Escherichia coli for producing 2 '-fucosyllactose and construction method and application thereof

Abstract

The invention relates to Escherichia coli for producing 2 '-fucosyllactose as well as a construction method and application of the Escherichia coli, and belongs to the technical field of genetic engineering. A CRISPR / Cas9 system is used for transforming a genome of escherichia coli MG1655, a wcaJ gene and a lacZ gene are knocked out, the utilization rate of lactose and GDP-L-fucose in a metabolic pathway is increased, a promoter of a lacY gene is replaced with PJ23119, an alpha-1, 2-fucosyltransferase mutant with a dissolution promoting tag GST added to the N end is heterologously introduced, the expression quantity of the alpha-1, 2-fucosyltransferase mutant is increased through multi-copy integration, and the expression quantity of the alpha-1, 2-fucosyltransferase mutant is increased. Meanwhile, a gmd gene, an fcl gene, a manB gene and a manC gene are co-expressed at proper sites, so that the synthesis route of the GDP-L-fucose is optimized. The strain obtained through transformation can efficiently convert lactose into 2 '-FL and has the advantages of being low in metabolic burden, sufficient in precursor supply, high in enzyme stability and the like, the construction method is clear in step and suitable for industrial production, the yield of 2'-FL can be remarkably increased through microbial agents and fermentation application, and the production cost is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to an Escherichia coli producing 2'-fucosyllactose, a construction method thereof and an application thereof. Background Art

[0002] 2'-Fucosyllactose (2'-FL) is the oligosaccharide with the highest content in breast milk, accounting for about 30% of the total HMOs, and is crucial for the health of infants, including promoting brain development, enhancing immunity, etc. With the increasing understanding of the benefits of 2'-FL, the international market demand for it is growing day by day. Although the chemical synthesis method is mature, it has problems such as multi-step reactions, high costs, complex purification steps and environmental pollution, which limit its large-scale production. The microbial fermentation method shows broader application potential due to its mild reaction conditions, low cost and environmental friendliness. At present, the construction of genome-integrated and dual-plasmid 2'-FL cell factories is mainly carried out, and the production of 2'-FL is improved by knocking out competing pathways, strengthening the expression of pathway enzymes and fucosyltransferases, etc. The key enzymes for the biosynthesis of 2'-FL are α-1,2-fucosyltransferases, which catalyze the transfer of fucose residues to lactose to synthesize 2'-FL.

[0003] However, the soluble expression level of the α-1,2-fucosyltransferase gene heterologously expressed in commercial strains such as Escherichia coli is extremely low, resulting in extremely poor enzyme catalytic efficiency, and the metabolic pathway of 2'-fucosyllactose is complex, also with multi-step reactions. Merely improving the efficiency of a certain step of the reaction has limitations in increasing the yield of 2'-FL. Therefore, in order to maximize the yield of 2'-FL to meet the requirements of industrial production, it is necessary to improve the soluble expression level of fucosyltransferase and comprehensively regulate the metabolic pathway as a whole. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides an Escherichia coli producing 2'-fucosyllactose, a construction method thereof and an application thereof. The genome of Escherichia coli MG1655 is modified using the CRISPR / Cas9 system, the wcaJ gene and the lacZ gene are knocked out, and the promoter of the lacY gene is replaced with P J23119 , the α-1,2-fucosyltransferase mutant is heterologously introduced, the GST solubility-enhancing tag is fused at the N-terminus, and the gmd gene and the fcl gene, as well as the manB gene and the manC gene are co-expressed at appropriate sites.

[0005] The first object of the present invention is to provide an Escherichia coli capable of producing 2'-fucosyllactose, in which the undecylphosphoglucose phosphotransferase gene wcaJ and the β-galactosidase-encoding gene lacZ are knocked out in the Escherichia coli genome, and the promoter of the lactose permease-encoding gene lacY is replaced with P J23119 promoter, heterologously introduce an α-1,2-fucosyltransferase mutant with an N-terminal fusion GST solubility-enhancing tag, co-express the GDP-mannose-4,6-dehydratase-encoding gene gmd and the GDP-L-fucose synthase-encoding gene fcl, and co-express the mannose-1-phosphate guanylyltransferase-encoding gene manB and the phosphomannomutase-encoding gene manC;

[0006] Among them, the amino acid sequence of the solubility-enhancing tag GST is as shown in SEQ ID NO.8, and the α-1,2-fucosyltransferase mutant is a mutant in which the valine at the 93rd position of the starting sequence with the amino acid sequence as shown in SEQ ID NO.7 is mutated to isoleucine.

