CN101870992A

CN101870992A - Method for synthesizing GDP-fucose by utilizing genetically engineered microorganism coupled fermentation

Info

Publication number: CN101870992A
Application number: CN 201010204353
Authority: CN
Inventors: 李玉; 贾红红; 王春霞; 黎明; 刘逸寒; 路福平
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2010-06-21
Filing date: 2010-06-21
Publication date: 2010-10-27

Abstract

The invention relates to a method for synthesizing GDP-fucose by utilizing genetically engineered microorganism coupled fermentation. The main content thereof includes an escherichia coli expression system BL21(DE3)/pET-22b is utilized to efficiently express related enzyme for catalyzing synthesis of GDP-fucose by taking GDP-mannose as substrate, two enzymes gmd and wcaG and two recombinant escherichia coli strains are constructed and produced, and metabolism control is applied to high efficiency synthesis of GDP-fucose. The strain construction method in the invention applies E. coli expression system, escherichia coli is taken as basis, genetic engineering recombination technology is applied to construct two genetic engineering strains, and mixed fermentation technology is applied, GDP-mannose is taken as fermentation substrate to synthesize GDP-fucose, high efficiency expression of various enzymes in metabolic pathway is realized, and further synthesis of massive GDP-fucose is achieved, thus not only providing an effective way for mass production of human milk oligosaccharide but also providing raw material for production of natural oligosaccharide medicine, thereby having strong fundamental research value, economic benefit, social benefit and wide market development prospect.

Description

Utilize the method for the synthetic GDP-Fucose of genetic engineering bacterium coupled fermentation

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 GDP-Fucose of genetic engineering bacterium coupled fermentation.

Background technology

Cow's milk especially infant formula powder plays the important and pivotal role to neonatal growing as a kind of gourmet food, but increasing clinical study shows that breast milk has the not replaceable physiological function of milk powder.Compare with not breast fed, breast-feeding can reduce the incidence of gastro-intestinal infection, otitis media, respiratory tract infection, microbemia, meningitis, necrotic colitis and other urinary system greatly.

Why breast milk can effectively reduce the morbidity degree and the death toll of ewborn infant, mainly contains panimmunity regulatory factors such as anti-infective, anti-inflammatory owing to it, mainly comprises secretor type antibody (sIgA), oligosaccharides, lactoferrin, white corpuscle, cytokine etc.Wherein its content of human milk oligosaccharides becomes the third-largest material in the human milk after lactose, lipid.Studies show that much human milk oligosaccharides not only has significantly anti-infective and some important non-specific immunity function, and can promote the propagation of bifidus bacillus in baby's digestive tube, and clostridium and enterococcal quantity are significantly reduced.The anti-microbial pathogen infection effect of human milk oligosaccharides is illustrated.The first step that pathogenic bacteria is invaded body also is that committed step is exactly the adhesion at mucomembranous epithelial cell, and it mainly combines with epithelial receptor at antigen by the pathogenic bacteria table.The anti-microbial pathogen adhesion function of human milk oligosaccharides has caused people's extensive concern, and many pharmaceuticals also begin to carry out exploitation and clinical study with the anti-adhesive medicine of human milk oligosaccharides and analogue thereof.

A large amount of acquisitions of human milk oligosaccharides also are not easy; in recent years; the synthetic glucide of chemical method has been obtained than much progress; can synthesize the above oligosaccharides of ten saccharide residues; but because the complexity of synthetic route and the costliness of glucosides donor; most of technologies still can't reach scale production; at present; utilizing genetic engineering technique structure metabolic engineering bacteria is the most promising method of scale preparation human milk oligosaccharides; but; how obtaining a large amount of cheap glycosyl donors also is vital for the preparation of oligosaccharides; human milk oligosaccharides is based on the fucosylation oligosaccharides; the GDP-Fucose is exactly important intermediate product in the fucosylation oligosaccharides building-up process, is an important intermediate in the oligosaccharides route of synthesis, and how can prepare the GDP-Fucose fast also is a kind of significant process of preparation oligosaccharides.

Summary of the invention

The objective of the invention is to overcome the deficiencies in the prior art part, a kind of method of utilizing the synthetic GDP-Fucose of genetic engineering bacterium coupled fermentation is provided, make the copy number of enzyme gene increase by the engineering strain that present method obtained, the expression amount of enzyme obviously improves, and it is very high to act on the synthetic GDP-Fucose efficient of substrate GDP-seminose.

