CN111471637A - 2' -fucosyllactose high-yield strain and preparation method and application thereof - Google Patents

2' -fucosyllactose high-yield strain and preparation method and application thereof Download PDF

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CN111471637A
CN111471637A CN202010379802.3A CN202010379802A CN111471637A CN 111471637 A CN111471637 A CN 111471637A CN 202010379802 A CN202010379802 A CN 202010379802A CN 111471637 A CN111471637 A CN 111471637A
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帅玉英
荣德明
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Jiangsu Huayan Group Co ltd
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Abstract

The invention discloses a 2' -fucosyllactose high-yield strain, a preparation method and application thereof, wherein the preservation number of the strain is CGMCC No. 19557. The invention uses Escherichia coliE.coliB L21 (DE3) is a production host, a synthetic route of 2'-F L is constructed by over-expressing phosphomannose mutase (manB), mannose-1-phosphoguanyltransferase (manC), GDP-mannose-4, 6-dehydratase (gmd), GDP-L-fucose synthase (flc) and α -1, 2-fucose transferase (futC), on the basis, the substrate supply is improved by over-expressing sucrose transporter and lactose transporter, the production capacity of 2' -F L is further improved, high-efficiency expression of 2'-F L in escherichia coli is realized, and a 2' -F L high-yield strain is obtained.

Description

2' -fucosyllactose high-yield strain and preparation method and application thereof
Technical Field
The invention relates to a 2' -fucosyllactose high-yield strain and a preparation method and application thereof.
Background
Human milk is a complex mixture of carbohydrates, fats, proteins, vitamins, minerals and trace elements. The main component is carbohydrates, which can be further divided into lactose and more complex oligosaccharides (human milk oligosaccharides, HMOs). Lactose, however, can be used by the human body as an energy source, and complex oligosaccharides cannot be metabolized by infants. Complex oligosaccharides consist of more than 200 different oligosaccharides, the content of which amounts to up to 20% of the total carbohydrate content. The presence and concentration of these complex oligosaccharides is unique to humans and has not been found in emulsions in other mammals.
About 200 structurally diverse HMOs have been identified to date, and many beneficial properties thereof have been reported. HMO is not digestible by breast-fed infants, but is available as Bifidobacterium in the gut: (Bifidobacteria) Lactobacillus (I) and (II)Lactobacillus) And Bacteroides genus (Bacteroides) And the carbon source and the energy source of the beneficial bacteria enable the bacteria to dominate the intestinal tract and overcome pathogens in competition, and can prevent the intestinal epithelial infection. Furthermore, HMOs can also bind directly to pathogenic bacteria, protozoa, and viruses, blocking pathogen-host interactions by mimicking glycan cell surface receptors, thereby protecting breastfed children from infectious diseases.
Among the numerous human milk oligosaccharides, the most important is 2' -fucosyllactose (2' -fucosylactose, 2' -F L.) other important HMOs present in human milk are 3-fucosyllactose, lacto-N-tetraose, lacto-N-neotetraose, and lacto-N-fucopentaose, in addition to these neutral oligosaccharides, acidic HMOs such as 3' -sialyllactose, 6' -sialyllactose, and sialyllacto-N-tetraose a, b, and c, or sialyllacto-N-fucopentaose II, etc. these structures are closely related to the epitope of the epithelial cell surface glycoconjugate (L ewis tissue blood group antigen), and structural homology of HMOs to epithelial epitopes suggests that they have protective properties against bacterial pathogens.
Due to their beneficial properties, it is advantageous to include HMOs as a component in infant formulas and other food products, which makes the mass production of HMOs (up to the ton grade) the gist of current oligosaccharide production. Due to the limited supply and the difficulty of obtaining the purity of individual human milk oligosaccharides, chemical routes to some of these complex molecules were developed. However, chemical and biocatalytic processes that have proven to be commercially unsustainable, and furthermore, the chemical synthetic routes of especially human milk oligosaccharides involve several toxic chemicals, which carries the risk of contaminating the final product.
Today, for several HMOs such as 2'-F L, 3' -fucosyllactose, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose I, lacto-N-difucohexose II, 3 '-sialyllactose and 6' -sialyllactose, related microbial fermentation processes have been developed, which mainly use genetically engineered bacterial strains such as recombinant escherichia coli (escherichia coli) ((r) ("b))Escherichia coli，E.coli)。
However, even the most efficient processes currently available based on bacterial fermentation, HMO titers in the fermentation broth of greater than 20 g/L cannot or are hardly achieved the industrial grade processes must usually exceed titers of 50 g/L, although 100 g/L is more desirable.
Disclosure of Invention
The object of the present invention is to provide a 2' -fucosyllactose high-producing strain that makes it possible to achieve high titer, and a method for preparing and use thereof.
The technical solution of the invention is as follows:
2' -fucosyllactose high-yield strain(s) ((Escherichia coli) The method is characterized in that: the preservation number is CGMCC No. 19557.
The 2' -fucosyllactose high-producing strain(s) ((Escherichia coli) The preparation method is characterized in that: comprises the following steps:
(1) construction ofE.coliBL21(DE3)-1：
To be provided withEscherichia coliMG1655, using genome as a template, designing a primer: an upstream primer:manB-F: CCGGAATTCATGATAAATATGGTCGTGTTGAGGA, downstream primer:manB-R: CGCGGATCCTTACAAAAGAGTAACTTTATGCAAA, respectively; PCR amplification of phosphomannose mutase (manB) expression genemanBA sequence; the PCR product was reacted with pCDF-Duet plasmidEcoRI/BamHI double enzyme digestion, enzyme digestion product is recovered and purified by glue, and the obtained product ismanBConnecting the gene fragment and the pCDF-Duet plasmid enzyme digestion fragment by using DNA ligase overnight; the ligation product is converted by heat shockE. coliJM109 is competent, transformants are screened and cultured by a streptomycin resistance plate, and plasmid extraction and enzyme digestion or PCR verification are carried out to obtain successfully constructed plasmid pCDF-Duet-manB；
To be provided withE. coliMG1655 genome is used as template, and upstream primer is designed:manC-F: CCCAAGCTTATGAGCTCACCTCTTATTCCGGTTA, downstream primer:manC-R: AAATATGCGGCCGCTCAATCTTCAAATCGAAGGATATCA, respectively; PCR amplification of mannose-1-phosphate guanylyltransferase (manC) expression GenemanCA sequence; the PCR product was mixed with pCDF-Duet-manBPlasmid carrying outHindIII/NotI double enzyme digestion, recovering and purifying the enzyme digestion product by glue, and then carrying out the purification on the obtained productmanCGene fragment and pCDF-Duet-manBCarrying out overnight connection on the plasmid digestion fragments by using DNA ligase; the ligation product is converted by heat shockE. coliJM109 is competent, transformants are screened and cultured by a streptomycin resistance plate, and plasmid extraction and enzyme digestion or PCR verification are carried out to obtain successfully constructed plasmid pCDF-Duet-manB-manC；