[0007] Furthermore, the nucleotide sequence of the solubility-enhancing tag GST is as shown in SEQ ID NO.9.

[0008] Furthermore, use P tac promoter to express the α-1,2-fucosyltransferase mutant.

[0009] Furthermore, use P tac promoter to express gmd and fcl.

[0010] Furthermore, use P tac promoter to express manB and manC.

[0011] Furthermore, perform a double-copy expression of the α-1,2-fucosyltransferase mutant.

[0012] Furthermore, the double-copy expression is respectively carried out at the betA locus and the nupG locus of the Escherichia coli genome.

[0013] Furthermore, perform a triple-copy expression of the α-1,2-fucosyltransferase mutant.

[0014] Furthermore, the triple-copy expression is respectively carried out at the betA locus, the nupG locus and the yeeL locus of the Escherichia coli genome. [[ID=~]] [[ID=~]]

[0015] The second object of the present invention is to provide a construction method of the above-mentioned Escherichia coli, and the construction is carried out using the CRISPR / Cas9 system, and the steps include S1-S5, S1-S6 or S1-S7:

[0016] Step S1: Knock out the undecylphosphoglucose phosphotransferase gene wcaJ and the β-galactosidase encoding gene lacZ in the starting Escherichia coli genome;

[0017] Step S2: Replace the promoter of the lactose permease encoding gene lacY on the genome of the strain obtained in S1 with the P J23119 promoter;

[0018] Step S3: Introduce the α-1,2-fucosyltransferase mutant at the betA locus of the genome of the strain obtained in S2;

[0019] Step S4: Co-express the GDP-mannose-4,6-dehydratase encoding gene gmd and the GDP-L-fucose synthase encoding gene fcl at the yhiM locus of the genome of the strain obtained in S3;

[0020] Step S5: Co-express the mannose-1-phosphate guanylyltransferase encoding gene manB and the phosphomannomutase encoding gene manC at the yeeL locus of the genome of the strain obtained in S4;

[0021] Step S6: Introduce the α-1,2-fucosyltransferase mutant at the nupG locus of the genome of the strain obtained in S5;

[0022] Step S7: Introduce the α-1,2-fucosyltransferase mutant at the ybbD locus of the genome of the strain obtained in S6.

[0023] Furthermore, the starting Escherichia coli is Escherichia coli MG1655.

[0024] The third object of the present invention is to provide a microbial inoculum containing the above Escherichia coli.

[0025] The fourth object of the present invention is to provide the application of the above Escherichia coli or the above microbial inoculum in the production of 2'-fucosyllactose.

[0026] The fifth object of the present invention is to provide a method for producing 2'-fucosyllactose, adding the above Escherichia coli or the above microbial inoculum to a fermentation system to produce 2'-fucosyllactose using lactose as a substrate.

[0027] In an embodiment of the present invention, the method for producing 2'-fucosyllactose is specifically to prepare the above Escherichia coli into a seed solution, the seed solution medium is LB medium, and then the seed solution is inoculated into the fermentation medium at an inoculation amount of 2% and cultured at 37°C and 220 r / min until OD 600 = 0.6, add 20 g / L lactose, and continue shake flask fermentation for 72 h.

[0028] In one embodiment of the present invention, the fermentation medium comprises 5 g of yeast extract, 3 g of potassium dihydrogen phosphate, 20 g of glycerol, 2 g of casein hydrolysate, 6.8 g of disodium hydrogen phosphate, 1 g of ammonium chloride, 0.5 g of sodium chloride. After dissolving with ultrapure water and making up the volume to 1 L, a 1.4 g / L MgSO4·7H2O solution treated with a sterile filter membrane is added.

