Construction method and application of recombinant Escherichia coli for efficiently synthesizing lactose-N-fucosylpentasaccharide I

Abstract

The present invention relates to a construction method and application of a recombinant Escherichia coli for efficiently synthesizing lactose-N-fucosylpentasaccharide I, by screening efficient β-1,3-acetylglucosamine transferase, β-1,4-galactosyl transferase and α-1,2-fucosyl transferase and heterologously introducing them into Escherichia coli BL21 (DE3), performing gene editing on multiple sites of its genome, and knocking out lacZ The expression of lactose-N-fucosyl pentasaccharide I is constructed to construct a complete pathway for producing lactose-N-fucosyl pentasaccharide I, thereby achieving the purpose of efficiently synthesizing lactose-N-fucosyl pentasaccharide I without the addition of exogenous plasmids and antibiotics, further improving the biosafety of the obtained product, and making it suitable for the preparation of food or medicine.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a construction method and application of a recombinant Escherichia coli capable of efficiently synthesizing lactose-N-fucosylpentasaccharide I. Background Art

[0002] Human milk oligosaccharides (HMOs) are the most abundant bioactive molecules in human milk, and the third most abundant component in breast milk after lactose and lipids. HMOs mainly include three categories: sialylated, fucosylated, and non-fucosylated. Among them, fucosylated lactose-N-fucosylpentaose I (Lacto-N-Fucopentaose I, LNFP I) is one of the most important HMOs, which has biological functions such as regulating intestinal flora, avoiding pathogen invasion, promoting nervous system development, and improving immune response, and plays an important role in infant health. Currently, the US Food and Drug Administration is evaluating the addition of LNFP I to infant formula and other foods, but there are still challenges in the efficient and economical production of LNFP I.

[0003] At present, the artificial synthesis of LNFP I mainly includes chemical synthesis, enzymatic synthesis and microbial synthesis. The chemical reaction steps involved in chemical synthesis are complex, costly and inefficient, while the substrates of enzymatic synthesis are expensive and cannot meet the needs of industrialization. In contrast, the microbial synthesis method has lower cost, higher yield and is environmentally friendly, which is suitable for industrial large-scale production of LNFP I. The main synthesis steps are β-1,3-N-acetylglucosamine transferase, β-1,3-galactosyltransferase and α-1,2-fucosyltransferase, which use uridine diphosphate-N-acetylglucosamine, uridine diphosphate-galactose and guanosine diphosphate-fucose as donors for glycosylation reaction. Therefore, screening out more efficient glycosyltransferases is the key to the microbial synthesis of LNFP I. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a construction method and application of a recombinant Escherichia coli for efficiently synthesizing lactose-N-fucosylpentasaccharide I.

[0005] The technical solution adopted by the present invention is: a recombinant Escherichia coli that efficiently synthesizes lactose-N-fucosylpentasaccharide I, using Escherichia coli BL21 (DE3) as the chassis host, and knocking out the gene encoding β-galactosidase in its genome lacZ , knocking out the gene encoding GDP-mannose hydrolase gmm , knocking out genes encoding tyrosine protein kinases wzc , knocking out the gene encoding dTDP-glucose 4,6-dehydratase rfbB , knocking out the gene encoding the dephosphorylase proteinwzb , knocking out the gene encoding N-acetylfucosamine transferase rffT , knock out the gene encoding dTDP deoxygalactose aminotransferase rffA , knocking out the gene encoding colanate phosphate flippase wzxC , knocking out genes encoding polysaccharide export proteins wza ;

[0006] Overexpression of the gene encoding UDP-glucose 4-epimerase galE , overexpression gmd-fcl Gene 、 manC-manB Gene; Heterologous integration of the gene encoding β-1,3-acetylglucosamine transferase NsplgtA , integration of the gene encoding β-1,3-galactosyltransferase Pbr3GalT ; Integration of the gene encoding α-1,2-fucosyltransferase MtFucT .

[0007] Preferably, MtFucT Derived from Methylobacter tundripaludum.

