A genetically engineered bacterium that produces lactose-N-tetrasaccharide and its applications

Abstract

This invention relates to a genetically engineered bacterium for producing lactose-N-tetrasaccharides, characterized in that the bacterium expresses a gene for β-1,3-galactosyltransferase, and the gene for expressing β-1,3-galactosyltransferase is derived from *Citrobacter freundii*. The genetically engineered bacterium for producing lactose-N-tetrasaccharides provided by this invention has advantages such as high yield and suitability for industrial-scale production.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, and in particular to a genetically engineered bacterium that produces lactose-N-tetrasaccharide and its applications. Background Technology

[0002] Breast milk not only provides infants with essential nutrients but also contains crucial factors that protect them from infection and inflammation. Human lactose oligosaccharides (HMOs), which are based on lactose, are a typical example of these factors and play an irreplaceable role in promoting infant health. HMOs, with a concentration of 5-25 g / L, are the third most abundant component of breast milk, after lactose and lipids. To date, over 200 structurally different HMOs have been identified in human milk, constituting a major difference between human and animal milk. Clinical studies have shown that formula with added HMOs is safe and exhibits growth patterns similar to those of breastfed infants. To date, at least seven different HMOs have received Generally Recognized As Safe (GRAS) status from the U.S. Food and Drug Administration (FDA), and six of these have been added to various brands of infant formula.

[0003] Lactose-N-tetrasaccharide (LNT) is composed of galactose, acetylglucosamine, and glucose, accounting for approximately 6% (w / w) of total HMOs. LNT possesses beneficial physiological functions, including prebiotic properties, anti-adhesion, antibacterial activity, and antiviral effects. Currently reported LNT synthesis methods include chemical synthesis, enzymatic synthesis, and microbial production. Chemical synthesis is typically accompanied by the formation of chemical waste and byproducts, requiring multiple steps of protection and deprotection of functional groups. In contrast to enzymatic synthesis, microbial synthesis based on metabolic engineering does not require expensive donor and acceptor substrates and can begin with simple and inexpensive materials through fermentation, making it an effective and feasible method for large-scale LNT production. Therefore, research on microbial LNT synthesis is increasing.

[0004] UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-galactose (UDP-Gal) are important precursors for the synthesis of LNTs in cells. After glucose enters the cell, it is converted into UDP-GlcNAc and UDP-Gal through two pathways, respectively. (1) Synthesis pathway of UDP-GlcNAc: Glucose-6-phosphate (Glc-6-P) is converted into fructose-6-phosphate (F6P) under the catalysis of glucose-6-phosphate isomerase encoded by pgi. F6P is then converted into UDP-GlcNAc under the catalysis of glutamine-fructose-6-phosphate aminotransferase encoded by glmS, phosphoglucosamine mutase encoded by glmM, and N-acetylglucosamine-1-phosphate uridine transferase / glucosamine-1-phosphate acetyltransferase encoded by glmU. (2) Synthesis pathway of UDP-Gal: Glc-6-P is converted into UDP-Gal under the catalysis of glucose-6-phosphate mutase encoded by pgm and UDP-glucose-4-isomerase encoded by galE.

[0005] Existing research has yielded strategies to increase LNT production. For example, Liu and McArthur identified WbgO and Cvβ3GalT as β-1,3-galactosyltransferases that produce LNTs; Zhu et al. attempted to screen for new LNT-producing β-1,3-galactosyltransferases from 14 hypothesized β-1,3-galactosyltransferases, and among the seven glycosyltransferases homologous to WbgO and Cvβ3GalT, only Pseudogulbenkiania ferrooxidans β-1,3-galactosyltransferase was screened as capable of producing LNTs, with a higher LNT yield than WbgO and Cvβ3GalT; Li et al. identified Pseudogulbenkiania ferrooxidans β-1,3-galactosyltransferase as a key enzyme in LNT production, using uridine... 5'-triphosphate (UTP) regeneration and enhanced UDP-Gal supply further enhanced the synthesis of microbial LNTs, with LNT titers in shake flask culture and fed-batch culture significantly increasing to 6.16 g / L and 57.5 g / L, respectively. Summary of the Invention

[0006] The problem the invention aims to solve

[0007] To address the shortcomings of existing technologies, this invention provides a genetically engineered bacterium for producing lactose-N-tetrasaccharides, which has advantages such as high yield and suitability for industrial production.

