CN102120999A - Method for synthesizing human milk fucosylation oligosaccharide by using genetic engineering strain through coupling and fermenting - Google Patents

Abstract

The invention relates to a method for synthesizing fucosylated oligosaccharides of human milk by coupled fermentation using genetic engineering strains. For the biosynthesis of lacto-fucosylated oligosaccharides, the engineering strain constructed by this method can increase the copy number of the enzyme gene and increase the expression of the enzyme so that it can efficiently synthesize human lacto-fucosylated oligosaccharides, which is low in industrialization. It not only has strong basic theoretical research value, but also has great economic and social benefits, and has broad market development prospects.

Description

Method for synthesizing human lacto-fucosylated oligosaccharides by coupled fermentation using genetically engineered strains

technical field

The invention belongs to the field of genetic engineering, and relates to the construction technology of genetic engineering strains, in particular to a method for synthesizing human milk fucosylated oligosaccharides by coupled fermentation using genetic engineering strains.

Background technique

In recent years, with the rapid development of food industry and biotechnology, the development of functional oligosaccharides has become an important topic in the field of international biotechnology. industry. Human milk oligosaccharides not only have physiological effects such as anti-viral infection, enhancing immune regulation, and reducing inflammation, but also have certain preventive effects on cancer and chronic recurrent colitis. As a class of carbohydrates with a special structure, it can act as a soluble receptor analog of epithelial cells and participate in the enhancement of the non-immune defense system of breastfed infants.

The content of human milk oligosaccharides is the third largest substance in human milk after lactose and lipids. The content of oligosaccharides in colostrum is 20-27g/L, and it is as high as 12-14g/L in mature milk. The content in other animal milk is very low. Bovine colostrum contains only 0.7-1.2g/L oligosaccharides, which is 20 times lower than that in human colostrum. In addition to rich content, the structure of human milk oligosaccharides is also very complex. According to mass spectrometry analysis It is estimated that human milk contains more than 900 oligosaccharides with different structures, and at least 130 oligosaccharides with different structures have been isolated, while cow milk only contains 18 oligosaccharides. Most of the human milk oligosaccharides were neutral, mainly fucosylated oligosaccharides, among which α1,2-fucosylated oligosaccharides accounted for an average of 73% of the total oligosaccharides in human milk. Fucose residues range from 1 to 15. This kind of fucosylated oligosaccharide, which is the most abundant in human milk, has not been detected in the mature milk of cattle, sheep, goats and horses, but it contains more neutral oligosaccharides in its colostrum . The differences in the structure of oligosaccharides between different mammalian milks may also suggest that human milk oligosaccharides have unique physiological functions. In addition, human milk oligosaccharides and their analogs have unique functions, are safe and effective, do not produce drug resistance, and can also resist mutant pathogenic bacteria resistant to antibiotics. Human milk oligosaccharides and their analogs are used as raw materials to prepare anti-adhesive drugs. Attached drugs have become another hot spot in the current drug development, and they will play a huge role in the field of prevention and treatment of bacterial diseases and infant nutrition and health care.

At present, the research on the function and preparation of human milk oligosaccharides has not been effectively carried out in my country. The development of functional oligosaccharides in my country mainly focuses on natural plant and microbial polysaccharides, which are degraded by acid, alkali, enzyme, oxidation and other chemical methods to prepare low molecular weight oligosaccharides, such as chitooligosaccharides and fructooligosaccharides. Human milk oligosaccharides are obtained by chemical synthesis. Due to the complexity of the synthetic route and the high cost of glycoside donors, most processes still cannot achieve large-scale production, which has become an insurmountable obstacle for chemical synthesis of oligosaccharides. With the continuous analysis of more and more glycosidases and their genes and the continuous elucidation of microbial metabolic regulation routes, as well as the rapid development of genetic engineering technology, the use of enzymes required for the production process of synthetic oligosaccharides to synthesize oligosaccharides in large quantities has become become possible.

Human milk oligosaccharides are mainly fucosylated oligosaccharides, and mannose is an important prerequisite for the synthesis of fucosylated oligosaccharides.

Contents of the invention

The purpose of the present invention is to provide a construction method for fermenting and synthesizing human lactosylated oligosaccharides using genetically engineered strains, using E.coli expression system to carry out related enzyme synthesis of fucosylated oligosaccharides using mannose as a substrate High-efficiency expression, using gene recombination technology to construct recombinant Escherichia coli containing related enzyme genes, so as to ferment and synthesize fucosylated oligosaccharides. The genetically engineered strain obtained by the method increases the copy number of the enzyme gene and significantly increases the expression level of the enzyme, so that it can act on the substrate mannose to synthesize fucosylated oligosaccharides.