To be provided withE. coliMG1655 genome is used as template, and upstream primer is designed:gmd-F: GGGAATTCCATATGTCAAAAGTCGCTCTCATCACCG, and the downstream primer:gmd-R: CGGGGTACCATGTCAAAAGTCGCTCTCATCACCG are provided. PCR amplification of GDP-mannose-4, 6-dehydratase (gmd) expression GenegmdA sequence; the PCR product was mixed with pCDF-Duet-manB-manCPlasmid carrying outNdeI/KpnI double enzyme digestion, recovering and purifying the enzyme digestion product by glue, and then carrying out the purification on the obtained productgmdGene fragment and pCDF-Duet-manB-manCCarrying out overnight connection on the plasmid digestion fragments by using DNA ligase; the ligation product is converted by heat shockE. coliJM109 is competent, transformants are screened and cultured by a streptomycin resistance plate, and plasmid extraction and enzyme digestion or PCR verification are carried out to obtain successfully constructed plasmid pCDF-Duet-manB-manC-gmd；
To be provided withE. coliMG1655 genome is used as template, and upstream primer is designed:flc-F: CCGGAATTCATGAGTAAACAACGAGTTTTTATTG, downstream primer:flcCGCGGATCCTTACCCCCGAAAGCGGTCTTGATTC, PCR amplification of GDP-L-fucose synthase (flc) expression GeneflcA sequence; the PCR product was reacted with pACYC-Duet plasmidEcoRI/BamHI double enzyme digestion, enzyme digestion product is recovered and purified by glue, and the obtained product isflcConnecting the gene fragment and the pACYC-Duet plasmid enzyme digestion fragment by using DNA ligase overnight; the ligation product is converted by heat shockE. coliJM109 is competent, transformants are screened and cultured by a chloramphenicol resistant plate, plasmid extraction and enzyme digestion or PCR verification are carried out, and the successfully constructed plasmid pACYC-Duet-flc；
Based onHelicobacter pyloriα -1, 2-fucosyltransferase (futC) sequence provided in genome, synthesized by Shanghai Productivity based on codon preference of Escherichia colifutCSequence, design of upstream primer:futC-F: GGGAATTCCATATGGCTTTTAAGGTGGTGCAAATTT, downstream primer:futCCGCGGATCCTTAAGCGTTATACTTTTGGGATTTC, PCR amplification α -1, 2-fucosyltransferase (futC) gene, and the PCR product and pACYC-Duet plasmidNdeI/KpnI double enzyme digestion, recovering and purifying the enzyme digestion product by glue, and then carrying out the purification on the obtained productfutCGene fragment and pACYC-Duet-flcCarrying out overnight connection on the plasmid digestion fragments by using DNA ligase; the ligation product is converted by heat shockE. coliJM109 is competent, transformants are screened and cultured by a chloramphenicol resistant plate, plasmid extraction and enzyme digestion or PCR verification are carried out, and the successfully constructed plasmid pACYC-Duet-flc-futC(ii) a The above plasmid pCDF-Duet-manB-manC-gmd、pACYC-Duet-flc-futCConstruction of transformed E.coli B L21 (DE3)E.coliBL21(DE3)-1；
(2) Constructing to obtain recombinant strainE.coliBL21(DE3)-3：
Using Bacillus subtilis (B), (B)Bacillus subtilis) 168 genome is used as a template, and an upstream primer is designed:scrB-F: CATGCCATGGATGACAGCACATGACCAGGAGCTTC, downstream primer:scrB-R: CGCGGATCCCTACATAAGTGTCCAAATTCCGACA, respectively; PCR amplification of scrB expression gene of one of sucrose transportersscrBA sequence; the PCR product was reacted with pET-Duet plasmidNcoI/BamHI double enzyme digestion, enzyme digestion product is recovered and purified by glue, and the obtained product isscrBConnecting the gene fragment and pET-Duet plasmid enzyme digestion fragment by using DNA ligase overnight; the ligation product is converted by heat shockE. coliJM109 competes, transformants are screened and cultured by an ampicillin resistance plate, and plasmid extraction and enzyme digestion or PCR verification are carried out to obtain the successfully constructed plasmid pET-Duet-scrB；
To be provided withE. coliThe K-12 genome is used as a template, and an upstream primer is designed:scrY-F: GGGAATTCCATATGATGTATAAAAAAACAACTTTGGCAG, downstream primer:scrY-R: CGGGGTACCTTAAAACCAGGTTTCCATTTGGACA are provided. PCR amplification of scrY expression gene of one of sucrose transportersscrYA sequence;
the PCR product was reacted with pET-Duet plasmidNdeI/KpnI double enzyme digestion, recovering and purifying the enzyme digestion product by glue, and then carrying out the purification on the obtained productscrYGene fragment and pET-Duet-scrBCarrying out overnight connection on the plasmid digestion fragments by using DNA ligase; the ligation product is converted by heat shockE. coliJM109 competes, transformants are screened and cultured by an ampicillin resistance plate, and plasmid extraction and enzyme digestion or PCR verification are carried out to obtain the successfully constructed plasmid pET-Duet-scrB-scrY. The plasmid pET-Duet-scrB - scrYTransformation of the plasmid intoE. coliB L21 (DE3) -1, recombinant strains were obtainedE.coliBL21(DE3)-2；
To be provided withE. coliB L21 (DE3) genome as template, designing sgRNA and homology arm primer for gene knockout, PCR amplifying lacZ gene upstream and downstream homology arms, obtaining pure knockout gene segment after recovery of glue, and graftingE. coliB L21 (DE3)/pKD46 was cultured at 30 ℃ and OD600When 0.1 was reached, L-arabinose was added at a final concentration of 0.2% to induce expression of pKD 46-lambda-red system when OD was reached600When the content of the compound (B) is 0.3 to 0.4, the feeling of preparation is obtainedCompetent, addition of a knock-out gene fragment to competence, after electrotransformation, knock-outlacZGene to obtain recombinant bacteriumE. coliBL21(DE3)-2/△lacZ；
The recombinant strain is prepared byE.coliBL21(DE3)-2/△lacZRespectively designing for starting strainslacY-F: GGGAATTCCATATGATGTACTATTTAAAAAACACAAACT, downstream primer:lacY-R: CGCGGATCCTTAAGCGACTTCATTCACCTGACGA, amplificationE. colilacY-expressing Gene in K-12lacYRespectively fusing the gene with a strong promoter PssrA in escherichia coli through fusion PCR to construct a high-efficiency expression reading coding frame of lacY; then, the expression cassette is integrated into the recombinant strain by using a homologous integration system in Escherichia coli, and the recombinant strain is constructedE.coliB L21 (DE3) -3, 2' -fucosyllactose high producing strainEscherichia coli。
The application of the 2'-fucosyllactose high-yield strain in improving the yield of 2' -F L is characterized in that a recombinant strain is usedE.coliB L21 (DE3) -3 was inoculated into seed medium and cultured overnight at 37 ℃ and OD was determined at 30 ℃6000.8 hours, 0.5 mM IPTG was added, and the fermentation time was 96 hours, under which conditions the concentrations of sucrose and fructose in the fermentation medium were 30 g/L and 20 g/L, respectively.
Recombinant bacteriaE.coliA single colony of B L21 (DE3) -3 strain is cultured in a seed solution with a liquid loading of 100 m L at 30 ℃ and 180r/min by a rotary shaking table to OD600The method is characterized by taking the seed solution with the concentration of 10.0 percent approximately, inoculating 60 m L of the seed solution into a fermentation culture medium with the working volume of 3L with the inoculation amount of 2.0 percent, fermenting at the fermentation temperature of 33 ℃ in a fermentation tank, stirring at the rotating speed of 600 r/min, the ventilation amount of 1vvm and the pH value of 6.8 (adding ammonia water for automatic control), adding IPTG with the concentration of 50 mg/m L in 1-2 m L in the logarithmic phase, adding sucrose after the logarithmic phase is ended until the concentration is 22 g/L, adding the initial 6h at the flow rate of 9 m L/h, then adding 115 h at the flow rate of 9 m L/h, and adding 100 g/L in the fermentation liquid 2' -F L after the fermentation is ended.
The invention uses Escherichia coliE.coliB L21 (DE3) as production host by overexpressing mannosylmutase phosphate (manB), mannose-1-phosphate guanylyltransferase ((R))manC), GDP-mannose-4, 6-dehydratase (gmd), GDP-L-fucose synthase (flc) and α -1, 2-fucose transferase (futC), a synthetic route of 2'-F L is constructed, on the basis, the supply of substrates is improved by over-expressing sucrose transporter and lactose transporter, the production capacity of 2' -F L is further improved, the 2'-F L is efficiently expressed in escherichia coli, and a 2' -F L high-yield strain is obtained.
The present invention will be further described with reference to the following examples.
