Construction method and application of recombinant escherichia coli for improving yield of lactoyl-N-tetrasaccharide

Abstract

The invention relates to a construction method and application of recombinant escherichia coli for increasing the yield of lactoyl-N-tetrasaccharide, and belongs to the field of microbial genetic engineering. According to the invention, three genes tauB, nikD and livM screened by using a transcriptome technology are proved to be capable of promoting the generation of LNT and LNTri II. An engineering strain MG-LNT-09 is used as a starting strain, the biosynthesis efficiency of endogenous transporter genes tauB, nikD, livM, LNT and LNTri II derived from Escherichia coli K-12 derivative bacteria MG1655 is remarkably improved through single plasmid overexpression, the yield of the LNT in shake-flask culture is as high as 7.77 g / L, and the yield of the LNTri II is as high as 3.03 g / L. The optimized strain MG3WB of which the tauB gene is integrated to the hlyE site obtains the maximum LNT titer of 40.35 g / L in a 5L bioreactor without using any antibiotics or plasmids, has a good industrial prospect, and lays a foundation for further industrial production and synthesis of other complex breast milk oligosaccharides.

Description

Technical Field

[0001] The present invention relates to a method for constructing a recombinant Escherichia coli for increasing the production of lactyl-N-tetraose and its application, belonging to the field of microbial genetic engineering. Background Art

[0002] Human milk oligosaccharides (HMOs) are bioactive components found in breast milk and are considered an irreplaceable nutritional source for developing infants. As indigestible carbohydrates, HMOs are the third most abundant component in breast milk after lactose and lipids, with more than 200 different structures. These identified HMOs can be roughly divided into three categories: neutral non-fucosylated (42 - 55%), neutral 3-fucosylated (35 - 50%), and acidic sialylated (12 - 14%) oligosaccharides. Lactyl-N-tetraose (LNT) is the most representative neutral non-fucosylated oligosaccharide in breast milk, accounting for 6% (w / w) of the total breast milk oligosaccharides (HMOs), with an average concentration of 0.79 to 1.04 g / L during lactation. Preclinical studies have found that LNT can be utilized by various probiotics (such as Bifidobacterium longum subsp. infantis, Bifidobacterium longum subsp. longum, Bifidobacterium bifidum, and Bifidobacterium breve), promoting the colonization of Bifidobacterium in the intestinal microenvironment, thereby reducing the risk of pathogen infection. In addition, studies have shown that LNT has a significant inhibitory effect on the growth of Streptococcus agalactiae, and the effect is superior to its isomer lacto-N-neotetraose (LNnT). LNT can also inhibit the invasion of the parasite Entamoeba histolytica. Among the 6 candidate HMOs, only LNT showed obvious protective effects, reducing the adhesion and cytotoxicity of amoeba to intestinal epithelial cells. At the same time, LNT effectively enhances the intestinal barrier function by reducing intestinal permeability caused by inflammatory factors, reducing the risk of intestinal-related diseases. Currently, LNT has been approved as a nutritional fortifier and is applied to infant formula and other health fields.

[0003] LNT has attracted increasing attention due to its significant health benefits and commercial value. Therefore, many researchers have attempted to efficiently produce LNT through chemical or enzymatic synthesis. With the growing focus on biosynthesis, the production strategy of LNT has gradually shifted towards biosynthesis due to its sustainability, biocompatibility, and low production cost. The biosynthesis of LNT requires the prior synthesis of lactosyl-N-triose II (LNTri II). Escherichia coli can endogenously synthesize UDP-GlcNAc. By introducing the gene lgtA encoding β-1,3-N-acetylglucosaminyltransferase from Neisseria meningitidis, it can catalyze UDP-GlcNAc as the donor and lactose as the acceptor to link the N-acetylglucosamine group to lactose via a β-1,3 glycosidic bond, generating LNTri II. Based on the biosynthesis of LNTri II, the gene encoding β-1,3-galactosyltransferase is further introduced to catalyze endogenous UDP-Gal and the already synthesized LNTri II, ultimately generating LNT (see literature DOI: 10.1016 / j.ijbiomac.2024.137987). However, the ability of this engineered bacterium to produce LNT is limited and there is room for improvement.

