Bioengineering method for efficiently synthesizing lactoyl-N-tetrasaccharide

Abstract

The invention discloses a bioengineering method for efficiently synthesizing lactoyl-N-tetrasaccharide, and belongs to the field of biotechnology and food fermentation engineering. The method comprises the following steps: firstly, carrying out combined regulation on lgtA, wbgO, galE, galT and galK genes in a lactoyl-N-tetrasaccharide synthesis pathway to optimize the expression dose of pathway genes, then, sequentially knocking out collateral metabolic pathway genes lacZ, wecB, ugd, nagB, pfkA, gloA and setA on a chromosome of escherichia coli BL21 (DE3) by utilizing a CRISPR-Cas9 technology, and respectively integrating a nucleotide glycometabolism key gene cluster galE-galT-galK at a yeeJ site, so as to obtain the lactoyl-N-tetrasaccharide synthesis pathway. A core glycosyl transferase gene lgtA-wbgO is integrated at a caiB site, and finally an engineering strain capable of efficiently synthesizing lactoyl-N-tetrasaccharide is constructed. The engineering bacterium can be used for realizing efficient synthesis of lactoyl-N-tetrasaccharide in a restrictive glycerol culture medium by taking lactose as a substrate. Under a shake-flask culture condition, the ability of the escherichia coli for synthesizing the lactoyl-N-tetrasaccharide is improved from 3.41 g / L to 11.15 g / L, and the yield of the lactoyl-N-tetrasaccharide reaches 125.35 g / L in fed-batch culture of a 3 L fermentation tank.

Description

Technical Field

[0001] This invention relates to a bioengineering method for the efficient synthesis of lactyl-N-tetrasaccharides, belonging to the fields of biotechnology and food fermentation engineering. Background Technology

[0002] Breast milk is the ideal food for infants, and its oligosaccharides play important physiological functions. According to Grand View Research, the global human milk oligosaccharide market is projected to maintain a CAGR of 13.8% between 2024 and 2030, reaching a market size of 5.35 billion yuan in 2030. Lactoyl-N-tetrasaccharide is one of the core structures of human milk oligosaccharides and can be modified into more complex oligosaccharides through fucylation or sialylation. Lactoyl-N-tetrasaccharide and its derivatives account for more than 60% of human milk oligosaccharides. Furthermore, lactoyl-N-tetrasaccharide plays multiple roles in infant growth and development, such as anti-pathogen adhesion and immune regulation. Based on its safety and nutritional value, lactoyl-N-tetrasaccharide has been approved by European and American food safety regulatory agencies as a nutritional fortifier in infant formula. For example, Nestlé's BEBA SUPREME Pre and Wyeth's Illuma® LUXA infant formula both contain lactoyl-N-tetrasaccharide. my country has also accepted applications for the use of lactyl-N-tetrasaccharides in food since 2024 (Approval No. 0090 of 2024 for Food Additives). Therefore, the preparation of large quantities of single-structure lactyl-N-tetrasaccharides is of great significance for their commercial application and food science research.

[0003] The process of isolating and purifying lactyl-N-tetrasaccharides from breast milk is complex and the sources are limited, restricting their application in formulated foods and health care. Therefore, artificial synthesis is the most suitable option for large-scale preparation of lactyl-N-tetrasaccharides. Compared with chemical and enzymatic synthesis, microbial synthesis of lactyl-N-tetrasaccharides does not require the addition of additional catalytic enzymes and nucleotide sugars. The catalytic enzymes can be heterologously expressed intracellularly by microorganisms, and the nucleotide sugars UDP-GlcNAc and UDP-Gal can be supplied by the strain's own metabolism, while the substrate lactose is supplemented by exogenous addition. During the synthesis of lactyl-N-tetrasaccharides, lactose is transported from the extracellular space to the intracellular space and reacts with UDP-GlcNAc to form the intermediate lactyl-N-trisaccharide under the catalysis of β-1,3-N-acetylglucosyltransferase. Lactyl-N-trisaccharide and UDP-Gal then generate lactyl-N-tetrasaccharide under the catalysis of β-1,3-galactosyltransferase. Therefore, microbial synthesis of lactyl-N-tetrasaccharides has the advantages of being green, efficient, and low-cost. Escherichia coli BL21(DE3) has advantages such as a known genome, short growth cycle, and simple culture conditions. Furthermore, the synthesis of lactyl-N-tetrasaccharides using E. coli BL21(DE3) meets the US GRAS and European QPS standards. Precise carbon flux regulation during lactyl-N-tetrasaccharide synthesis is crucial for reducing byproduct accumulation, increasing lactyl-N-tetrasaccharide yield, and maintaining robust host cell growth.

[0004] However, the carbon flux regulation mechanism in the biosynthesis of lactyl-N-tetrasaccharides remains unclear, leading to uneven carbon flux utilization and limited optimization of metabolic pathways, thus restricting further increases in lactyl-N-tetrasaccharide yield. These issues are not only a core bottleneck for the industrial application of biosynthesized lactyl-N-tetrasaccharides but also a key common scientific problem in the field of microbial synthesis of human milk oligosaccharides, urgently requiring in-depth research and breakthroughs. Therefore, how to construct engineered strains that efficiently synthesize lactyl-N-tetrasaccharides through functional gene and carbon flux analysis, overcoming interference from other metabolic carbon fluxes, is not only crucial for achieving efficient, stable, and sustainable biosynthesis of lactyl-N-tetrasaccharides but also an important scientific and engineering problem that urgently needs to be solved in the field of food synthetic biology. Summary of the Invention

[0005] How to construct recombinant Escherichia coli that can efficiently synthesize lactyl-N-tetrasaccharides using bioengineering methods is a key technical problem that urgently needs to be solved.

