CN106636023A - Method for enhancing expression intensity of zwf gene promoter - Google Patents
Method for enhancing expression intensity of zwf gene promoter Download PDFInfo
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- CN106636023A CN106636023A CN201710012859.8A CN201710012859A CN106636023A CN 106636023 A CN106636023 A CN 106636023A CN 201710012859 A CN201710012859 A CN 201710012859A CN 106636023 A CN106636023 A CN 106636023A
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Abstract
The invention belongs to the technical field of gene engineering and in particular relates to a method for enhancing the expression intensity of a glucose-6-phosphate dehydrogenase (G6PDH) coding gene zwf promoter by adding betaine. According to the method provided by the invention, the activity of G6PDH is improved, and the method has the characteristics of low cost, simplicity and feasibility; the effect of enhancing the expression intensity of the zwf promoter by 0.98 times can be realized under the condition that the adding amount is 1g/L; the method can be applied to fermented products including amino acids and the like and other genes needing to be subjected to expression intensity enhancement; references are provided for breeding fermented product strains including the amino acids and the like in the future.
Description
Technical field：
The invention belongs to gene engineering technology field, and in particular to one kind strengthens G6P by addition glycine betaine
The method of dehydrogenase coding genes zwf promoter expression intensities.
Background technology：
Promoter is the DNA sequence dna of RNA polymerase specific recognition and combination.Promoter is a composition portion of gene
Point, the initial time of gene expression (transcription) and the degree of expression are controlled, just as " switch ", determine the activity of gene.Promoter
Itself Gene Activity is not controlled, but combined by this protein with referred to as transcription factor and control Gene Activity.
Promoter is located at the section of DNA sequence of structural gene 5' end upstream, and holoenzyme (holoenzyme) can be instructed correctly to tie with template
Close, activate RNA polymerase, promotor gene transcription.
At present conventional promoter and enhancer has constitutive promoter (gene can be made to start expression in all cells
Promoter) and inducible promoter (under some specific physically or chemically stimulations of signal, the promoter of this type can
So that the transcriptional level of gene is significantly increased).The regulation and control of constitutive promoter are not affected by external condition, institute's promotor gene
Expression there is continuation, but do not show Space-time speciality；And inducible promoter can pass through extraneous physiochemical signal
The expression of artificial adjusted and controlled gene.
Constitutive promoter of the prior art, expression intensity does not have Space-time speciality, it is not possible to artificial regulation and control, in reality
Do not possess operability in the application of border；And adding the allogenic materials such as IPTG, lactose in inducible promoter, its is relatively costly, and to bacterium
Body has in itself certain side effect；Thermoinducible promoter is then more sensitive to temperature, higher to equipment requirement, increases the consumption of the energy.
As patent CN101148679A discloses a kind of extraneous sources IPTG and O simultaneously2The expression vector of concentration regulation and control, this
It is bright to can be used for using the expression of various genes for photosynthetic system in hydrogenlike silicon ion research photosynthetic bacteria and in hydrogenlike silicon ion
The expression of artificial adjustment foreign protein and purifying provide effective instrument in expression system；Patent CN102978232A,
CN101914603A and CN104357423A individually disclose one kind and realize that recombinant protein is high using IPTG, lactose or D- lactitols
The method of effect abduction delivering；Patent CN102952817A provides a kind of method using temperature-induced production foreign protein, leads to
Cross regulation and control of the control ambient temperature realization to expression of structural gene；Patent CN102978209A can be in large intestine bar there is provided one kind
The constitutive promoter of express express target protein in bacterium.
It is an object of the invention to improve existing promoter regulation technology, there is provided it is a kind of by external source add glycine betaine come
The method for strengthening zwf promoter expression intensities.During the fermented products such as amino acid can be applied to, and other need to strengthen
The gene of expression intensity.The present invention can provide reference for the breeding of the fermented product bacterial strain such as later amino acid.
