CN102120999A - Method for synthesizing human milk fucosylation oligosaccharide by using genetic engineering strain through coupling and fermenting - Google Patents
Method for synthesizing human milk fucosylation oligosaccharide by using genetic engineering strain through coupling and fermenting Download PDFInfo
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Abstract
The invention relates to a method for synthesizing human milk fucosylation oligosaccharide by using a genetic engineering strain through coupling and fermenting. A metabolic engineering strain is constructed by mainly using a genetic engineering technology so that efficient expression of a key enzyme in a metabolic pathway is realized and biosynthesis of the human milk fucosylation oligosaccharide is realized in vitro. Due to the adoption of the engineering strain constructed by using the method, the copy number of enzyme genes can be increased, and the expression quantity of the enzyme can be improved, therefore, the human milk fucosylation oligosaccharide can be efficiently expressed. The invention provides a feasible path for industrially synthesizing the human milk oligosaccharide at low cost, and has stronger fundamental theoretical research value, larger economic and social benefits and wide market development prospect.
Description
Technical field
The invention belongs to the genetically engineered field, relate to the constructing technology of engineering strain, especially a kind of method of utilizing the synthetic human milk fucosylation oligosaccharides of coupling and fermenting gene engineering strains.
Background technology
In recent years, along with the fast development of foodstuffs industry and biotechnology, the exploitation of function oligosaccharides has become the important topic of international biological technical field, and the oligosaccharides industry has now become an emerging important industry that is applied to industries such as food, feed, medicine, chemical industry.Human milk oligosaccharides not only has anti-virus infection, physiological actions such as inflammation are regulated, reduced to strengthening immunity, and for cancer and chronic recurrent colitis etc. certain prophylactic effect is arranged.It can be used as epithelial soluble receptors analogue as the glucide of a class special construction, participates in the enhancing of the non-immune defense system of breast feeding babies.
Its content of human milk oligosaccharides is after lactose, lipid, become the third-largest material in the human milk, oligosaccharide content is 20-27g/L in first Ruzhong, in ripe Ruzhong also up to 12-14g/L, and content is very low in other animal milk, oligosaccharides only contains 0.7-1.2g/L in the bovine coloctrum, be lower than 20 times more than of people's colostrums, except that content is abundant, the human milk oligosaccharides structure is also very complicated, according to mass spectroscopy, estimates to contain in the human milk oligosaccharides of more than 900 kind of different structure, at present isolate more than the 130 kind of oligosaccharides that structure is different at least, and only contain 18 kinds of oligosaccharides in the cow's milk.It is neutral that the human milk oligosaccharides great majority are, and based on the fucosylation oligosaccharides, wherein α 1, and 2-fucosylation oligosaccharides accounts for the ratio average out to 73% of total oligosaccharides in the tested person Ruzhong, and fucosyl residues is 1-15 not to be waited.This fucosylation oligosaccharides that content enriches the most in human milk does not but detect at the mature milk of animals such as ox, sheep, goat and horse, but just contains more neutral oligosaccharides in Ruzhong at it.Difference between the different Mammals milk on the oligosaccharide structure also may point out human milk oligosaccharides to have special physiological properties.In addition, human milk oligosaccharides and analogue thereof are because the function uniqueness, safety, effective, do not develop immunity to drugs, and can also resist the variation pathogenic bacteria that microbiotic is produced resistance, with human milk oligosaccharides and analogue is the another big focus that feedstock production anti-adhesive medicine becomes current drug development, and it will bring into play enormous function aspect prevention and treatment bacteriosis field and the infant nutrition health care.
Effectively do not carried out yet in China about the function and the preparation research of human milk oligosaccharides at present.China mainly concentrates on natural phant and microbial polysaccharide to the exploitation of function oligosaccharides class material, adopts chemical processes such as acid, alkali, enzyme, oxidation to degrade and prepares low-molecular-weight oligosaccharides, as oligochitosan, Nutriflora P etc.Obtain human milk oligosaccharides by chemical synthesis, because the complexity of synthetic route and the costliness of glucosides donor, most of technologies still can't reach scale production, become the obstacle that the chemical method synthesis of oligose is difficult for going beyond.Along with the continuous parsing of increasing Glycosylase and gene thereof and constantly illustrating of microorganism metabolism controlling route, and the developing rapidly of genetic engineering technique, use the required enzyme of oligosaccharide synthesis production process and come a large amount of oligosaccharide synthesis to become possibility.