[0006] To address the application research problems of existing microbially synthesized lactyl-N-tetrasaccharides, this invention provides a highly efficient bioengineering method for synthesizing lactyl-N-tetrasaccharides.

[0007] The purpose of this invention is to provide a genetically engineered bacterium that produces lactyl-N-tetrasaccharides. This bacterium knocks out the genes encoding β-galactosidase (lacZ), UDP-N-acetylglucosamine-2-epomerase (wecB), UDP-glucose-6-dehydrogenase (ugd), glucosamine-6-phosphate deaminase (nagB), fructose-6-phosphate kinase (pfkA), lactylglutathione lyase (gloA), and sugar efflux transporter (setA) in the *E. coli* genome. It also integrates and expresses the genes encoding UDP-glucose-4-epomerase (galE), galactose-1-phosphotransferase (galT), and galactokinase (galK) in the *E. coli* genome, and integrates and expresses the genes encoding β-1,3-N-acetylglucosamine transferase (lgtA) and β-1,3-galactosyltransferase (wbgO) in the *E. coli* genome (CaiB).

[0008] In one embodiment, the genetically engineered bacteria also overexpress the UDP-glucose-4-epiisomerase gene galE, the galactose-1-phosphate uracil transferase gene galT, and / or the galactokinase gene galK.

[0009] In one embodiment, the genetically engineered bacteria also overexpress the β-1,3-N-acetylglucosamine transferase gene lgtA and the β-1,3-galactosyltransferase gene wbgO.

[0010] In one embodiment, the β-1,3-N-acetylglucosamine transferase gene lgtA is derived from Neisseria meningitidis, and the β-1,3-galactosyltransferase gene wbgO is derived from Escherichia coli O55:H7.

[0011] In one embodiment, β-1,3-N-acetylglucosamine transferase LgtA has the NCBI accession number AAF25877.1, and β-1,3-galactosyltransferase WbgO has the NCBI accession number CAD5494137.1.

[0012] In one embodiment, the UDP-glucose-4-epiisomerase gene galE, the galactose-1-phosphate uracil transferase gene galT, and the galactokinase gene galK are all derived from Escherichia coli K-12.

[0013] In one embodiment, the UDP-glucose-4-epiisomerase GalEzai1 has the NCBI sequence number NP_415280.3, the galactose-1-phosphate uracil transferase GalT has the NCBI sequence number NP_415279.1, and the galactokinase GalK has the NCBI sequence number NP_415278.1.

[0014] In one embodiment, the genetically engineered bacteria uses Escherichia coli BL21(DE3) as a host.

[0015] In one embodiment, the genetically engineered bacteria express the genes galE, galT, galK, lgtA and / or wbgO using the plasmids pCOLDuet-1, pACYDuet-1, pCDFDuet-1, pETDuet-1 or pRSFDuet-1.

[0016] In one embodiment, the genetically engineered bacteria express lgtA and wbgO using pRSFDuet-1 and express genes galE, galT, and galK using pETDuet-1 plasmid.

[0017] In one embodiment, the genetically engineered bacteria express lgtA and wbgO using pRSFDuet-1, and express genes galE, galT, and galK using pCDFDuet-1 plasmid.

[0018] In one embodiment, the genetically engineered bacteria express lgtA and wbgO using pRSFDuet-1 and express genes galE, galT, and galK using pACYDuet-1 plasmid.

[0019] In one embodiment, the genetically engineered bacteria express lgtA and wbgO using pRSFDuet-1 and express genes galE, galT, and galK using pCOLDuet-1 plasmid.

[0020] In one embodiment, the genetically engineered bacteria express lgtA and wbgO using pETDuet-1 and express genes galE, galT, and galK using pRSFDuet-1 plasmid.

[0021] In one embodiment, the genetically engineered bacteria express lgtA and wbgO using pETDuet-1 and express genes galE, galT, and galK using pCDFDuet-1 plasmid.

[0022] In one embodiment, the genetically engineered bacteria express lgtA and wbgO using pETDuet-1 and express genes galE, galT, and galK using pACYDuet-1 plasmid.

[0023] In one embodiment, the genetically engineered bacteria express lgtA and wbgO using pETDuet-1 and express genes galE, galT, and galK using pCOLDuet-1 plasmid.

[0024] In one embodiment, the genetically engineered bacteria express lgtA and wbgO using pCDFDuet-1 and express genes galE, galT, and galK using pRSFDuet-1 plasmid.

[0025] In one embodiment, the genetically engineered bacteria express lgtA and wbgO using pCDFDuet-1 and express genes galE, galT, and galK using pETDuet-1 plasmid.

[0026] In one embodiment, the genetically engineered bacteria express lgtA and wbgO using pCDFDuet-1 and express genes galE, galT, and galK using pACYDuet-1 plasmid.

[0027] In one embodiment, the genetically engineered bacteria express lgtA and wbgO using pCDFDuet-1, and express genes galE, galT, and galK using pCOLDuet-1 plasmid.

[0028] In one embodiment, the genetically engineered bacteria express lgtA and wbgO using pACYDuet-1 and express genes galE, galT, and galK using pRSFDuet-1 plasmid.

[0029] In one embodiment, the genetically engineered bacteria express lgtA and wbgO using pACYDuet-1, and express genes galE, galT, and galK using pETDuet-1 plasmid.

[0030] In one embodiment, the genetically engineered bacteria express lgtA and wbgO using pACYDuet-1 and express genes galE, galT, and galK using pCDFDuet-1 plasmid.

[0031] In one embodiment, the genetically engineered bacteria express lgtA and wbgO using pACYDuet-1 and express genes galE, galT, and galK using pCOLDuet-1 plasmid.