The objective of the invention is to be achieved through the following technical solutions:

A kind of method of utilizing the synthetic GDP-Fucose of genetic engineering bacterium coupled fermentation, step is as follows:

(1) gene amplification: according to GDP-seminose 4 in the e. coli k-12 E.coli-K12 genome, 6-dehydratase and GDP-4-ketone-6-deoxymannose 3, the sequences Design primer of 5-mutarotase/4-reductase enzyme, clone the GDP-seminose 4 in the intestinal bacteria, 6-dehydratase and GDP-4-ketone-6-deoxymannose 3,5-mutarotase/4-reductase gene;

(2) construction of recombinant plasmid: the method for cutting connection with enzyme links to each other above-mentioned two kinds of goal gene respectively with carrier pET-22b, obtain carrying the recombinant expression vector of goal gene, amplification back enzyme is cut checking, and checking correctly obtains plasmid pET-22b-gmd or pET-22b-wcaG;

(3) structure of engineering strain: the recombinant expression vector that above-mentioned checking is correct transforms among the host strain BL21 (DE3), obtains containing engineering strain BL21 (the DE3)/pET-22b-gmd and BL21 (the DE3)/pET-22b-wcaG of recombinant plasmid;

(4) the synthetic GDP-Fucose of mixed fermentation: with two engineering strains mixed fermentation in substratum that step (3) obtains, being inductor during the fermentation with IPTG, is the synthetic GDP-Fucose of substrate with the GDP-seminose.

And amplification is adopted in the described step (2) is plasmid to be transformed in the competent cell of bacillus coli DH 5 alpha increase, and amplification back enzyme is cut checking, is used for the conversion of step (3) after correct.

And the moiety of described step (4) substratum is: BL21 (DE3)/pET-22b-gmd 25g/L; BL21 (DE3)/pET-22b-wcaG 25g/L; GDP-seminose 30g/L; Phytic acid 5g/L; KH ₂PO ₄25g/L; MgSO ₄.7H ₂O 5g/L; ATP 5g/L; Nymeen S-2154g/L.

Advantage of the present invention and positively effect are:

1, bacterial classification construction process among the present invention is used the E.coli expression system, based on intestinal bacteria, utilization genetically engineered recombinant technology makes up BL21 (DE3)/pET-22b-gmd engineering strain and BL21 (DE3)/pET-22b-wcaG engineering strain, the application mix fermentation technique, with the GDP-seminose is the synthetic GDP-Fucose of fermentation substrate, various enzymes efficiently expresses in the realization pathways metabolism, and then reach a large amount of synthetic of GDP-Fucose, not only provide valid approach for producing human milk oligosaccharides in batches, also supply raw materials simultaneously for natural oligosaccharides medicine production, have stronger fundamental research value and economic and social benefit, the prospect of marketing is wide.

The production method synthetic route of the GDP-Fucose that 2, the present invention relates to is simple, does not relate to expensive intermediate product and complicated accurate equipment, effectively reduces production cost, has improved production efficiency, has high economic benefit.

Description of drawings:

Fig. 1 is an amplification electrophorogram of the present invention: wherein:

band

1,2,3 is GDP-4-ketone-6-deoxymannose 3,5-mutarotase/4-reductase enzyme (wcaG) gene; Band 4 is 1kb DNA marker;

Band

5,6,7 is a GDP-seminose 4,6-dehydratase (gmd) gene;

Fig. 2 is the structure schema of recombinant expression plasmid pET-gmd of the present invention;

Fig. 3 is the structure schema of recombinant expression plasmid pET-wcaG of the present invention;

Fig. 4 is pET-gmd of the present invention, the protein expression electrophorogram of pET-wcaG; Wherein: band 1 is BL21/pET-gmd; Band 2 is BL21/pET-wcaG; Band 3 is BL21/pET-22b;

Fig. 5 utilizes the fermentation schema of the synthetic GDP-Fucose system of engineering strain mixed fermentation for the present invention.

Embodiment

The present invention is further described below in conjunction with accompanying drawing and specific embodiments, and its specific embodiments only is construed as to illustrating, and is not determinate, can not limit protection scope of the present invention with following illustrating.