[0029] Advantages of the present invention:

[0030] In the Escherichia coli genome for producing 2'-fucosyllactose provided by the present invention, wcaJ and lacZ are knocked out, reducing polysaccharide by-products and lactose consumption, and improving substrate utilization rate; the lacY promoter is replaced to strengthen lactose transport and promote substrate accumulation; the introduction and multi-copy expression of the α-1,2-fucosyltransferase mutant significantly improve the catalytic efficiency and stability; the fusion expression of the solubilizing tag GST significantly increases the soluble expression level of the α-1,2-fucosyltransferase. The co-expression of the gmd-fcl and manB-manC gene modules efficiently supplies GDP-L-fucose to promote 2'-FL synthesis. Through gene editing and multi-copy integration strategies, high-yield production of 2'-FL is achieved, and the shake-flask yield of strain MGP7 reaches 6.5 g / L. The regulation of the metabolic pathway and the comprehensive application of various metabolic engineering strategies are beneficial to the improvement of the 2'-FL yield and promote its large-scale industrial production process. Description of the Drawings

[0031] In order to make the content of the present invention easier to be clearly understood, the following further details the present invention according to specific embodiments of the present invention in combination with the drawings, wherein

[0032] Figure 1 is a schematic diagram for constructing a recombinant strain in an embodiment of the present invention;

[0033] Figure 2 is the SDS-PAGE gel diagram of FutC V93I in Example 1 of the present invention;

[0034] Figure 3 is a schematic diagram of the 2'-fucosyllactose yield of the recombinant strain in shake-flask fermentation in Example 5 of the present invention. Detailed Embodiments

[0035] The following further illustrates the present invention in combination with the drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the examples given are not intended to limit the present invention.

[0036] The media involved in the examples are as follows:

[0037] (1) LB liquid medium: 5 g / L of yeast extract, 10 g / L of peptone, 10 g / L of sodium chloride.

[0038] (2) M9CA liquid medium: 5 g of yeast extract, 3 g of potassium dihydrogen phosphate, 20 g of glycerol, 2 g of casein hydrolysate, 6.8 g of disodium hydrogen phosphate, 1 g of ammonium chloride, 0.5 g of sodium chloride. After dissolving with ultrapure water and making up the volume to 1 L, add a 1.4 g / L MgSO4·7H2O solution treated with a sterile filter membrane.

[0039] (3) Lactose mother liquor: 200 g / L lactose.

[0040] The detection methods and transformation methods involved in the examples are as follows:

[0041] (1) Detection of 2'-fucosyllactose:

[0042] To detect the yield of 2'-FL synthesized by the recombinant Escherichia coli and the consumption of the substrate lactose described in the present invention, high performance liquid chromatography (HPLC) system was used for determination.

[0043] HPLC detection conditions: through a high performance liquid chromatography (HPLC) system (Waters e2695); chromatographic column: Rezex ROA-Organic acid H + (8%); detector: Waters 2414 RIDetector differential refractometer; mobile phase: 5 mM H2SO4; flow rate: 0.6 mL / min; column temperature: 60 °C; injection volume: 10 μL.

[0044] (2) Fusion PCR

[0045] First, perform amplification on the target fragments to be fused. Then, purify and recover the products obtained by PCR amplification. Subsequently, start the small cycle step, add the obtained DNA fragments in equimolar amounts to the reaction system (requiring the total amount of DNA in the system to exceed 1,000 ng). This reaction system contains an equal volume of enzyme, set the annealing temperature at 55 °C, and the number of cycles at 13 times. Finally, take an appropriate amount of the PCR product after the small cycle as a template and carry out the amplification of the fusion fragment according to the amplification method of the target fragment.

[0046] (3) Preparation of electrocompetent E. coli and electrotransformation method

[0047] Preparation of competent cells: First, pick a single colony of the target strain from a fresh plate and inoculate it into an LB medium containing the corresponding resistance for overnight culture. Then, transfer it to a liquid LB medium containing the corresponding antibiotic and 1 mol / L arabinose (containing the Cas9 plasmid) and continue to culture until the OD of the bacteria 600≈0.7. After centrifugation for 30 min in an ice bath, the cells were collected. The cells were washed once with pre-cooled sterile ultrapure water (50 mL) and sterile 10% glycerol, and finally centrifuged at 4,500×g for 10 min at 4 °C to collect the cells. The cells were resuspended in 450 μL of ice-bathed sterile 10% glycerol to obtain electrocompetent cells, which were stored at -80 °C.