[0032] In one embodiment, the genetically engineered bacteria express lgtA and wbgO using pCOLDuet-1 and express genes galE, galT, and galK using pRSFDuet-1 plasmid.

[0033] In one embodiment, the genetically engineered bacteria express lgtA and wbgO using pCOLDuet-1 and express genes galE, galT, and galK using pETDuet-1 plasmid.

[0034] In one embodiment, the genetically engineered bacteria express lgtA and wbgO using pCOLDuet-1 and express genes galE, galT, and galK using pCDFDuet-1 plasmid.

[0035] In one embodiment, the genetically engineered bacteria express lgtA and wbgO using pCOLDuet-1 and express genes galE, galT, and galK using pACYDuet-1 plasmid.

[0036] This invention provides a method for constructing the recombinant *E. coli*, which involves first knocking out the following genes in the *E. coli* genome: lacZ (encoding β-galactosidase), wecB (encoding UDP-N-acetylglucosamine-2-epimerase), ugd (encoding UDP-glucose-6-dehydrogenase), nagB (encoding glucosamine-6-phosphate deaminase), pfkA (encoding 6-phosphofructokinase), gloA (encoding lactylglutathione lyase), and setA (encoding a glucose efflux transporter). Then, the following genes are overexpressed: lgtA (encoding β-1,3-acetylglucosamine transferase), wbgO (encoding β-1,3-galactosyltransferase), galE (UDP-glucose-4-epimerase), galT (galose-1-phosphotransferase), and galK (galkinase).

[0037] In one embodiment of the present invention, the pTargetF plasmid is used to knock out the following genes: lacZ (encoding β-galactosidase), wecB (encoding UDP-N-acetylglucosamine-2-epimerase), ugd (encoding UDP-glucose-6-dehydrogenase), nagB (encoding glucosamine-6-phosphate deaminase), pfkA (encoding 6-phosphofructokinase), gloA (encoding lactylglutathione lyase), and setA (encoding a glucose efflux transporter).

[0038] In one embodiment of the present invention, the recombinant Escherichia coli integrates and expresses genes galE, galT and galK at gene locus yeeJ, and integrates and expresses genes lgtA and wbgO at gene locus CaiB.

[0039] The present invention also provides the application of the above-mentioned genetically engineered bacteria in the production of lactoyl-N-tetrasaccharide and products containing lactoyl-N-tetrasaccharide.

[0040] The present invention also provides a method for producing lactyl-N-tetrasaccharide, wherein lactose and glycerol are used as carbon sources, and the above-mentioned genetically engineered bacteria are used as fermentation strains to produce lactyl-N-tetrasaccharide.

[0041] In one embodiment, the genetically engineered bacteria are inoculated into a fermentation medium and cultured until OD (Organic Degree) reaches 100%.600 The concentration is 14-20, and lactose and IPTG are added to a final concentration of 20 g / L.

[0042] In one embodiment, after the initial carbon source is consumed, 750-850 g / L glycerol and 15-25 g / L MgSO4·7H2O are added. After the initial lactose is consumed, 200 g / L lactose aqueous solution is added to maintain the final lactose concentration at 3-10 g / L.

[0043] In one embodiment, the fermentation conditions are as follows: culture temperature of 24~38℃, stirring speed of 250~850 r / min, aeration rate of 0.8~1.2 vvm, pH of 6.5~7.0, and fermentation time of 15~55 h.

[0044] In one embodiment, the fermentation medium consists of: 10-20 g / L glycerol, 10-15 g / L potassium dihydrogen phosphate, 2-6 g / L diammonium hydrogen phosphate, 1-2 g / L citric acid, 1-2 g / L magnesium sulfate heptahydrate, and 7.5-12.5 mL / L trace metal elements, with the remainder being water.

[0045] In one embodiment, the trace metal elements are composed of: 8~12 g / L ferrous sulfate, 2~2.5 g / L zinc sulfate heptahydrate, 0.5~1.5 g / L anhydrous copper sulfate and 1.5~2.5 g / L calcium chloride dihydrate.

[0046] The beneficial effects of this invention are:

[0047] This invention constructs an engineered strain capable of efficiently synthesizing lactoyl-N-tetrasaccharides by combining and regulating the expression of lgtA, wbgO, galE, galT, and galK genes in the lactoyl-N-tetrasaccharide synthesis pathway, further knocking out the side-branch metabolic pathway genes lacZ, wecB, ugd, nagB, pfkA, gloA, and setA in the lactoyl-N-tetrasaccharide synthesis pathway of *E. coli*, and optimizing the integrated expression of core genes. In shake-flask experiments, the ability of *E. coli* to synthesize lactoyl-N-tetrasaccharides increased from 3.41 g / L to 11.15 g / L. In fed-batch culture in a 3 L fermenter, the yield of lactoyl-N-tetrasaccharides reached 125.35 g / L. This invention provides a key technological foundation for the efficient manufacturing of lactoyl-N-tetrasaccharides and lays an important foundation for the large-scale, industrialized production of high-value-added functional oligosaccharides. Attached Figure Description

[0048] Figure 1 This is a diagram of the lactyl-N-tetrasaccharide metabolic pathway.

[0049] Figure 2High-performance liquid chromatograms of lactyl-N-tetrasaccharide standard and lactyl-N-tetrasaccharide product samples Detailed Implementation

[0050] The following examples and accompanying drawings further illustrate the specific implementation of the present invention. The plasmids, PCR reagents, restriction endonucleases, plasmid extraction kits, DNA gel recovery kits, etc. used in the following examples are commercial products, and the specific operations are performed in accordance with the kit instructions.

