CN103923864A - Strain of high-flavonoid compound tolerant engineered escherichia coli as well as construction method and application thereof - Google Patents
Strain of high-flavonoid compound tolerant engineered escherichia coli as well as construction method and application thereof Download PDFInfo
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- CN103923864A CN103923864A CN201410169822.2A CN201410169822A CN103923864A CN 103923864 A CN103923864 A CN 103923864A CN 201410169822 A CN201410169822 A CN 201410169822A CN 103923864 A CN103923864 A CN 103923864A
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Abstract
The invention discloses a strain of high-flavonoid compound tolerant engineered escherichia coli as well as a construction method and an application thereof, and belongs to the genetic engineering field. An escherichia coli producing strain applicable to the production of flavonoids is taken as a receptor and then the malE gene of the escherichia coli producing strain is silenced to obtain the strain of high-flavonoid compound tolerant engineered escherichia coli; the tolerance of E.coli to flavonoid compounds can be remarkably improved, exogenous flavonoid compounds are added to stimulate the growth of E.coli, and compared with the original bacteria, the maximum specific growth rate of the engineered bacteria is increased by 2.07 times.
Description
Technical field
The present invention relates to a kind of high flavonoid compound tolerance colibacillus engineering and construction process and application, particularly a kind of intestinal bacteria that flavonoid compound tolerance is improved that obtain by reticent malX gene.
Background technology
Flavonoid compound (Flavonoids) is the polyphenolic plant secondary metabolite of nature most species, is extensively present in vegitabilia, and the kind having been found that has at present exceeded kind more than 8000.Flavonoid compound has pharmacy value widely, have stronger antibacterial, anti-inflammatory, anti-oxidant, antitumor, remove the various biological effects such as free radical, immunomodulatory, anticancer, antiviral, hypoglycemic, radioprotective and reducing blood-fat.Along with progressively going deep into Physiological Activities of Flavonoid research, also more and more to its exploitation, also increasing to its demand, in recent years, the market of flavonoid compound increases more than 30% every year, on medicine and nutrient chemistry product field, has broad application prospects.But the acquisition of natural flavonoid material is subject to the restriction in time and region, and content is low in plant, be unfavorable for research and Clinical Laboratory rapidly, and chemical structure is very complicated, very complicated by chemical synthesis, is unfavorable for large-scale production.Therefore be more and more subject to people's attention with microorganisms producing flavonoid compound.
Along with the range of application of flavonoid compound is more and more wider, its demand, also in continuous increase, is extracted and is had various problems from plant, therefore, has great application prospect by microorganism fermentative production flavonoid compound.At present, the biosynthetic pathways metabolism of flavonoid compound is perfectly clear, the basic framework of flavonoid compound is synthetic under the catalysis of chalcone synthase (CHS) by 3 malonyl coenzyme As (malonylCoA) and 1 coumaric acyl coenzyme A (coumaroylCoA), and then the flavones of other kinds is to generate by chemical reactions such as hydroxyl, methoxy substitution or alkylations on the basis of parent nucleus.Intestinal bacteria genetic background is clear, and molecule manipulation technology is perfect, is the first-selection of biotechnology production platform, and therefore someone produces flavonoid compound by transforming E.coli associated metabolic approach very early.Much study at present successfully and realized multiple flavonoid compound synthesizing in E.coli by technique means such as genetically engineereds, as naringenin, Quercetin, trans-resveratrol etc.But flavonoid compound itself is to E.coli, growth has certain restraining effect, is mainly by following several mechanism: 1. destroy the integrity of cell walls and cytolemma, change cell membrane permeability, and then destroy the function of cytolemma, affect the growth of bacterium; 2. suppress the synthetic of nucleic acid, Quercetin, 4',5,7-trihydroxyflavone, pentahydroxyflavones etc. can suppress the activity of DNA gyrase and then the synthesizing of inhibition DNA of E.coli.Rutin can promote the DNA cleavage of E.coli, causes SOS reaction to occur, thereby suppresses the growth of thalline; 3. suppress energy metabolism, by suppressing the vigor of atp synthase and then suppressing the content of ATP in born of the same parents, affect the growth of thalline.Therefore utilize E.coli to produce flavonoid compound and must consider the toxic action of flavonoid compound to E.coli.