[0048] Electroporation method: Take out the electroporation cuvette stored in absolute ethanol and place it on the ultra-clean workbench with ultraviolet lamp on to air dry. Mix the transformant with the thawed electrocompetent cells and transfer them into the air-dried electroporation cuvette. After ice-bathing for 30 min, perform electroporation (conditions: 1800 V, 200 Ω, 200 uF, 1 mm); after electroporation, add 1 mL of antibiotic-free LB liquid medium and gently pipette to mix the cells evenly, then culture at 37 °C and 220 rpm for 2 h. After the culture, spread the cells on the LB plate containing the corresponding antibiotic and incubate at 37 °C in an inverted position for 12 h.

[0049] (4) Identification of recombinant transformants

[0050] Pick a single colony from the above plate into the prepared Taq PCR system for colony PCR. Verify the correct colonies and inoculate them into 3 - 5 mL of LB medium containing spectinomycin or chloramphenicol. After shaking culture in a 37 °C constant temperature incubator for 8 - 12 h, extract the plasmid and send it to Genewiz for sequencing. The colonies with correct sequences are positive transformants. The Taq PCR system and PCR reaction program are shown in the following tables.

[0051] Table 1 Taq PCR system

[0052] Component Volume (μL) 2×Rapid Taq Master Mix 10 Primer 1 1 Primer 2 1 Sterile water 8

[0053] Table 2 Taq PCR reaction program

[0054]

[0055] (5) Elimination of antibiotic plasmid

[0056] pTarget plasmid elimination experiment: Pick a single colony and inoculate it into fresh LB medium, add 20 μL of 10 mM rhamnose with a final concentration of 50 mg / L kanamycin. After culturing at 37 °C for 12 h, streak the bacterial solution on the LB plate containing kanamycin and continue to culture for 12 h. Pick the single colonies grown on the plate and spot-inoculate them on the double-antibody LB plate containing kanamycin and spectinomycin, and the single-antibody LB plate containing only kanamycin. The colonies that grow on the single-antibody plate but not on the double-antibody plate have successfully eliminated the pTarget plasmid.

[0057] Cas9 plasmid elimination experiment: Pick colonies that have eliminated the pTarget plasmid and inoculate them into fresh LB liquid medium containing 5 g / L glucose. After culturing at 37 °C for 12 h, streak the bacterial solution on an LB plate supplemented with 10 g / L sucrose and 5 g / L glucose and continue culturing for 12 h. Pick single colonies grown on the plate and spot-inoculate them onto a monoclonal LB plate containing kanamycin and an antibiotic-free LB plate. Colonies that grow on the antibiotic-free plate but not on the double-antibiotic plate have successfully eliminated the Cas9 plasmid. The strains and their genotypes involved in the examples are as follows:

[0058] Table 3 Strains and Genotypes

[0059]

[0060] Example 1: Recombinant Escherichia coli expressing fucosyltransferase

[0061] First, construct a plasmid for recombinant protein expression. Using the plasmid pET28a stored in the laboratory as a template, obtain a linearized plasmid vector by PCR amplification. Using FutC V93I as a template, obtain the DNA fragment of the mutant protein FutC V93I , and then use a seamless cloning kit to ligate the DNA sequence and the linearized plasmid vector to obtain the plasmid pET28a-FutC V93I . Using the plasmid pET28a-FutC V93I as a template, continue to obtain a linearized plasmid vector by PCR amplification. Using the GST tag stored in the laboratory as a template, obtain the DNA sequence of GST by PCR amplification, and then use a seamless cloning kit to ligate the DNA sequence and the linearized plasmid to obtain the plasmid pET28a-GST-FutC V93I . Transfer the plasmids pET28a-FutC V93I and pET28a-GST-FutC V93I into Escherichia coli MG1655 respectively to obtain recombinant Escherichia coli M1 and M2.

[0062] Secondly, perform the expression of recombinant proteins. Strains M1 and M2 are shaken (220 rpm) overnight in LB medium at 37 °C. Next, transfer 4 mL of fresh cells grown in LB into 200 mL of fresh TB medium in a 500 mL baffled flask and incubate until the OD 600 reaches 0.6. Add IPTG with a final concentration of 0.3 mM to the medium to induce the expression of the target protein. After incubating at 20 °C for 20 h, centrifuge at 8000×g for 10 min to collect the recombinant cells.