[0004] Summary of the Invention

[0005] Object of the Invention

[0006] In the present invention, genes tauB, nikD, and livM that can improve LNT are obtained by screening 29 genes, which can improve the LNT biosynthesis efficiency in recombinant Escherichia coli. Specifically:

[0007] To address this problem, the present invention screens 29 genes obtained from RNA sequencing and transcriptome analysis, and finally screens out three endogenous transporters TauB (GenBank: AUG15154.1), LivM (GenBank: AAC76481.1), and NikD (GenBank: AAC76504.1). Overexpressing the genes tauB, nikD, and livM using the pCDFDuet-1 vector significantly improves the biosynthesis efficiency of LNT and LNTri II in recombinant Escherichia coli; finally, by genetically overexpressing the endogenous sugar transporter in the Escherichia coli genome, high-titer synthesis of lactosyl-N-tetraose is achieved in plasmid-free strains.

[0008] Technical Solution

[0009] A recombinant bacterium, which uses Escherichia coli as the starting strain, and the following genetic engineering operations are performed on the starting strain: knocking out the β-galactosidase encoding gene lacZ, UDP-glucose lipid carrier transferase encoding gene wcaJ, UDP-N-acetylglucosamine-2-epimerase wecB, and glucosamine-6-phosphate deaminase nagB on the genome of the starting strain; using promoter P J23119 to replace the promoter of the β-galactoside permease encoding gene lacY on the genome; using promoter P tac70 to replace the promoter of the UDP-glucose 4-epimerase gene galE on the genome; integrating the gene lgtA encoding β-1,3-acetylglucosaminyltransferase derived from Neisseria meningitidis and the gene wbgO encoding β-1,3-galactosyltransferase derived from Escherichia coli O55:H7 into the genome of Escherichia coli; overexpressing the endogenous transport genes tauB, nikD or livM derived from Escherichia coli K-12 derivative MG1655..

[0010] In one embodiment, the Escherichia coli includes Escherichia coli MG1655.

[0011] In one embodiment, the Gene IDs of the gene lacZ, gene wcaJ, gene wecB, gene nagB, gene lacY, and gene galE are: 945006, 946583, 944789, 45290, 949083, and 945354, respectively.

[0012] In one embodiment, the nucleotide sequence of the promoter P J23119 is shown in SEQ ID No.3.

[0013] In one embodiment, the nucleotide sequence of the promoter P tac70 is shown in SEQ ID No.4.

[0014] In one embodiment, the lgtA gene is derived from Neisseria meningitidis, its nucleotide sequence is shown in SEQ ID NO.1, and the integrated copy number is three copies.

[0015] In one embodiment, the wbgO gene is derived from Escherichia coli O55:H7, its nucleotide sequence is shown in SEQ ID NO.2, and the integrated copy numbers are two copies and three copies.

[0016] In one embodiment, the recombinant bacterium uses the pCDFDuet-1 plasmid to express the endogenous transport gene tauB, and the endogenous transport gene tauB is cloned into the first multiple cloning site of the pCDFDuet-1 plasmid vector.

[0017] In one embodiment, the recombinant bacterium uses the pCDFDuet-1 plasmid to express the endogenous transport gene nikD, and the endogenous transport gene nikD is cloned into the first multiple cloning site of the pCDFDuet-1 plasmid vector.

[0018] In one embodiment, the recombinant bacterium uses the pCDFDuet-1 plasmid to express the endogenous transport gene livM, and the endogenous transport gene livM is cloned into the first multiple cloning site of the pCDFDuet-1 plasmid vector.

[0019] In one embodiment, the recombinant bacterium integrates at the hlyE locus on the Escherichia coli genome and uses the strong promoter P J23119 to initiate the expression of the endogenous transport gene tauB derived from the Escherichia coli K-12 derivative MG1655.

[0020] In one embodiment, the recombinant bacterium integrates at the hlyE locus on the Escherichia coli genome and uses the strong promoter P J23119 to initiate the expression of the endogenous transport gene nikD derived from the Escherichia coli K-12 derivative MG1655.

[0021] In one embodiment, the recombinant bacterium integrates at the hlyE locus on the Escherichia coli genome and uses the strong promoter P J23119 to initiate the expression of the endogenous transport gene livM derived from the Escherichia coli K-12 derivative MG1655.