[0008] Solution for solving the problem

[0009] On one hand, the present invention provides a genetically engineered bacterium that produces lactose-N-tetrasaccharide, wherein the genetically engineered bacterium expresses a gene for β-1,3-galactosyltransferase, and the gene for β-1,3-galactosyltransferase is derived from Citrobacter freundii.

[0010] Preferably, the genetically engineered bacteria have the β-galactosidase gene lacZ and / or the UDP-N-acetylglucosamine-2-epimerase gene wecB and / or the glucosamine-6-phosphate deaminase gene nagB knocked out.

[0011] Preferably, the genetically engineered bacteria express the β-1,3-acetylglucosamine transferase gene lgtA.

[0012] More preferably, the β-1,3-acetylglucosamine transferase gene lgtA is derived from Neisseria meningitidis.

[0013] Preferably, the genetically engineered bacteria also express the UDP-glucose-4-epimerase gene galE.

[0014] More preferably, the UDP-glucose-4-epimerase gene galE is derived from Escherichia coli.

[0015] Preferably, the amino acid sequence of the β-1,3-galactosyltransferase derived from Citrobacter freundii has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO.1, and is more preferably SEQ ID NO.1.

[0016] Preferably, the nucleotide sequence of the β-1,3-galactosyltransferase derived from Citrobacter freundii has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO.2, and is more preferably SEQ ID NO.2.

[0017] Preferably, the nucleotide sequence of the β-1,3-acetylglucosamine transferase gene lgtA derived from Neisseria meningitidis has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO.3, and is more preferably SEQ ID NO.3.

[0018] Preferably, the nucleotide sequence of the UDP-glucose-4-epimerase gene galE derived from Escherichia coli has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO.4, and is more preferably SEQ ID NO.4.

[0019] On the one hand, the present invention provides a biological agent comprising the genetically engineered bacteria described in any of the above claims.

[0020] On the one hand, the present invention provides an application of the above-mentioned genetically engineered bacteria or the above-mentioned biological agents in the production of lactose-N-tetrasaccharide.

[0021] On the one hand, the present invention provides a method for producing lactose-N-tetrasaccharide, using glucose as a carbon source and lactose as a substrate, and using the above-mentioned genetically engineered bacteria or the above-mentioned biological agents to ferment and produce lactose-N-tetrasaccharide.

[0022] The effects of the invention

[0023] This invention achieves efficient and high-yield synthesis of lactose-N-tetrasaccharide by introducing and expressing the β-1,3-galactosyltransferase gene from *Citrobacter freundii* into a genetically engineered bacterium. The preparation of lactose-N-tetrasaccharide using this genetically engineered bacterium also offers advantages such as simple culture medium, readily available and inexpensive substrates, rapid strain growth, genetic stability, and high expression levels, demonstrating significant potential for industrial-scale production. Detailed Implementation

[0024] Microbial metabolic engineering synthesis is considered the most efficient route for large-scale production of LNTs. Studies have confirmed that β-1,3-galactosyltransferase is the limiting step in the galactose transfer reaction from LNT II to LNT during microbial synthesis. To date, only three β-1,3-galactosyltransferases have been identified capable of producing LNTs. Research has shown that in in vitro enzymatic reactions, LNTs can be successfully synthesized from LNT II using β-1,3-galactosyltransferase (wbgO) from Escherichia coli O55:H7 and the gene encoding β-1,3-galactosyltransferase (Cvβ3GalT) from C. violaceum. However, heterologous expression in E. coli presents challenges such as difficulty in soluble expression and easy degradation. However, β-1,3-galactosyltransferase derived from Pseudogulbenkiania ferrooxidans can efficiently produce LNT from LNT II through metabolic engineering, and it has good solubility and high yield. Although it has the highest yield in existing studies, it is still far from enough for large-scale practical production applications.

[0025] Through extensive and in-depth research and screening, the inventors unexpectedly discovered that introducing the β-1,3-galactosyltransferase gene from *Citrobacter freundii* into the starting strain, while simultaneously knocking out the *lacZ*, *wecB*, and *nagB* genes, and introducing β-1,3-acetylglucosamine transferase (lgtA) from *Neisseria meningitidis* and UDP-glucose 4 epimerase (galE) from *Escherichia coli*, significantly improved LNT production capacity. Based on this discovery, the inventors completed this invention.

[0026] To make the technical solution and beneficial effects of the present invention more apparent and understandable, a detailed description is provided below by listing specific embodiments. Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application pertains.