[0063] Next, the recombinant protein was purified and detected. The cell pellet was resuspended in lysis buffer and sonicated on ice, with 1 s on and 2 s off for 20 min. The lysed mixture was centrifuged at 8000 g for 10 min and filtered through a 0.45 μm filter to obtain a crude enzyme solution. Protein purification was performed by nickel affinity chromatography based on the HIS tag, and the concentration of the purified enzyme was determined by SDS-PAGE.

[0064] The protein sample obtained above was mixed with Thermo Fisher NuPAGE TM 4×LDS sample buffer, then denatured in a metal bath at 98 °C for 10 min and cooled to room temperature before use. The protein precast gel was fixed in the protein electrophoresis tank, an appropriate amount of the treated protein sample and Protein Maker were pipetted into the protein gel wells, and the voltage was set to 120 V for 120 min. The protein gel was placed in a gel box containing an appropriate amount of Coomassie Brilliant Blue staining solution and stained for 30 min. After staining, the staining solution was poured back into the bottle, and water or decolorizing solution was added for decolorization. When the protein bands were clearly visible, they were observed in a gel imaging system.

[0065] Comparing the protein expression levels of M1 and M2, it was found that the soluble protein expression level of M2 with the added GST solubility tag was significantly increased. The protein purification result of M1 is shown as Figure 2 a in the figure. The theoretical size of FutC V93I is 38 kDa. Bands were present between 35 kDa and 45 kDa in the precipitate, crude enzyme, and elution with 30% Buffer B, indicating that the expression system successfully expressed FutC V93I . However, due to the poor soluble expression of this protein and its poor binding ability to the nickel column, the concentration of the purified protein was low, and most of the protein was lost in the precipitation solution. The result of M2 with the GST protein tag linked to the N-terminus of the target protein sequence through a protein linker is shown as Figure 2 b in the figure. Except for no target band in the control group, the molecular weight of FutC V93I (containing the GST tag) was approximately 61 kDa, which was consistent with the theoretical size band, and the concentration of the target protein band was significantly higher than that of M1.

[0066] Example 2: Recombinant strain of Escherichia coli MG1655 with wcaJ, lacZ knocked out and the original promoter of lacY replaced

[0067] The undecylphosphoglucose phosphotransferase gene wcaJ and the β-galactosidase-encoding gene lacZ in the Escherichia coli genome were knocked out using the CRISPR-Cas9 gene editing system, and the original promoter of the lactose permease-encoding gene lacY was replaced with the P J23119 promoter. The specific steps are as follows:

[0068] (1) Knockout of wcaJ

[0069] According to the Escherichia coli sequence information, a fragment containing the upstream and downstream homologous arms of wcaJ was constructed using fusion PCR. The nucleotide sequence of the wcaJ gene is shown in SEQ ID NO.1;

[0070] The pTarget plasmid was constructed to express the sgRNA required for gene editing. Using pTarget as a template, a linear plasmid fragment with an introduced N20 sequence was obtained by circular PCR. These fragments were separately transformed into Escherichia coli JM109, and the obtained transformants were sequenced to obtain the plasmid pTarget-wcaJ;

[0071] The plasmid pCas9 expressing the Cas9 protein was transformed into competent Escherichia coli MG1655 cells, and the successfully transformed strains were screened. The strains transformed with pCas9 were prepared into electrocompetent cells. The fragment containing the upstream and downstream homologous arms of wcaJ and the pTarget-wcaJ plasmid were co-transformed into the competent Escherichia coli MG1655 cells carrying the pCas9 plasmid, and plated on a plate containing kanamycin and spectinomycin, and cultured inverted at 37°C. The transformants were verified by colony PCR, and the obtained positive transformants were sequenced. The obtained recombinant Escherichia coli strains were cured of the plasmids pTarget and pCas9 to obtain the plasmid-free recombinant Escherichia coli MG1655ΔwcaJ.

[0072] (2) Knockout of lacZ and replacement of the original promoter of lacY

[0073] According to the Escherichia coli sequence information, a fusion PCR method was used to construct an integration fragment containing the upstream and downstream homologous arms of lacZ and the P J23119 promoter. The nucleotide sequence of the fragment for replacing the original promoter of lacY is shown in SEQ ID NO.2.