[0051] The embodiments of the present invention are not limited thereto; other unspecified experimental operations and process parameters shall be carried out in accordance with conventional techniques.

[0052] The sequencing of plasmids and DNA products was outsourced to Shanghai Sangon Biotech Co., Ltd.

[0053] Preparation of competent Escherichia coli cells: reagent kit from Shanghai Sangon Biotech Co., Ltd.

[0054] LB liquid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride.

[0055] LB solid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 17 g / L agar powder.

[0056] The method for determining lactyl-N-tetrasaccharide described in this embodiment of the invention uses high-performance liquid chromatography (HPLC). Specifically, 1 mL of fermentation broth is centrifuged at 12000 g for 10 min, and the supernatant is filtered through a 0.22 μm membrane. The amount of lactyl-N-tetrasaccharide produced is then detected using HPLC. HPLC detection conditions: Agilent 1260 Infinity II RID differential refractive index detector; Rezex R0A-ORGAN 00A-0138-E0 column (Agilent Technologies, USA); column temperature 30ºC; mobile phase 70% acetonitrile aqueous solution; flow rate 1.0 mL / min; injection volume 10 μL.

[0057] The shake-flask fermentation method is as follows:

[0058] The constructed engineered bacteria were inoculated into LB liquid medium and cultured in shake flasks at 37°C and 200 rpm for 12 h to obtain seed culture. The seed culture was then inoculated into 50 mL of fermentation medium at an inoculation rate of 2 mL / 100 mL and cultured in shake flasks at 37°C and 200 rpm until OD reached. 600 The concentration was 0.6; IPTG was added to a final concentration of 0.4 mM, and lactose was added at a concentration of 10 g / L. The mixture was induced and cultured at 25℃ and 200 rpm for 96 h.

[0059] Fermentation medium: 20 g / L glycerol, 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 trace metal elements (10 mg / L ferrous sulfate, 2.25 mg / L zinc sulfate heptahydrate, 1.0 mg / L anhydrous copper sulfate, 2.0 mg / L calcium chloride dihydrate), pH 6.8.

[0060] pRSFDuet-1: Novagen (WI, USA)

[0061] pETDuet-1: Novagen (WI, USA)

[0062] pCDFDuet-1: Novagen (WI, USA)

[0063] pACYDuet-1: Novagen (WI, USA)

[0064] pCOLDuet-1: Novagen (WI, USA)

[0065] Example 1: Genetic manipulation of E. coli BL21(DE3) chromosome genes lacZ, wecB, ugd, nagB, pfkA, gloA, and setA, and the integration of genes galE-galT-galK and lgtA-wbgO into the yeeJ and CaiB gene loci, respectively.

[0066] The lacZ, wecB, ugd, nagB, pfkA, gloA, and setA genes in the *E. coli* BL21(DE3) genome were knocked out using the CRISPR-Cas9 gene knockout system, and the galE-galT-galK and lgtA-wbgO genes were integrated into the yeeJ and CaiB gene loci, respectively. The specific steps are as follows (primer sequences involved are shown in Table 1):

[0067] (1) Using the Escherichia coli BL21(DE3) genome as a template, upstream and downstream fragments of lacZ, ugd and wecB were amplified by PCR using primer pairs lacZ-up-F / R and lacZ-down-F / R, wecB-up-F / R and wecB-down-F / R, ugd-up-F / R and ugd-down-F / R, nagB-up-F / R and nagB-down-F / R, pfkA-up-F / R and pfkA-down-F / R, gloA-up-F / R and setA-down-F / R, setA-up-F / R and setA-down-F / R, and the fragments were recovered by gel. Using upstream and downstream fragments of lacZ, wecB, ugd, nagB, pfkA, gloA, and setA as templates, complete homologous templates for lacZ, wecB, ugd, nagB, pfkA, gloA, and setA were obtained through overlap PCR using primers lacZ-up-F / lacZ-down-R, wecB-up-F / wecB-down-R, nagB-up-F / nagB-down-R, pfkA-up-F / pfkA-down-R, gloA-up-F / gloA-down-R, and setA-up-F / setA-down-R. DNA fragments were then recovered via gel electrophoresis. Colony PCR amplification of *E. coli* BL21(DE3) was performed using two primer pairs, yeeJ-up-F / R and yeeJ-down-F / R, and the upstream and downstream fragments of the gene were obtained after gel electrophoresis. Using the previously constructed plasmid pCDF-galETK as a template (Table 2), primer pair P was used. T7 -galETK-F / R amplified the inserted gene fragment P via PCR. T7 -galE-galT-galK, gel recovery. Then, using the upstream and downstream fragments of yeeJ and the inserted gene fragment P respectively. T7 Using -galE-galT-galK as a template, overlap extension PCR was used to amplify P containing the insert fragment. T7 The homology repair template for -galE-galT-galK was used, followed by gel extraction to obtain the correct band and DNA fragments. Colony PCR amplification of *E. coli* B-L21(DE3) was performed using two primer pairs, yeeJ-up-F / R and yeeJ-down-F / R, and the upstream and downstream fragments of the gene were obtained by gel extraction. Further, using the previously constructed plasmid pCDF-lgtA-wbgO as a template (Table 2), primer pair P... T7 -WO-F / R amplifies the inserted gene fragment P via PCR. T7 -lgtA-P T7-wbgO, gel recovery. Then, using the upstream and downstream fragments of caiB and the inserted gene fragment P respectively. T7 Using -lgtA-wbgO as a template, overlap extension PCR was used to amplify P containing the insert fragment. T7 -lgtA-P T7 -wbgO is the homology repair template, and then the correct band is recovered by gel electrophoresis to obtain the DNA fragment.