Human milk oligosaccharides is based on the fucosylation oligosaccharides, and seminose is exactly important prerequisite material of fucosylation oligosaccharides synthetic.
Summary of the invention
The purpose of this invention is to provide a kind of construction process that utilizes the synthetic human milk glycosylation oligosaccharides of engineering strain fermentation, use the E.coli expression system, to being that the relevant enzyme of the synthetic fucosylation oligosaccharides of substrate efficiently expresses with the seminose, utilize gene recombination technology to make up and contain the recombination bacillus coli of relative enzyme gene, thus the method for the synthetic fucosylation oligosaccharides that ferments.Make the copy number of enzyme gene increase by the engineering strain that present method obtained, the expression amount of enzyme obviously improves, thereby can act on the synthetic fucosylation oligosaccharides of substrate seminose.
The objective of the invention is to be achieved through the following technical solutions:
A kind of method of utilizing the synthetic human milk fucosylation oligosaccharides of coupling and fermenting gene engineering strains comprises following step:
(1) gene amplification: according to hexokinase glk among the intestinal bacteria E.coli K12, mannose-phosphate mutase manB, seminose-1 guanosine 5-monophosphate transferring enzyme manC, GDP-seminose 4,6-dehydratase gmd, GDP-4-ketone-6-deoxymannose 3, α-1 among 5-mutarotase/4-reductase enzyme wcaG and the Hp Helicobacter pylori HPAG1, the sequences Design primer of 2 fucosyl transferase gene fuct, with intestinal bacteria E.coli-K12 and HPAG1 chromosomal DNA is that template is carried out pcr amplification to the above-mentioned purpose gene respectively, the corresponding target gene that amplification obtains;
(2) construction of recombinant plasmid: the method for cutting connection with enzyme links to each other the corresponding target gene that amplification obtains with carrier pET-22b, obtain carrying the recombinant expression vector pET-glk-manB-manC of goal gene, pET-gmd-wcaG and pET-fuct, and carry out the double digestion checking;
(3) structure of engineering strain: the recombinant expression vector pET-glk-manB-manC that above-mentioned checking is correct, pET-gmd-wcaG and pET-fuct transform respectively among the host strain BL21 (DE3), obtain containing engineering strain BL21 (DE3)/pET-glk-manB-manC, B2:BL21 (the DE3)/pET-gmd-wcaG and B3:BL21 (the DE3)/pET-fuct of recombinant plasmid;
(4) the synthetic human milk fucosylation sugar of coupled fermentation: will go on foot three strain gene engineering bacterial strain mixed fermentations in substratum that (3) obtain, be synthetic substrate with seminose, synthetic human milk fucosylation oligosaccharides.
And the fermenting process of step (4) adds Nymeen S-215.
And fermention medium is that LB cultivates in the described step (4), and to add other composition be g/L:B1 weight in wet base 30; B2 weight in wet base 25; B3 weight in wet base 25; Seminose 30; Phytic acid 5; KH
2 PO
425; MgSO
4.7H
2O 5; Fructose 25; NymeenS-215 4.
Advantage of the present invention and positively effect are:
1, the present invention is based on intestinal bacteria, utilization genetically engineered recombinant technology makes up B1, B2, three kinds of engineering strains of B3, enzyme efficiently expresses in the realization pathways metabolism, and application of fermentation technology, gene engineering strain is acted on seminose, carry out coupled fermentation, the feasible way that provides of a large amount of suitability for industrialized production human milk oligosaccharides at a low price is provided synthetic then human milk fucosylation oligosaccharides, this production method has reduced production cost, improve production efficiency, not only had economic benefit, also had the certain social benefit.