[0022] The second object of the present invention is to provide a method for producing lactyl-N-tetraose, and the method uses the above-mentioned recombinant bacterium to ferment and produce lactyl-N-tetraose.

[0023] In one embodiment, the method includes:

[0024] (1) Inoculate the recombinant bacterium into a seed medium and culture it at 37 °C for 8 - 14 h to obtain a primary seed solution;

[0025] (2) Transfer the primary seed solution obtained in step (1) to a fermentation medium at a transfer volume of 1% - 2% and culture it at 30 - 40 °C for 3 - 6 h to obtain a secondary seed solution;

[0026] (3) Inoculate the secondary seed solution obtained in step (2) into a fermentation medium at an inoculation amount of 5% - 15% and culture it until OD 600It is 30 - 50, lactose is added, and lactosyl-N-tetraose is produced by fermentation at 25 - 35 °C.

[0027] In one embodiment, the formulation of the seed medium includes: 1 - 10 g / L of yeast extract, 5 - 20 g / L of peptone, and 5 - 20 g / L of sodium chloride. Optionally, the formulation of the seed medium includes: 5 g / L of yeast extract, 10 g / L of peptone, and 10 g / L of sodium chloride.

[0028] In one embodiment, the formulation of the fermentation medium includes: 10 - 30 g / L of glycerol, 1 - 10 g / L of yeast extract, 5 - 20 g / L of potassium dihydrogen phosphate, 1 - 10 g / L of diammonium hydrogen phosphate, 0.5 - 3 g / L of citric acid, 0.5 - 3 g / L of magnesium sulfate heptahydrate, 1 - 20 ml / L of trace metal elements, and 1 - 10 mg / L of thiamine. Optionally, the formulation of the fermentation medium includes: 20 g / L of glycerol, 5 g / L of yeast extract, 13.5 g / L of potassium dihydrogen phosphate, 4.0 g / L of diammonium hydrogen phosphate, 1.7 g / L of citric acid, 1.4 g / L of magnesium sulfate heptahydrate, 10 ml / L of trace metal elements, and 4.5 mg / L of thiamine.

[0029] In one embodiment, the formulation of the trace metal elements includes: 5 - 20 g / L of ferrous sulfate, 1 - 5 g / L of zinc sulfate heptahydrate, 0.5 - 2 g / L of anhydrous copper sulfate, 0.1 - 1 g / L of manganese sulfate monohydrate, 0.1 - 1 g / L of sodium borate decahydrate, 0.1 - 1 g / L of ammonium molybdate, and 0.5 - 5 g / L of calcium chloride dihydrate. Optionally, the formulation of the trace metal elements includes: 10 g / L of ferrous sulfate, 2.25 g / L of zinc sulfate heptahydrate, 1.0 g / L of anhydrous copper sulfate, 0.35 g / L of manganese sulfate monohydrate, 0.23 g / L of sodium borate decahydrate, 0.11 g / L of ammonium molybdate, and 2.0 g / L of calcium chloride dihydrate.

[0030] In one embodiment, the lactose concentration in the fermentation system is maintained at 2 - 5 g / L by feeding.

[0031] In one embodiment, the glycerol concentration in the fermentation system is maintained at 3 - 10 g / L by feeding.

[0032] In one embodiment, the fermentation time is not less than 60 h.

[0033] The third object of the present invention is to provide the application of the above recombinant bacteria or the above method in the fields of medicine, food or chemical industry.

[0034] In one embodiment, the application includes using the above recombinant bacteria or the method to produce lactosyl-N-tetraose or products containing lactosyl-N-tetraose.

[0035] Table 1 Literature reports and functional introductions of tauB, nikD and livM

[0036]

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] Three genes tauB, nikD and livM screened by the transcriptome technology of the present invention are proved to be able to promote the production of LNT and LNTri II. When these genes are expressed separately in the pCDFDuet-1 vector of the engineering strain, the biosynthesis efficiency of LNT and LNTri II is significantly improved. The yield of LNT in shake flask culture is as high as 7.77 g / L, and the yield of LNTri II is as high as 3.03 g / L. Without using any antibiotics or plasmids, when the optimized strain MG3WB integrating the tauB gene into the genomic hlyE locus was used in a 5L bioreactor, the maximum LNT titer of 40.35 g / L was obtained. The gene tauB significantly increased the yield of LNT. Compared with the control strain MG-LNT-09, the optimized strain MG3WB achieved a 1.28-fold increase in the bioreactor, showing good industrial application prospects. Description of the Drawings

[0039] Figure 1 It is a schematic diagram of the metabolic pathway for the synthesis of lactoyl-N-tetrasaccharide by recombinant Escherichia coli.