[0068] (2) Using the original pTargetF plasmid (Addgene: #62226) as a template, and lacZ-sg-F / R, wecB-sg-F / R, ugd-sg-F / R, nagB-sg-F / R, pfkA-sg-F / R, gloA-sg-F / R, setA-sg-F / R, yeeJ-sg-F / R, and caiB-sg-F / R as primers, PCR amplification was performed to replace the N20 sequence on the original plasmid with N20 sequences complementary to the sequences of lacZ, wecB, ugd, nagB, pfkA, gloA, setA, yeeJ, and caiB, respectively, to obtain the pTargetF plasmid targeting lacZ. The following plasmids were used: pTargetF targeting wecB, pTargetF targeting ugd, pTargetF targeting nagB, pTargetF targeting pfkA, pTargetF targeting gloA, pTargetF targeting setA, pTargetF targeting yeeJ, and pTargetF targeting caiB (i.e., pTargetF plasmids with N20 specific sequences for lacZ, wecB, ugd, nagB, pfkA, gloA, setA, yeeJ, and caiB). These plasmids were transformed into *E. coli* DH5α competent cells, plated on LB agar plates (containing spectinomycin), cultured at 30°C, and plasmids were extracted and sequenced.

[0069] (3) Take pCas plasmid (Addgene: #60847) and Escherichia coli BL21(DE3) competent cells, place them on ice for 5 min until the competent cells thaw, take 5 μL of plasmid and add it to 100 μL of competent cells, and mix gently. Incubate on ice for 30 min, heat shock at 42℃ for 90 s, and immediately place on ice for 5 min. Add 1 mL of LB medium and incubate at 30℃ and 180 rpm for 1 h. Take 200 μL of concentrated bacterial solution and spread it evenly on LB agar plates (containing kanamycin), and incubate upside down at 30℃ overnight until single colonies of Escherichia coli BL21(DE3) / pCas grow.

[0070] (4) Single colonies of *E. coli* BL21(DE3) / pCas were picked and cultured in LB medium at 30°C for 1.0 h. L-arabinose was added to a final concentration of 30 mM to induce pCas-λ-red system expression. When OD 600 When the concentration reaches 0.6-0.8, Escherichia coli BL21(DE3) / pCas competent cells are prepared.

[0071] (5) 200 ng of the target plasmid pTargetF with the lacZ-specific N20 sequence constructed in step (2) and 1000 ng of donor DNA fragment (i.e. the complete lacZ template obtained in step (1)) were electroporated into E. coli BL21(DE3) / pCas competent cells prepared in step (4), plated on LB plates (kanamycin and spectinomycin), and cultured at 30℃ for 24 h. Positive colonies on the plates were picked and cultured in LB for 10 h, and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing verification.

[0072] (6) Pick the positive clones that were successfully knocked out in step (5) into 4 mL LB liquid tubes, add IPTG to a final concentration of 1 mM and kanamycin to 30 mg / L, and incubate at 30℃ for 8-16 h to remove pTargetF plasmid. Then incubate at 42℃ for 12 h to remove pCas plasmid and obtain Escherichia coli BL21(DE3) ΔlacZ with the lacZ gene knocked out.

[0073] (7) Using Escherichia coli BL21(DE3) ΔlacZ as the host bacterium, the same method was used to combine the target plasmid pTargetF with the wecB-specific N20 sequence obtained in step (2) and 1000 ng of donor DNA fragment (i.e. the complete wecB template obtained in step (1)) to knock out the wecB gene in the genome of Escherichia coli BL21(DE3) ΔlacZ, so as to obtain the corresponding lacZ gene of Escherichia coli BL21(DE3) and the wecB gene knockout strain Escherichia coli BL21(DE3) ΔlacZΔwecB.

[0074] (8) Using Escherichia coli BL21(DE3) ΔlacZΔwecB as the host bacterium, the same method was used to combine the target plasmid pTargetF with the ugd-specific N20 sequence obtained in step (2) and 1000 ng of donor DNA fragment (i.e. the complete ugd template obtained in step (1)) to knock out the ugd gene in the genome of Escherichia coli BL21(DE3) ΔlacZΔwecBΔugd.

[0075] (9) Using Escherichia coli BL21(DE3) ΔlacZΔwecBΔugd as the host bacterium, the same method was used to combine the target plasmid pTargetF with the nagB-specific N20 sequence obtained in step (2) and 1000 ng of donor DNA fragment (i.e. the complete nagB template obtained in step (1)) to knock out the nagB gene in the genome of Escherichia coli BL21(DE3) ΔlacZΔwecBΔugd, so as to obtain the corresponding Escherichia coli BL21(DE3) lacZ, wecB, ugd and nagB gene knockout strain Escherichia coli BL21(DE3) ΔlacZΔwecBΔugdΔnagB.

[0076] (10) Using Escherichia coli BL21(DE3) ΔlacZΔwecBΔugdΔnagB as the host bacterium, the same method was used to combine the target plasmid pTargetF with the pfkA-specific N20 sequence obtained in step (2) and 1000 ng of donor DNA fragment (i.e. the complete pfkA template obtained in step (1)) to knock out the pfkA gene in the genome of Escherichia coli BL21(DE3) ΔlacZΔwecBΔugdΔnagB, so as to obtain the corresponding Escherichia coli BL21(DE3) lacZ, wecB, ugd, nagB and pfkA gene knockout strain Escherichia coli BL21(DE3) ΔlacZΔwecBΔugdΔnagBΔpfkA.