Do not have at home at present about the report that increases the associated protein of E.coli to flavonoid compound tolerance.
Summary of the invention
The technical problem to be solved in the present invention is to find out can improve the associated protein of E.coli to flavonoid compound tolerance and provide a kind of to flavonoid compound height endurability colibacillus engineering, by producing malX gene in bacterial strain and carry out silence acquisition can be used for intestinal bacteria that flavones produces.
The plasmid that described engineering bacteria contains reticent manX gene.
Described manX gene nucleotide series and upstream sequence thereof are as shown in SEQ ID NO.1.
Described manX gene clone is in E.coli BL21 (DE3) bacterial strain, and construction recombination plasmid pRSF-PTasRNA-manX proceeding in E.coli BL21 (DE3).
Described flavonoid compound is rutin, naringenin or trans-resveratrol.
The basis of proposition of the present invention and pre-stage test:
1) in the time of E.coli stationary phase, add after flavonoid compound stimulates and collect somatic cells, extract membranin, logical
Cross the technique means of proteomics, find that the expression amount of ManX albumen increases significantly.
2) improve the mRNA level of this albumen of qPCR technical identification in this physiological process.
3) find the nucleotide sequence design primer of this gene and clone by ncbi.
4) gene is connected and obtains recombinant expression vector with carrier;
5) will after the recombinant expression vector Transformed E .coli BL21 (DE3) obtaining, obtain recombinant bacterial strain;
6) recombinant bacterial strain carries out verifying by qPCR whether its mRNA level declines is whether gene is silenced after IPTG induction;
7) by the mensuration of growth curve under the environment existing at flavonoid compound, verify whether the overexpression of this albumen has improved the tolerance of E.coli to flavonoid compound.
The specific descriptions of technical solution of the present invention below:
The discovery of ManX albumen
The single bacterium colony of picking E.coli BL21 (DE3) is with after the activation of LB substratum, be inoculated in 25mL LB substratum by 1% inoculum size, cultivate and add 1g/L flavonoid compound (rutin stationary phase (10h), naringenin, trans-resveratrol) collect somatic cells after stimulating 3h.According to FOCUS
tMglobal Fractionation specification sheets extracts membranin, and remove the ion in protein sample with 2-D Clean-Up test kit, finally use the concentration in non-interfering determination of protein concentration kit measurement sample, the last concentration aquation loading that contains 800ng according to 450 μ L, use the adhesive tape of the pH4-7 of 24cm to carry out IEF, then the SDS-PAGE of twice rear use 13.5% of balance carry out second to, after end with the R250 12h that dyes, after decolouring, with ImageScanner III instrument, glue is taken pictures, acquisition is known after image and is carried out atlas analysis with PDQuest, digging out the point differing greatly decolours, the steps such as enzymolysis, then utilize MALDI-TOF/MS mass spectrum to carry out identification of proteins to peptide section sample, by retrieval Matrix Science database, determine albumen kind.
qPCR
Collect the method for thalline the same, utilize the RNAprep Pure Cell/Bacteria test kit of Tian Gen company to extract total RNA of strain cell, utilize TaKaRa company
rNA reverse transcription is become cDNA construction cDNA library by RTreagent test kit, utilize SYBR Premix ExTaq test kit and the LightCycler480II Real Time PCR instrument of TaKaRa company to carry out qPCR reaction, specific procedure is as follows: 95 DEG C of denaturation 30s; 95 DEG C of C5s → 55 DEG C 20s, 40 circulations (PCR reaction); 95 DEG C of 10s → 65 DEG C 1min → 95 DEG C 10s (solubility curve analysis).For the circulation ratio of warranty test, 3, each sample is parallel, then averages as the basis of calculating.
The structure of plasmid and recombinant bacterium
The gene order design primer of the manX finding from ncbi is increased, be connected respectively to cloning vector pMD19-T order-checking, obtain and carry out double digestion after correct transformant and be connected on the pRSF-PTasRNA plasmid that this laboratory builds, structure obtains recombinant plasmid pRSF-PTasRNA-manX, by the recombinant expression vector Transformed E .coli JM109 building, bacterium colony PCR verifies positive transformant, extracts the recombinant plasmid that builds and proceeds to and in E.coli BL21 (DE3) bacterium, obtained the recombinant bacterial strain of manX gene reticent.