[0040] Figure 2 It is a comparison chart of the yields of recombinant Escherichia coli expressing 29 endogenous transporters by plasmid.

[0041] Figure 3 It is a comparison chart of the yields of recombinant Escherichia coli expressing 3 endogenous transporters by plasmid combination.

[0042] Figure 4 It is a comparison chart of the yields of recombinant Escherichia coli with different endogenous sugar transporters integrated into the genome.

[0043] Figure 5 It is the LC-MS spectrum result of the engineering strain, where (a) is the LC-MS spectrum result of the LNT standard, (b) is the LC-MS spectrum result of the fermentation product of the strain MG-LNT-09 in shake flask, and (c) is the LC-MS spectrum result of the fermentation product of the strain MG3 WB in shake flask. Detailed Embodiments

[0044] Furthermore, the present invention is described in more detail by specific implementation examples.

[0045] For commercial products such as plasmids, restriction enzymes, PCR enzymes, column DNA extraction kits, and DNA gel extraction kits used in the following examples, the specific operations were carried out according to the kit instructions. Conventional operation methods such as colony PCR, nucleic acid agarose gel electrophoresis, heat shock transformation, electroporation, preparation of competent cells, and extraction and preservation of bacterial genomes were carried out according to Molecular Cloning: A Laboratory Manual (Fourth Edition). The sequencing of plasmids and DNA products was entrusted to Genewiz (Suzhou).

[0046] The media involved in the fermentation process of LNT are as follows:

[0047] (1) LB liquid medium: 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride.

[0048] (2) LB solid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar powder.

[0049] (3) Fermentation medium: 20 g / L glycerol, 5 g / L yeast extract, 13.5 g / L potassium dihydrogen phosphate, 4.0 g / L diammonium hydrogen phosphate, 1.7 g / L citric acid, 1.4 g / L magnesium sulfate heptahydrate, and 10 ml / L trace metal elements (10 g / L ferrous sulfate, 2.25 g / L zinc sulfate heptahydrate, 1.0 g / L anhydrous copper sulfate, 0.35 g / L manganese sulfate monohydrate, 0.23 g / L sodium borate decahydrate, 0.11 g / L ammonium molybdate, 2.0 g / L calcium chloride dihydrate, dissolved in 5 M hydrochloric acid). The pH of the medium was adjusted to 6.8 with sodium hydroxide, sterilized at 115 °C for 30 min, and 4.5 mg / L thiamine was added before use.

[0050] (4) Antibiotic concentrations: ampicillin 100 mg / L (liquid medium), ampicillin 200 mg / L (solid medium), kanamycin 50 mg / L, streptomycin 50 mg / L, chloramphenicol 34 mg / L.

[0051] (5) Inducer concentrations: During flask fermentation, the final concentration of isopropyl-β-D-thiogalactopyranoside (IPTG) added was 0.5 mM. In the CRISPR-related transposase system, the final concentrations of IPTG added were 0.1 mM and 1 mM (if needed), the final concentration of rhamnose added was 10 mM, and the final concentration of sucrose added was 10 g / L.

[0052] (6)Fed-batch fermentation feeding solution: glycerol 600 g / L, magnesium sulfate heptahydrate 20 g / L, thiamine 0.2 g / L, ampicillin 100 mg / L, streptomycin 50 mg / L. pH regulation: 50% ammonia water (v / v).

[0053] (7)Shake flask fermentation production of LNT: Inoculate the engineered strain into 4 mL of LB liquid medium containing specific antibiotics, and culture at 37 °C and 200 rpm for 10 - 12 h to prepare the primary seed solution. Inoculate the seed solution into 20 mL of modified DM medium (containing 5 g / L yeast extract) at an inoculation amount of 2%. When OD 600 increases to 0.6 - 0.8, add lactose with a final concentration of 5 g / L and IPTG with a final concentration of 0.5 mM, and continue to culture at 30 °C and 200 rpm for 96 h.