[0027] On one hand, the present invention provides a genetically engineered bacterium that produces lactose-N-tetrasaccharide, wherein the genetically engineered bacterium expresses a gene for β-1,3-galactosyltransferase, and the gene for β-1,3-galactosyltransferase is derived from Citrobacter freundii.

[0028] In some embodiments, the genetically engineered strain is selected from genetically engineered Escherichia coli.

[0029] In some embodiments, the genetically engineered strain is selected from the genetically engineered Escherichia coli (E. coli) BL21(DE3) strain.

[0030] In some implementations, chassis strains are constructed using genetic engineering knockout techniques to reduce the metabolic flux of the UDP-GlcNAc bypass pathway.

[0031] In some embodiments, the genetically engineered bacteria have the β-galactosidase gene lacZ knocked out.

[0032] In some embodiments, the nucleotide sequence of the lacZ is as described in SEQ ID NO.5.

[0033] SEQ ID NO.5

[0034]

[0035] In some implementations, the UDP-N-acetylglucosamine-2-epimerase gene wecB.

[0036] In some embodiments, the nucleotide sequence of the wecB is as described in SEQ ID NO.6.

[0037] SEQ ID NO.6

[0038]

[0039] In some embodiments, it is the glucosamine-6-phosphate deaminase gene nagB.

[0040] In some embodiments, the nucleotide sequence of said nagB is as set forth in SEQ ID NO. 7.

[0041] SEQ ID NO. 7

[0042] ATGAGACTGATCCCCCTGACTACCGCTGAACAGGTCGGCAAATGGGCTGCTCGCCATATCGTCAATCGTATCAATGCGTTCAAACCGACTGCTGATCGTCCGTTTGTTCTGGGCCTGCCGACTGGCGGCACGCCGATGACCACCTATAAAGCGTTAGTCGAAATGCATAAAGCAGGCCAGGTCAGCTTTAAGCACGTTGTCACCTTCAACATGGACGAATATGTCGGTCTGCCGAAAGAGCATCCGGAAAGCTACTACAGCTTTATGCACCGTAATTTCTTCGATCACGTTGATATTCCAGCAGAAAACATCAACCTTCTCAACGGCAACGCCCCGGATATCGACGCCGAGTGCCGCCAGTATGAAGAAAAAATCCGTTCTTACGGAAAAATTCATCTGTTTATGGGCGGTGTAGGAAACGACGGTCATATTGCATTTAACGAACCGGCGTCCTCTCTGGCTTCTCGTACTCGTATCAAAACCTTGACTCATGACACTCGCGTCGCAAACTCTCGTTTCTTTGATAACGATGTCAATCAGGTGCCAAAATATGCCCTGACTGTCGGTGTTGGTACATTGCTGGATGCCGAAGAAGTGATGATTCTGGTGCTGGGTAGCCAGAAAGCACTGGCACTGCAGGCCGCCGTTGAAGGTTGCGTAAACCACATGTGGACCATCAGCTGTCTGCAACTGCATCCAAAAGCGATCATGGTGTGCGATGAACCTTCCACCATGGAGCTGAAAGTTAAGACTTTAAGATATTTCAATGAATTAGAAGCAGAAAATATCAAAGGTCTGTAA

[0043] In some embodiments, the genetically engineered bacteria express the β-1,3-acetylglucosamine transferase gene lgtA.

[0044] In some embodiments, the β-1,3-acetylglucosamine transferase gene lgtA is derived from Neisseria meningitidis.

[0045] In some embodiments, the genetically engineered bacteria also express the UDP-glucose-4-epimerase gene galE.

[0046] In some embodiments, the UDP-glucose-4-epimerase gene galE is derived from Escherichia coli.

[0047] In some embodiments, the UDP-glucose-4-epimerase gene galE is derived from Escherichia coli MG1655.

[0048] In some embodiments, the amino acid sequence of the β-1,3-galactosyltransferase derived from Citrobacter freundii has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO.1.

[0049] In some embodiments, the amino acid sequence of the β-1,3-galactosyltransferase derived from Citrobacter freundii is shown in SEQ ID NO.1.