The purpose of the present invention is achieved through the following technical solutions:

A method for synthesizing human lactofucosylated oligosaccharides by coupled fermentation using genetically engineered strains, comprising the following steps:

(1) Gene amplification: based on the hexokinase glk, phosphomannose mutase manB, mannose-1 phosphoguanosyltransferase manC, GDP-mannose 4,6-dehydratase gmd in E. coli K12 , GDP-4-keto-6-deoxymannose 3, 5-mutrotase/4-reductase wcaG and Helicobacter pylori HPAG1 α-1,2 fucosyltransferase gene fuct sequence design primers , using Escherichia coli E.coli-K12 and HPAG1 chromosomal DNA as templates to perform PCR amplification on the above target genes respectively, and amplify the corresponding target genes;

(2) Construction of recombinant plasmids: The amplified corresponding target genes were connected to the vector pET-22b by restriction endonuclease ligation to obtain recombinant expression vectors pET-glk-manB-manC, pET-gmd- wcaG and pET-fuct, and double enzyme digestion verification;

(3) Construction of genetically engineered strains: Transform the above-mentioned correct recombinant expression vectors pET-glk-manB-manC, pET-gmd-wcaG and pET-fuct into the host strain BL21(DE3) to obtain genes containing recombinant plasmids Engineering strains BL21(DE3)/pET-glk-manB-manC, B2:BL21(DE3)/pET-gmd-wcaG and B3:BL21(DE3)/pET-fuct;

(4) Coupling fermentation to synthesize human milk fucosylated sugars: the three genetically engineered strains obtained in step (3) were mixed and fermented in the medium, and mannose was used as a synthetic substrate to synthesize human milk fucosylated sugars. oligosaccharides.

And, the fermentation process of step (4) adds Nymeen S-215.

Moreover, the fermentation medium in the step (4) is LB culture, and other ingredients are added as g/L: B1 wet weight 30; B2 wet weight 25; B3 wet weight 25; mannose 30; phytic acid 5; KH ₂ PO ₄ 25 ; MgSO ₄ .7H ₂ O 5 ; Fructose 25 ; Nymeen S-215 4 .

Advantage and positive effect of the present invention are:

1. The present invention is based on Escherichia coli, and uses genetic engineering recombination technology to construct three genetically engineered strains B1, B2, and B3 to realize high-efficiency expression of enzymes in the metabolic pathway, and applies fermentation technology to act on the genetically engineered strains on mannose, Carrying out coupled fermentation and then synthesizing human milk fucosylated oligosaccharides provides a feasible way for a large amount of low-cost industrial production of human milk oligosaccharides. This production method reduces production costs and improves production efficiency. It is not only economical It also has certain social benefits.

2. The present invention utilizes GTP-producing bacteria C.ammoniagenes to provide energy for the entire catalytic reaction, combined fermentation to produce GDP-fucose, and then achieves a large amount of synthesis of human milk fucosylated oligosaccharides, which not only enables mass production of human milk oligosaccharides Sugar becomes possible, and it also provides raw materials for the production of natural oligosaccharide drugs, which has strong basic theoretical research value and social and economic benefits, and has broad market development prospects.

Description of drawings:

Fig. 1 is the amplified electrophoresis figure of the present invention, wherein:

a: 1. Hexokinase glk; 2. Mannose phosphomutase gene manB; 3. Mannose-1-phosphate guanylyltransferase gene manC;

b: 1-3 is the GDP-4-keto-6-deoxymannose 3,5-mutatase/4-reductase (wcaG) gene; band 4 is 1kb DNAmarker; band 5-7 is GDP-mannose Sugar 4,6-dehydratase (gmd) gene;

c: 1, 1kb DNA marker; 2, α-1,2 fucosyltransferase gene (fuct) PCR product;

Fig. 2 is pET-glk-manB-manC of the present invention, pET-gmd-wcaG and pET-fuct, the structural diagram of the recombinant expression plasmid, wherein

a: Construction of recombinant plasmid pET-glk-manB-manC;

b: construction of recombinant plasmid pET-gmd-wcaG;

c: construction of recombinant plasmid pET-fuct;

Fig. 3 is the protein expression electrophoresis figure of pET-glk-manB-manC of the present invention, pET-gmd-wcaG and pET-fuct; Wherein:

a: M is protein marker, 1-2 is pET-glk-manB-manC/BL21;

b: M is protein marker, 1-2 is pET-gmd-wcaG/BL21;

c: M is protein marker, 1 is pET-22b(+)/BL21; 2 is pET-fuct/BL21;

Fig. 4 is a flowchart of the construction of the human milk fucosylated oligosaccharide system synthesized by mixed fermentation of genetically engineered strains in the present invention.