[0054] (8)Fed-batch fermentation production of LNT: Add 2 L of fermentation medium to a 5 L bioreactor and sterilize at 115 °C for 30 minutes. After sterilization, gradually reduce the temperature of the bioreactor to 30 °C using a condenser. Before inoculation, adjust the pH of the DM medium to approximately 6.8 using 50% (v / v) NH₃·H₂O. Maintain the dissolved oxygen level in the bioreactor between 25% - 35%. Inoculate the prepared seed solution into the bioreactor at an inoculation amount of 10%. After culturing for 18.5 hours, OD 600 reaches approximately 40, start feeding lactose to the medium, sample at regular intervals, monitor the remaining carbon source concentration and lactose concentration, and maintain the glycerol concentration at 3 - 10 g / L and the lactose concentration at 2 - 5 g / L by feeding.

[0055] (9)Detection of the content of various substances (LNT, LNTri II, lactose, and glycerol) in the fermentation broth: Take 1 mL of the fermentation broth, centrifuge at 10,000 rpm for 10 min, take the supernatant, filter the supernatant through a 0.22 μm aqueous filter, and determine the content by HPLC.

[0056] (10)HPLC detection conditions: High-performance liquid chromatography (HPLC) system (Waters e2695); chromatographic column: Rezex ROA-organic acid H⁺ (8%); detector: Waters 2414 RIDetector differential refractive index detector; mobile phase: 5 mM H 2 SO 4 ₄; flow rate: 0.6 mL / min; column temperature: 60 °C; injection volume: 10 μL.

[0057] The primer sequences involved in the following examples are shown in Table 2.

[0058] Table 2: Primer sequences and targeted plasmid information

[0059]

[0060]

[0061] The PCR reaction system involved in the following examples is shown in Table 3.

[0062] Table 3: PCR System

[0063]

[0064] PCR reaction system conditions: Pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 56°C for 30 s, extension at 72°C at 2 kb / min; 32 cycles, preservation at 4°C, and gel recovery of PCR products.

[0065] The Gibson assembly reaction system involved in the following examples is shown in Table 4.

[0066] Table 4: Gibson Assembly Reaction System

[0067]

[0068]

[0069] Gibson assembly reaction conditions: 50°C, 30 min.

[0070] The amounts of linearized vector and inserted fragment can be calculated according to the formula for X / Y:

[0071] X = (0.02 × number of base pairs of cloning vector / concentration of corresponding DNA fragment recovered by gel) ng;

[0072] Y = (0.02 × number of base pairs of inserted fragment / concentration of corresponding DNA fragment recovered by gel) ng;

[0073] The overlap extension PCR system involved in the following examples is shown in Table 5.

[0074] Table 5: Overlap Extension PCR System

[0075]

[0076] Overlap extension PCR reaction system conditions: Pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 56°C for 30 s, extension at 72°C at 2 kb / min; 32 cycles, preservation at 4°C, taking part of the PCR products for nucleic acid electrophoresis verification, then using demethylase DPN1 for enzymatic digestion to remove the template plasmid, and then carrying out transformation verification.

[0077] Example 1: Construction of Recombinant Plasmid Vector

[0078] RNA sequencing and transcriptome analysis were performed on three strains with different lacto-N-tetraose yields, namely MG3A, MG-LNT-08, and MG-LNT-09 (shown in Table 6). The three strains were disclosed in the literature Qian, Q.; Yang, L.; Zhao, C.; Tao, M.; Zhang, W.; Zhu, Y.; Mu, W. Highly efficient production of lacto-N-tetraose in plasmid-free Escherichia coli through chromosomal integration of multicopy key glycosyltransferase genes. Int J Biol Macromol. 2025, 284(Pt 1), 137987. With the integration of two-copy and three-copy wbgO into the genome, significant changes occurred in the RNA transcription level. Combining GO classification enrichment analysis and KEGG pathway analysis, a total of 29 candidate genes related to transporter function were found to be significantly upregulated compared with the control group, and all of them belong to the members of the ATP-binding cassette (ABC) transporter superfamily.