[0077] (11) Using Escherichia coli BL21(DE3) ΔlacZΔwecBΔugdΔnagBΔpfkA as the host bacterium, the same method was used to combine the target plasmid pTargetF with the gloA-specific N20 sequence obtained in step (2) and 1000 ng of donor DNA fragment (i.e. the complete gloA template obtained in step (1)) to knock out the gloA gene in the genome of Escherichia coli BL21(DE3) ΔlacZΔwecBΔugdΔnagBΔpfkA, so as to obtain the corresponding Escherichia coli BL21(DE3) lacZ, wecB, ugd, nagB, pfkA and gloA gene knockout strain Escherichia coli BL21(DE3) ΔlacZΔwecBΔugdΔnagBΔpfkAΔgloA.

[0078] (12) Using Escherichia coli BL21(DE3) ΔlacZΔwecBΔugdΔnagBΔpfkAΔgloA as the host bacterium, the same method was used to combine the target plasmid pTargetF with the setA-specific N20 sequence obtained in step (2) and 1000 ng of donor DNA fragment (i.e. the complete setA template obtained in step (1)) to knock out the setA gene in the genome of Escherichia coli BL21(DE3) ΔlacZΔwecBΔugdΔnagBΔpfkAΔgloA, so as to obtain the corresponding Escherichia coli BL21(DE3) lacZ, wecB, ugd, nagB, pfkA, gloA and setA gene knockout strain Escherichia coli BL21(DE3) ΔlacZΔwecBΔugdΔnagBΔpfkAΔgloAΔsetA.

[0079] (13) Using the same method, combine the target plasmid pTargetF with the yeeJ-specific N20 sequence obtained in step (2) with 800 ng of donor DNA fragment (i.e., the complete P obtained in step (1)). T7 -galE-galT-galK homology repair template), integrating P T7 -galE-galT-galK was incorporated into the yeeJ gene locus of the *E. coli* BL21(DE3) genome ΔlacZΔwecBΔugdΔnagBΔpfkAΔgloAΔsetA, resulting in the knockout of the corresponding *E. coli* BL21(DE3) genes lacZ, wecB, ugd, nagB, pfkA, gloA, and setA, as well as the integration of P into the yeeJ gene locus. T7 -galE-galT-galK gene fragment of E. coli BL21(DE3) ΔlacZΔwecBΔugdΔnagBΔpfkAΔgloAΔsetAΔyeeJ::P T7 -galE-galT-galK.

[0080] (14) Using the same method, combine the target plasmid pTargetF with the caiB-specific N20 sequence obtained in step (2) with 800 ng of donor DNA fragment (i.e., the complete P obtained in step (1)). T7 -lgtA-P T7 -wbgO's homology repair template), integrating P T7 -lgtA-P T7 -wbgO to E. coli BL21(DE3) ΔlacZΔwecBΔugdΔnagBΔpfkAΔgloAΔsetAΔyeeJ::P T7The caiB gene locus of the -galE-galT-galK genome was used to obtain the corresponding lacZ, wecB, ugd, nagB, pfkA, gloA, and setA gene knockouts in E. coli BL21(DE3), and the integration of P into the yeeJ and caiB gene loci, respectively. T7 -galE-galT-galK and P T7 -lgtA-P T7 E. coli BL21(DE3) with the -wbgO gene fragment ΔlacZΔwecBΔugdΔnagBΔpfkAΔgloAΔsetAΔyeeJ::P T7 -galE-galT-galKΔcaiB::P T7 -lgtA-P T7 -wbgO.

[0081] Table 1. Primer sequences for gene knockout and primer sequences for integration into the yeeJ and caiB gene sites.

[0082]

[0083] Example 2: Construction and screening of recombinant plasmids of lactoyl-N-tetrasaccharide engineered bacteria

[0084] The specific steps for constructing recombinant plasmids are as follows (the primer sequences involved are shown in Table 2):

[0085] (1) Construction of galE-galT-galK gene expression plasmids: Using the genome of Escherichia coli K-12 as a template, and galETK-F / galETK-R as primers, the galE-galT-galK gene fragment was amplified by PCR. The DNA fragment was recovered by gel extraction. The recovered galE-galT-galK gene fragment was ligated between the BamHI and SaiI restriction sites of vectors pRSFDuet-1, pETDuet-1, pCDFDuet-1, pACYDuet-1 and pCOLDuet-1 respectively using a seamless cloning kit (Nanjing Novizan Biotechnology Co., Ltd.) to obtain plasmids pRSF-galETK, pET-galETK, pCDF-galETK, pACY-galETK and pCOL-galETK.

[0086] (2) Construction of the lgtA-wbgO gene expression plasmid: The lgtA gene from Neisseria meningitidis and the wbgO gene from Escherichia coli O55:H7 were synthesized by Shanghai Sangon Biotech Co., Ltd. The synthesized lgtA gene fragment was amplified by PCR using lgtA-F / R as primers. The DNA fragment was recovered by gel cloning and ligated between the BamHI and SaiI restriction sites of the vector pCDF-lgt-1 using a seamless cloning kit (Nanjing Novizan Biotechnology Co., Ltd.) to obtain the plasmid pCDF-lgtA. The synthesized wbgO gene fragment was amplified by PCR using wbgO-F / R as primers. The DNA fragment was recovered by gel cloning and ligated between the BgiII and XhoI restriction sites of the vector pCDF-lgtA using a seamless cloning kit (Nanjing Novizan Biotechnology Co., Ltd.) to obtain the plasmid pCDF-lgtA-wbgO. Using lgtA-F / wbgO-R as primers, the lgtA-wbgO gene fragment was amplified by PCR. The DNA fragment was recovered by gel extraction, and the recovered lgtA-wbgO gene fragment was ligated to the BamHI and XhoI restriction sites of vectors pRSFDuet-1, pETDuet-1, pACYDuet-1, and pCOLDuet-1 respectively using a seamless cloning kit (Nanjing Novizan Biotechnology Co., Ltd.) to obtain plasmids pRSF-lgtA-wbgO, pET-lgtA-wbgO, pACY-lgtA-wbgO, and pCOL-lgtA-wbgO.