[0008] Preferably, NsplgtA The amino acid sequence is shown in SEQ ID NO.1, Pbr3GalT The amino acid sequence is shown in SEQ ID NO.2, MtFucT The amino acid sequence is shown in SEQ ID NO.3.

[0009] Preferably, gene editing is performed by a two-step homologous recombination method to achieve a traceless knockout; based on the chassis host Escherichia coli, the knockout lacZ and rffA , and integrated at the corresponding sites Pbr3GalT ; Knockout gmm and rffT , and integrated at the corresponding sites NsplgtA ; Knockout wzc , and overexpressed at this site galE ; Knockout wzxC , and overexpressed at this site gmd- fcl ; Knockout rfbB , and overexpressed at this site manC-manB ; Knockout wzb and wza , and heterologously expressed at the corresponding sites MtFucT ; Finally, recombinant Escherichia coli that can efficiently synthesize lactose-N-fucosylpentasaccharide I was obtained.

[0010] Preferably, NsplgtA Gene 、Pbr3GalTGenes and MtFucT The number of copies of the gene integrated into the chassis host cell is single copy, double copy, triple copy or quadruple copy.

[0011] A method for preparing lactose-N-fucosyl pentasaccharide I is provided, wherein the lactose-N-fucosyl pentasaccharide I is obtained by culturing and fermenting recombinant Escherichia coli that efficiently synthesizes lactose-N-fucosyl pentasaccharide I.

[0012] Preferably, the recombinant Escherichia coli with increased difucosyllactose production is cultured and fermented using glycerol as a carbon source and lactose as a substrate.

[0013] Preferably, the fermentation medium comprises: 2 g / L tryptone, 4 g / L yeast extract, 20 g / L glycerol, 12-14 g / L potassium dihydrogen phosphate, 1.3-1.5 g / L diammonium hydrogen phosphate, 1.6-1.8 g / L citric acid, 1.4-1.6 g / L magnesium sulfate heptahydrate, and 8-12 ml / L trace metal elements.

[0014] Preferably, the specific steps are:

[0015] Step 1: culturing the recombinant Escherichia coli to obtain a seed solution, preferably, the seed culture medium is 2YT medium;

[0016] Step 2: Inoculate the seed solution into the fermentation tank to form a fermentation system; culture at 37°C until the bacterial OD 600 When the pH reaches 12, add lactose to 10 g / L, adjust the temperature to 30°C, add 10 g / L lactose and 20 g / L glycerol every 12 h, and control the pH to 6.8;

[0017] Preferably, the fermentation process has an aeration rate of 2-6 vvm and an agitation speed of 300-900 r / min.

[0018] The invention discloses a method for preparing lactose-N-fucosylpentasaccharide I and its application in preparing food or medicine containing lactose-N-fucosylpentasaccharide I.

[0019] The advantages and positive effects of the present invention are: by modifying the chassis host Escherichia coli BL21 (DE3), a complete synthesis pathway of lactose-N-fucosylpentasaccharide I is constructed, and efficient β-1,3-acetylglucosamine transferase is screened out. NsplgtA , β-1,3-galactosyltransferase Pbr3GalT and α-1,2-fucosyltransferase MtFucT Heterologous introduction into E. coli and enhancement galE Gene 、gmd-fcl Gene 、manC-manBThe gene is expressed, and the receptor lactose is added externally to construct a strain that can synthesize lactose-N-fucosyl pentasaccharide I, thereby achieving the goal of high yield of lactose-N-fucosyl pentasaccharide I without the need for the addition of exogenous plasmids and antibiotics during the fermentation process, and can further improve the biosafety of the obtained product; the constructed recombinant Escherichia coli with improved lactose-N-fucosyl pentasaccharide I can efficiently produce lactose-N-fucosyl pentasaccharide I, and has good prospects for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The metabolic pathway for the synthesis of lactose-N-fucosylpentasaccharide I by recombinant Escherichia coli;

[0021] Figure 2 Comparison of the yield of lactose-N-fucosylpentasaccharide I in shake flask fermentation after sequential transformation of different recombinant bacteria;

[0022] Figure 3 It is the dynamic changes of products and substrates and growth curve of recombinant Escherichia coli in a 5L fermenter;

[0023] Figure 4 Different sources in E5 strain MtFucT Capacity to produce lactose-N-fucosylpentaose I and by-products. DETAILED DESCRIPTION

[0024] The embodiments of the present invention are described below with reference to the accompanying drawings.