With deepening continuously for life science and medical research, researchers can express in the urgent need to one kind in live body
And be easy to detect reporter gene.Existing reporter gene mainly has：Secreting type placenta phosphate (SEAP), beta galactose glycosides
Enzyme (LacZ), beta-glucuronidase (GUS), firefly luciferase (LUC) etc., but the detection method of these genes and pay no attention to
Think, they are required for substrate and confactor, thus the application in live body is restricted.Recently, a kind of brand-new non-enzymatic
Reporter gene-green fluorescent protein (Green fluorescent protein, GFP) is attracted attention, the albumen
Self-catalysis cyclisation fluorescence can be produced after translation.Used as reporter gene, GFP is currently the only to express in living cells
Luminescent protein and in addition to oxygen molecule do not need other any substrates or co-factor；Used as fluorescent tag molecule, GFP both had
Sensitive mark verification and measurement ratio, again without radioactive harm, thus is widely used in life studies field.The present invention is adopted
GFP as reporter gene, to the expression intensity of fast verification promoter.
Glucose-6-phosphate dehydrogenase (G6PDH, encoding gene is zwf) is pentose phosphate pathway (PPP) in Escherichia coli
Key enzyme in approach, and PPP is the main path for synthesizing reducing power NADPH, the synthesis of many amino acid is all relied on
NADPH, is supplied by the vigor of the intensity reinforcement G6PDH of raising zwf gene promoters so as to increase the metabolic flux of PPP approach
NADPH is answered to be a kind of approach for improving Amino acid synthesis.
It is contemplated that strengthening the expression intensity of zwf promoters by adding glycine betaine, the vigor of G6PDH is improved, increased
The metabolic flux of PPP approach provides more NADPH, and the method is applied to into the fermentation such as various amino acid, organic acid
In the production process of product.
The content of the invention：
In order to achieve the above object, the present invention provides a kind of method of enhancing zwf gene promoter expression intensities, the side
Method is to realize zwf genes by adding glycine betaine in the microorganism expression system containing zwf gene promoters or fermentation system
Promoter expression intensity strengthens 0.2-0.98 times；
Addition manner is that disposable addition or stream add；
The addition concentration of the glycine betaine is 0.5-1.0g/L；
The stream plus the mode of glycine betaine are：Starting stage, without glycine betaine in culture medium, purpose product produce into
Add 60% when entering peak period, remainder equivalent addition when the purpose product production peak period stage point, 2-3 was inferior；
Beneficial effect：
1st, the present invention realizes that zwf promoters expression intensity strengthens by adding glycine betaine in expression system or fermentation system
Purpose, so as to improve the vigor of G6PDH, with low cost, and it is simple and easy to do the characteristics of, under conditions of addition 1g/L,
Can reach zwf promoters expression intensity strengthens 0.98 times of effect；
What the 2nd, the present invention was provided adds method of the glycine betaine to strengthen zwf promoter expression intensities by external source, can be by
It is applied in the fermented products such as amino acid, and other need the gene of Enhanced expressing intensity, the fermentation such as amino acid after being
The breeding of product bacterial strain provides reference.
Description of the drawings：
Fig. 1 plasmid pUC19L-Pzwf- gfp builds flow chart；
Fig. 2 E.coli DH5 α pUC19L-PzwfThe each stage PCR proof diagrams of-gfp strain constructions
Wherein, M is DNA marker；
Swimming lane 1 is Inverse PCR amplification pUC19-L fragments (2252bp) in A；
Swimming lane 2 is that PCR expands P in BzwfFragment (485bp)；
Swimming lane 3 is PCR amplifications gfp fragments (720bp) in C；
Swimming lane 4 is that over-lap PCR expands P in Dzwf- gfp fragments (1205bp)；
Swimming lane 5 is PCR identifications positive strain (2208bp) in E；
Impact of Fig. 3 variable concentrations glycine betaine to unit thalline fluorescence intensity；
Enzyme activity of Fig. 4 variable concentrations glycine betaine to G6PDH；
The impact to Fungal biodiversity of Fig. 5 glycine betaines；
Impact of Fig. 6 glycine betaines to the G6PDH activity of THRD；
Impact of Fig. 7 glycine betaines addition manner to enzyme activity.