[0079] According to the screening results of transcriptome analysis, 29 candidate genes related to transporter function (amplified from the Escherichia coli genome) were cloned into the vector pCDFDuet-1 for further screening and confirmation.

[0080] Among them: The primers used for plasmid construction are listed in Table 2.

[0081] Taking the construction of pCDF-tauB (MCS1) plasmid as an example, the specific steps are as follows:

[0082] Using the pCDFDuet-1 plasmid vector as a template, a vector fragment was amplified with a pair of primers V-pCD-1-F / R; using the genome of Escherichia coli K-12 derivative MG1655 as a template, a tauB gene fragment was amplified with a pair of primers pCD-1-tauB-F / R. The two fragments were recovered by gel electrophoresis respectively, and the two fragments were ligated with the Gibson kit (NEB reagent company, USA) to obtain the plasmid pCDF-tauB (MCS1). The information of the constructed plasmid is shown in Table 7 below.

[0083] Table 6 Information of engineering strains for transcriptome analysis

[0084]

[0085]

[0086] Plasmids constructed in Table 7

[0087]

[0088]

[0089] Example 2: Screening of 29 genes by the method of producing lactosyl-N-tetraose through plasmid expression and shake flask fermentation

[0090] The plasmids constructed in Example 1 were respectively transferred into the Escherichia coli recombinant strain MG-LNT-09 to obtain strains MG3WS-1 to MG3WS-29. MG3WS-0 (carrying the plasmid pCDFDuet-1, an empty vector) was used as a control to compare the effects of strains containing different genes on the yield of lactosyl-N-tetraose.

[0091] Fermentation method: After the above recombinant strains were grown overnight on a resistant agar LB plate, they were inoculated into 4 mL of LB medium with the corresponding antibiotics as the initial seed solution and cultured at 37 °C and 200 rpm for 8 - 12 h. 400 μL of the seed solution was taken and inoculated into 20 mL of fermentation medium, and cultured at 37 °C and 200 rpm until the OD 600 reached 0.6 - 0.8. Lactose with a final concentration of 5 g / L and 0.5 mM IPTG were added, and the induction culture was continued at 30 °C and 200 rpm for 96 h. 1 mL of the fermentation broth was taken, centrifuged at 12,000 rpm for 10 min, and the supernatant was taken for HPLC determination.

[0092] Results:

[0093] As shown in Table 8, screening of 29 different genes was carried out. Not all transport genes could improve the yield of lactosyl-N-tetraose, and even some genes led to a decrease in the yield of lactosyl-N-tetraose (such as MG3WS-1, etc.); only MG3WS-3, MG3WS-18, and MG3WS-21 increased the yield of lactosyl-N-tetraose in the strain. The corresponding genes were tauB, livM, and nikD respectively. Among them, the strain MG3WS-3 had the highest yield of lactosyl-N-tetraose, which was 7.77 g / L. Further, the above three genes were combined in pairs, such as MG3WD-1 - MG3WD-4, to investigate the yield of lactosyl-N-tetraose. It was found that there were also differences among the combinations, and they were lower than that of a single gene.

[0094] Table 8 Shake flask fermentation information of engineering strains for LNT production

[0095]

[0096]

[0097] Example 3: Construction of Recombinant Strains (Integration of Transport Genes into the Genome) and Investigation of Lactoyl-N-tetraose Production by Genome Expression Flask Fermentation

[0098] 1. Integrate the endogenous transport genes tauB, livM, and nikD into the genome of the strain respectively. The primers used for genome integration are shown in Table 2. Taking the integration of the endogenous transport gene tauB into the hlyE gene in Escherichia coli using pEcCpf1 / pcrEG as an example (the dual-plasmid gene editing system pEcCpf1 / pcrEG was published in the literature: Zhu X, Wu Y, Lv X. Combining CRISPR–Cpf1 and Recombineering Facilitates Fast and Efficient Genome Editing in Escherichia coli[J]. ACS Synthetic Biology, 2022(5):11.), the specific steps are as follows:

[0099] (1) Transform the pEcCpf1 plasmid into the MG-LNT-09 strain. After a single colony grows on the plate, inoculate it into a test tube containing 4 mL of LB medium with kanamycin resistance and culture for 12 h, then prepare electrocompetent cells.