[0025] The present invention relates to a recombinant Escherichia coli for efficiently synthesizing lactose-N-fucosylpentasaccharide I, using Escherichia coli BL21 (DE3) as a chassis host cell, and knocking out the bypass gene β-galactosidase lacZ β-1,3-acetylglucosamine transferase NsplgtA , β-1,3-galactosyltransferase Pbr3GalT and α-1,2-fucosyltransferase MtFucT Heterologous introduction into Escherichia coli BL21 (DE3) was performed for gene editing at multiple sites in the genome and enhanced galE Gene 、gmd-fcl Gene 、 manC-manB The expression of the gene is achieved without the need for heterologous expression or the addition of antibiotics, and the gene is stably expressed. A strain that efficiently produces lactose-N-fucosyl pentasaccharide I is constructed. The receptor lactose is added exogenously, and glycerol and lactose are used as substrates to culture and ferment the recombinant engineered bacteria, so that lactose-N-fucosyl pentasaccharide I can be efficiently obtained.

[0026] Using Escherichia coli BL21 (DE3) as the host, the gene encoding β-galactosidase was knocked out lacZ, knocking out the gene encoding GDP-mannose hydrolase gmm (WP_000479835.1), knockout of the gene encoding tyrosine protein kinase wzc (WP_000137133.1), knocking out the gene encoding colanate phosphate flippase wzxC (WP_000058488), knockout of the gene encoding dTDP-glucose 4,6-dehydratase rfbB (AXC25041.1), knockout of the gene encoding the dephosphorylase protein wzb (AKK12342.2), knockout of the gene encoding N-acetylfucosamine transferase rffT (WP_000217220), knockout of the gene encoding dTDP deoxygalactose aminotransferase rffA (WP_083582921), knocking out the gene encoding the polysaccharide export protein wza (EHM9396083.1).

[0027] Integration of the gene encoding β-1,3-acetylglucosamine transferase NsplgtA (WP_304703337.1), integrating the gene encoding UDP-glucose 4-epimerase galE (NP_415280.3), integrating the gene encoding β-1,3-galactosyltransferase Pbr3GalT (WP_230643796.1), integrating the gene encoding α-1,2-fucosyltransferase MtFucT (WP_031437198.1).

[0028] In certain embodiments of the present invention, the chassis host Escherichia coli is used as the starting strain, and the knockout lacZ and rffA , and integrated at the corresponding sites Pbr3GalT ; Knockout gmm and rffT , and integrated at the corresponding sites NsplgtA ; Knockout wzc , and overexpressed at this site galE ; Knockout wzxC , and overexpressed at this site gmd-fcl ; Knockout rfbB , and overexpressed at this site manC-manB ; Knockout wzb and wza , and heterologously expressed at the corresponding sites MtFucT Finally, a recombinant Escherichia coli capable of efficiently synthesizing lactose-N-fucosylpentasaccharide I was obtained. gmd-fclThe genes include the gene encoding GDP-D-mannose-4,6-dehydratase gmd (WP_005128297.1) and the gene encoding GDP-L-fucose synthase fcl (NP_416556.1); manC-manB Mannosyl-1-phosphate guanosyltransferase gene manC (NP_310881.1) and the gene encoding phosphomannose mutase manB (WP_001590293.1).

[0029] Among them, the enzyme encoding β-1,3-acetylglucosamine transferase NsplgtA The optimized amino acid sequence is shown in SEQ ID NO.1; the gene encoding the β-1,3-galactosyltransferase Pbr3GalT The optimized amino acid sequence is shown in SEQ ID NO.2; encoding α-1,2-fucosyltransferase MtFucT The optimized amino acid sequence is shown in SEQ ID NO.3.