The 2' -fucosyllactose high-yield strain has a preservation number of CGMCC No. 19557. And (3) classification and naming: escherichia coli, latin literature name:Escherichia coliand the preservation unit: china general microbiological culture Collection center, address: xilu l No. 3, Beijing, Chaoyang, Beijing, on the north of the Chaoyang district, with a preservation date of 2020, 4 months and 7 days.
Detailed Description
Culture medium:
the seed culture medium comprises sucrose 5.0 g/L, nitrogen source and trace elements 1.0 g/L yeast extract, and 2.0 g/L NH4Cl，10.0 g/L Na2HPO4·7H2O，3.0 g/L KH2PO4，0.5 g/L NaCl，0.25 g/L MgSO4·7H2O，15.0 mg/L CaCl2·2H2O and 10 mg/L vitamin B1.
The fermentation medium comprises sucrose 22.0 g/L and sucrose 13.5 g/L KH2PO4，4.0 g/L (NH4)2H2PO41.7 g/L citric acid, 1.4 g/L MgSO4·7H2O, 10 mg/L solution of trace elements (10 g/L ferric citrate, 2.25 g/L ZnSO)4·7H2O，10 g/L CuSO4·5H2O，0.35 g/L MnSO4·H2O，0.23 g/L Na2B4O7·10H2O，0.11g/L (NH4)6Mo7O24，2.0 g/L CaCl2·2H2O ）
Analytical method
In the following, concentration detection was performed using a spectrophotometer (Biomate 5, Thermo, n.y., USA), intracellular FucT2 enzyme protein was detected using 12% SDS-PAGE, after 3 h addition of 0.1 mM IPTG, the cells were collected and adjusted to a concentration of 7.2 g/L, resuspended and sonicated using 50 mM phosphate buffer (pH 7.0), followed by centrifugation at 15000 g for 20min to pellet the supernatant, and then analyzed by gel electrophoresis.
The 2-F L yields were analyzed using high performance liquid chromatography HP L C (Agilent Technologies 1200 Series) equipped with a Rezex ROA organic acid column H + (Phenomenex, Torrance, Calif. USA) and an RI detector (Agilent, Palo Alto, Calif., USA). The column was tested using 0.01M sulfuric acid at a flow rate of 0.6M L/min, at a column temperature of 50 ℃.
To confirm the purity of 2' -F L, the fermentation broth was collected and assayed by L C/MS, where L C (Agilent technologies 1100 Series) was equipped with an Agilent Zorbax Eclipse ZDB-C8 (4.6 x150 mM, 5 micron) column and an Agilent L C/MSD capture XCT Plus detector at a column flow rate of 0.4 ml/min, with the elution procedure being 95% (v/v) eluent A (15 mM ammonium acetate) and 5% eluent B (acetonitrile) for 1 min, increasing the B concentration from 5% to 95% over 6 min, followed by 95% B wash for 10 min, where MS was assayed in the range of 70-600 (m/z).
2' -fucosyllactose high-yield strain(s) ((Escherichia coli) The preparation method comprises the following specific steps:
(1) construction ofE.coliBL21(DE3)-1：
To be provided withEscherichia coliMG1655 genome is used as template, designing primer: an upstream primer:manB-F: CCGGAATTCATGATAAATATGGTCGTGTTGAGGA, downstream primer:manB-R: CGCGGATCCTTACAAAAGAGTAACTTTATGCAAA, respectively; PCR amplification of phosphomannose mutase (manB) expression genemanBA sequence; the PCR product was reacted with pCDF-Duet plasmidEcoRI/BamHI double enzyme digestion, enzyme digestion product is recovered and purified by glue, and the obtained product ismanBConnecting the gene fragment and the pCDF-Duet plasmid enzyme digestion fragment by using DNA ligase overnight; the ligation product is converted by heat shockE. coliJM109 is competent, transformants are screened by streptomycin resistant plates and cultured, and plasmid extraction and enzyme digestion or PCR verification are carried out to obtain the structureSuccessfully constructed plasmid pCDF-Duet-manB；
To be provided withE. coliMG1655 genome is used as template, and upstream primer is designed:manC-F: CCCAAGCTTATGAGCTCACCTCTTATTCCGGTTA, downstream primer:manC-R: AAATATGCGGCCGCTCAATCTTCAAATCGAAGGATATCA, respectively; PCR amplification of mannose-1-phosphate guanylyltransferase (manC) expression GenemanCA sequence; the PCR product was mixed with pCDF-Duet-manBPlasmid carrying outHindIII/NotI double enzyme digestion, recovering and purifying the enzyme digestion product by glue, and then carrying out the purification on the obtained productmanCGene fragment and pCDF-Duet-manBCarrying out overnight connection on the plasmid digestion fragments by using DNA ligase; the ligation product is converted by heat shockE. coliJM109 is competent, transformants are screened and cultured by a streptomycin resistance plate, and plasmid extraction and enzyme digestion or PCR verification are carried out to obtain successfully constructed plasmid pCDF-Duet-manB-manC；
To be provided withE. coliMG1655 genome is used as template, and upstream primer is designed:gmd-F: GGGAATTCCATATGTCAAAAGTCGCTCTCATCACCG, and the downstream primer:gmd-R: CGGGGTACCATGTCAAAAGTCGCTCTCATCACCG are provided. PCR amplification of GDP-mannose-4, 6-dehydratase (gmd) expression GenegmdA sequence; the PCR product was mixed with pCDF-Duet-manB-manCPlasmid carrying outNdeI/KpnI double enzyme digestion, recovering and purifying the enzyme digestion product by glue, and then carrying out the purification on the obtained productgmdGene fragment and pCDF-Duet-manB-manCCarrying out overnight connection on the plasmid digestion fragments by using DNA ligase; the ligation product is converted by heat shockE. coliJM109 is competent, transformants are screened and cultured by a streptomycin resistance plate, and plasmid extraction and enzyme digestion or PCR verification are carried out to obtain successfully constructed plasmid pCDF-Duet-manB-manC-gmd；
To be provided withE. coliMG1655 genome is used as template, and upstream primer is designed:flc-F: CCGGAATTCATGAGTAAACAACGAGTTTTTATTG, downstream primer:flcCGCGGATCCTTACCCCCGAAAGCGGTCTTGATTC, PCR amplification of GDP-L-fucose synthase (flc) expression GeneflcA sequence; the PCR product was reacted with pACYC-Duet plasmidEcoRI/BamHI double enzyme digestion, enzyme digestion product is recovered and purified by glue, and the obtained product isflcGene fragment and pACYC-DueCarrying out overnight connection on the t plasmid enzyme digestion fragments by using DNA ligase; the ligation product is converted by heat shockE. coliJM109 is competent, transformants are screened and cultured by a chloramphenicol resistant plate, plasmid extraction and enzyme digestion or PCR verification are carried out, and the successfully constructed plasmid pACYC-Duet-flc；