[0087] Table 2. Primers for plasmid construction

[0088]

[0089] (3) Based on the key genes in the lactyl-N-tetrasaccharide synthesis pathway, the plasmids pRSF-galETK, pET-galETK, pCDF-galETK, pACY-galETK, pCOL-galETK, pRSF-lgtA-wbgO, pET-lgtA-wbgO, pCDF-lgtA-wbgO, pACY-lgtA-wbgO, and pCOL-lgtA-wbgO obtained in step (1) were combined and transformed into Escherichia coli BL21(DE3) ΔlacZ in Example 1. A total of 20 different engineered bacteria were obtained, which are represented as B1, B2, B3, B4, B5, B6, B7, B8, B9, B10, B11, and B12, respectively. Table 3 shows the specific combinations of key gene plasmids in the lactoyl-N-tetrasaccharide synthesis pathway carried by strains B13, B14, B15, B16, B17, B18, B19, and B20, as well as the lactoyl-N-tetrasaccharide yield after fermentation of the engineered strains. The plasmids pRSFDuet-1, pETDuet-1, pCDFDuet-1, pACYCDuet-1, and pCOLADuet-1 are known to have the highest, higher, medium, lower, and lowest copy numbers, respectively. The changes in lactoyl-N-tetrasaccharide yield after fermentation of the engineered strains were identified by high-performance liquid chromatography (HPLC). Figure 2 As shown in Table 3, all strains produced lactyl-N-tetrasaccharide, but the yields varied considerably. The highest lactyl-N-tetrasaccharide yield (3.41 g / L) was observed with the pCDF-lgtA-wbgO and pET-galETK plasmid combination. These results suggest that relatively high expression levels of the lgtA-wbgO and galE-galT-galK genes may lead to a burden on cell growth, while low expression levels of the galE-galT-galK gene may result in insufficient supply of the lactyl-N-tetrasaccharide precursor nucleotide sugar UDP-Gal, thus hindering the efficient metabolic synthesis of lactyl-N-tetrasaccharide.

[0090] Table 3. Detailed information on each engineered microorganism

[0091]

[0092] Example 3: Verification of knockout of catabolic genes wecB, nagB, ugd, pfkA, gloA, and setA in the lactoyl-N-tetrasaccharide synthesis pathway

[0093] To investigate the effect of blocking competitive carbon flux in the lactoyl-N-tetrasaccharide synthesis pathway, this embodiment was based on the engineered strain of Escherichia coli BL21(DE3) ΔlacZ. Six genes (wecB, nagB, ugd, pfkA, gloA, and setA) related to the lactoyl-N-tetrasaccharide side metabolic pathway were knocked out in sequence to obtain single-gene mutants and multi-gene combination mutants. The wecB gene is involved in the conversion of UDP-N-acetylglucosamine to UDP-N-acetylmmannosamine, serving as a branch node in the cell wall O-antigen biosynthesis pathway. ugd encodes UDP-glucose-6-dehydrogenase, catalyzing the oxidative conversion of UDP-glucose to UDP-glucuronic acid, acting as a precursor supply node in the cell wall lipopolysaccharide and extracellular polysaccharide biosynthesis pathways; its knockout reduces competitive consumption of UDP-glucose. nagB encodes N-acetylglucosamine-6-phosphate deaminase, converting GlcNAc-6-P to fructose-6-phosphate. pfkA encodes phosphofructokinase I, a key rate-limiting enzyme in the glycolysis pathway. gloA encodes lactylglutathione lyase, involved in methylglyoxal bypass metabolism. setA encodes an H⁺ / hexose backbone derivative reverse sugar transporter, which can export key intermediates such as glucose-6-phosphate and glucose-1-phosphate extracellularly; its knockout effectively prevents leakage of key intracellular glycosyl donor precursors, increasing the production of UDP-Glc and UDP-Gal. Availability with UDP-GlcNAc. The highest quality granule combination screened in Example 3 was transformed into the knockout strains constructed in Example 1, resulting in strains B21, B22, B23, B24, B25, and B26, respectively. The lactyl-N-tetrasaccharide yields of these strains after shake-flask fermentation are shown in Table 4. The results showed that, based on the *E. coli* BL21(DE3)ΔlacZ engineered strain, strain B26, which had six side-branch metabolic pathway genes knocked out sequentially, had the highest yield (4.38 g / L). This indicates that blocking side-branch metabolic pathways helps improve the conversion of lactyl-N-tetrasaccharide and increase its titer.

[0094] Table 4. Detailed information on each engineered microorganism

[0095]

[0096] Example 4: Validation of the synthesis of lactyl-N-tetrasaccharide by integrating metabolic pathway genes galE-galT-galK and lgtA-wbgO