Detailed ways

Below in conjunction with the examples, the present invention is further described, the following examples are illustrative, not limiting, and the protection scope of the present invention cannot be limited by the following examples.

The method for constructing human lactofucosylated oligosaccharide system by coupling fermentation with genetic engineering strains involved in the present invention is to use the E. coli expression system BL21/pET-22b to catalyze the synthesis of human milk rock with mannose as a substrate Cocosylated oligosaccharide-related enzyme genes such as hexokinase gene, phosphomannose mutase gene, mannose-1-phosphate guanylyltransferase gene, GDP-mannose 4,6-dehydratase, GDP -4-keto-6-deoxymannose 3,5-mutrotase/4-reductase and α-1,2 fucosyltransferase genes (glk, manB, manC, gmd, wcaG, fuct) for efficient Expression and construction of three recombinant E. coli strains containing different target genes, and efficient synthesis of human milk fucosylated oligosaccharides through metabolic regulation. The enzyme used in the present invention is derived from E.coli K12 and Helicobacter pylori HPAG1, and the host cell of the recombinant vector in the present invention is Escherichia coli BL21 (DE3). The constructed recombinant expression vector not only includes the DNA sequence encoding the enzyme, but also has the control elements required for expressing the gene.

The steps to build the method are:

1. Amplification of the target gene

(1) Extract the genomic DNA of Escherichia coli (E.coli K12), and design the following primers:

Pglk1: 5'-GGAATTC TCTAGA ATGACAAAGTATGCATTAGTCGGT-3' restriction site is XbaI;

Pglk2: 5'-CGC GCCCGGGC TTACAGAATGTGACCTAAGGTCTG-3' restriction site is SrfI.

PmanB1: 5'-GGAATTC GGTACC ATGAAAAAATTAACCTGCTTT-3' restriction site is KpnI;

PmanB2: 5'-CCC AGTACT TTACTCGTTCAGCAACGTC-3' restriction site is ScaI.

PmanC1: 5'-CATG AGTACT ATATGACAAAGTATGCATTAGTCGGT-3' restriction site is ScaI;

PmanC2: 5'-CCC CATGG TTACAGAATGTGACCTAAGGTCTG-3' restriction site is NcoI.

Pgmd1: 5'-CATG GCCCGGGC ATATGTCAAAAAGTCTCTCATC-3' restriction site is SrfI;

Pgmd2: 5'-CCC GGTACC TTATGACTCCAGCGCGATC-3' restriction site is KpnI.

PwcaG1: 5'-CATG GAGCTC GCATGAGTAAACAACGAGTTTTTATTG-3' restriction site is SacI;

PwcaG2: 5'-CCC CTCGAG TTACCCCCGAAAGCGGT-3' restriction site is XhoI.

(2) Extract Helicobacter pylori (Helicobacter pylori HPAG1) genomic DNA, and design the following primers:

Pfuct1: 5'-CATG CCATGG ATCCGATGGCTTTTAAAAGTGTGCAA-3' restriction site is BamHI;

Pfuct2: 5'-CCC AAGCTT GAGCTCTTAAGCGTTATACTTTTGGGATTT-3' restriction site is HindIII.

Use Escherichia coli E.coli-K12 and HPAG1 chromosomal DNA as templates to carry out PCR amplification of the target gene respectively, and mix them in a sterile EP tube in the following order,

a. Use 20 μL of PCR amplification system:

b. PCR conditions for amplifying the target gene:

The amplified product of gained is carried out agarose gel electrophoresis detection, detects amplified product 0.9kb (glk), 1.3kb (manB), 1.4kb (manC), 1.1kb (gmd), 1.0 (wcaG) and 0.9 ( fuct), the results are shown in Figure 1. It can be seen that specific bands appear at the corresponding band size positions, and their size is completely consistent with the size of the target gene. The amplified target genes glk, gmd, and fuct are connected to pET-22b On the vector, construct recombinant plasmids pET-glk, pET-gmd and pET-fuct, then connect manB and manC to pET-glk one by one, construct recombinant plasmid pET-glk-manB-manC, connect wcaG to pET-gmd , construct the recombinant plasmid pET-gmd-wcaG, and sequence it to know (entrusted by Shanghai Sangong) to amplify hexokinase gene, phosphomannose mutase gene, mannose-1-phosphate guanylyltransferase gene DNA of , GDP-mannose 4,6-dehydratase, GDP-4-keto-6-deoxymannose 3,5-mutrotase/4-reductase and α-1,2 fucosyltransferase genes The sequence is shown in the table below.