Based onHelicobacter pyloriα -1, 2-fucosyltransferase (futC) sequence provided in genome, synthesized by Shanghai Productivity based on codon preference of Escherichia colifutCSequence, design of upstream primer:futC-F: GGGAATTCCATATGGCTTTTAAGGTGGTGCAAATTT, downstream primer:futCCGCGGATCCTTAAGCGTTATACTTTTGGGATTTC, PCR amplification α -1, 2-fucosyltransferase (futC) gene, and the PCR product and pACYC-Duet plasmidNdeI/KpnI double enzyme digestion, recovering and purifying the enzyme digestion product by glue, and then carrying out the purification on the obtained productfutCGene fragment and pACYC-Duet-flcCarrying out overnight connection on the plasmid digestion fragments by using DNA ligase; the ligation product is converted by heat shockE. coliJM109 is competent, transformants are screened and cultured by a chloramphenicol resistant plate, plasmid extraction and enzyme digestion or PCR verification are carried out, and the successfully constructed plasmid pACYC-Duet-flc-futC(ii) a The above plasmid pCDF-Duet-manB-manC-gmd、pACYC-Duet-flc-futCConstruction of transformed E.coli B L21 (DE3)E.coliBL21(DE3)-1；
(2) Constructing to obtain recombinant strainE.coliBL21(DE3)-3：
Using Bacillus subtilis (B), (B)Bacillus subtilis) 168 genome is used as a template, and an upstream primer is designed:scrB-F: CATGCCATGGATGACAGCACATGACCAGGAGCTTC, downstream primer:scrB-R: CGCGGATCCCTACATAAGTGTCCAAATTCCGACA, respectively; PCR amplification of scrB expression gene of one of sucrose transportersscrBA sequence; the PCR product was reacted with pET-Duet plasmidNcoI/BamHI double enzyme digestion, enzyme digestion product is recovered and purified by glue, and the obtained product isscrBConnecting the gene fragment and pET-Duet plasmid enzyme digestion fragment by using DNA ligase overnight; the ligation product is converted by heat shockE. coliJM109 was competent, transformants were selected on ampicillin-resistant plates and cultured, and plasmid extraction andthe plasmid pET-Duet-scrB；
To be provided withE. coliThe K-12 genome is used as a template, and an upstream primer is designed:scrY-F: GGGAATTCCATATGATGTATAAAAAAACAACTTTGGCAG, downstream primer:scrY-R: CGGGGTACCTTAAAACCAGGTTTCCATTTGGACA are provided. PCR amplification of scrY expression gene of one of sucrose transportersscrYA sequence;
the PCR product was reacted with pET-Duet plasmidNdeI/KpnI double enzyme digestion, recovering and purifying the enzyme digestion product by glue, and then carrying out the purification on the obtained productscrYGene fragment and pET-Duet-scrBCarrying out overnight connection on the plasmid digestion fragments by using DNA ligase; the ligation product is converted by heat shockE. coliJM109 competes, transformants are screened and cultured by an ampicillin resistance plate, and plasmid extraction and enzyme digestion or PCR verification are carried out to obtain the successfully constructed plasmid pET-Duet-scrB-scrY. The plasmid pET-Duet-scrB - scrYTransformation of the plasmid intoE. coliB L21 (DE3) -1, recombinant strains were obtainedE.coliBL21(DE3)-2；
To be provided withE. coliB L21 (DE3) genome as template, designing sgRNA and homology arm primer for gene knockout, PCR amplifying lacZ gene upstream and downstream homology arms, obtaining pure knockout gene segment after recovery of glue, and graftingE. coliB L21 (DE3)/pKD46 was cultured at 30 ℃ and OD600When 0.1 was reached, L-arabinose was added at a final concentration of 0.2% to induce expression of pKD 46-lambda-red system when OD was reached600When the gene reaches 0.3-0.4, preparing competence, adding a knockout gene segment into the competence, and after electrotransformation, knocking out the knockout gene segmentlacZGene to obtain recombinant bacteriumE. coliBL21(DE3)-2/△lacZ；
The recombinant strain is prepared byE.coliBL21(DE3)-2/△lacZRespectively designing for starting strainslacY-F: GGGAATTCCATATGATGTACTATTTAAAAAACACAAACT, downstream primer:lacY-R: CGCGGATCCTTAAGCGACTTCATTCACCTGACGA, amplificationE. colilacY-expressing Gene in K-12lacYRespectively fusing the gene with a strong promoter PssrA in escherichia coli through fusion PCR to construct a high-efficiency expression reading coding frame of lacY; subsequently, homologous integration in E.coli was usedA system for integrating the expression frame into the recombinant strain to construct a recombinant strainE.coliB L21 (DE3) -3, 2' -fucosyllactose high producing strainEscherichia coli。
The application of the 2'-fucosyllactose high-yield strain in improving the yield of 2' -F L is to use a recombinant strainE.coliB L21 (DE3) -3 is inoculated into seed culture medium, cultured overnight at 37 deg.C, transferred to fermentation medium at 1%, 5% and 10% inoculum concentration respectively, and treated with OD600Increasing to 0.6-0.8, adding 0.1, 0.5 and 1mM IPTG, cooling on ice for 20min, inducing fermentation at 20, 25, 30 and 37 deg.C for 120 hr, sampling at 12 hr intervals, measuring the yield of 2-F L, and determining OD at 30 deg.C6000.8 hour, adding 0.5 mM IPTG, and taking fermentation time of 96 hours as an optimal fermentation condition, wherein under the condition, the concentrations of sucrose and fructose in a fermentation medium are respectively 30 g/L and 20 g/L, the thallus concentration can reach 18 g/L, and the yield of 2' -F L can reach 35 g/L.
Based on the fermentation conditions, the fermentation conditions of Escherichia coli 2-F L are optimized in a 3L fermentation tank, and the recombinant bacteria are subjected to fermentationE.coliA single colony of B L21 (DE3) -3 strain is cultured in a seed solution with a liquid loading of 100 m L at 30 ℃ and 180r/min by a rotary shaking table to OD600The method is characterized by taking the seed solution with the concentration of 10.0 percent approximately, inoculating 60 m L of the seed solution into a fermentation culture medium with the working volume of 3L with the inoculation amount of 2.0 percent, fermenting at the fermentation temperature of 33 ℃ in a fermentation tank, stirring at the rotating speed of 600 r/min, the ventilation amount of 1vvm and the pH value of 6.8 (adding ammonia water for automatic control), adding IPTG with the concentration of 50 mg/m L in 1-2 m L in the logarithmic phase, adding sucrose after the logarithmic phase is ended until the concentration is 22 g/L, adding the initial 6h at the flow rate of 9 m L/h, then adding 115 h at the flow rate of 9 m L/h, and adding 100 g/L in the fermentation liquid 2' -F L after the fermentation is ended.