[0030] SEQ ID NO.1:

[0031] MPSEAFRRHRAYRENKLQPLVSVLICAYNVEKYFAQSLAAVVNQTWRNLEILIVDDGSTDGTLAIAKDFQKRDSRIKILAQAQNSGLIPSLNIGLDELAKSGGGEYIARTDADDIAAPDWIEKIVGEMEKDRSIIAMGAWLEVLSEEKDGNRLARHHRHGAIWDKPTRHEDIVAVFPFGN PIHNNTMIMRRSVIDGGLRYNTERDWAEDYQFWYDVSKLGRLAYYPEALVKYRLHANQVSSKHSVRQHEIAQGIQKTVRNDFLQSMGFKIRFDSLEYRQTKAAAYELLEKLPEEDFERARRFLYQCFKRTDTPPAGAWLDFAADGRMRRLFTLRQYFSILHRLIKNRRQARSDSAGKEQEI

[0032] SEQ ID NO.2:

[0033] MINHSLVSVIIPVNKHNPFLKEAIESIQNQSYSNIEVIIIANGCSDLFFNSLQEFSDEKTKIIRTELSFLPFSLNLGIHIANGEFIARMDSDDIADVNRIAKQVAYMIAHSDVTVIGSNVKFINE HGIITGMSDYPTSHRNIKKRMMYNCCVAPTVMMRRDVIVKAGGYMYGSLSEDYDLWLRLLQDKNVVFHNIKEPLLQYRIHANQATGKKSLYKIFIYDLCLKLRFFLLYPNVSYFLGCIRGFLSY IYCRYIKK

[0034] SEQ ID NO.3:

[0035] MVITHLIGGLGNQMFQYAAGRAVSLERGVSLSLDISGFANYGLHQGFELQRIFNCTAEIANEADVRGILGWQSSPRIRQLLSRQNMAIFRREGFVVEPHFHYWQGIKSVPRDCYLTGYWQSEQYFLEAAAQIRADFTFKLPLDNQN IELAKQINAVNAVSLHVRRGDYANTPETTATHGLCSLDYYRVAIRHIAEQVQQPHFFVFSDDIAWVKNNLSIDFPCQYVDHNQGAESYNDMRLMSMCRHHIIANSSFSWWGAWLNPNVNKIVVAPSRWFAKQTDVRDLLPQGWIKQ

[0036] When constructing recombinant E. coli, a two-step homologous recombination method was used for gene editing to achieve seamless knockout. The pKD46 plasmid was introduced into the host bacteria BL21 (DE3) to induce the expression of the recombinase, and two-step homologous recombination was performed in the strain containing the recombinase. The chloramphenicol resistance gene and the sucrose sensitivity gene were used sacB The fusion was performed by overlap extension PCR. cat - sacB The fragments were used to replace the gene to be deleted, and after electroporation, the colonies that could grow on the chloramphenicol and gentamicin double-resistance plates were screened for PCR identification. sacB The sucrose polymerization lethality of genes was screened out without cat-sacB The strains with the fragments were identified by PCR and sequenced again to obtain the strains with successful integration of the target gene.

[0037] Using recombinant Escherichia coli as a fermentation strain, glycerol as a carbon source, and lactose as a substrate for cultivation and fermentation can efficiently synthesize lactose-N-fucosylpentasaccharide I. The recombinant Escherichia coli was fermented in a shake flask system and a 5L fermenter, respectively. DM medium was used for the fermentation of recombinant Escherichia coli, wherein the DM medium contained: 2 g / L tryptone, 4 g / L yeast extract, 20 g / L glycerol, 12-14 g / L potassium dihydrogen phosphate, 1.3-1.5 g / L diammonium hydrogen phosphate, 1.6-1.8 g / L citric acid, 1.4-1.6 g / L magnesium sulfate heptahydrate, and 8-12 ml / L trace metal elements; the trace metal elements contained: 2.25 g / L zinc sulfate hydrate, 10 g / L ferrous sulfate, 0.35 g / L manganese sulfate monohydrate, 1.0 g / L anhydrous copper sulfate, 0.23 g / L sodium borate decahydrate, 2.0 g / L calcium chloride dihydrate, and 0.11 g / L ammonium molybdate.