Specific embodiment：
Embodiment 1：Structure (1) comprising zwf gene promoters and the recombinant vector of egfp expression gene gfp
The transformation of carrier pUC19
Carrier pUC19 is transformed, it is whole to remove its by designing reverse amplimer pUC19-F/pUC19-R
Lac operators (PCR proof diagrams such as Fig. 2-A), improved carrier segments are named as pUC19-L (SEQ ID No.1), concrete structure
Build flow process as shown in Figure 1；
(2) structure of the amplification of zwf gene promoters and gfp genes and overlapping fragmentses
Designed as masterplate with the zwf promoter sequences (SEQ ID No.2) in E. coli MG1655 genomes
Amplimer Pzwf- up and Pzwf-down；With gfp (SEQ ID No.3) gene orders as masterplate design amplimer gfp-up and
Gfp-down, primer PzwfOverlap containing 20bp in the middle of-down and gfp-up, primer PzwfThe 5 ' of-up and gfp-down
End is respectively containing the homologous sequence that cloning site upstream and downstream is treated with carrier.
Zwf gene promoter P are expanded respectively using high-fidelity enzyme PrimeSTAR HS DNA PolymerasezwfAnd gfp
Gene, electrophoresis checking obtains purpose band (see Fig. 2-B, 2-C), flies upward the OMEGA's of bioengineering Co., Ltd using Guangzhou
PCR primer purification kit carries out recovery and obtains P to PCR primerzwfWith gfp fragments；
PCR system is as follows：
Again by PrimeSTAR HS DNA Polymerase enzymes to PzwfFragment and gfp fragments carry out over-lap PCR amplification,
Electrophoresis checking obtains purpose band (see Fig. 2-D), carries out recovery using kit and obtains Pzwf- gfp is stand-by；
PCR system is as follows：
(3) carrier construction pUC19L-Pzwf-gfp
Using ClonExpress-II One Step Cloning Kit kits by carrier pUC19-L and overlapping fragmentses
Pzwf- gfp carries out restructuring connection；
Restructuring linked system is as follows：
(4) connection product of recombinating is converted：
1. by ice bath 5min after restructuring 37 DEG C of water-bath 30min of connection product, add to E.coli DH5 α impressions
In state, mixed with pipettor, continue ice bath 20min；
2. EP pipes are positioned in 42 DEG C of water-baths, heat shock 60s；
3. ice bath 2min, adds 800 μ L SOC resuscitation fluids, and 37 DEG C, 200rpm is incubated 1h；
4. 8000rpm centrifugations 2min, stays 100 μ L liquid, resuspended thalline to coat containing ampicillin
In the LB flat boards of resistance, 37 DEG C of overnight incubations.