2, the present invention utilizes GTP to produce bacterium C.ammoniagenes and provides energy for whole catalyzed reaction, combined ferment is produced the GDP-Fucose, and then reach a large amount of synthetic of human milk fucosylation oligosaccharides, this makes that not only producing human milk oligosaccharides in batches becomes possibility, also supply raw materials simultaneously for natural oligosaccharides medicine production, have stronger fundamental research value and economic results in society, the prospect of marketing is wide.
Description of drawings:
Fig. 1 is an amplification electrophorogram of the present invention, wherein:
A:1, hexokinase glk; 2, mannose-phosphate mutase gene manB; 3, mannose-1-phosphate guanosine acyltransferase gene manC;
B:1-3 is GDP-4-ketone-6-deoxymannose 3,5-mutarotase/4-reductase enzyme (wcaG) gene; Band 4 is 1kb DNAmarker; Band 5-7 is a GDP-seminose 4,6-dehydratase (gmd) gene;
C:1,1kb DNA marker; 2, α-1,2 fucosyl transferase gene (fuct) PCR product;
Fig. 2 is pET-glk-manB-manC of the present invention, pET-gmd-wcaG and pET-fuct, the structure iron of recombinant expression plasmid, wherein
A: the structure of recombinant plasmid pET-glk-manB-manC;
B: the structure of recombinant plasmid pET-gmd-wcaG;
C: the structure of recombinant plasmid pET-fuct;
Fig. 3 is pET-glk-manB-manC of the present invention, the protein expression electrophorogram of pET-gmd-wcaG and pET-fuct; Wherein:
A:M is albumen marker, and 1-2 is pET-glk-manB-manC/BL21;
B:M is albumen marker, and 1-2 is pET-gmd-wcaG/BL21;
C:M is albumen marker, and 1 is pET-22b (+)/BL21; 2 is pET-fuct/BL21;
Fig. 4 utilizes the structure schema of the synthetic human milk fucosylation oligosaccharides system of engineering strain mixed fermentation for the present invention.
Embodiment
Below in conjunction with embodiment, the present invention is further described, and following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.
The construction process that utilizes the synthetic human milk fucosylation oligosaccharides system of coupling and fermenting gene engineering strains involved in the present invention, be to utilize escherichia expression system BL21/pET-22b, to catalysis is the gene such as the hexokinase gene of the relevant enzyme of the synthetic human milk fucosylation oligosaccharides of substrate with the seminose, the mannose-phosphate mutase gene, mannose-1-phosphate guanosine acyltransferase gene, GDP-seminose 4, the 6-dehydratase, GDP-4-ketone-6-deoxymannose 3,5-mutarotase/4-reductase enzyme and α-1,2 fucosyl transferase gene (glk, manB, manC, gmd, wcaG, fuct) efficiently express, make up three reorganization large intestine bacterial strains that comprise the various objectives gene respectively, carry out the efficient synthetic of human milk fucosylation oligosaccharides body by the metabolic regulation means.The used enzyme source of the present invention is in E.coli K12 and Helicobacter pylori HPAG1, among the present invention recombinant vectors host cell be e. coli bl21 (DE3).Constructed recombinant expression vector not only comprises the dna sequence dna of codase, also has the required controlling elements of this gene of expression.
The step of construction process is:
One, the amplification of goal gene
(1) genomic dna of extraction intestinal bacteria (E.coli K12), design following primer:
Pglk1:5 '-GGAATTC
TCTAGAATGACAAAGTATGCATTAGTCGGT-3 ' restriction enzyme site is XbaI;
Pglk2:5 '-CGC
GCCCGGGCTTACAGAATGTGACCTAAGGTCTG-3 ' restriction enzyme site is SrfI.
PmanB1:5 '-GGAATTC
GGTACCATGAAAAAATTAACCTGCTTT-3 ' restriction enzyme site is KpnI;
PmanB2:5 '-CCC
AGTACTTTACTCGTTCAGCAACGTC-3 ' restriction enzyme site is ScaI.