[0074] The pTarget plasmid was constructed to express the sgRNA required for gene editing. Using pTarget as a template, a linear plasmid fragment with an introduced N20 sequence was obtained by circular PCR. These fragments were separately transformed into Escherichia coli JM109, and the obtained transformants were sequenced to obtain the plasmid pTarget-lacZ;

[0075] The plasmid pCas9 expressing Cas9 protein was transformed into competent cells of Escherichia coli MG1655ΔwcaJ, and the strains with successful transformation were screened. The strains transformed with pCas9 were prepared into electrocompetent cells, and the integration fragment and pTarget-lacZ plasmid were co-transformed into the competent cells of Escherichia coli MG1655ΔwcaJ carrying the pCas9 plasmid, and then spread on a plate containing kanamycin and spectinomycin and cultured upside down at 37°C. The transformants were verified by colony PCR, and the obtained positive transformants were sequenced. The obtained recombinant Escherichia coli strains were cured of plasmid pTarget and plasmid pCas9 to obtain plasmid-free recombinant Escherichia coli MG1655ΔwcaJ / ΔlacZ / P J23119 -lacY.

[0076] Example 3: Recombinant strains of Escherichia coli MG1655 overexpressing gmd-fcl and manC-manB

[0077] The CRISPR-Cas9 gene editing system was used to overexpress the genes encoding GDP-mannose-4,6-dehydratase (gmd), GDP-L-fucose synthase (fcl), phosphomannomutase (manC), and mannosyl-1-phosphate guanylyltransferase (manB) in the Escherichia coli genome. The specific steps are as follows:

[0078] (1) Overexpression of gmd-fcl

[0079] According to the Escherichia coli sequence information, upstream and downstream homologous arms containing the insertion site yhiM were constructed by fusion PCR, as well as the integration fragment P tac expressing gmd-fcl driven by the promoter, P tac -gmd-fcl, and the nucleotide sequence of the fragment P tac -gmd-fcl is shown in SEQ ID NO.3;

[0080] The pTarget plasmid was constructed to express the sgRNA required for gene editing. Using pTarget as a template, linear plasmid fragments introduced with the N20 sequence were obtained by circular PCR, and these fragments were separately transformed into Escherichia coli JM109, and the obtained transformants were sequenced to obtain the plasmid pTarget-yhiM;

[0081] The plasmid pCas9 expressing Cas9 protein was transformed into the competent cells of Escherichia coli MG1655ΔwcaJ / ΔlacZ / P J23119 -lacY, and the strains with successful transformation were screened. The strains transformed with pCas9 were prepared into electrocompetent cells, and the upstream and downstream homologous arms of the site yhiM, P tac-The gmd-fcl and pTarget-yhiM plasmids were co-transformed into the competent cells of Escherichia coli MG1655ΔwcaJ carrying the pCas9 plasmid, and the mixture was spread on a plate containing kanamycin and spectinomycin, and cultured inverted at 37°C. The transformants were verified by colony PCR, and the obtained positive transformants were sequenced. The obtained recombinant E. coli strain was cured of the plasmids pTarget and pCas9 to obtain a plasmid-free recombinant E. coli MG1655ΔwcaJ / ΔlacZ / P tac -lacY / yhiM::P tac -gmd-fcl. (2) Overexpression of manC-manB

[0082] According to the E. coli sequence information, the upstream and downstream homologous arms containing the insertion site yeeL, and P tac The integration fragment P tac -manC-manB, P tac -The nucleotide sequence of the manC-manB fragment is shown in SEQ ID NO.4;

[0083] Construct the pTarget plasmid to express the sgRNA required for gene editing. The pTarget plasmid contains the sgRNA of the N20 sequence of the insertion site yeeL, named pTarget-yeeL;

[0084] The plasmid pCas9 expressing the Cas9 protein was transformed into Escherichia coli MG1655ΔwcaJ / ΔlacZ / P tac -lacY / yhiM::P tac -gmd-fcl competent cells, and the successfully transformed strains were screened. The strain transformed with pCas9 was prepared into electrocompetent cells, and the upstream and downstream homologous arms of the site yeeL, P tac -manC-manB and the pTarget-yeeL plasmid were co-transformed into Escherichia coli MG1655ΔwcaJ / ΔlacZ / P tac -lacY / yhiM::P tac -gmd-fcl competent cells to construct the recombinant Escherichia coli MGP1 (MG1655ΔwcaJ / ΔlacZ / P tac -lacY / yhiM::P tac -gmd-fcl / yeeL::P tac -manC-manB).