[0038] The recombinant E. coli was cultured in a 5L fermenter containing DM medium at 37°C. 600 Reach 12, add lactose content to 10 g / L, adjust the temperature to 30 degrees; add 10 g / L lactose and 20 g / L glycerol every 12 hours, and then add NH4OH to stabilize the pH at 6.8, ventilation (2 vvm ~ 6 vvm) and stirring speed 300-900 r / min, and culture and ferment the recombinant engineered bacteria. It has been verified that in the shake flask fermentation experiment, the recombinant Escherichia coli can produce lactose-N-fucosyl pentose I to 8.7 g / L, and in the 5L fermentation tank experiment, the yield of lactose-N-fucosyl pentose I can reach 48g / L, which lays the foundation for the industrial production of lactose-N-fucosyl pentose I.

[0039] The scheme of the present invention is described below in conjunction with the accompanying drawings, wherein the experimental methods without specific operating steps are all carried out in accordance with the corresponding product instructions, and the instruments, reagents, and consumables used in the examples can all be purchased from commercial companies unless otherwise specified.

[0040] The plasmids, PCR enzymes, column DNA extraction kits, DNA gel recovery kits, bacterial genome extraction kits and other commercial products used in the examples were specifically operated according to the instructions in the kits. Colony PCR, agarose gel electrophoresis, electrotransformation, preparation of competent cells, etc. were performed according to conventional operating methods. The construction of the plasmid was completed by the company, and the DNA product sequencing and PCR primer synthesis were completed by Anshengda (Tianjin) Company. Preparation of competent E. coli: E. coli was cultured in 100 mL LB medium. When the bacterial OD 600When it reaches 0.4-0.6, place it on ice for 30 minutes, centrifuge the bacterial solution at 4°C at 5500rpm for 5 minutes to collect the bacteria, and then wash the bacterial precipitate three times with a 10% glycerol aqueous solution pre-cooled at 4°C. Inducer concentration: The final concentration of L-arabinose added during the preparation of E. coli sensing is 5%. Antibiotic concentration: ampicillin 100 mg / L, streptomycin 50 mg / L, gentamicin 25 mg / L, chloramphenicol 25 mg / L. LB liquid medium: yeast extract 5g / L, tryptone 10g / L, sodium chloride 10g / L; LB solid medium: yeast extract 5g / L, tryptone 10g / L, sodium chloride 10g / L, agar 15g / L.

[0041] Example 1: Construction of recombinant Escherichia coli for efficient synthesis of lactose-N-fucosylpentasaccharide I

[0042] Overlap extension PCR amplification

[0043] NsplgtA , Pbr3GalT、MtFucT Obtaining gene fragments: Download the gene sequence from NCBI, entrust a company to synthesize the plasmid, use the synthetic plasmid as a template, and design primers for PCR amplification.

[0044] galE Obtaining gene fragments: Using plasmid PET-galE as a template, primers were designed for amplification.

[0045] gmm, wzc Acquisition of gene fragments: Using the Escherichia coli BL21 genome as a template, primers were designed for amplification.

[0046] Gene knockout and integration

[0047] To knock out a gene using λ-Red homologous recombination, it is necessary to introduce the pKD46 plasmid into the host bacteria BL21 (DE3) to induce the expression of the recombinase, and then perform two-step homologous recombination in the strain containing the recombinase. sacB The fusion was performed by overlap extension PCR. cat - sacB The fragments were used to replace the gene to be deleted, and after electroporation, the colonies that could grow on the chloramphenicol and gentamicin double-resistance plates were screened for PCR identification. sacB The sucrose polymerization lethality of genes was screened out without cat-sacB The strains with the fragments were identified by PCR and sequenced again to obtain the strains with successful integration of the target gene.

