is forwarded in 250mL triangular flask by primary seed solution by 5% inoculum size, containing 50mL M9 substratum, adds 200g/L MgSO during switching seed liquor ₄.7H ₂o 100 μ L, 500g/L glucose 2mL, 1000 × micro-50 μ L, 50 μ g/mL kantlex, 50 μ g/mL paraxin are Double, 37 DEG C, 180rpm cultivates 8-12h.

3) secondary seed solution is forwarded in 5L fermentor tank by 5% inoculum size, ferment containing in 2-3L fermention medium, be cultured to OD600 under the condition of culture temperature 37 DEG C, stirring velocity 400-800rpm, pH 6.0-8.0 and dissolved oxygen more than 18% and be about 12, add inductor IPTG to final concentration 100uM/L, 50%-80% glucose liquid storage continues fed-batch fermentation 12 hours, centrifuging and taking supernatant, measures Phloroglucinol content.

The content detection of 4.3 Phloroglucinols

The assay of Phloroglucinol is according to embodiment 2.3

According to the present embodiment operation steps, on the basis containing two kinds of recombinant plasmid pET-phlD-mar and pACYC-accADBC, shaking flask level, the PG output of wild control strain is 0.30g/L, and the PG output of mutant strain ZG1949 is 0.87g/L; In fed-batch fermentation tank level, the PG output of wild control strain is 3.2g/L, and the PG output of mutant strain ZG1949 is 9.8g/L.Under identical fermentation condition, on the basis containing two kinds of recombinant plasmid pET-phlD-mar and pACYC-accADBC, shaking flask and fermentation tank level, engineering strain ZG1949 provided by the invention, yield increased bacterial strain Escherichiacoli BL21 (DE3) of Phloroglucinol improves 2.06 times.

The present embodiment institute column data is repeatedly the mean values of revision test.

Although present invention has disclosed exemplary exemplary scheme, but those skilled in the art should be appreciated that, under the condition not deviating from the spirit and scope of the present invention defined by accompanying claim, the change of various forms and details can be carried out, the arbitrary combination of various embodiment can be carried out.

Claims

1. one kind is improved the genetic modification method of Phloroglucinol output, it is characterized in that, it is the mutant strain that mode by knocking out or inserting obtains starting strain global regulation factor arcA gene inactivation, import the recombinant plasmid containing polyketide synthases gene phlD, multiple resistance incitant marA, acetyl CoA carboxylase Gene A CCase after again mutant strain being transferred to competence, obtain reconstitution cell.

2. method described in claim 1, is characterized in that, step is as follows:

2) preparation process 2) competent cell of gained mutant strain;

3. method described in claim 2, is characterized in that, step 1) described starting strain is intestinal bacteria.

4. method described in claim 3, is characterized in that, described intestinal bacteria, is E. coli BL21 (DE3).

5. method described in claim 2, is characterized in that, step 3) described polyketide synthases gene phlD, derive from Pseudomonas fluorescens; Described multiple resistance incitant marA, derives from intestinal bacteria; Described acetyl CoA carboxylase Gene A CCase, derives from intestinal bacteria.

6. method described in claim 5, is characterized in that, described polyketide synthases gene phlD, GeneBank ID are 11830552; Described multiple resistance incitant marA, GeneBank ID are 6060688; Described acetyl CoA carboxylase Gene A CCase, wherein, the GeneBank ID of subunit accA is 6062185, and the GeneBank ID of subunit accB is 6058890, and the GeneBank ID of subunit accC is 6058863, and the GeneBank ID of subunit accD is 6059083.

7. method described in claim 2, is characterized in that, concrete steps are as follows:

Described arcA gene knock out sequence as shown in SEQ ID NO.1;

2) preparation process 2) competent cell of gained mutant strain;

Described polyketide synthases gene phlD, derives from Pseudomonas fluorescens, Genebank ID:11830552; Described multiple resistance incitant marA, derives from intestinal bacteria, Genebank ID:6060688; Described acetyl CoA carboxylase Gene A CCase, derives from intestinal bacteria, wherein, the Genebank ID:6062185 of subunit accA, the Genebank ID:6059083 of the Genebank ID:6058863 of the Genebank ID:6058890 of subunit accB, subunit accC, subunit accD;

8. method described in claim 1-7, is characterized in that, for obtaining the genetic engineering bacterium of high yield Phloroglucinol and producing Phloroglucinol.

9. method described in claim 8, is characterized in that, step is as follows:

Described arcA gene knock out sequence as shown in SEQ ID NO.1;

2) preparation process 2) competent cell of gained mutant strain;

10. method described in claim 9, is characterized in that, step 5) described cultivation, the inoculum size of reconstitution cell seed liquor is the 1%-5% of culture volume, and culture temperature is 37 DEG C, and stirring velocity is 400-800rpm, pH6.0-8.0, is cultured to OD under the condition of dissolved oxygen more than 18% ₆₀₀for 8-12, then add inductor IPTG to final concentration 100 μMs, terminate after utilizing the glucose liquid storage of massfraction 50-80% to continue fed-batch fermentation 12-24h.