Method general introduction of the present invention and principle are as follows: the method for the system of utilizing coupling and fermenting gene engineering strains GDP-Fucose involved in the present invention, it to the effect that utilizes escherichia expression system BL21 (DE3)/pET-22b, is that the relevant enzyme of the synthetic GDP-Fucose of substrate efficiently expresses to catalysis with the GDP-seminose, make up two kinds of enzyme (GDP-seminoses 4,6-dehydratase (gmd), GDP-4-ketone-6-deoxymannose 3,5-mutarotase/4-reductase enzyme (wcaG)) two reorganization large intestine bacterial strains are used the metabolic regulation means and are carried out the efficient synthetic of GDP-Fucose.

The host bacterium of the source of two used enzyme genes and recombinant vectors is respectively E.coli-K12 and e. coli bl21 (DE3) among the present invention, constructed recombinant expression vector not only comprises the dna sequence dna of codase, also has the required controlling elements of this gene of expression.

One, the structure of genetic engineering bacterium

1, the GDP-seminose 4,6-dehydrase gene (gmd) and GDP-4-ketone-6-deoxymannose 3, the amplification of 5-mutarotase/4-reductase gene (wcaG)

Extract the genome of intestinal bacteria E.coli-K12, design following primer:

Pgmd1:5 '-CATG CCATGGATATGTCAAAAGTCTCTCATC-3 ' restriction enzyme site is Nco I.

Pgmd2:5 '-CCC AAGCTTTTATGACTCCAGCGCGATC-3 ' restriction enzyme site is HindIII

PwcaG1:5 '-CATG CCATGGGCATGAGTAAACAACGAGTTTTTATTG-3 ' restriction enzyme site is Nco I.

PwcaG2:5 '-CCC AAGCTTTTACCCCCGAAAGCGGT-3 ' restriction enzyme site is HindIII.

Amplification system: the pcr amplification system that adopts 20 μ l:

Amplification condition: the PCR condition that is used for amplifying target genes:

95℃ 5min

72℃ 5min

The amplified production of gained is carried out the agarose gel electrophoresis detection, detect amplified production 1.1kb (gmd), 1.0 (wcaG), the result as shown in Figure 1, can see at about 1.0kb and 1.0kb place and a specific band occur, its size fits like a glove with the goal gene size, be connected on the pET-22b carrier, obtain pET-gmd, pET-wcaG with its order-checking as can be known (entrust Shanghai give birth to worker) increase (GDP-seminose 4, the 6-dehydratase, GDP-4-ketone-6-deoxymannose 3, the dna sequence dna of 5-mutarotase/4-reductase gene is as the back table.

2, construction of recombinant plasmid

(1) preparation of expression vectors

Carry 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, 12000r/min, centrifugal 30s collect thalline, abandon supernatant, control dried raffinate.Precipitation is resuspended in solution 1 (50mmol sucrose, 25mmol Tris, the 10mmol EDTA of 100 μ L precoolings, PH8.0) in, mix, (0.2molNaOH 1%SDS) covers the tight mouth of pipe to add the new solution 2 that disposes of 200 μ L, shake up gently, place that 1-2min is limpid to liquid on ice, (3mol acetate first PH4.8) is rotated centrifuge tube gently to add the solution 3 of 150 μ L precoolings, 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 liquor in another centrifuge tube again, the dehydrated alcohol that adds 2-2.5 times of volume, mixing, ice bath (or-20 ℃) is placed 30min, 12000r/min, centrifugal 5min, collection plasmid DNA precipitation precipitates 2-3 time with 70% washing with alcohol, discards raffinate, air drying 10-20min is with the distilled water dissolution precipitation of 20 μ L.

The pET-22b that is obtained promptly can be used as the carrier that connects amylase gene.

(2) the GDP-seminose 4,6-dehydratase (gmd) gene and GDP-4-ketone-6-deoxymannose 3, the structure of 5-mutarotase/4-reductase enzyme (wcaG) expression vector

1. to carrier pET-22b and gmd, the PCR product of wcaG carries out double digestion, and 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.

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 gmd, wcaG 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 purifying after product is used for electrotransformation transformed into escherichia coli DH5 α.