[0100] (2) Use overlapping extension PCR to generate homologous fragments, which include 500-bp regions upstream and downstream of the hlyE gene and the tauB gene expression cassette.

[0101] (3) Perform PCR amplification of the pcrEG plasmid using the primers hlyE-N23-F / R. The obtained linear DNA is circularized into the pcrEG-ΔhlyE plasmid targeting hlyE by single-fragment one-step cloning.

[0102] (4) Take the prepared electrocompetent cells containing the pEcCpf1 plasmid on ice. After thawing, add the pcrEG-ΔhlyE plasmid in (3) and the homologous fragment in (2). Immediately after electroporation, add 1 mL of LB medium that has been fully pre-cooled. Gently pipette and mix evenly, then transfer it to a sterile 1.5 mL ep tube. Incubate at 37 °C and 200 r / min for 2 h. Subsequently, spread all the bacteria on a double-antibody plate (final concentration of 50 μg / L kanamycin and 50 μg / L spectinomycin), and incubate it upside down in a 37 °C incubator for 12 - 15 h.

[0103] (5) Verify the single colonies grown by colony PCR. Send the colonies that have passed the colony PCR verification to Suzhou Genewiz Biotechnology Co., Ltd. for sequencing. The colonies that match the theoretical sequence are the colonies in which tauB has been successfully integrated.

[0104] (6) Inoculate the bacteria that have passed the above verification into a 4 mL test tube and culture for 12 h (add 50 μg / L kanamycin and rhamnose with a final concentration of 10 mM during inoculation) to remove the pcrEG-ΔhlyE plasmid. Further inoculate the target bacteria into a liquid LB medium containing 5 g / L glucose, and then streak the bacterial liquid on a plate containing 5 g / L glucose and 15 g / L sucrose to remove the pEcCpf1 plasmid, obtaining strain MG3WB.

[0105] The construction steps of strain MG3WD and strain MG3WM are the same as above.

[0106] 2. Investigation on the production of lactosyl-N-tetraose by genomic expression shake flask fermentation

[0107] Ferment the constructed Escherichia coli recombinant strains MG3WB, MG3WD, and MG3WM to verify the lactosyl-N-tetraose production of the strains.

[0108] Fermentation method: After the above recombinant strains grow overnight on an LB plate respectively, inoculate them into 4 mL of LB medium as the initial seed liquid, and culture at 37 °C and 200 rpm for 8 - 12 h. Take 400 μL of the seed liquid and inoculate it into 20 mL of fermentation medium, and culture at 37 °C and 200 rpm until the OD 600 reaches 0.6 - 0.8, add lactose with a final concentration of 5 g / L, and continue to induce and culture at 30 °C and 200 rpm for 96 h. Take 1 mL of the fermentation broth, centrifuge at 12,000 rpm for 10 min, and take the supernatant for HPLC determination.

[0109] Results

[0110] As shown in Table 8, compared with the control strain MG-LNT-09 (no genes involved in transporter protein function are overexpressed in the genome), the integration of endogenous transporter genes tauB, livM, and nikD (genes involved in transporter protein function are overexpressed in the genome) all increase the lactosyl-N-tetraose production to varying degrees. Among them, the plasmid-free strain MG3WB (containing tauB) has the highest lactosyl-N-tetraose production, which is 6.13 g / L.

[0111] Table 8 Shake flask fermentation information of engineered strains for LNT production

[0112]

[0113] Example 4: Fed-batch fermentation for the production of LNT

[0114] To further verify the scale-up production effect of the genetically modified strains, the engineered strains MG-LNT-09 (which does not overexpress any genes involved in transporter functions) and MG3WB (which overexpresses the gene tauB involved in transporter functions in the genome) were selected for fed-batch fermentation experiments in a 5 L fermenter.