Growth curve checking
With M9 substratum (13.3g/L M9CA Broth, 0.2% glucose, 1mM magnesium sulfate, 0.5 μ g/mLthiamine-HCl) activation engineering bacteria (add 1% paraxin) and original bacterium, inoculate into containing respectively 1g/L rutin according to 1% inoculum size, naringenin or trans-resveratrol, IPTG (0,100,200,400mM) in M9 substratum, 30 DEG C of 200r/min cultivate, and survey OD every 1h
600, until strain growth is to stationary phase.Obtain the growth curve chart of bacterial strain, then according to formula μ=d
x/ x*d
tthe specific growth rate figure that calculates bacterial strain, then draws maximum specific growth rate μ
max.
The resistance to flavonoid compound ability of engineering bacteria provided by the invention improves, and external source is added flavonoid compound and stimulated E.coli growth, and compared to original bacterium, the maximum specific growth rate of engineering bacteria has improved 2.05 times compared to original bacterium.
Embodiment
The discovery of embodiment 1ManX
With inoculating needle on E.coli BL21 (DE3) flat board picking list colony inoculation in 25mL LB substratum, activation 12h.Inoculum size according to 1% is inoculated in respectively 4 bottles of 25mL LB substratum, being placed in 37 DEG C of shaking tables (200rpm) was cultured to after stationary phase, wherein three bottles of to add respectively concentration that 25 μ L DMSO dissolve be 0.1g/L rutins, naringenin and trans-resveratrol (final concentration is 1g/L), other one bottle is added 25 μ L DMSO in contrast, cultivate 8000rpm low-temperature centrifugation after 3h and collect thalline, wash 3 times with deionized water after according to FOCUS
tMglobal Fractionation specification sheets extracts membranin, and remove the ion in protein sample with 2-D Clean-Up test kit, finally use the concentration in non-interfering determination of protein concentration kit measurement sample, the last concentration aquation loading that contains 800ng according to 450 μ L, use the adhesive tape of the pH4-7 of 24cm to carry out IEF, then the SDS-PAGE of twice rear use 13.5% of balance carry out second to, after end with the R250 12h that dyes, after decolouring, with ImageScanner III instrument, glue is taken pictures, acquisition is known after image and is carried out atlas analysis with PDQuest, one of them glue point is having rutin, in the sample of naringenin and trans-resveratrol, all there is more than 2 times downwards compared to control group, this point is dug out and decoloured, dehydration, the steps such as enzymolysis, then utilize MALDI-TOF/MS mass spectrum to carry out identification of proteins to peptide section sample, show that by retrieval MatrixScience database this point is the ManX albumen that E.coli belongs to.
The structure of embodiment 2 engineering bacterias of resistance to flavonoid compound
The 40bp design primer of 140bp and upstream before the gene of the manX finding from ncbi is increased, be connected respectively to cloning vector pMD19-T order-checking, obtain and carry out double digestion after correct transformant and be connected on plasmid pRSF-PTasRNA, structure obtains recombinant plasmid pRSF-PTasRNA-manX, the expression vector building is transformed into E.coli JM109, be applied to (yeast extract paste 5g/L on the LB substratum that contains paraxin, peptone 10g/L, NaCl10g/L, solid medium adds 20g/L agar, 121 DEG C of sterilizing 15min), the transformant that picking transforms on rear plate carries out PCR checking, obtain correct single bacterium colony and carry out fermented extracted plasmid, plasmid is proceeded to E.coli BL21 (DE3) competence, be applied on the LB substratum that contains paraxin, the transformant that picking transforms on rear plate carries out PCR checking, obtain correct single bacterium colony and be recombinant bacterial strain, also obtain E.coli BL21 (DE3) engineering bacteria of high tolerance flavonoid compound.