[0050] SEQ ID NO.1

[0051] MIIDETESAQSSYPVVSVILPVNKKNPFFDEAINSILSQTFSLFEIIIVANCCTDDFFNELKCKVNDKIKLVRTDIAYLPYSLNKAIDLSRGEFIARMDSDDISHPDRFTKQVSFLKNNPHVDVVGTNAIFID EKGLEISKTKLPEENLDIVKTLPYKCCIVHPSVMFRKEVIASMGGYMFSNYSEDYELWNRLSLAKIRFQNLPEYLFYYRLHEGQSTDKKNLYIVMVNDLAIKMKCFFVTGNIHYIFGGIRTIASVIYCKYIK*

[0052] In some embodiments, the nucleotide sequence of the β-1,3-galactosyltransferase derived from Citrobacter freundii has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO. 2.

[0053] In some embodiments, the nucleotide sequence of the β-1,3-galactosyltransferase derived from Citrobacter freundii is shown in SEQ ID NO.2.

[0054] SEQ ID NO.2

[0055] ATGATTATCGATGAGACAGAAAGCGCCCAAAGCTCCTATCCGGTCGTGTCCGTTATTCTTCCGGTGAACAAGAAGAATCCGTTCTTTGATGAAGCAATCAATTCAATTTTATCGCAGACCTTCAGCTTGTTCGAAATCATCATTGTAGCTAACTGCTGCACGGATGACTTCTTCAATGAATTGAAATGCAAAGTAAACG ATAAAATTAAACTGGTGCGTACGGATATCGCATACTTACCGTATTCCCTGAACAAAGCCATCGACCTGTCGCGTGGCGAATTCATCGCGCGTATGGACAGCGATGACATCAGCCACCCGGATCGCTTTACTAAACAAGTTTCGTTCCTGAAGAATAACCCGCATGTTGATGTGGTTGGCACGAATGCGATCTTTATTGAT GAGAAAGGTCTGGAAATCTCTAAAACGAAACTGCCGGAAGAGAATCTGGATATTGTTAAGACCTTGCCGTACAAATGTTGCATCGTGCACCCGTCCGTCATGTTCCGGAAAGAAGTCATCGCCTCGATGGGCGGCTACATGTTCTCTAACTATTCTGAAGATTACGAACTCTGGAACCGCCTGTCACTTGCCAAAATCC GCTTCCAGAACCTGCCAGAATACCTCTTCTACTATCGCCTGCACGAAGGTCAATCAACTGATAAGAAGAACCTGTACATTGTAATGGTTAACGACCTGGCTATCAAGATGAAATGCTTCTTTGTGACCGGTAACATTCACTATATCTTTGGTGGCATTCGCACCATTGCGTCCGTGATCTATTGCAAGTATATTAAATAG

[0056] In some embodiments, the nucleotide sequence of the β-1,3-acetylglucosamine transferase gene lgtA derived from Neisseria meningitidis has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO.3.

[0057] In some embodiments, the nucleotide sequence of the β-1,3-acetylglucosamine transferase gene lgtA derived from Neisseria meningitidis is shown in SEQ ID NO.3.

[0058] SEQ ID NO.3

[0059]

[0060] In some embodiments, the nucleotide sequence of the UDP-glucose-4-epimerase gene galE derived from Escherichia coli has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO.4.

[0061] In some embodiments, the nucleotide sequence of the UDP-glucose-4-epimerase gene galE derived from Escherichia coli is shown in SEQ ID NO.4.

[0062] SEQ ID NO.4

[0063]

[0064] In some implementations, the actual sequence used is the same as or similar to the above-mentioned sequence, as well as the amino acid sequence translated from the nucleic acid sequence, and the codons can be optimized according to different expression strains before use.

[0065] This invention uses *Escherichia coli* as the starting strain and, by knocking out its Laz, wecB, and nagB genes, efficiently produces lactose-N-tetrasaccharide through plasmid overexpression of β-1,3-galactosyltransferase from *Citrobacter freundii*, β-1,3-acetylglucosamine transferase (lgtA) from *Neisseria meningitidis*, and UDP-glucose 4-epimerase (galE) from *E. coli*. The genetically engineered bacteria provided by this invention achieve a lactose-N-tetrasaccharide yield of 107.3 g / L in a 3L fermenter, showing great promise for large-scale industrial production. Furthermore, when the β-1,3-galactosyltransferase from *Citrobacter freundii* is heterologously expressed in the genetically engineered bacteria provided by this invention, the enzyme exhibits good soluble expression, high catalytic efficiency, and substrate specificity.

[0066] On the one hand, the present invention provides a biological agent comprising the genetically engineered bacteria described in any of the above claims.