[0115] After culturing the above strains MG-LNT-09 and MG3WB overnight in solid LB medium at 37 °C, three medium-sized monoclonal colonies were inoculated into 4 mL of LB liquid medium and grown in an orbital shaker set at 30 °C and 200 rpm for 9 hours as the primary seed culture; 1 mL of the primary seed culture was inoculated into 100 mL of fermentation medium (DM, 30 g / L glycerol, 5 g / L yeast extract) and expanded at 37 °C and 200 rpm for 3 - 6 h until the OD 600 reached 1.8 to obtain the secondary seed culture. Then, 10% of the above medium was added to a 5 L bioreactor containing 2 L of fermentation medium (DM, 30 g / L glycerol) and cultured at 30 °C. The rotational speed was set at 300 rpm, the dissolved oxygen was set at 30%, the aeration was set at 2.5 vvm, and the pH was set at 6.7 ± 0.5. The pH was monitored using a pH sensor and maintained at 6.7 ± 0.5 by periodically adding 50% (v / v) ammonia solution. Meanwhile, lactose with a final concentration of 1 - 2 g / L was slowly supplemented to the DM medium. Importantly, the concentrations of glycerol and lactose should be controlled within the range of 0 - 5 g / L to prevent excessive accumulation in the bioreactor. In addition, the glycerol supplement consisted of 600 g / L glycerol, 20 g / L magnesium sulfate heptahydrate, and 0.2 g / L thiamine. The concentration of the lactose stock solution was 200 g / L.

[0116] When the fermentation reached 63 h, MG3WB obtained a maximum LNT titer of 40.35 g / L in the 5 L bioreactor, and the concentration of the retained LNTri II reached 11.17 g / L without using any antibiotics or plasmids. While the LNT accumulation rate of MG-LNT-09 was slower, and the maximum yield at the end of fermentation was 31.48 g / L. Compared with the control strain MG-LNT-09, the strain MG3WB achieved a 1.28-fold increase in the bioreactor.

[0117] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A lactoyl-producing N - a recombinant bacterium expressing tetrasaccharide, wherein the recombinant bacterium has knocked out the β -Galactosidase encoding gene lacZ , UDP-glucose lipid carrier transferase encoding gene wxya 、UDP- N -acetylglucosamine-2-epimerase wxya and glucosamine-6-phosphate deaminase nagB ; Using promoter P J23119 Replace the genome β -galactoside permease encoding gene lacY promoter; using promoter P tac70 Replace the UDP-glucose 4-epimerase gene on the genome galE promoter; multiple copies of the coding sequence are integrated into the genome of Escherichia coli β -1,3-acetylglucosamine transferase gene oeLh and encoding β -1,3-galactosyltransferase gene wxya ; It is characterized in that, Overexpression of endogenous transporter genes alone or in combination tauB , nikD and livM .

2. The recombinant bacterium according to claim 1, characterized in that The Escherichia coli is Escherichia coli MG1655.

3. The recombinant bacterium according to claim 2, characterized in that The recombinant bacteria are in Escherichia coli E Site integration using a strong promoter P J23119 Start Expression tauB , nikD or livM .

4. A method for producing lactoyl- N -tetrasaccharide method, characterized in that The recombinant bacteria of claims 1-3 are inoculated into a seed culture medium, cultured at 30-40°C for 8-14 h to obtain a seed solution, and the seed solution is transferred to a fermentation medium and cultured until OD 600 When the temperature is 0.6-0.8, lactose is added to reduce the fermentation temperature to 20-28°C, and the fermentation produces lactoyl- N -Tetrasaccharide.

5. The method according to claim 4, characterized in that The method comprises the following steps: (1) inoculating the recombinant bacteria into a seed culture medium, and culturing at 30-40° C. for 8-14 h to obtain a primary seed solution; (2) transferring the first-level seed solution obtained in step (1) into a fermentation medium at a transfer rate of 0.5%-2%, and culturing at 30-40° C. for 3-6 h to obtain a second-level seed solution; (3) Inoculate the secondary seed solution obtained in step (2) into the fermentation medium at an inoculum rate of 5%-15% and culture until OD 600 30-50, add lactose, ferment to produce lactoyl- N -Tetrasaccharide.

6. The method according to claim 5, characterized in that The fermentation production temperature in step (3) is 25-35°C.

7. The method according to claim 4, characterized in that The concentration of glycerol in the fermentation system was maintained at 3-10 g / L, and the concentration of lactose was maintained at 2-5 g / L by feeding.

8. The method according to claim 4, characterized in that The fermentation time is not less than 60 h.

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