[0048] The above gene editing method was used to knock out the genes in Escherichia coli BL21 (DE3) lacZ, gmm, wzc, wzxC, rfbB, wzb, rffT, rffA, wzaGenes are integrated and expressed in sequence Pbr3GalT,NsplgtA , galE , gmd- fcl,manC-manB,MtFucT,NsplgtA , Pbr3GalT,MtFucT。 Finally, pKD46 plasmid was eliminated at 37℃ to obtain the final high-yield strain. The specific steps are as follows:

[0049] Using BL21(DE3) as the starting strain, knock out the gene lacZ , reorganization Pbr3GalT , strain E0 was obtained; knockout gene gmm , reorganization NsplgtA , strain E1 was obtained; knockout gene wzc , reorganization galE , strain E2 was obtained; knockout gene wzxC , reorganization gmd-fcl , strain E3 was obtained; knockout gene rfbB , reorganization manC-manB , strain E4 was obtained; knockout gene wzb , reorganization MtFucT , strain E5 was obtained; knockout gene rffT , reorganization NsplgtA , strain E6 was obtained; knockout gene rffA , reorganization Pbr3GalT , strain E7 was obtained; knockout gene wza , reorganization MtFucT , strain E8 was obtained. The composition of the obtained strains E0-E8 is shown in Table 1, and the primers used to construct each strain are shown in Table 2.

[0050] Table 1 Construction of recombinant Escherichia coli with high production of lactose-N-fucosylpentasaccharide I

[0051]

[0052] Table 2 Primers used to construct recombinant Escherichia coli with high production of lactose-N-fucosylpentasaccharide I

[0053]

[0054]

[0055]

[0056] Example 2: Production of lactose-N-fucosylpentasaccharide I by recombinant bacterial fermentation

[0057] 2.1 Strain culture and fermentation

[0058] The strains E0-E8 constructed in Example 1 were cultured and fermented respectively. Single colonies were picked from the corresponding plates and added to 50 ml conical flasks containing 20 ml 2YT medium without antibiotics. The culture was carried out at 37°C overnight for 12-14 h. Then, 5 ml of seed solution was transferred to a 250 ml shake flask containing 50 ml DM medium and cultured at 37°C and 200 rpm. When the bacterial OD 600 When the pH value reached 1.8-2.2, the substrate lactose was added to a final concentration of 10 g / L. After adding lactose, the culture conditions were changed to 30°C, 200 rpm, and cultured continuously for 72 h.

[0059] In the 5 L fermentation tank experiment, the initial DM medium was 2 L, the seed inoculation volume was 10% (v / v), and the seed solution was transferred to the fermentation tank at the beginning, the temperature was 37 °C, and when the bacterial OD 600 When the fermentation time reaches 10-12 hours, add the substrate lactose to a concentration of 10 g / L, adjust the temperature to 30°C, and add 10 g / L lactose and 20 g / L glycerol every 12 hours. During the fermentation, 14% ammonia water is added through a constant flow pump to stabilize the pH of the culture medium at around 6.8, and the aeration volume is 2 vvm-6 vvm and the stirring speed is 300-900 r / min to maintain the dissolved oxygen content at 20%-30%. When the glycerol in the tank is consumed to 3 g / L, 30 ml of 600 g / L glycerol is added through a constant flow pump. When the lactose concentration is lower than 3 g / L, 30 ml of 200 g / L lactose is added through a constant flow pump.

[0060] The formula of DM culture medium is: 2 g / L tryptone, 4 g / L yeast extract, 20 g / L glycerol, 12-14 g / L potassium dihydrogen phosphate, 1.3-1.5 g / L diammonium hydrogen phosphate, 1.6-1.8 g / L citric acid, 1.4-1.6 g / L magnesium sulfate heptahydrate, 8-12 ml / L trace metal elements; trace metal elements contain: 2.25 g / L zinc sulfate hydrate, 10 g / L ferrous sulfate, 0.35 g / L manganese sulfate monohydrate, 1.0 g / L anhydrous copper sulfate, 0.23 g / L sodium borate decahydrate, 2.0 g / L calcium chloride dihydrate, and 0.11 g / L ammonium molybdate.