3, the amplification of plasmid and checking μ L

The competent cell of bacillus coli DH 5 alpha: 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 is 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 that installs to precooling, place-70 ℃ of preservations then by each part 40 μ L;

During use competent cell placed on ice and melt, the connection product that in the competent cell of a pipe 40 μ L, adds the above-mentioned purification process of 4 μ L, the electricity that adds precooling behind the mixing transforms in the cup, touch liquid to guarantee that bacterium and DNA suspension are positioned at electricity and transform the cup bottom, open electric conversion instrument, adjust to the Ecl shelves, promptly aim at intestinal bacteria and transform one grade that is provided with; 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, be inoculated in and contain in penbritin (100 μ g/mL) the liquid LB substratum, cultivate 12-18h in 37 ℃, extract plasmid DNA then in a small amount, carry out double digestion with corresponding restriction enzyme and identify that the recombinant plasmid structure is seen Fig. 2, Fig. 3.

4, the abduction delivering of genetic engineering bacterium

(1) prepares the competent cell of e. coli bl21 according to the method for describing in the step 3, and utilize a small amount of plasmid pET-22b-gmd or pET-22b-wcaG that extracts the correct genetic engineering bacterium of plasmid checking, carry out transformation experiment according to aforesaid method;

(2) recombinant bacterial strain reorganization BL21 (DE3) 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 0.1mmoL/L;

4. induce 4h under 20 ℃, 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-HCl(1.5M)pH8.8 2.5 -

Tris-HCl(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

The 1mL nutrient solution is placed small-sized centrifuge tube, in 4 ℃ of centrifugal 3min of following 12000r/min; Remove supernatant, make sedimentary thalline " drying " as far as possible; Resuspended thalline is in 100 μ L1 * SDS-PAGE electrophoresis sample-loading buffer, and carries out thorough mixing; 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. 4.

Two, the synthetic GDP-Fucose of the mixed fermentation of recombinant strain

Carry out in the LB substratum that shakes 30mL in the bottle of 250ml, its moiety is (g/L): BL21 (DE3)/pET-22b-gmd25 (thalline weight is weight in wet base); BL21 (DE3)/pET-22b-wcaG (thalline weight is weight in wet base) 25; GDP-seminose 30; Phytic acid 5; KH ₂PO ₄25; MgSO ₄.7H ₂O 5; ATP 5; Nymeen S-2155 (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, is inductor with IPTG in reaction process, and making final concentration is the recombinate abduction delivering of bacterium of 0.1mmol/L, and fermenting process as shown in Figure 5.

Earlier BL21 (the DE3)/pET-22b-gmd and BL21 (the DE3)/pET-22b-wcaG engineering strain that build are cultivated in the LB substratum respectively before the reaction, the thalli growth amount is reached OD ₆₀₀=0.5～0.6 o'clock bacterium is as the preparation bacterium of the synthetic GDP-Fucose of mixed fermentation.

Reaction mixture is centrifugal under 4 ℃, 4000r/min, and 15min removes thalline, gets supernatant and carries out the HPLC analysis, and chromatographic condition is: chromatographic column: Hypersil NH ₂, 4.6mm * 150mm, packing material size 5 μ m; Detector: differential refraction detector; Moving phase acetonitrile, water=80,20 (v/v); Column temperature: 25 ℃; Sample size: 5 μ L; Flow velocity: 0.8mL/min.

The output of calculating the GDP-Fucose with external standard method is 10mg/l, and the regression equation of GDP-seminose is as follows:

Y=0.001+12.7x (R=0.9998) wherein does equation of linear regression with peak area y to sample concentration x.

The mixed fermentation of two engineering strains makes the product of engineering strain act on the synthetic GDP-Fucose of GDP-seminose by changing the cell permeability.

Claims

1. method of utilizing the synthetic GDP-Fucose of genetic engineering bacterium coupled fermentation, it is characterized in that: step is as follows:

2. the method for utilizing the synthetic GDP-Fucose of genetic engineering bacterium coupled fermentation according to claim 1, it is characterized in that: amplification is adopted in the described step (2) is plasmid to be transformed in the competent cell of bacillus coli DH 5 alpha increase, amplification back enzyme is cut checking, is used for the conversion of step (3) after correct.

3. the method for utilizing the synthetic GDP-Fucose of genetic engineering bacterium coupled fermentation according to claim 1, it is characterized in that: the moiety of described step (4) substratum is: BL21 (DE3)/pET-22b-gmd 25g/L; BL21 (DE3)/pET-22b-wcaG 25g/L; GDP-seminose 30g/L; Phytic acid 5g/L; KH ₂PO ₄25g/L; MgSO ₄.7H ₂O 5g/L; ATP 5g/L; Nymeen S-215 4g/L.