The related sequences are as follows:
Seq1-ManB: GTGATAAATATGGTCGTGTTGAGGATAAATAATTATATGAATATTACCAATGATGTAATTAAAAATAGCGGAATTGCATTTGGTACAAGTGGTGCTCGTGGATTAGTGAAAGATTTTACACCCAACGTGTGTGCTGCATTCACTTATGCTTTTATCGATGTAATGATGAAGGATTTTTCTTTTAAAGAGGTTGCGCTCGCAATAGATAATCGCCCAAGCAGCTATGCTATGGCACAAGCTTGCGCAGCAGCATTGTTGGAATGTAGTATTAAACCTATTTACTACGGTGTAATTCCAACTCCAGCTTTGGCAAATCAAGCGATTGCCGATGGGATTCCTGCAATAATGGTTACTGGAAGTCATATTCCATTTGATAGAAATGGTTTGAAATTCTATCGTCCAGACGGGGAAATTTCAAAAGAAAATGAGAACAGCATTATACATGCTGAAAAAGAGTATCCTGATGTAACTGTATTGCCAGAATTACAATGTTCAAAGCGTGCCGCTGATACTTATATCGAGAGAAACACTTCAATTTTTTCAAATATATTTAAAGGTAAACGTATCGGGATTTATGAACATTCAAGTGCGGGACGGGATCTTTATTCAGAAATATTCAGTAAGCTTGGTGCTGAAGTTGTAAGTATTGGTAGAAGTGATGAGTTTGTGCCTATTGATACTGAAGCAGTATCAAAAGAAGATGAAGAAAAAGCTTTACACTGGTCATCTGAGTATGACCTCGATATGATTTTTTCTACCGATGGTGATGGCGATCGTCCTTTGGTTGCTGACGAAAATGGCGTTTGGTTACGGGGTGATATATTAGGACTATTTTGCTCAAAGGCAATGAATATTGAAGCGGTAGCTGTACCTGTAAGTTGTAATACGGTTATTCAGACTTGCGGGTGGTTTAATCATGTAACGCTGACTAAAATTGGTTCACCATACGTCATTTCAGCGTTTGATTCTCTGAATAATAGTTTTGATAGGGTTGCAGGGTTTGAGGCTAACGGTGGGTATCTTTTAGGTAGCGATGTAAATTATAACAGCGGTATGATTAAAGCGCTTCCAACAAGAGATGCTGTTTTGCCTGCTTTAATGGTACTGGCACTGGCTATAAAAAATAATGTGACTATTTCAAAACTCTTGGCGGAGTTGCCACCACGATTTACCTACTCAAATAGAGTTCAAAATTTTGCAACAAGCAAAAGCAAAAGCAAAAGCATTATTGCGTCAGCTCAAGAAAATCCTCAAGAATTTATCGAAAGAATAGGCTTTGTCGATCTTCATGTACAATCGGTGGATACAATTGACGGGTTAAGATTGACACTAAGTGATGGTTCAATAATCCATTTGCGACCGTCAGGCAACGCACCTGAATTGCGATGCTATGCAGAAGCAGATAACATAATAAAAGCTGAAAAATTAGTGTTAGAAGTTTTGCATAAAGTTACTCTTTTGTAA
Seq2-manC: ATGAGCTCACCTCTTATTCCGGTTATATTAAGTGGTGGTAATGGTACTAGGTTATGGCCACTATCTAGAGAGGAATATCCTAAGCAGTTTTTAAAACTAACCGACTCAATATCAATGCTGCAATCAACAATATCTCGGTTAGACTCATTAAATACTTCCTCTCCAGTTGTAATATGCAATGAATTACACAGATTTATTGTTGCAGAACAACTCAGGCACTTAAATAAATTAGATAATAATATTATTTTAGAACCATCTGGTCGCAATACTGCACCTGCTATTTGTATTGCTGCTTTAATTTTAAAAATGAAGCATCCAAATGAAAATCCACTTATGCTCGTTCTTCCAGCCGATCACTCCGTAAAAAAAGTCAAAACTTTTTGTAATACAATAAAAAGTGCTATTCCCTTCGCTGAAGCTGGTAATTTGGTTTCTTTTGGTATTAAACCTACTCATCCTGAGACGGGGTATGGATATATACAAAAAGGCAAAGTGTTATCTGATTCTGATATATATGAGGTCAGTGAAGTTAGAACTTTTGTTGAAAAGCCTAATCTTAAAACAGCAGAAAGCTTTATAGAAAAAGATGAGTATTATTGGAATAGTGGTATGTATTTGTTCAGTGTTGAACGTTACTTACAAGAGTTATCATTATACCGACCAGACATAGTTAAAGTATGCCAGGAAACTGTTAAAAATATTCATTATGATATGGATTTTATTAGATTGGACGATAAAATATTTCGGAACTGTCCACAGGAGTCTATTGATTATGCTGTAATGGAGAAAACAAAGGATGCTGTAGTTGCTACAATGGATATCGGTTGGAATGATGTAGGAGCATGGTCTTCGCTTTGGGAATTAGGGAAAAAAGACTCCTCTGGTAATGTTATCACGGGAGACATCGTTTGCCACGAGACAGAAAATAGTTATATTTATACTGAGTCTGGATTGGTAGCAACTATTGGTATTCAAGATCTTGTTATTATTCATACTAAAGATTCATTACTGGTTTCCAGACGCGATTCAGTACAAAATGTAAAAAATATTGTTCAGCATCTTGATTTGTCAGGACGTAAAGAACATAAAGAACATAGGGAAGTATTCAAGTCATGGGGACGATGTGACTCCATAGATAGTAGTGAAAAGTACCACTATCAGGTCAAACGAATAACAGTTAATCCAAGTGAAAAATTATCGTTGCAATTACATCATCACCGTGCGGAACATTGGGTTGTTGTAATGGGGATTGCTAAACTTACAGTTGCAGAAGAAATAAAAATTTTAAAAGAGAATGAGTCTGTATATATTCCTGCAGGTATTAAGCATAGTTTGGAAAATATTGGGACAATACCACTTGTGTTAATAGAAGTTTGGACCGGTTCTTATCTTGCTGATGATGATATCCTTCGATTTGAAGATTGA
Seq3-gmd:
ATGTCAAAAGTCGCTCTCATCACCGGTGTAACCGGACAAGACGGTTCTTACCTGGCAGAGTTTCTGCTGGAAAAAGGTTACGAGGTGCATGGTATTAAGCGTCGCGCATCGTCATTCAACACCGAGCGCGTGGATCACATTTATCAGGATCCGCACACCTGCAACCCGAAATTCCATCTGCATTATGGCGACCTGAGTGATACCTCTAACCTGACGCGCATTTTGCGTGAAGTACAGCCGGATGAAGTGTACAACCTGGGCGCAATGAGCCACGTTGCGGTCTCTTTTGAGTCACCAGAATATACCGCTGACGTCGACGCGATGGGTACGCTGCGCCTGCTGGAGGCGATCCGCTTCCTCGGTCTGGAAAAGAAAACTCGTTTCTATCAGGCTTCCACCTCTGAACTGTATGGTCTGGTGCAGGAAATTCCGCAGAAAGAGACCACGCCGTTCTACCCGCGATCTCCGTATGCGGTCGCCAAACTGTACGCCTACTGGATCACCGTTAACTACCGTGAATCCTACGGCATGTACGCCTGTAACGGAATTCTCTTCAACCATGAATCCCCGCGCCGCGGCGAAACCTTCGTTACCCGCAAAATCACCCGCGCAATCGCCAACATCGCCCAGGGGCTGGAGTCGTGCCTGTACCTCGGCAATATGGATTCCCTGCGTGACTGGGGCCACGCCAAAGACTACGTAAAAATGCAGTGGATGATGCTGCAGCAGGAACAGCCGGAAGATTTCGTTATCGCGACCGGCGTTCAGTACTCCGTGCGTCAGTTCGTGGAAATGGCGGCAGCACAGCTGGGCATCAAACTGCGCTTTGAAGGCACGGGCGTTGAAGAGAAGGGCATTGTGGTTTCCGTCACCGGGCATGACGCGCCGGGCGTTAAACCGGGTGATGTGATTATCGCTGTTGACCCGCGTTACTTCCGTCCGGCTGAAGTTGAAACGCTGCTCGGCGACCCGACCAAAGCGCACGAAAAACTGGGCTGGAAACCGGAAATCACCCTCAGAGAGATGGTGTCTGAAATGGTGGCTAATGACCTCGAAGCGGCGAAAAAACACTCTCTGCTGAAATCTCACGGCTACGACGTGGCGATCGCGCTGGAGTCATAA
Seq4-flc:
ATGAGTAAACAACGAGTTTTTATTGCTGGTCATCGCGGGATGGTCGGTTCCGCCATCAGGCGGCAGCTCGAACAGCGCGGTGATGTGGAACTGGTATTACGCACCCGCGACGAGCTGAACCTGCTGGACAGCCGCGCCGTGCATGATTTCTTTGCCAGCGAACGTATTGACCAGGTCTATCTGGCGGCGGCGAAAGTGGGCGGCATTGTTGCCAACAACACCTATCCGGCGGATTTCATCTACCAGAACATGATGATTGAGAGCAACATCATTCACGCCGCGCATCAGAACGACGTGAACAAACTGCTGTTTCTCGGATCGTCCTGCATCTACCCGAAACTGGCAAAACAGCCGATGGCAGAAAGCGAGTTGTTGCAGGGCACGCTGGAGCCGACTAACGAGCCTTATGCTATTGCCAAAATCGCCGGGATCAAACTGTGCGAATCATACAACCGCCAGTACGGACGCGATTACCGCTCAGTCATGCCGACCAACCTGTACGGGCCACACGACAACTTCCACCCGAGTAATTCGCATGTGATCCCAGCATTGCTGCGTCGCTTCCACGAGGCGACGGCACAGAATGCGCCGGACGTGGTGGTATGGGGCAGCGGTACACCGATGCGCGAATTTCTGCACGTCGATGATATGGCGGCGGCGAGCATTCATGTCATGGAGCTGGCGCATGAAGTCTGGCTGGAGAACACCCAGCCGATGTTGTCGCACATTAACGTCGGCACGGGCGTTGACTGCACTATCCGCGAGCTGGCGCAAACCATCGCCAAAGTGGTGGGTTACAAAGGCCGGGTGGTTTTTGATGCCAGCAAACCGGATGGCACGCCGCGCAAACTGCTGGATGTGACGCGCCTGCATCAGCTTGGCTGGTATCACGAAATCTCACTGGAAGCGGGGCTTGCCAGCACTTACCAGTGGTTCCTTGAGAATCAAGACCGCTTTCGGGGGTAA
Seq5-futC:
ATGGCTTTTAAGGTGGTGCAAATTTGCGGGGGGCTTGGGAATCAAATGTTTCAATACGCTTTCGCTAAAAGTTTGCAAAAACACTCTAATACGCCTGTGCTGTTAGATATTACTTCTTTTGATTGGAGCGATAGGAAAATGCAATTAGAGCTTTTCCCTATTGATTTGCCCTATGCGAGTGAAAAAGAAATCGCTATAGCTAAAATGCAACACCTCCCCAAGCTAGTAAGAGACGCGCTCAAATGCATGGGGTTTGATAGGGTGAGCAAAGAAATCGTTTTTGAATACGAGCCTGAATTGTTAAAACCAAGCCGCTTGACTTATTTTTATGGTTACTTTCAAGATCCACGATATTTTGATGCTATATCTCCTTTAATCAAGCAAACCTTCACCCTACCCCCCCCCCCCCCCGAAAATGGAAATAATAAAAAAAAAGAGGAAGAATACCACCGCAAACTTGCTTTGATTTTAGCCGCTAAAAACAGCGTGTTTGTGCATATAAGAAGAGGGGATTATGTGGGGATTGGCTGTCAGCTTGGCATTGATTATCAAAAAAAGGCACTTGAGTATATGGCAAAGCGCGTGCCAAACATGGAGCTTTTTGTGTTTTGCGAAGACTTAACATTCACGCAAAACCTTGATCTGGGCTACCCTTTTATGGACATGACCACTAGGGATAAAGAAGAAGAGGCGTATTGGGACATGCTGCTCATGCAATCTTGTCAGCATGGCATTATCGCTAATAGCACTTATAGCTGGTGGGCGGCTTATTTGATAAACAATCCAGAAAAAATCATTATTGGCCCTAAACACTGGCTTTTTGGGCATGAGAATATCCTTTGTAAGGAATGGGTGAAAATAGAATCCCATTTTGAGGTGAAATCCCAAAAGTATAACGCTTAA
Seq6-scrB:
ATGACAGCACATGACCAGGAGCTTCGTCGCCGGGCTTATGAAGAAGTGGAGAAAAAAGAGCCCATCGCTAACAGCGATCCGCACCGCCAGCATTTTCATATCATGCCGCCGGTTGGGCTGCTGAATGACCCGAATGGCGTGATTTATTGGAAGGGCAGCTATCATGTATTCTTTCAGTGGCAGCCGTTTCAGACGGGGCACGGCGCAAAATTTTGGGGGCATTATACGACACAGGATGTTGTGAATTGGAAGCGGGAAGAGATTGCGCTGGCTCCGAGTGATTGGTTTGATAAAAACGGCTGCTACTCGGGCAGCGCTGTCACGAAAGACGATCGGCTCTATCTTTTTTACACAGGAAATGTCAGGGATCAGGATGGAAATCGGGAAACGTATCAATGCCTTGCTGTTTCTGACGACGGGCTGTCCTTTGAGAAAAAGGGTGTCGTCGCCCGCCTTCCGGAAGGATATACGGCGCATTTTCGCGATCCGAAGGTATGGGAGCATGAAGGCACATGGTATATGGTGATTGGTGCGCAAACAGAGAATTTGAAAGGGCAGGCTGTGTTGTTTGCTTCTGATAACCTGACAGAGTGGAGATTTCTTGGCCCGATAACCGGCGCGGGCTTCAACGGGCTGGACGATTTTGGATACATGTGGGAATGCCCTGATTTGTTTTCCCTTCAAGGATCGGATGTGCTGATTGTTTCGCCTCAAGGGCTTGAGGCTGACGGTTTCCGTTATCAGAACGTATATCAATCAGGTTATTTTGTCGGCCGCCTCGATTATAACAAGCCTGAACTGAAGCATGGTGAATTTACGGAGCTTGATCAAGGTTTTGATTTTTACGCGCCGCAAACACTTGAAGACGATCAGGGAAGGCGGATTTTATTTGCATGGATGGCGGTGCCTGATCAGGATGAAGGGTCCCATCCGACCATTGACTGCCACTGGATTCACTGCATGACGCTGCCGAGACAGCTGACGCTTTCAGGACAGAAGCTGATTCAGCAGCCGCTGCCTGAGCTAAAAGCCATGCGCAGAAATGAGAAAAAAATACACATCAACATGCATGGATCATCTGGTGCGCTTCCAGTGGAAAAACCTGAAAGAACTGAGATTCTACTGGAAGACATTCATACGGAGTCTGGCTTTTCAATCAGTATCCGCGGAACGGCTACGTTTTCCTTCCATAAAGACGAGGGGATTGTTACGCTGGAACGAAAGAGCTTTGACGGAAAAAGAACAGAAGCGAGACATTGCCGCATCAAGGATTTGCATACCGTACACATGTTTCTCGACGCGTCATCTGTGGAAATCTTTATCAATAACGGAGAAGAGGTCTTGAGTGCAAGATATTTTCCTTTCCCGGGAAATCATGAAGTAACAGCCAGTGCGACCGGGAAATCTGAAATGAATGTCGGAATTTGGACACTTATGTAG
Seq7-scrY:
ATGTATAAAAAAACAACTTTGGCAGTGTTAATTGCTTTGCTGACCGGTGCTACAACGGTACATGCGCAAACGGATATTAGCAGTATCGAATCTCGACTGGCGGCGTTGGAACAACGTTTAAAAAATGCGGAATCCCGCGCCCAGGCGGCAGAAGCAAGGGCCAAAACAGCTGAATTACAGGTTCAGAAACTGGCTGAAACACAACAACAAAATCAGCTAACAACTCAAGAAGTAGCACAGAGAACAGTTCAGCTCGAACAGAAATCCGCAGAAAACAGTGGTTTTGAGTTTCATGGCTATGCCCGTTCCGGGTTACTGATGAATGATGCCGCTTCCAGTAGCAAAAGTGGGCCGTATCTGACTCCCGCAGGTGAAACTGGTGGAGCTGTTGGCCGTCTGGGAAATGAAGCCGATACCTATGTCGAGTTAAATGTAGAACATAAACAAACACTGGATAACGGTGCGACCACACGCTTTAAAGCAATGTTGGCTGACGGACAAAGAGATTACAACGACTGGACTGGCGGCTCCAGTAACCTGAATATCCGACAGGCTTTTGCCGAACTGGGCGCATTACCAAGTTTTACCGGAGCATTCAAAGACAGTACTGTCTGGGCTGGTAAACGCTTTGATCGCGACAATTTTGATATTCACTGGTTAGACTCCGATGTCGTATTTTTAGCGGGAACGGGCGGCGGTATCTATGACGTAAAATGGAACGATACATTCCGCAGTAACTTTTCTCTCTACGGACGTAATTTCGGCGATCTTGATGATATCGACAATAACGTTCAGAACTACATCCTCACCATGAATCATTATGCAGGCCCCTTCCAGTTGATGGTTAGCGGATTACGGGCAAAAGATAATGATGATCGAAAAGATGCCAATGGTGATCTCATTCAAACTGATGCTGCAAATACTGGCGTACATGCGTTAGTTGGTCTGCACAATGACACTTTCTATGGCCTGCGTGAAGGGACGGCAAAAACAGCACTGCTATATGGCCATGGCCTGGGTGCGGAAGTCAAAGGGATTGGCTCCGATGGCGCTCTGCTGTCTGAGGCGAATACCTGGCGCTTCGCATCTTACGGCACAACACCTCTGGGAAGCGGTTGGTATGTTGCGCCAGCAATTCTCGCACAAAGCAGTAAAGATCGTTACGTCAAAGGCGATAGCTACGAATGGGTGACCTTCAATACACGTCTGATCAAAGAGGTAACACAGAATTTTGCTCTGGCCTTTGAGGGTAGCTATCAATATATGGATCTGAAGCCAAAGGGGTATCAAAACCACAACGCCGTAAACGGCAGCTTCTATAAACTCACCTTTGCTCCAACTCTAAAAGCTAACGATATCAATAATTTCTTTAGCCGTCCGGAGCTTCGCCTGTTTGCCACCTGGATGGACTGGAGCAGCAAACTTGATGATTTTGCCAGCAATGACGCTTTCGGCAGCAGTGGTTTCAATACTGGCGGAGAGTGGAATTTTGGTGTCCAAATGGAAACCTGGTTTTAA
Seq8-lacY:
ATGTACTATTTAAAAAACACAAACTTTTGGATGTTCGGTTTATTCTTTTTCTTTTACTTTTTTATCATGGGAGCCTACTTCCCGTTTTTCCCGATTTGGCTACATGACATCAACCATATCAGCAAAAGTGATACGGGTATTATTTTTGCCGCTATTTCTCTGTTCTCGCTATTATTCCAACCGCTGTTTGGTCTGCTTTCTGACAAACTCGGGCTGCGCAAATACCTGCTGTGGATTATTACCGGCATGTTAGTGATGTTTGCGCCGTTCTTTATTTTTATCTTCGGGCCACTGTTACAATACAACATTTTAGTAGGATCGATTGTTGGTGGTATTTATCTAGGCTTTTGTTTTAACGCCGGTGCGCCAGCAGTAGAGGCATTTATTGAGAAAGTCAGCCGTCGCAGTAATTTCGAATTTGGTCGCGCGCGGATGTTTGGCTGTGTTGGCTGGGCGCTGTGTGCCTCGATTGTCGGCATCATGTTCACCATCAATAATCAGTTTGTTTTCTGGCTGGGCTCTGGCTGTGCACTCATCCTCGCCGTTTTACTCTTTTTCGCCAAAACGGATGCGCCCTCTTCTGCCACGGTTGCCAATGCGGTAGGTGCCAACCATTCGGCATTTAGCCTTAAGCTGGCACTGGAACTGTTCAGACAGCCAAAACTGTGGTTTTTGTCACTGTATGTTATTGGCGTTTCCTGCACCTACGATGTTTTTGACCAACAGTTTGCTAATTTCTTTACTTCGTTCTTTGCTACCGGTGAACAGGGTACGCGGGTATTTGGCTACGTAACGACAATGGGCGAATTACTTAACGCCTCGATTATGTTCTTTGCGCCACTGATCATTAATCGCATCGGTGGGAAAAACGCCCTGCTGCTGGCTGGCACTATTATGTCTGTACGTATTATTGGCTCATCGTTCGCCACCTCAGCGCTGGAAGTGGTTATTCTGAAAACGCTGCATATGTTTGAAGTACCGTTCCTGCTGGTGGGCTGCTTTAAATATATTACCAGCCAGTTTGAAGTGCGTTTTTCAGCGACGATTTATCTGGTCTGTTTCTGCTTCTTTAAGCAACTGGCGATGATTTTTATGTCTGTACTGGCGGGCAATATGTATGAAAGCATCGGTTTCCAGGGCGCTTATCTGGTGCTGGGTCTGGTGGCGCTGGGCTTCACCTTAATTTCCGTGTTCACGCTTAGCGGCCCCGGCCCGCTTTCCCTGCTGCGTCGTCAGGTGAATGAAGTCGCTTAA
Seq10-PssA:
GTAAGAAACAGCACGATAAACGTTCAGATATCAAAGAGCGCGAATGGCAGGTGGATAAAGCACGTATCATGAAAAACGCCCACCGTTAAACCTGCACTCCAATTATTGACCAGTTCCTCACCGCGCCTCCCTCTCCGGCGGCGCGAATGAACATCTTATTGGCTATCACATCCGACACAAATGTTGCCATCCCATTGCTTAATCGAATAAAAATCAGGCTACATGGGTGCTAAATCTTTAACGATAACGCCATTGAGGCTGGTCATGGCGCTCATAAATCTGGTATACTTACCTTTACACATTG。

Claims (4)