(5) checking of positive colony：
Bacterium colony PCR system is as follows：
Tweezers clamp toothpick used in super-clean bench, and the tip below toothpick is quickly passed through into flame 2～3 times, have in length
With toothpick tip one single bacterium colony of accurate picking on the flat board of single bacterium colony, first in the flat board for pulled grid line gently
Standardized road, then toothpick is put in corresponding PCR pipe rinse several times.Electrophoresis verify, obtain positive strain (see Fig. 2-E,
Due to identifying primer on plasmid, so the band that PCR amplifications are obtained is bigger than purpose band), by correct numbering in flat board
Bacterial strain accesses to LB and shakes in pipe, and 37 DEG C of overnight incubations, glycerol tube is preserved in -80 DEG C, is named as E.coli DH5 α pUC19L-
Pzwf-gfp。
The fluoroscopic examination of embodiment 2 and the enzyme activity determination of G6PDH
(1) the E.coli DH5 α pUC19L-P of 20 μ L are drawn from glycerol tubezwf- gfp bacterium solutions are extremely anti-containing ampicillin
Property 5mL LB shake in pipe, 37 DEG C, 200rpm culture 12h；
(2) shake in pipe from 5mL and be forwarded in the LB culture mediums of 30mL by 0.1% inoculum concentration, 37 DEG C, 200rpm cultures
8h；
(3) be seeded in the LB culture mediums of 50mL by 2% inoculum concentration (add final concentration of 0.05 in experimental group respectively,
0.1st, 0.5,0.75,1g/L glycine betaines；Without glycine betaine in control group), 37 DEG C, 200rpm cultures, take 6 respectively, 9,12h
Sample determination OD600；
(4) fluorescent value of the GFP of the F-7000 type fluorescence spectrophotometer measurement samples of Hitachi companies of Japan is used.
Excitation wavelength is set as 491nm, launch wavelength is 511nm；
As a result see the table below and Fig. 3, it is known that the addition of glycine betaine can strengthen PzwfExpression, and in 1g/L concentration ranges, with
Glycine betaine adds the increase of concentration, PzwfExpression intensity is in raising trend；
(5) glycine betaine addition is taken for 0.5,1g/L, the vigor of the sample determination G6PDH of 12h；
The enzyme activity of G6PDH according to Suzhou Ke Ming Bioisystech Co., Ltd glucose-6-phosphate dehydrogenase (G6PDH)
Kit is operated, and as a result sees Fig. 4, and as seen from the figure, the addition of glycine betaine can improve the G6PDH enzyme activity of thalline；
(6) impact of the cultivating system of addition glycine betaine to Fungal biodiversity is determined, Fig. 5 is as a result seen, it is known that addition beet
Alkali is for the growth (OD of thalline600) have a certain impact, the inhibitory action of high concentration is obvious, glycine betaine pair in comprehensive Fig. 3
PzwfThe impact of expression, it is determined that the addition of glycine betaine is 0.5-1g/L in expression or fermentation system, can give birth in purpose product
Produce peak period and supplement glycine betaine by way of stream adds, impact of the glycine betaine to thalli growth so can have both been reduced, while can
P is improved to reachzwfThe effect of expression intensity.
The glycine betaine of embodiment 3 produces the impact of the G6PDH activity of bacterium THRD to threonine
THRD is the Escherichia coli of the production threonine comprising zwf genes in a pnca gene group, using THRD as experiment
Bacterial strain, determine glycine betaine affects on its G6PDH enzyme activity, and experimentation is as follows：
(1) ring threonine production bacterium-E.coli THRD (THRD preserving number CGMCC are taken from glycerol tube with oese
No.11074) bacterium solution carries out line of gathering, 37 DEG C of cultures 8h (generation inclined-plane) into activated inclined plane culture medium；
(2) take a ring nectar cloth from generation inclined-plane to line in an other inclined-plane, 37 DEG C of cultures 8h (two generation inclined-plane)；
(3) take a ring bacterium from two generation inclined-planes to be forwarded in the seed culture medium of 50mL, 37 DEG C, 200rpm culture 8h；
(4) it is seeded in fermentation medium containing 30mL by 20% inoculum concentration and (adds final concentration of 0.5g/L in experimental group
Glycine betaine；Without glycine betaine in control group), 37 DEG C, 200rpm cultures adjust pH6.7-7.0 with ammoniacal liquor, take 6,12,18h
The vigor of sample determination G6PDH；
(5) enzyme activity of G6PDH according to Suzhou Ke Ming Bioisystech Co., Ltd glucose-6-phosphate dehydrogenase
(G6PDH) kit is operated, and as a result sees Fig. 6, and as seen from the figure 0.5g/L glycine betaines during the fermentation can be by THRD
G6PDH enzyme activity is improved to 2.7 times or so.