PmanC1:5 '-CATG
AGTACTATATGACAAAGTATGCATTAGTCGGT-3 ' restriction enzyme site is ScaI;
PmanC2:5 '-CCC
CATGGTTACAGAATGTGACCTAAGGTCTG-3 ' restriction enzyme site is NcoI.
Pgmd1:5 '-CATG
GCCCGGGCATATGTCAAAAGTCTCTCATC-3 ' restriction enzyme site is SrfI;
Pgmd2:5 '-CCC
GGTACCTTATGACTCCAGCGCGATC-3 ' restriction enzyme site is KpnI.
PwcaG1:5 '-CATG
GAGCTCGCATGAGTAAACAACGAGTTTTTATTG-3 ' restriction enzyme site is SacI;
PwcaG2:5 '-CCC
CTCGAGTTACCCCCGAAAGCGGT-3 ' restriction enzyme site is XhoI.
(2) extract Hp (Helicobacter pylori HPAG1) genomic dna, design following primer:
Pfuct1:5 '-CATG
CCATGGATCCGATGGCTTTTAAAAGTGTGCAA-3 ' restriction enzyme site is BamHI;
Pfuct2:5 '-CCC
AAGCTTGAGCTCTTAAGCGTTATACTTTTGGGATTT-3 ' restriction enzyme site is HindIII.
With intestinal bacteria E.coli-K12 and HPAG1 chromosomal DNA is that template is carried out pcr amplification to goal gene respectively, in the following order, in sterilization EP pipe, mix,
A. adopt the pcr amplification system of 20 μ L:
B. the PCR condition that is used for amplifying target genes:
The amplified production of gained is carried out the agarose gel electrophoresis detection, detect amplified production 0.9kb (glk), 1.3kb (manB), 1.4kb (manC), 1.1kb (gmd), 1.0 (wcaG) with 0.9 (fuct), the result as shown in Figure 1, can see in corresponding stripe size position and specific band occur, its size fits like a glove with the goal gene size, with amplifying target genes glk, gmd, fuct is connected on the pET-22b carrier, construction recombination plasmid pET-glk, pET-gmd and pET-fuct, subsequently with manB, manC one by one be connected to pET-glk, construction recombination plasmid pET-glk-manB-manC, wcaG is connected on the pET-gmd, construction recombination plasmid pET-gmd-wcaG, (entrusting Shanghai to give birth to the worker) as can be known its order-checking increases to such an extent that be the hexokinase gene, the mannose-phosphate mutase gene, mannose-1-phosphate guanosine acyltransferase gene, GDP-seminose 4, the 6-dehydratase, GDP-4-ketone-6-deoxymannose 3, the dna sequence dna of 5-mutarotase/4-reductase enzyme and α-1,2 fucosyl transferase gene is as the back table.
Two, construction of recombinant plasmid
1, preparation of expression vectors
(1) carries the e. coli jm109 bacterial strain (available from precious biotech firm) of plasmid pET-22b at the LB inoculation of medium of penbritin (50 μ g/mL), spend the night in 37 ℃ of shaking culture, 1.5mL bacterium liquid is changed in the Eppendorf tube, the 12000r/min clock, centrifugal 30s collects thalline, abandon supernatant, control dried raffinate.(2) (10mmol EDTA PH8.0), mixes for 50mmol sucrose, 25mmol Tris precipitation to be resuspended in the solution 1 of 100 μ L precoolings.(3) (0.2molNaOH 1%SDS) covers the tight mouth of pipe, shakes up gently, places that 1-2min is limpid to liquid on ice to add the solution 2 of the new configuration of 200 μ L.(4) solution 3 (the 3mol acetate first of adding 150 μ L precoolings, PH4.8) rotate centrifuge tube gently, solution 3 is mixed in the heavy-gravity bacterial lysate, ice bath 3-5min, 12000r/min, centrifugal 5min, supernatant is transferred in another pipe, the 12000r/min clock, centrifugal 5min moves on to supernatant r in another centrifuge tube again.(5) dehydrated alcohol of 2-2.5 times of volume of adding, mixing, ice bath (or-20 ℃) is placed 30min.(6) 12000r/min, centrifugal 5min collects the plasmid DNA precipitation.(7) with 70% washing with alcohol precipitation 2-3 time, discard raffinate, air drying 10-20min is the pET-22b plasmid vector with the distilled water dissolution precipitation of 20 μ L.The carrier pET-22b that is obtained promptly can be used as the carrier of ligase enzyme gene.Subsequently plasmid is transformed, with original multiple clone site BamHI and SacI on the plasmid is restriction enzyme site, give birth to the synthetic minigene fragment that contains special multiple clone site SrfI and KpnI of worker company by Shanghai and insert between the selected restriction enzyme site, make up and contain the more the more carrier pET-22b (+) of cloning site.