[0085] Example 4: Recombinant strain of Escherichia coli MG1655 integrating multiple copies of futC

[0086] Construct P tac The integration fragment P composed of GST-futC expressed by the promoter tac -GST-futC and P tac GST-futC expressed by the promoter V93I The integration fragment P composed tac -GST-futC V93I wherein the nucleotide sequence of the futC fragment is as shown in SEQ ID NO.5, the nucleotide sequence of the futC V93I fragment is as shown in SEQ ID NO.6, the amino acid sequence of the GST solubilizing tag is as shown in SEQ ID NO.8, and the nucleotide sequence is as shown in SEQ ID NO.9.

[0087] According to the Escherichia coli sequence information, upstream and downstream homologous arms containing the insertion sites betA, nupG and ybbD were constructed respectively;

[0088] The plasmid pCas9 expressing the Cas9 protein was transformed into Escherichia coli MGP1 competent cells, and the successfully transformed strains were screened. The strains transformed with pCas9 were prepared into electrocompetent cells, and the upstream and downstream homologous arms of the site betA, P tac -GST-futC fragment and pTarget-betA plasmid were co-transformed into Escherichia coli MGP1 competent cells carrying the pCas9 plasmid to construct recombinant Escherichia coli MGP2.

[0089] The plasmid pCas9 expressing the Cas9 protein was transformed into Escherichia coli MGP1 competent cells, and the successfully transformed strains were screened. The strains transformed with pCas9 were prepared into electrocompetent cells, and the upstream and downstream homologous arms of the site betA, P tac -GST-futC V93I fragment and pTarget-betA plasmid were co-transformed into Escherichia coli MGP1 competent cells carrying the pCas9 plasmid to construct recombinant Escherichia coli MGP5.

[0090] The plasmid pCas9 expressing the Cas9 protein was transformed into Escherichia coli MGP2 competent cells, and the successfully transformed strains were screened. The strains transformed with pCas9 were prepared into electrocompetent cells, and the upstream and downstream homologous arms of the site nupG, P tac -GST-futC and pTarget-nupG plasmid were co-transformed into Escherichia coli MGP2 competent cells carrying the pCas9 plasmid to construct recombinant Escherichia coli MGP3.

[0091] The plasmid pCas9 expressing Cas9 protein was transformed into competent Escherichia coli MGP2 cells, and the successfully transformed strains were screened. The strains transformed with pCas9 were prepared into electrocompetent cells, and the upstream and downstream homologous arms of the site nupG, P tac -GST-futC V93I and the plasmid pTarget-nupG were co-transformed into the competent Escherichia coli MGP2 carrying the plasmid pCas9 to construct recombinant Escherichia coli MGP6.

[0092] The plasmid pCas9 expressing Cas9 protein was transformed into competent Escherichia coli MGP3 cells, and the successfully transformed strains were screened. The strains transformed with pCas9 were prepared into electrocompetent cells, and the upstream and downstream homologous arms of the site ybbD, P tac -GST-futC and the plasmid pTarget-ybbD were co-transformed into the competent Escherichia coli MGP3 carrying the plasmid pCas9 to construct recombinant Escherichia coli MGP4.

[0093] The plasmid pCas9 expressing Cas9 protein was transformed into competent Escherichia coli MGP3 cells, and the successfully transformed strains were screened. The strains transformed with pCas9 were prepared into electrocompetent cells, and the upstream and downstream homologous arms of the site ybbD, P tac -GST-futC V93I and the plasmid pTarget-ybbD were co-transformed into the competent Escherichia coli MGP3 carrying the plasmid pCas9 to construct recombinant Escherichia coli MGP7.

[0094] Example 5: Shake-flask fermentation for the production of 2'-fucosyllactose

[0095] The above-mentioned Escherichia coli MGP1 - MGP7 were made into seed solutions, and the seed solution medium was LB medium; the method for preparing the seed solution was as follows: Single colonies on a fresh plate were picked into the seed medium and cultured for 12 h. Then the seed solution was inoculated into the M9CA medium at an inoculation amount of 2% and cultured at 37 °C and 220 r / min until OD 600 = 0.6, and 20 g / L lactose was added, and the shake-flask fermentation was continued until 72 h.