2. Construction of recombinant plasmids

1. Preparation of expression vector

(1) Inoculate the Escherichia coli JM109 strain carrying the plasmid pET-22b (purchased from Treasure Biotech Co., Ltd.) in LB medium of ampicillin (50 μg/mL), culture it with shaking at 37°C overnight, and transfer 1.5 mL of the bacterial liquid into micropipette In a centrifuge tube, centrifuge at 12000r/min for 30s to collect the bacteria, discard the supernatant, and dry the residual liquid. (2) Resuspend the pellet in 100 μL of pre-cooled solution 1 (50 mmol sucrose, 25 mmol Tris, 10 mmol EDTA, pH 8.0), and mix well. (3) Add 200 μL of newly prepared solution 2 (0.2 mol NaOH, 1% SDS) to cover the tube tightly, shake gently, and place on ice for 1-2 min until the liquid becomes clear. (4) Add 150 μL pre-cooled solution 3 (3mol methyl acetate, pH 4.8) and gently rotate the centrifuge tube to mix solution 3 evenly in the viscous bacterial lysate, ice bath for 3-5min, 12000r/min, centrifuge 5min, transfer the supernatant to another tube, centrifuge at 12000r/min for 5min, then transfer the supernatant r to another centrifuge tube. (5) Add 2-2.5 times the volume of absolute ethanol, mix well, and place in an ice bath (or -20°C) for 30 minutes. (6) Centrifuge at 12000r/min for 5min to collect the plasmid DNA precipitate. (7) Wash the precipitate with 70% ethanol for 2-3 times, discard the residual liquid, dry in the air for 10-20 min, dissolve the precipitate with 20 μL of double distilled water, and obtain the pET-22b plasmid vector. The obtained vector pET-22b can be used as a carrier of the ligase gene. Subsequently, the plasmid was transformed, using the original multiple cloning sites BamHI and SacI on the plasmid as restriction sites, and a small gene fragment containing specific multiple cloning sites SrfI and KpnI was synthesized by Shanghai Sangon Company and inserted into the selected enzymes Between the cutting sites, the vector pET-22b(+) containing more multiple cloning sites was constructed.

2. Construction of recombinant expression vector

(1) Carry out double digestion of the vector pET-22b and the PCR product with the corresponding restriction endonuclease, then electrophoresis and gel cutting to recover the digested plasmid and PCR product, all using a 50 μL enzyme digestion system:

a.1) The amplified target genes (glk, gmd, fuct) were respectively digested with the vector pET-22b, and the restriction endonucleases used were the same as those of the restriction endonucleases carried in the upstream and downstream primers.

a.2) Target genes (manB, wcaG) were digested and ligated with plasmids pET-glk and pET-gmd respectively.

a.3) Digest the target gene (manC) with the plasmid pET-glk-manB

b.

The above-mentioned digested fragments were purified with a DNA purification kit (TaKaBa Company), and the obtained linearly purified pET-22b and PCR products could be used to construct recombinant plasmids.

(2) The 10 μL ligation system is used for the ligation of the digested vector and the target gene:

The obtained ligation mixture was purified using a DNA purification kit (TaKaRa Company), and the purified products pET-glk-manB-manC, pET-gmd-wcaG and pET-fuct were used to transform Escherichia coli DH5α by electroporation.

3. Construction of genetically engineered strains

Prepare Escherichia coli DH5α competent cells according to the following method: inoculate E. coli DH5α slant strains into 5 mL LB medium, shake and culture at 37°C for 2-3 hours, and make the cells reach the logarithmic growth phase (OD ₆₀₀ =0.5- 0.7); transfer the triangular flask to ice for 20 minutes, centrifuge at 4000r/min for 15min at 4°C, and collect the cells; suspend the cells with 300 μL of 10% glycerol, centrifuge at 4000r/min for 15 minutes at 4°C, and repeat this process once; finally Suspend the cells in 300 μL of 10% glycerol, aliquot each 40 μL into pre-cooled centrifuge tubes, and store at -70°C.