The high tolerance flavonoid compound E.coli BL21 (DE3) of embodiment 3 engineering bacteria growth curve is measured
Picking engineering bacteria and the single colony inoculation of E.coli BL21 (DE3) that contains empty plasmid pRSF-PTasRNA are to the M9 substratum that contains 1% kantlex (13.3g/L M9CA Broth, 0.2% glucose, 1mM magnesium sulfate, 0.5 μ g/mL thiamine-HCl) middle activation after 10h, contain 1% kantlex by 1% inoculum size access, 1g/L flavonoid compound (rutin, naringenin, trans-resveratrol), and the IPTG (0,100 of different concns, 200,400 μ M) M9 substratum in, 30 DEG C of 200rpm cultivate, and survey an OD every 1h
600until strain growth is to stationary phase.According to formula μ=d
x/ x*d
tthe specific growth rate figure that calculates bacterial strain, then draws maximum specific growth rate μ
max, the μ of discovery engineering bacteria in the environment that has 1g/L rutin
maxbe 0.37, and original strain is 0.23, growth velocity has promoted 1.61 times; The μ of engineering bacteria in the naringenin environment of 1g/L
maxbe 0.39, and original strain is 0.22, growth velocity has promoted 1.77 times; The μ of engineering bacteria in the trans-resveratrol environment of 1g/L
maxbe 0.29, and original strain is 0.14, growth velocity has promoted 2.07 times.
Although the present invention with preferred embodiment openly as above; but it is not in order to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, therefore protection scope of the present invention should be with being as the criterion that claims were defined.
Claims (6)
1. a plant height flavonoid compound tolerance colibacillus engineering, is characterized in that the intestinal bacteria that can be used for flavones production are produced to malX gene in bacterial strain carries out silence.
2. colibacillus engineering claimed in claim 1, is characterized in that described malE gene nucleotide series and upstream sequence thereof are as shown in SEQ ID NO.1.
3. the colibacillus engineering described in claim 1 or 2, other are being E.coli BL21 (DE3) the described intestinal bacteria that can be used for flavones production.
4. the method described in claim 1 or 2, is characterized in that described flavonoid compound is rutin, naringenin or trans-resveratrol.
5. the construction process of colibacillus engineering claimed in claim 1, is characterized in that comprising the steps:
1) clone according to the front 140bp of manX gene in looked into E.coli BL21 (DE3) and the 30bp primers of upstream;
2) gene is connected and obtains recombinant expression vector with carrier;
3) will after the recombinant expression vector Transformed E .coli obtaining, obtain recombinant bacterial strain.
6. the application of the colibacillus engineering that claim 1 obtains in flavonoid compound is produced.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2620387A1 (en) * | 2005-09-20 | 2007-04-12 | Basf Plant Science Gmbh | Methods for controlling gene expression using ta-sirna |
| EP1811030A1 (en) * | 2004-10-29 | 2007-07-25 | INTERNATIONAL FLOWER DEVELOPMENTS Pty. Ltd. | Method of stabilizing, and bluing, of anthocyanin pigment using gene coding for transferase of aromatic acyl to 3 -position of anthocyanin |
| CN102286420A (en) * | 2011-06-27 | 2011-12-21 | 江南大学 | High-glucosamine-yield engineering bacterium with manX knocked out by homologous recombination and construction method thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1811030A1 (en) * | 2004-10-29 | 2007-07-25 | INTERNATIONAL FLOWER DEVELOPMENTS Pty. Ltd. | Method of stabilizing, and bluing, of anthocyanin pigment using gene coding for transferase of aromatic acyl to 3 -position of anthocyanin |
| CA2620387A1 (en) * | 2005-09-20 | 2007-04-12 | Basf Plant Science Gmbh | Methods for controlling gene expression using ta-sirna |
| CN102286420A (en) * | 2011-06-27 | 2011-12-21 | 江南大学 | High-glucosamine-yield engineering bacterium with manX knocked out by homologous recombination and construction method thereof |
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Inventor after: Zhou Jingwen Inventor after: Chen Jian Inventor after: Wang Kui Inventor after: Li Jianghua Inventor after: Du Guocheng Inventor before: Chen Jian Inventor before: Zhou Jingwen Inventor before: Wang Kui Inventor before: Li Jianghua Inventor before: Du Guocheng |
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Address after: 214122 Jiangsu city of Wuxi Province District Liangxi No. 898 South Road 7 layer beam Creek area of food science and Technology Park Patentee after: Jiangnan University Address before: 1800 No. 214122 Jiangsu city of Wuxi Province Li Lake Avenue Patentee before: Jiangnan University |