2, the structure of recombinant expression vector
(1) carrier pET-22b and PCR product are carried out double digestion with corresponding restriction enzyme, electrophoresis, plasmid and PCR product after cutting glue and reclaiming enzyme and cut then, all select for use 50 μ L enzymes to cut system:
A.1) (glk, gmd fuct) carry out enzyme with carrier pET-22b respectively and cut Kuo Zeng goal gene, and the restriction endonuclease of the restriction enzyme site that the restriction enzyme of use is the same, carry in the downstream primer is identical.
A.2) (manB wcaG) carries out enzyme with plasmid pET-glk and pET-gmd respectively and cuts and be connected goal gene.
A.3) goal gene (manC) and plasmid pET-glk-manB carry out enzyme and cut
b.
Fragment after with DNA purification kit (TaKaBa company) above-mentioned enzyme being cut is carried out purifying, and the pET-22b of the linear purifying that is obtained and PCR product promptly can be used to construction recombination plasmid.
(2) being connected of carrier after enzyme is cut and goal gene, all select 10 μ L linked systems for use:
Gained connects mixture and adopts DNA purification kit (TaKaRa company) to carry out purifying, and the product pET-glk-manB-manC behind the purifying, pET-gmd-wcaG and pET-fuct are used for electrotransformation transformed into escherichia coli DH5 α.
Three, the structure of engineering strain
The competent cell for preparing bacillus coli DH 5 alpha as follows: connect E.coli DH5 α slant strains and be inoculated in the 5mL LB substratum, 37 ℃ of shaking culture 2-3h make cell reach logarithmic phase (OD
600=0.5-0.7); Triangular flask transferred to place 20min on ice, 4000r/min, 4 ℃ of centrifugal 15min, collecting cell; With 300 μ L, 10% glycerine suspension cell, 4000r/min, 4 ℃ of centrifugal 15min, this process repeats once; At last with cell suspension at 300 μ L, in 10% the glycerine, divide the centrifuge tube install to precooling by each part 40 μ L, place-70 ℃ of preservations then.
During use competent cell placed on ice and melt, connection product (the pET-glk-manB-manC that in the competence of a pipe 40 μ L, adds the above-mentioned purification process of 4 μ L, pET-gmd-wcaG, pET-fuct), the electricity that adds precooling behind the mixing transforms in the cup, touches liquid to guarantee that bacterium and DNA suspension are positioned at electricity and transform the cup bottom; (ETM399 BTX-USA), adjusts to the Ecl shelves, promptly aims at intestinal bacteria and transforms one grade that is provided with to open electric conversion instrument; Dry water of condensation and fog that electricity transforms the cup outside, put in the electric conversion instrument, by the shelves of above-mentioned setting, the electricity that starts pair cell transforms; After transforming end, fast as far as possible taking-up electricity revolving cup adds 600 μ L SOC nutrient solutions, changes over to behind the mixing in the 1.5ml centrifuge tube, and in 37 ℃, 180r/min shakes 1h slowly; Be applied on the LA flat board that contains penbritin (100 μ g/mL) by each dull and stereotyped 100 μ L, be inverted overnight incubation (16-20h) for 37 ℃; The single bacterium colony of picking from the flat board is inoculated in and contains in penbritin (100 μ g/mL) the liquid LB substratum, cultivates 12-18h in 37 ℃, extracts plasmid DNA then in a small amount, carries out the double digestion evaluation with corresponding restriction enzyme, and the recombinant plasmid structure is seen Fig. 2.