[0061] 2.2 Production detection of lactose-N-fucosylpentasaccharide I

[0062] The lactose-N-fucosyl pentasaccharide I in the fermentation broth of strains E0-E8 was detected respectively. The fermentation broth was centrifuged at 12000 rpm for 5 min, the supernatant was aspirated with a sterile syringe, filtered with a 0.22 μm water filter, and the yield of lactose-N-fucosyl pentasaccharide I in the fermentation broth was detected by high performance liquid chromatography. HPLC (Waterse2695); Column: Carbohydrate Analysis column (Rezex ROA-organic acid H + (8%) 300×7.8 mm); mobile phase: 5 mM H2SO4; flow rate: 0.5 mL / min; detector: differential detector; column temperature 60℃; injection volume: 10 μL.

[0063] The expression of recombinant E. coli E0-E8 strains with high production of lactose-N-fucosylpentaose I in shake flask system and fermentation tank system is shown in Figure 2 and Figure 3 As shown in the data, it can be seen that the ability of recombinant E. coli to express lactose-N-fucosyl pentasaccharide I can be significantly improved by modifying E. coli, especially strain E8. The lactose-N-fucosyl pentasaccharide I production of the recombinant strain in a 5L fermenter reached 48 g / L. Figure 3 Shown are the dynamic changes of products and substrates as well as the growth curve of strain E8 in a 5L fermenter. It can be seen from the figure that the recombinant engineered bacteria can continuously consume the substrates glycerol and lactose, and as the OD value of the strain increases, the expression level of the target lactose-N-fucosyl pentasaccharide I is significantly increased, proving that the constructed recombinant Escherichia coli can efficiently produce lactose-N-fucosyl pentasaccharide I.

[0064] Example 3: Effect of α-1,2-fucosyltransferase from different sources on the efficiency of recombinant bacterial fermentation to produce lactose-N-fucosylpentasaccharide I

[0065] The E4 strain was constructed according to the construction method of Example 1. When constructing the E5 strain from the E4 strain, α-1,2-fucosyltransferases from different sources were used, which were BbFucT Gene, SbFucT Gene, MtFucT Gene, MspFucT Genes and HpFucT Gene. BbFucT Derived from Burkholderiales bacterium; SbFucT Derived from Sphingobacteriaceae bacterium; MtFucTDerived from Methylobacter teff (Methylobactertundripaludum); MspFucT Derived from Methylobacter sp.; HpFucT Derived from Helicobacter pylori. The different similar E5 strains constructed were cultured and fermented respectively, and the yield of the byproduct 2-fucosyllactose and the target product lactose-N-fucosyllpentasaccharide I were detected. The results are shown in Figure 4 As shown in the figure, it can be seen that MtFucT Compared with α-1,2-fucosyltransferases from other sources, it has fewer by-products and can efficiently produce lactose-N-fucosylpentaose I.

[0066] The embodiments of the present invention are described in detail above, but the contents described are only preferred embodiments of the present invention and cannot be considered to limit the scope of implementation of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims

1. A recombinant Escherichia coli for synthesizing lactose-N-fucosylpentasaccharide I, characterized in that: Using Escherichia coli BL21 (DE3) as the host, the gene encoding β-galactosidase in its genome was knocked out lacZ , knocking out the gene encoding GDP-mannose hydrolase gmm , knocking out genes encoding tyrosine protein kinases wxya , knocking out the gene encoding dTDP-glucose 4,6-dehydratase rf , knocking out the gene encoding the dephosphorylase protein wxya , knocking out the gene encoding N-acetylfucosamine transferase r , knock out the gene encoding dTDP deoxygalactose aminotransferase r , knocking out the gene encoding colanate phosphate flippase wxya , knocking out genes encoding polysaccharide export proteins wxya ; Overexpression of the gene encoding UDP-glucose 4-epimerase galE , overexpression gmd-fcl Gene 、manC- manB Gene; Heterologous integration of the gene encoding β-1,3-acetylglucosamine transferase NsplgtA , integration of the gene encoding β-1,3-galactosyltransferase Pbr3GalT ; Integration of the gene encoding α-1,2-fucosyltransferase FqV ; Gene editing is performed by a two-step homologous recombination method to achieve scarless knockout; based on the chassis host Escherichia coli, knockout lacZ and r , and integrated at the corresponding sites Pbr3GalT ; Knockout gmm and r , and integrated at the corresponding sites NsplgtA ; Knockout wxya , and overexpressed at this site galE ; Knockout wxya , and overexpressed at this site gmd-fcl ; Knockout rf , and overexpressed at this site manC-manB ; Knockout wxya and wxya , and heterologously expressed at the corresponding sites FqV ; Finally, the recombinant Escherichia coli synthesizing lactose-N-fucosylpentasaccharide I was obtained; Among them, the gene of GDP-mannose hydrolase gmm The amino acid sequence encoded is shown in WP_000479835.1, the gene of tyrosine protein kinase wxya The amino acid sequence encoded is shown in WP_000137133.1, the gene of kolanate phosphate flippase wxya The amino acid sequence encoded is shown in WP_000058488, the gene for dTDP-glucose 4,6-dehydratase rf The amino acid sequence encoded is shown in AXC25041.1, the gene for dephosphorylase protein wxya The amino acid sequence encoded is shown in AKK12342.2, the gene of N-acetylfucosamine transferase r The amino acid sequence encoded is shown in WP_000217220, the gene of dTDP deoxygalactose aminotransferase r The amino acid sequence encoded is shown in WP_083582921, the gene for polysaccharide export protein wxya The amino acid sequence encoded is shown in EHM9396083.1, the gene of β-1,3-acetylglucosamine transferase NsplgtA The amino acid sequence encoded is shown in WP_304703337.1, the gene of UDP-glucose 4-epimerase galE The amino acid sequence encoded is shown in NP_415280.3, the gene for β-1,3-galactosyltransferase Pbr3GalT The amino acid sequence encoded is shown in WP_230643796.1, the gene of α-1,2-fucosyltransferase FqV The amino acid sequence encoded by the gene is shown in WP_031437198.1, the amino acid sequence encoded by the GDP-D-mannose-4,6-dehydratase gene gmd is shown in WP_005128297.1, and the GDP-L-fucose synthase gene fcl The amino acid sequence encoded is shown in NP_416556.1, mannose-1-phosphate guanosine transferase gene manC The amino acid sequence encoded is shown in NP_310881.1, the phosphomannose mutase gene manB The encoded amino acid sequence is shown in WP_001590293.

1.

2. A method for preparing lactose-N-fucosylpentasaccharide I, characterized in that: The method is obtained by culturing and fermenting the recombinant Escherichia coli that synthesizes lactose-N-fucosylpentasaccharide I according to claim 1.

3. The method for preparing lactose-N-fucosylpentasaccharide I according to claim 2, characterized in that: The recombinant Escherichia coli with improved difucosyllactose production was cultured and fermented using glycerol as a carbon source and lactose as a substrate.

4. The method for preparing lactose-N-fucosylpentasaccharide I according to claim 3, characterized in that: The fermentation medium includes: 2 g / L tryptone, 4 g / L yeast extract, 20 g / L glycerol, 12-14 g / L potassium dihydrogen phosphate, 1.3-1.5 g / L diammonium hydrogen phosphate, 1.6-1.8 g / L citric acid, 1.4-1.6 g / L magnesium sulfate heptahydrate, and 8-12 ml / L trace metal elements.

5. The method for preparing lactose-N-fucosylpentasaccharide I according to claim 4, characterized in that: The specific steps are: Step 1: obtaining seed solution by seed culture of the recombinant Escherichia coli, wherein the seed culture medium is 2YT culture medium; Step 2: Inoculate the seed solution into the fermentation tank to form a fermentation system; culture at 37°C until the bacterial OD 600 When the pH reaches 12, lactose content is added to 10 g / L, the temperature is adjusted to 30°C, 10 g / L lactose and 20 g / L glycerol are added every 12 h, and the pH is controlled at 6.

8.

6. The method for preparing lactose-N-fucosylpentasaccharide I according to claim 5, characterized in that: The ventilation volume during the fermentation process is 2-6 vvm and the stirring speed is 300-900 r / min.

7. Use of the method for preparing lactose-N-fucosylpentasaccharide I according to any one of claims 2 to 6 in preparing foods or medicines containing lactose-N-fucosylpentasaccharide I.

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