[0067] On the one hand, the present invention provides an application of the above-mentioned genetically engineered bacteria or the above-mentioned biological agents in the production of lactose-N-tetrasaccharide.

[0068] On the one hand, the present invention provides a method for producing lactose-N-tetrasaccharide, using glucose as a carbon source and lactose as a substrate, and using the above-mentioned genetically engineered bacteria or the above-mentioned biological agents to ferment and produce lactose-N-tetrasaccharide.

[0069] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Conditions for experimental methods not specifically described in the following embodiments are generally in accordance with the conventional conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions described in the Journal of Microbiology: Laboratory Manual (edited by James Cappuccino and Natalie Sherman of Pearson Education Press), or the manufacturer's recommended conditions. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0070] Unless otherwise stated, all materials used in the examples are commercially available products.

[0071] In this specific embodiment, three different sources of β-1,3-galactosyltransferase disclosed in the prior art were selected for comparative experiments with the β-1,3-galactosyltransferase from Citrobacter freundii of the present invention. Information on the different sources of β-1,3-galactosyltransferase involved in the present invention is shown in Table 1.

[0072]

[0073] In this invention, lgtA was synthesized from N. meningitidis (lgtA) through codon optimization; the required gene ec-wbgO was synthesized from Escherichia coli O55:H7 through codon optimization; the required gene pf-wbgO was synthesized from Pseudogulbenkiania ferrooxidans through codon optimization; the required gene se-wbgO was synthesized from Salmonella enterica through codon optimization; and the required gene cf-wbgO was synthesized from Citrobacter freundii through codon optimization. All of the above gene synthesis was completed by Genewiz.

[0074] The galE gene was cloned directly from Escherichia coli MG1655.

[0075] The cloning host strain (Escherichia coli DH5α) and expression host strain (Escherichia coli BL21(DE3)) and Escherichia coli MG1655 of the present invention were all purchased from Shanghai Weidi Biotechnology.

[0076] The vectors pTargetF, pCas, pCOLADuet-1, and pACYCDuet-1 were all purchased from Tolo Biotech.

[0077] High-fidelity enzymes, common amplification enzymes, markers, and DNA gel recovery kits were purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0078] GelRed nucleic acid dye and 50×TAE were purchased from Biosharp.

[0079] The bacterial genomic DNA extraction kit and plasmid rapid extraction kit were purchased from Tiangen Biotech (Beijing) Co., Ltd.

[0080] Glycerin, spectinomycin, kanamycin, L-arabinose, and IPTG (isopropanol-β-D-thiogalactoside) were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0081] Seamless ligase, LB, agar, and agarose were purchased from Shanghai Sangon Biotech Co., Ltd.

[0082] Glucose, citric acid, KH₂PO₄, (NH₄)₂PO₄, MgSO₄·7H₂O, ferric citrate, ZnSO₄·7H₂O, CuSO₄·5H₂O, MnSO₄·H₂O, Na₂B₄O₇·2H₂O, (NH₄)₆Mo₇O 24 CaCl2·2H2O, lactose, and IPTG (isopropanol-β-D-thiogalactoside) were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0083] LB was purchased from Shanghai Sangon Biotech Co., Ltd., H2SO4 was purchased from Sinopharm Reagent, and lactose-N-tetrasaccharide (LNT) was purchased from Shanghai Zhenzhun Biotechnology Co., Ltd.

[0084] LB liquid medium: LB 20.0 g / L, autoclaved.

[0085] LB solid medium: Add 1.5% agar to LB liquid medium and autoclave.

[0086] 0.4M IPTG: Accurately weigh 9.532g of IPTG and dissolve it in deionized water to 400mL. Filter through a 0.22μm filter membrane and dispense into sterile 15mL centrifuge tubes and 1.5mL EP tubes. Store at -80℃ for later use.

[0087] Glucose medium (pH = 6.80): Glucose 15.07 g / L, Citric acid 1.7 g / L, KH₂PO₄ 13.5 g / L, (NH₄)₂PO₄ 4 g / L, MgSO₄·7H₂O 1.4 g / L, trace metal solution 10 mL / L (ferric citrate 10 g / L, ZnSO₄·7H₂O 2.25 g / L, CuSO₄·5H₂O 1.0 g / L, MnSO₄·H₂O 0.35 g / L, Na₂B₄O₇·2H₂O 0.23 g / L, (NH₄)₆Mo₇O₇ 24 0.11g / L, CaCl2·2H2O 2.0g / L).