Four, the abduction delivering of genetic engineering bacterium
1, the competent cell for preparing e. coli bl21 according to the method for describing in the step 3, and utilize the plasmid that extracts the correct genetic engineering bacterium of plasmid checking in a small amount, carry out transformation experiment according to aforesaid method.
2, recombinant bacterial strain BL21 (DE3's) induces
1. the single bacterium colony of picking from the flat board is inoculated in and contains in penbritin (100 μ g/mL) the liquid LB substratum, cultivates 12-18h in 37 ℃.
2. taking over the night culture by 1% inoculum size contains in the LB substratum of 60 μ g/mL penbritins in 30mL.
3. 37 ℃ of shaking culture are worked as OD
600Add IPTG during=0.4-0.6, final concentration is to 0.1mM.
4. under 20 ℃, induce 4h.Sampling detects.
3, SDS-PAGE expression analysis
1. preparing gel
Solution composition 12% separation gel (10mL) mL 5% concentrates glue (4mL) mL
30% acrylamide 4.0 0.67
Tris-Cl(1.5M)pH8.8 2.5 -
Tris-Cl(1.0M)pH6.8 - 0.5
10％SDS 0.1 0.04
10% ammonium persulphate 0.1 0.04
TEMED 0.004 0.004
Deionized water 3.3 0.7
2. sample preparation
A) the 1mL nutrient solution is in small-sized centrifuge tube, in 4 ℃ of centrifugal 3min of following 12000r/min.
B) remove supernatant, make sedimentary thalline " drying " as far as possible.
C) resuspended thalline is in 100 μ L1 * SDS-PAGE electrophoresis sample-loading buffer, and carries out thorough mixing.
D) boil 5min under 100 ℃, the centrifuging and taking supernatant, sample retention in-20 ℃ up to carrying out the protein electrophoresis analysis, electrophoresis result is seen Fig. 3
Five, the synthetic fucosylation oligosaccharides of the mixed fermentation of recombinant strain
With B1:BL21 (the DE3)/pET-glk-manB-manC that builds; B2:BL21 (DE3)/pET-gmd-wcaG; B3:BL21 (DE3)/pET-fuct engineering strain is cultivated in the LB substratum respectively, when the thalli growth amount reaches OD
600The preparation bacterium of synthesizing human milk fucosylation oligosaccharides during=0.4-0.6 as mixed fermentation.
The synthetic GDP-seminose of mixed fermentation carries out in the LB substratum that shakes 30mL in the bottle of 250mL, and to add other composition be g/L:B1 weight in wet base 30; B2 weight in wet base 25; B3 weight in wet base 25; Seminose 30; Phytic acid 5; KH
2PO
425; MgSO
4.7H
2O5; ATP 5; Nymeen S-215 4 (tensio-active agent, the perviousness of increase bacterium surface); Reaction system maintains 7.2 with the NaOH of 4N with the pH value, is reflected at 32 ℃, carries out 22h under the condition of 900r/min.
Reaction mixture is centrifugal under 4 ℃, 4000r/min, and 15min removes thalline, gets supernatant and carries out the HPLC analysis.Chromatographic condition is: chromatographic column: Aminex HOX-87H (Bio-Red) 7.8mm * 300mm, detector: differential refraction detector; Moving phase 40mmol/LH
2SO
4Column temperature: room temperature; Sample size: 5 μ L; Flow velocity: 1.0mL/min.
The output of calculating the fucosylation oligosaccharides with external standard method is 4.4mg/L, the regression equation of fucosylation oligosaccharides:
Y=0.001+12.7x (R=0.9998) wherein does equation of linear regression with peak area y to sample concentration x.
Thalline is induced fermentation, make the product of engineering strain act on the synthetic fucosylation oligosaccharides of seminose by changing the cell permeability.