1. 2' -fucosyllactose high-yield strain(s) ((Escherichia coli) The method is characterized in that: the preservation number is CGMCC No. 19557.
2. A2' -fucosyllactose high-producing strain(s) according to claim 1 (a)Escherichia coli) The preparation method is characterized in that: comprises the following steps:
(1) construction ofE.coliBL21(DE3)-1：
To be provided withEscherichia coliMG1655 genome is used as template, designing primer: an upstream primer:manB-F: CCGGAATTCATGATAAATATGGTCGTGTTGAGGA, downstream primer:manB-R: CGCGGATCCTTACAAAAGAGTAACTTTATGCAAA, respectively; PCR amplification phosphomannose mutase expression genemanBA sequence; the PCR product was reacted with pCDF-Duet plasmidEcoRI/BamHI double enzyme digestion, enzyme digestion product is recovered and purified by glue, and the obtained product ismanBConnecting the gene fragment and the pCDF-Duet plasmid enzyme digestion fragment by using DNA ligase overnight; the ligation product is converted by heat shockE. coliJM109 is competent, transformants are screened and cultured by a streptomycin resistance plate, and plasmid extraction and enzyme digestion or PCR verification are carried out to obtain successfully constructed plasmid pCDF-Duet-manB；
To be provided withE. coliMG1655 genome is used as template, and upstream primer is designed:manC-F: CCCAAGCTTATGAGCTCACCTCTTATTCCGGTTA, downstream primer:manC-R: AAATATGCGGCCGCTCAATCTTCAAATCGAAGGATATCA, respectively; PCR amplification of mannose-1-phosphate guanylyltransferase expression GeneDue to the fact thatmanCA sequence; the PCR product was mixed with pCDF-Duet-manBPlasmid carrying outHindIII/NotI double enzyme digestion, recovering and purifying the enzyme digestion product by glue, and then carrying out the purification on the obtained productmanCGene fragment and pCDF-Duet-manBCarrying out overnight connection on the plasmid digestion fragments by using DNA ligase; the ligation product is converted by heat shockE. coliJM109 is competent, transformants are screened and cultured by a streptomycin resistance plate, and plasmid extraction and enzyme digestion or PCR verification are carried out to obtain successfully constructed plasmid pCDF-Duet-manB-manC；
To be provided withE. coliMG1655 genome is used as template, and upstream primer is designed:gmd-F: GGGAATTCCATATGTCAAAAGTCGCTCTCATCACCG, and the downstream primer:gmd-R: CGGGGTACCATGTCAAAAGTCGCTCTCATCACCG, respectively; PCR amplification of GDP-mannose-4, 6-dehydratase expression genegmdA sequence; the PCR product was mixed with pCDF-Duet-manB-manCPlasmid carrying outNdeI/KpnI double enzyme digestion, recovering and purifying the enzyme digestion product by glue, and then carrying out the purification on the obtained productgmdGene fragment and pCDF-Duet-manB-manCCarrying out overnight connection on the plasmid digestion fragments by using DNA ligase; the ligation product is converted by heat shockE. coliJM109 is competent, transformants are screened and cultured by a streptomycin resistance plate, and plasmid extraction and enzyme digestion or PCR verification are carried out to obtain successfully constructed plasmid pCDF-Duet-manB-manC-gmd；
To be provided withE. coliMG1655 genome is used as template, and upstream primer is designed:flc-F: CCGGAATTCATGAGTAAACAACGAGTTTTTATTG, downstream primer:flcCGCGGATCCTTACCCCCGAAAGCGGTCTTGATTC, PCR amplification of GDP-L-fucose synthase expression GeneflcA sequence; the PCR product was reacted with pACYC-Duet plasmidEcoRI/BamHI double enzyme digestion, enzyme digestion product is recovered and purified by glue, and the obtained product isflcConnecting the gene fragment and the pACYC-Duet plasmid enzyme digestion fragment by using DNA ligase overnight; the ligation product is converted by heat shockE. coliJM109 is competent, transformants are screened and cultured by a chloramphenicol resistant plate, plasmid extraction and enzyme digestion or PCR verification are carried out, and the successfully constructed plasmid pACYC-Duet-flc；
Based onHelicobacter pyloriProvided in the genomeα -1, 2-fucosyltransferase, designing an upstream primer:futC-F: GGGAATTCCATATGGCTTTTAAGGTGGTGCAAATTT, downstream primer:futCCGCGGATCCTTAAGCGTTATACTTTTGGGATTTC, PCR amplification α -1, 2-fucosyltransferase gene, and the PCR product and pACYC-Duet plasmidNdeI/KpnI double enzyme digestion, recovering and purifying the enzyme digestion product by glue, and then carrying out the purification on the obtained productfutCGene fragment and pACYC-Duet-flcCarrying out overnight connection on the plasmid digestion fragments by using DNA ligase; the ligation product is converted by heat shockE. coliJM109 is competent, transformants are screened and cultured by a chloramphenicol resistant plate, plasmid extraction and enzyme digestion or PCR verification are carried out, and the successfully constructed plasmid pACYC-Duet-flc-futC(ii) a The above plasmid pCDF-Duet-manB-manC-gmd、pACYC-Duet-flc-futCConstruction of transformed E.coli B L21 (DE3)E.coliBL21(DE3)-1；
(2) Constructing to obtain recombinant strainE.coliBL21(DE3)-3：