[0088] Example 1 Plasmid Construction

[0089] 1. Synthesis of plasmid pAC-lgtA

[0090] The lgtA gene from *N. meningitidis* (Genbank ID: AAC44084.1) was obtained using NCBI, optimized according to the codon preference of *E. coli*, and named the gene lgtA. The lgtA gene was synthesized by Suzhou Genewiz Company and ligated into the expression vector pACYCDuet-1 to obtain the plasmid pAC-lgtA.

[0091] 2. Synthesis of plasmids pCO-ec-wbgO, pCO-pf-wbgO, pCO-se-wbgO, and pCO-cf-wbgO

[0092] (1) The β1,3-GalT gene from Escherichia coli O55:H7 (Genbank ID:WP_000582563.1) was obtained using NCBI and optimized according to the codon preference of E. coli. It was named the gene ec-wbgO (its corresponding amino acid sequence is shown in SEQ ID NO.8). The ec-wbgO gene was synthesized by Suzhou Genewiz Company and the synthesized gene was ligated into the expression vector pCOLADuet-1 to obtain the plasmid pCO-ec-wbgO.

[0093] SEQ ID NO.8

[0094] MIIDEAESAESTHPVVSVILPVNKKNPFLDEAINSILSQTFSSFEIIIVANCCTDDFYNELKHKVNDKIKLIRTNIAYLPYSLNKAIDLSNGEFIARMDSDDISHPDRFTKQVDFLKNNPYVDVVGTNAIFID DKGREINKTKLPEENLDIVKNLPYKCCIVHPSVMFRKKVIASIGGYMFSNYSEDYELWNRLSLAKIKFQNLPEYLFYYRLHEGQSTAKKNLYMVMVNDLVIKMKCFFLTGNINYLFGGIRTIASFIYCKYIK*

[0095] (2) The β1,3-GalT gene from *Pseudogulbenkiania ferrooxidans* (Genbank ID: WP_008952439.1) was obtained using NCBI. Optimized according to the codon preference of *E. coli*, the gene was named pf-wbgO (its corresponding amino acid sequence is shown in SEQ ID NO. 9). The pf-wbgO gene was synthesized by Suzhou Genewiz Company, and the synthesized gene was ligated into the expression vector pCOLADuet-1 to obtain the plasmid pCO-pf-wbgO.

[0096] SEQ ID NO.9

[0097] MDKIKQGSASLVVGDQQEKHPVVSVLLPVNRVDRFFIPAVESILTQTLQDFELIIIANGCSTEHLNKIRLTYGDHNRVRILNTEIKGLPFALNLGVHNARGLYIARMDADDISIPERLEKQLNTLEQNKKIGVVSSGV DFIDENDQAIREGKFPELTDKDHRRLLPLICCIAHPTVMVRKEIINKLGGYSFGSFSEDYDLWLRIMRELPEVEFYRIPESLLKYRRHGNQATSSKNIKKIRAYNSALKIRELFLSRKLKFIIGIILPARMVTLWRK*

[0098] (3) The β1,3-GalT gene from Salmonella enterica (Genbank ID: EFS9643522.1) was obtained using NCBI and optimized according to the codon preference of E. coli. It was named the gene se-wbgO (its corresponding amino acid sequence is shown in SEQ ID NO.10). The se-wbgO gene was synthesized by Suzhou Genewiz and the synthesized gene was ligated into the expression vector pCOLADuet-1 to obtain the plasmid pCO-se-wbgO.

[0099] SEQ ID NO.10

[0100] MLTEFRPVSTTKPLVSVILPVNKFNPYLDRAIHSILSQSYPSIELIIIANNCTNDFFDALKKRECETIKVLRTNIAYLPYCLNKGLDLCNGDFVARMDSDDISHPERIDRQVDFLINNPDIDVVGTNAVYID EDDIELEKSNLPENNDAIRKMLPYKCCLVHPSVMFRKNVVISSGGYMFANYSEDYELWNRLAVEGRSFYNLSEYLLYYRLHNNQSTSKNNLFMVMVNDVAIKVKYFLLTKKVSYLFGIIRTVFSVFYCKYIK*

[0101] (4) The β1,3-GalT gene from *Citrobacter freundii* (Genbank ID: WP_187258760.1) was obtained using NCBI, optimized according to the codon preference of *E. coli*, and named the gene cf-wbgO (its corresponding amino acid sequence is shown in SEQ ID NO.1). The cf-wbgO gene was synthesized by Suzhou Genewiz Company, and the synthesized gene was ligated into the expression vector pCOLADuet-1 to obtain the plasmid pCO-cf-wbgO.