Culture medium prescription：
Activated inclined plane culture medium (g/L)：Glucose 1, peptone 10, beef extract 5, dusty yeast 5, NaCl2.5, agar strip
20, adjust pH 7.0-7.2,121 DEG C of sterilizing 20min.
Seed culture medium (g/L)：Sucrose 25, dusty yeast 10, peptone 6, KH2PO41.2, MgSO4·7H2O0.5,
FeSO4·7H2O 10mg/L, MnSO4·H2O 10mg/L, VB11.3mg/L, VH 0.3mg/L, adjusts pH 7.0-7.2, froth breaking
Agent 1-2 drops, 115 DEG C of sterilizing 15min.
Fermentation medium (g/L)：Glucose 30, dusty yeast 2, peptone 4, sodium citrate 1, KH2PO42, MgSO4·7H2O
0.7, FeSO4·7H2O 100mg/L, MnSO4·H2O 100mg/L, VB10.8mg/L, VH 0.2mg/L, phenol red 2ml/
100ml, adjusts pH 7.0-7.2, defoamer 1-2 drops, 115 DEG C of sterilizing 15min.
Impact of the glycine betaine addition manner of embodiment 4 to zwf promoters
(1) the carrier pUC19-P that will be built in embodiment 1zwf- gfp is converted into E.coli THRD, builds THRD
pUC19L-Pzwf- gfp bacterial strains；
(2) a ring THRD pUC19L-P are taken from glycerol tube with oesezwf- gfp bacterium solutions are extremely anti-containing ampicillin
Property activated inclined plane culture medium in, carry out gather line, 37 DEG C culture 12h (generation inclined-plane)；
(3) take a ring nectar cloth from generation inclined-plane to line in an other inclined-plane, 37 DEG C of cultures 8h (two generation inclined-plane)；
(4) take a ring bacterium from two generation inclined-planes to be forwarded in the seed culture medium of 50mL, 37 DEG C, 200rpm culture 8h, respectively
Culture medium is with embodiment 3；
(5) it is seeded in fermentation medium containing 30mL by 20% inoculum concentration, 37 DEG C, 200rpm cultures are adjusted with ammoniacal liquor
PH6.7-7.0, experiment is divided into 3 groups, and 1 group is added final concentration 1g/L glycine betaines without glycine betaine, in 2 groups of culture mediums, and 3 tissue cultures are supported
Start in base without glycine betaine, stream plus glycine betaine in sweat, stream plus process are as follows：Glycine betaine is configured to into high concentration female
Liquid (is dissolved in deionized water, 115 DEG C of high-temperature sterilization 10min), and its concentration is 60g/L, and thalline produces acid peak period (14h-
22h), wherein disposably adding the beet alkali liquor of 0.3mL in 14h, addition 0.1mL (the 18h and 22h respectively additions one per 4h later
It is secondary), take 16 respectively, 20, the sample determination fluorescent value of 24h (the addition time with Time Inconsistency is measured by sampling, it is therefore an objective to allow beet
Alkali first plays certain effect in fermentation system).
Experimental result is shown in Fig. 7, as seen from the figure, in the case of adding same concentrations glycine betaine in fermentation system, fed-batch mode
For the enhancing effect of promoter is better than disposable addition, and effect is more apparent after addition a period of time, disposably adds during 24h
Plus the glycine betaine of 1g/L is 1.36 times without zwf promoters expression intensity during glycine betaine, the glycine betaine of stream plus 1g/L is not
1.98 times of zwf promoters expression intensity during addition glycine betaine.
(note：The glycine betaine of 1g/L is with the addition of in experimental group 2, volume is 30mL, so the glycine betaine total amount of addition is 1mg/
ML*30mL=30mg；Because addition is high concentration mother liquor into the glycine betaine in culture medium, the Volume Changes that it is brought are very
It is little, it is negligible；In experimental group 3, the total amount of adding of glycine betaine is：60mg/mL*0.5mL=30mg).