Using Bacillus subtilis (B), (B)Bacillus subtilis) 168 genome is used as a template, and an upstream primer is designed:scrB-F: CATGCCATGGATGACAGCACATGACCAGGAGCTTC, downstream primer:scrB-R: CGCGGATCCCTACATAAGTGTCCAAATTCCGACA, respectively; PCR amplification of scrB expression gene of one of sucrose transportersscrBA sequence; the PCR product was reacted with pET-Duet plasmidNcoI/BamHI double enzyme digestion, enzyme digestion product is recovered and purified by glue, and the obtained product isscrBConnecting the gene fragment and pET-Duet plasmid enzyme digestion fragment by using DNA ligase overnight; the ligation product is converted by heat shockE. coliJM109 competes, transformants are screened and cultured by an ampicillin resistance plate, and plasmid extraction and enzyme digestion or PCR verification are carried out to obtain the successfully constructed plasmid pET-Duet-scrB；
To be provided withE. coliThe K-12 genome is used as a template, and an upstream primer is designed:scrY-F: GGGAATTCCATATGATGTATAAAAAAACAACTTTGGCAG, downstream primer:scrY-R: CGGGGTACCTTAAAACCAGGTTTCCATTTGGACA, respectively; PCR amplification of scrY expression gene of one of sucrose transportersscrYA sequence;
the PCR product was reacted with pET-Duet plasmidNdeI/KpnI double enzyme digestion, recovering and purifying the enzyme digestion product by glue, and then carrying out the purification on the obtained productscrYGene fragment and pET-Duet-scrBCarrying out overnight connection on the plasmid digestion fragments by using DNA ligase; the ligation product is converted by heat shockE. coliJM109 competes, transformants are screened and cultured by an ampicillin resistance plate, and plasmid extraction and enzyme digestion or PCR verification are carried out to obtain the successfully constructed plasmid pET-Duet-scrB-scrY(ii) a The plasmid pET-Duet-scrB - scrYTransformation of the plasmid intoE. coliB L21 (DE3) -1, recombinant strains were obtainedE.coliBL21(DE3)-2；
To be provided withE. coliB L21 (DE3) genome as template, designing sgRNA and homology arm primer for gene knockout, PCR amplifying lacZ gene upstream and downstream homology arms, obtaining pure knockout gene segment after recovery of glue, and graftingE. coliB L21 (DE3)/pKD46 was cultured at 30 ℃ and OD600When 0.1 was reached, L-arabinose was added at a final concentration of 0.2% to induce expression of pKD 46-lambda-red system when OD was reached600When the gene reaches 0.3-0.4, preparing competence, adding a knockout gene segment into the competence, and after electrotransformation, knocking out the knockout gene segmentlacZGene to obtain recombinant bacteriumE. coliBL21(DE3)-2/△lacZ；
The recombinant strain is prepared byE.coliBL21(DE3)-2/△lacZRespectively designing for starting strainslacY-F: GGGAATTCCATATGATGTACTATTTAAAAAACACAAACT, downstream primer:lacY-R: CGCGGATCCTTAAGCGACTTCATTCACCTGACGA, amplificationE. colilacY-expressing Gene in K-12lacYRespectively fusing the gene with a strong promoter PssrA in escherichia coli through fusion PCR to construct a high-efficiency expression reading coding frame of lacY; then, the expression cassette is integrated into the recombinant strain by using a homologous integration system in Escherichia coli, and the recombinant strain is constructedE.coliB L21 (DE3) -3, 2' -fucosyllactose high producing strainEscherichia coli。
3. The application of the 2'-fucosyllactose high-producing strain of claim 1 in improving the yield of 2' -F L is characterized in that a recombinant strain is usedE.coliB L21 (DE3) -3 inoculation into seed MediumCulturing at 37 deg.C overnight; determining OD at 30 ℃6000.8 hours, 0.5 mM IPTG was added, and the fermentation time was 96 hours, under which conditions the concentrations of sucrose and fructose in the fermentation medium were 30 g/L and 20 g/L, respectively.
4. The application of the 2'-fucosyllactose high-producing strain of claim 1 in improving the yield of 2' -F L is characterized in that a recombinant strain is usedE.coliA single colony of B L21 (DE3) -3 strain is cultured in a seed solution with a liquid loading of 100 m L at 30 ℃ and 180r/min by a rotary shaking table to OD600The method is characterized by taking the seed liquid with the concentration of 10.0 percent approximately as the seed liquid, inoculating 60 m L of the seed liquid into a fermentation culture medium with the working volume of 3L with the inoculation amount of 2.0 percent, fermenting at the fermentation temperature of 33 ℃ in a fermentation tank, stirring at the rotating speed of 600 r/min, ventilating at the volume of 1vvm and pH of 6.8, adding IPTG with the concentration of 50 mg/m L in 1-2 m L during the logarithmic growth phase, feeding cane sugar after the logarithmic phase is ended until the concentration is 22 g/L, feeding at the initial 6h at the feeding rate of 9 m L/h, then feeding at the rate of 9 m L/h for 115 h, and feeding the fermentation liquid 2' -F L with the concentration of 100 g/L after the fermentation is ended.
CN202010379802.3A 2020-05-08 2020-05-08 2' -fucosyllactose high-yield strain and preparation method and application thereof Pending CN111471637A (en)

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CN116218892A (en) * 2023-02-13 2023-06-06 天津科技大学 Construction method and application of CLM24 (DE 3) strain
CN116479070A (en) * 2023-06-20 2023-07-25 山东合成远景生物科技有限公司 Fermentation production method for improving yield of fucosyllactose of escherichia coli
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