[0097] To improve the sugar donor generation capacity of engineered bacteria under lactose substrate conditions, thereby enhancing the synthesis efficiency of lactyl-N-tetrasaccharides, this embodiment integrates and expresses the metabolic pathway genes galE-galT-galK and lgtA-wbgO in the engineered strain *E. coli* BL21(DE3)ΔlacZΔwecBΔugdΔnagBΔpfkAΔgloAΔsetA, constructing a stable lactyl-N-tetrasaccharide synthesis module. Specifically, galE-galT-galK constitutes an efficient regeneration pathway for the lactose-galactosyl donor UDP-Gal: galK encodes galactokinase, phosphorylating galactose to Gal-1-P; galT encodes galactose-1-phosphoururidine transferase, converting Gal-1-P to UDP-Gal; and galE encodes UDP-galactose-4-epimerase, which can interconvert UDP-Gal and UDP-Glc, thus ensuring a sufficient supply of UDP-Gal donors. Meanwhile, lgtA and wbgO, as key glycosyltransferases for the bonding of lactyl groups to oligosaccharides in the lactyl-N-tetrasaccharide structure, catalyze the stepwise transfer of galactose and N-acetylglucosamine residues from donors such as UDP-Gal and UDP-GlcNAc to the acceptor substrate, ultimately completing the assembly of the lactyl-N-tetrasaccharide backbone. To avoid plasmid burden and metabolic instability, the galE-galT-galK module was integrated into the yeeJ gene locus of the *E. coli* chromosome according to Example 1, and the plasmid containing the lgtA-wbgO module was transformed into the above-mentioned strain to construct strain B27. In addition, the galE-galT-galK module and the lgtA-wbgO module were integrated into the yeeJ and caiB gene loci of the *E. coli* chromosome, respectively, according to Example 1, to construct strain B28. After shake-flask fermentation, the lactyl-N-tetrasaccharide yields of strains B27 and B28 reached 5.41 g / L and 11.15 g / L, respectively (see Table 4). Compared to strain B26, the yields were increased by 24.5% and 154.6%, respectively. These results indicate that constructing expression systems with no or low plasmid burden by integrating pathway genes can significantly enhance the biosynthetic capacity of lactyl-N-tetrasaccharide, providing important technical basis and strategy verification for the subsequent construction of high-yield engineered strains and optimization of metabolic pathways.

[0098] Example 5: Validation of high-efficiency engineered bacteria for batch feed production of lactyl-N-tetrasaccharides in a 3 L fermenter

[0099] To further verify the effectiveness of the lactyl-N-tetrasaccharide synthesis method and improve the yield of lactyl-N-tetrasaccharide, the constructed genetically engineered strain B28 was inoculated into LB liquid medium and cultured in shake flasks at 37℃ and 200 rpm for 12 h to obtain seed culture. The seed culture was then inoculated into a 1 L working volume fermentation medium at a volume ratio of 10%. The fermentation temperature in the fermenter was 37℃, the stirring speed was 900 rpm, the aeration rate was 1 vvm, and the pH was 6.8 (ammonia supplementation was automatically controlled). Fermentation was carried out for 12 h (OD... 600 Approximately 18), lactose was added to a final concentration of 20 g / L and 0.5 mM IPTG, and the fermentation temperature was adjusted to 25℃. To maintain cell growth and the synthesis of lactyl-N-tetrasaccharides, after the initial carbon source was consumed to a concentration of 3 g / L, 800 g / L glycerol (containing 20 g / L MgSO4·7H2O) was added to replenish the glycerol to a concentration of 20 g / L. After the initial lactose was consumed to a concentration of 3 g / L, 200 g / L of lactose aqueous solution was added to maintain the lactose concentration in the system at approximately 10 g / L until the end of fermentation. After the entire culture process reached 116 h, the cell OD... 600 The yield reached 138.6 g / L, with the highest yield of lactoyl-N-tetrasaccharide reaching 125.35 g / L. Table 5 shows the dynamic changes in lactoyl-N-tetrasaccharide synthesis during fermentation.

[0100] Table 5. Dynamic changes in lactyl-N-tetrasaccharide synthesis during fermentation

[0101]

[0102] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

[0103] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A genetically engineered bacterium producing lactosyl-N-tetraose, characterized by, The recombinant E. coli has the genes related to the decomposition of substrate lactose and key intermediate metabolites knocked out, and has the genes galE, galT and galK coding UDP-glucose-4-epimerase, galactose-1-phosphate uridylyltransferase and galactokinase integrated at the yeeJ gene of the E. coli genome, and has the genes lgtA and wbgO coding β-1,3-N-acetylglucosamine transferase and β-1,3-galactosyltransferase integrated at the CaiB gene of the E. coli genome; The knocking out specifically refers to knocking out the genes lacZ coding β-galactosidase, wecB coding UDP-N-acetylglucosamine-2-epimerase, ugd coding UDP-glucose-6-dehydrogenase, nagB coding glucosamine-6-phosphate deaminase, pfkA coding 6-phosphofructokinase, gloA coding lactoylglutathione lyase and setA coding sugar efflux transporter. 2.The genetically engineered bacteria according to claim 1, characterized in that, The genes galE, galT and galK coding UDP-glucose-4-epimerase, galactose-1-phosphate uridylyltransferase and galactokinase are all derived from E. coli K-12, the gene lgtA coding β-1,3-N-acetylglucosamine transferase is derived from Neisseria meningitidis, and the gene wbgO coding β-1,3-galactosyltransferase is derived from E. coli O55:H7.

3. The genetically engineered bacteria according to claim 1 or 2, characterized in that, The host is E. coli BL21 (DE3).

4. The genetically engineered bacterium according to any one of claims 1-3 for use in the production of lacto-N-tetraose and products containing lacto-N-tetraose.

5. A bioengineering method for efficient synthesis of lacto-N-tetraose, characterized by, The genetically engineered bacterium according to any one of claims 1-3 is used for the fermentation production of lacto-N-tetraose with lactose and glycerol as carbon sources and 0.2-1.0 mM IPTG as inducer.

6. The method of claim 5, wherein, The fermentation conditions are as follows: the culture temperature is 24-38℃, the stirring speed is 250-900 r / min, the aeration amount is 0.8-1.2 vvm, and the pH is 6.5-7.0.

Images