Claims (3)
1. method of utilizing the synthetic human milk fucosylation oligosaccharides of coupling and fermenting gene engineering strains is characterized in that: comprise following step:
(1) gene amplification: according to hexokinase glk among the intestinal bacteria E.coli K12, mannose-phosphate mutase manB, seminose-1 guanosine 5-monophosphate transferring enzyme manC, GDP-seminose 4,6-dehydratase gmd, GDP-4-ketone-6-deoxymannose 3, α-1 among 5-mutarotase/4-reductase enzyme wcaG and the Hp Helicobacter pylori HPAG1, the sequences Design primer of 2 fucosyl transferase gene fuct, with intestinal bacteria E.coli-K12 and HPAG1 chromosomal DNA is that template is carried out pcr amplification to the above-mentioned purpose gene respectively, the corresponding target gene that amplification obtains;
(2) construction of recombinant plasmid: the method for cutting connection with enzyme links to each other the corresponding target gene that amplification obtains with carrier pET-22b, obtain carrying the recombinant expression vector pET-glk-manB-manC of goal gene, pET-gmd-wcaG and pET-fuct, and carry out the double digestion checking;
(3) structure of engineering strain: the recombinant expression vector pET-glk-manB-manC that above-mentioned checking is correct, pET-gmd-wcaG and pET-fuct are transformed into respectively among the host strain BL21 (DE3), obtain containing engineering strain BL21 (DE3)/pET-glk-manB-manC, B2:BL21 (the DE3)/pET-gmd-wcaG and B3:BL21 (the DE3)/pET-fuct of recombinant plasmid;
(4) the synthetic human milk fucosylation sugar of coupled fermentation: with three strain gene engineering bacterial strain mixed fermentations in substratum that step (3) obtains, be synthetic substrate with seminose, synthetic human milk fucosylation oligosaccharides.
2. the method for utilizing the synthetic human milk fucosylation oligosaccharides of coupling and fermenting gene engineering strains according to claim 1 is characterized in that: the fermenting process of step (4) adds Nymeen S-215.
3. the method for utilizing the synthetic human milk fucosylation oligosaccharides of coupling and fermenting gene engineering strains according to claim 2, it is characterized in that: the moiety of fermention medium is the LB substratum in the described step (4), and to add other composition be g/L:B1 weight in wet base 30; B2 weight in wet base 25; B3 weight in wet base 25; Seminose 30; Phytic acid 5; KH
2PO
425; MgSO
4.7H
2O 5; Fructose 25; Nymeen S-215 4
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| CN104023560A (en) * | 2011-08-29 | 2014-09-03 | 雅培制药有限公司 | Human Milk Oligosaccharides For Preventing Injury And/Or Promoting Healing Of The Gastrointestinal Tract |
| US20170152538A1 (en) * | 2012-12-20 | 2017-06-01 | Won-Heong Lee | Biosynthesis of Oligosaccharides |
| CN110172486A (en) * | 2019-05-14 | 2019-08-27 | 天津科技大学 | A method of synthesis 2'-Fucosyl lactose |
| CN110804577A (en) * | 2019-11-28 | 2020-02-18 | 江南大学 | Escherichia coli engineering strain for producing 2' -fucosyllactose |
| CN114276971A (en) * | 2022-01-07 | 2022-04-05 | 天津科技大学 | Recombinant escherichia coli for synthesizing 2' -fucosyllactose by utilizing mannose and application thereof |
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| US9944965B2 (en) * | 2012-12-20 | 2018-04-17 | The Board Of Trustees Of The University Of Illinois | Biosynthesis of oligosaccharides |
| US10907137B2 (en) | 2012-12-20 | 2021-02-02 | Board Of Trustees Of The University Of Illinois | Biosynthesis of oligosaccharides |
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| CN110804577A (en) * | 2019-11-28 | 2020-02-18 | 江南大学 | Escherichia coli engineering strain for producing 2' -fucosyllactose |
| CN110804577B (en) * | 2019-11-28 | 2021-05-28 | 江南大学 | Construction method and application of recombinant bacteria for efficiently producing 2' -fucosyllactose |
| CN114276971A (en) * | 2022-01-07 | 2022-04-05 | 天津科技大学 | Recombinant escherichia coli for synthesizing 2' -fucosyllactose by utilizing mannose and application thereof |
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