[0096] At the end of fermentation, the total amount of extracellular 2'-FL in the cells and the remaining amount of lactose in the fermentation broth were measured by a high-performance liquid chromatograph (HPLC). First, 2 mL of the fermentation broth was centrifuged at 12,000 rpm for 15 min, and the supernatant was collected, and the concentrations of extracellular 2'-FL and lactose were detected by HPLC.

[0097] The detection results are as Figure 3As shown, the MG7 had the highest 2'-fucosyllactose production, reaching 6.5 g / L, which was 141% higher than the average production of 2.7 g / L of the single-copy original protein expression strain MGP2 (one copy of futC integrated into the genome of the chassis strain MGP1), and 51% higher than the average production of 4.3 g / L of the triple-copy original protein expression strain MGP4.

[0098] Obviously, the above embodiments are merely examples for clear illustration and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. An Escherichia coli capable of producing 2'-fucosyllactose, characterized in that: Knock out the undecylphosphoglucose phosphotransferase gene wcaJ and the β-galactosidase encoding gene lacZ in the Escherichia coli genome, replace the promoter of the lactose permease encoding gene lacY with the P J23119 promoter, heterologously introduce an α-1,2-fucosyltransferase mutant with a solubility-enhancing tag GST fused to the N-terminus, co-express the GDP-mannose-4,6-dehydratase encoding gene gmd and the GDP-L-fucose synthase encoding gene fcl, and co-express the mannose-1-phosphate guanylyltransferase encoding gene manB and the phosphomannomutase encoding gene manC; Among them, the amino acid sequence of the solubility-enhancing tag GST is shown in SEQ ID NO.8, and the α-1,2-fucosyltransferase mutant is obtained by mutating the valine at position 93 of the starting sequence with the amino acid sequence shown in SEQ ID NO.7 to isoleucine.

2. The Escherichia coli according to claim 1, characterized in that: The α-1,2-fucosyltransferase mutant is expressed in a double-copy manner.

3. The Escherichia coli according to claim 2, characterized in that: The double-copy expression is carried out at the betA locus and the nupG locus of the Escherichia coli genome respectively.

4. The Escherichia coli according to claim 1, characterized in that: The α-1,2-fucosyltransferase mutant is expressed in a triple-copy manner.

5. The Escherichia coli according to claim 4, characterized in that: The triple-copy expression is carried out at the betA locus, the nupG locus and the yeeL locus of the Escherichia coli genome respectively.

6. The construction method of Escherichia coli according to any one of claims 1-5, characterized in that, The construction is carried out using the CRISPR / Cas9 system, and the steps include S1-S5, S1-S6 or S1-S7: Step S1: Knock out the undecylphosphoglucose phosphotransferase gene wcaJ and the β-galactosidase-encoding gene lacZ in the starting Escherichia coli genome. Step S2: Replace the promoter of the lactose permease encoding gene lacY on the genome of the strain obtained in S1 with promoter P J23119 Promoter Step S3: Introduce the α-1,2-fucosyltransferase mutant into the betA locus of the genome of the strain obtained in S2. Step S4: Co-express the GDP-mannose-4,6-dehydratase-encoding gene gmd and the GDP-L-fucose synthase-encoding gene fcl at the yhiM locus of the genome of the strain obtained in S3. Step S5: Co-express the mannose-1-phosphate guanylyltransferase-encoding gene manB and the phosphomannomutase-encoding gene manC at the yeeL locus of the genome of the strain obtained in S4. Step S6: Introduce the α-1,2-fucosyltransferase mutant into the nupG locus of the genome of the strain obtained in S5. Step S7: Introduce the α-1,2-fucosyltransferase mutant into the ybbD locus of the genome of the strain obtained in S6.

7. The construction method according to claim 6, characterized in that: The starting Escherichia coli is Escherichia coli MG1655.

8. A microbial inoculant comprising the Escherichia coli according to any one of claims 1-5.

9. Use of the Escherichia coli according to any one of claims 1-5 or the microbial inoculant according to claim 8 in the production of 2'-fucosyllactose.

10. A method for producing 2'-fucosyllactose, characterized in that: Add the Escherichia coli according to any one of claims 1-5 or the microbial inoculant according to claim 8 to a fermentation system to produce 2'-fucosyllactose using lactose as a substrate.

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