When using, put the competent cells on ice to thaw, add 4 μL of the above-mentioned purified ligation products (pET-glk-manB-manC, pET-gmd-wcaG, pET-fuct) to a 40 μL tube of competent cells, and mix well Then add it to the pre-cooled electrotransformation cup, tap the liquid to ensure that the bacteria and DNA suspension are at the bottom of the electrotransformation cup; turn on the electrotransformer (ETM399, BTX-USA), and adjust to the Ecl file, which is specially designed for Escherichia coli transformation The first gear is set; wipe off the condensed water and mist outside the electroporation cup, put it into the electroporation instrument, and start the electroporation of cells according to the above-mentioned gear; after the transformation is completed, take out the electroporation cup as soon as possible, Add 600 μL of SOC culture solution, mix well, transfer to a 1.5ml centrifuge tube, shake slowly at 37°C, 180 r/min for 1 hour; spread 100 μL of each plate on the LA plate containing ampicillin (100 μg/mL), 37 Invert overnight at ℃ (16-20h); pick a single colony from the plate, inoculate it in liquid LB medium containing ampicillin (100 μg/mL), culture at 37°C for 12-18h, then extract a small amount of plasmid DNA, use The corresponding restriction endonucleases were identified by double digestion, and the structure of the recombinant plasmid is shown in Figure 2.

4. Induced expression of genetically engineered bacteria

1. Prepare competent cells of Escherichia coli BL21 according to the method described in step 3, and use a small amount of plasmid extraction to verify the correct plasmid of the genetically engineered bacteria, and perform transformation experiments according to the above method.

2. Induction of recombinant strain BL21(DE3)

①Pick a single colony from the plate, inoculate it in liquid LB medium containing ampicillin (100 μg/mL), and incubate at 37°C for 12-18 hours.

②Put the overnight culture at 1% inoculum size in 30 mL LB medium containing 60 μg/mL ampicillin.

③ Shaking culture at 37°C, when OD ₆₀₀ = 0.4-0.6, add IPTG to a final concentration of 0.1 mM.

④Induce at 20°C for 4h. Sampling for testing.

3. SDS-PAGE expression analysis

①Gel preparation

Solution Composition 12% Separating Gel (10mL)mL 5% Concentrating Gel (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 persulfate 0.1 0.04

TEMED 0.004 0.004

Deionized water 3.3 0.7

②Sample processing

a) 1 mL of the culture solution was placed in a small centrifuge tube and centrifuged at 12000 r/min for 3 min at 4°C.

b) Remove the supernatant and make the precipitated cells as "dry" as possible.

c) Resuspend the bacteria in 100 μL 1×SDS-PAGE electrophoresis loading buffer and mix thoroughly.

d) Boil at 100°C for 5 minutes, centrifuge to take the supernatant, store the sample at -20°C until protein electrophoresis analysis, the electrophoresis results are shown in Figure 3

5. Synthesis of fucosylated oligosaccharides by mixed fermentation of recombinant engineered strains

The constructed B1: BL21(DE3)/pET-glk-manB-manC; B2: BL21(DE3)/pET-gmd-wcaG; B3: BL21(DE3)/pET-fuct genetic engineering strains were respectively in LB medium Culture in , and when the growth of the bacterium reached OD ₆₀₀ =0.4-0.6, it was used as a preparation bacterium for mixed fermentation to synthesize human milk fucosylated oligosaccharides.

Synthesis of GDP-mannose by mixed fermentation was carried out in 30mL LB medium in a 250mL shake flask, and other ingredients were added at g/L: B1 wet weight 30; B2 wet weight 25; B3 wet weight 25; mannose 30; Phytic acid 5; KH ₂ PO ₄ 25; MgSO ₄ .7H ₂ O5; ATP 5; Nymeen S-215 4 (surfactant, increase the permeability of the bacterial surface); the reaction system uses 4N NaOH to maintain the pH value at 7.2 , the reaction was carried out at 32°C and 900r/min for 22h.

The reaction mixture was centrifuged at 4° C. at 4000 r/min for 15 min to remove bacteria, and the supernatant was taken for HPLC analysis. The chromatographic conditions are: chromatographic column: Aminex HOX-87H (Bio-Red) 7.8mm×300mm, detector: differential refractive index detector; mobile phase 40mmol/LH ₂ SO ₄ ; column temperature: room temperature; injection volume: 5μL; flow rate : 1.0 mL/min.

The output of fucosylated oligosaccharides calculated by external standard method is 4.4mg/L, the regression equation of fucosylated oligosaccharides:

y=0.001+12.7x (R=0.9998), where the peak area y is used to perform a linear regression equation on the sample concentration x.

The bacterial cells are induced to ferment, and the products of the engineered strains act on mannose to synthesize fucosylated oligosaccharides 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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