SEQUENCE LISTING
<110>University Of Science and Technology Of Tianjin
<120>A kind of method of enhancing zwf gene promoter expression intensities
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<170> PatentIn version 3.5
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<213>Artificial sequence
<400> 6
ggcagtgagc gcaacgcaat tcggttcgct aacattggct 40
<210> 7
<211> 37
<212> DNA
<213>Artificial sequence
<400> 7
tgctcaccat gtcattctcc ttaagttaac taacccg 37
<210> 8
<211> 28
<212> DNA
<213>Artificial sequence
<400> 8
ggagaatgac atggtgagca agggcgag 28
<210> 9
<211> 43
<212> DNA
<213>Artificial sequence
<400> 9
gggtgtcggg gctggcttaa ttacttgtac agctcgtcca tgc 43
<210> 10
<211> 20
<212> DNA
<213>Artificial sequence
<400> 10
agcaccgcct acatacctcg 20
<210> 11
<211> 25
<212> DNA
<213>Artificial sequence
<400> 11
gcatttatca gggttattgt ctcat 25
Claims (6)
1. a kind of method of enhancing zwf gene promoter expression intensities, it is characterised in that methods described is by containing zwf
Add glycine betaine in the microorganism expression system or fermentation system of gene promoter, realize that zwf gene promoters expression intensity increases
It is strong 0.2-0.98 times.
2. a kind of method of enhancing zwf gene promoter expression intensities as claimed in claim 1, it is characterised in that described sweet
The addition manner of dish alkali is that disposable addition or stream add.
3. a kind of method of enhancing zwf gene promoter expression intensities as claimed in claim 2, it is characterised in that described sweet
The concentration of dish alkali addition is 0.5-1.0g/L.
4. a kind of method of enhancings zwf gene promoter expression intensities as claimed in claim 2, it is characterised in that stream plus just
Formula is：Starting stage, without glycine betaine in culture medium, add 60% when purpose product production enters peak period, its remaining part
Divide the equivalent addition when the purpose product production peak period stage point, 2-3 was inferior.
5. a kind of method of enhancing zwf gene promoter expression intensities as claimed in claim 1, it is characterised in that the zwf
Gene promoter is as shown in sequence table SEQ ID No.2.
6. it is a kind of strengthen destination gene expression intensity method, it is characterised in that the promoter of genes of interest is replaced with into zwf bases
After because of promoter, by adding the expression intensity that glycine betaine improves genes of interest in expression system or fermentation system.
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| CN116102640A (en) * | 2022-10-13 | 2023-05-12 | 浙江双糖生物科技有限公司 | Recombinant lactoferrin derived peptides and their use in enhancing immunity |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104531745A (en) * | 2014-12-09 | 2015-04-22 | 江南大学 | Construction of novel plasmid with resistance and application of plasmid to riboflavin producing strain |
-
2017
- 2017-01-09 CN CN201710012859.8A patent/CN106636023B/en active Active
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104531745A (en) * | 2014-12-09 | 2015-04-22 | 江南大学 | Construction of novel plasmid with resistance and application of plasmid to riboflavin producing strain |
Non-Patent Citations (2)
| Title |
|---|
| BARRA L.等: "Glucose 6-phosphate dehydrogenase is required for sucrose and trehalose to be effcient osmoprotectants in Sinorhizobium meliloti", 《FEMS MICROBIOLOGY LETTERS》 * |
| CARDI M.等: "The effects of salt stress cause a diversion of basal metabolism in barley roots: Possible different roles for glucose-6-phosphate dehydrogenase isoforms", 《PLANT PHYSIOLOGY AND BIOCHEMISTRY》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116102640A (en) * | 2022-10-13 | 2023-05-12 | 浙江双糖生物科技有限公司 | Recombinant lactoferrin derived peptides and their use in enhancing immunity |
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