[0102] 3. Construction of plasmids pCO-ec-wbgO-galE, pCO-pf-wbgO-galE, pCO-se-wbgO-galE, and pCO-cf-wbgO-galE

[0103] (1) Extraction of E. coil MG1655 genome. Primers pCO-HP-F / R (see Table 2) were designed using plasmids pCO-ec-wbgO, pCO-pf-wbgO, pCO-se-wbgO, and pCO-cf-wbgO as gene templates, and primers galE-F / R (see Table 2) using E. coil MG1655 genome as template for PCR reaction. Linear vectors pCO-ec-wbgO, pCO-pf-wbgO, pCO-se-wbgO, and pCO-cf-wbgO, as well as the galE gene fragment, were obtained. The size of the amplified fragments was determined by gel electrophoresis, and then the linear fragments were recovered, seamlessly cloned, and assembled with the vectors.

[0104] (2) The seamless cloning reaction solution was transformed into the cloning host *Escherichia coli* DH5α. After overnight culture at 37°C, single clones were selected for colony PCR verification (pCO-CX-F / R, primer sequences are shown in Table 2), and sequence-positive transformants were further identified by sequencing, and plasmids were extracted. The successfully constructed plasmids pCO-ec-wbgO-galE, pCO-pf-wbgO-galE, pCO-se-wbgO-galE, and pCO-cf-wbgO-galE were obtained.

[0105] Table 2

[0106]

[0107]

[0108] 4. Construction of plasmid pTargetF-ΔlacZ

[0109] (1) Based on the lacZ knockout site of Escherichia coli BL21(DE3), gRNA1 (CGCCACGATTGCCGCAAGTG) and gRNA2 (CGGGATGTTAATCGGCTATG) with high specificity and cleavage efficiency scores were selected using the website (http: / / crispor.tefor.net / ) and the designed 20bp sequences were introduced into pTargetF. Primers lacZ-N20-F1 / R1 and lacZ-N20-F2 / R2 (primers are shown in Table 3) were designed, and lacZ.pTargetF1 and lacZ.pTargetF2 plasmids, which have been replaced with gRNA1 and gRNA2 respectively, were obtained using pTargetF as a vector.

[0110] (2) Extraction of Escherichia coli BL21(DE3) genome. Primers PF1-F / R and PF2-F / R (primers are shown in Table 3) were designed and used as templates for PCR to obtain the pTargetF1 linear vector and sgRNA2. Primers lacZ-HL-F / R and lacZ-HR-F / R (primers are shown in Table 3) were used as templates for PCR to obtain the upstream and downstream homologous arms. The size of the amplified fragment was determined by gel electrophoresis, the linear fragment was recovered, and the fragment and vector were assembled using a seamless cloning method.

[0111] (3) The seamless cloning reaction solution was transferred into the cloning host *Escherichia coli* DH5α. After overnight culture at 37°C, single clones were selected for colony PCR verification, and sequence-positive transformants were further identified and plasmids were extracted. The successfully constructed plasmid pTargetF-ΔlacZ was obtained.

[0112] Table 3

[0113]

[0114] Note: Primers PF1-F / R and PF2-F / R are common primers used in the construction of the pTargetF recombinant plasmid, and their purpose is to amplify the pTargetF plasmid backbone.

[0115] 5. Construction of plasmid pTargetF-ΔwecB

[0116] The plasmid pTargetF-ΔwecB was constructed using the same method as pTargetF-ΔlacZ. The selected gRNA1 and gRNA2 were GACTGTATTTGGTACGCGCC and AGCCGCGCCCATAACCCGTA, respectively. The primers used are shown in Table 4.

[0117] Table 4

[0118]

[0119]

[0120] 6. Construction of plasmid pTargetF-ΔnagB

[0121] The plasmid pTargetF-ΔnagB was constructed using the method for constructing plasmid pTargetF-ΔlacZ. The selected gRNA1 and gRNA2 were CGCTGAACAGGTCGGCAAAT and TTAGAAGCAGAAAATATCAA, respectively. The primers used are shown in Table 5.

[0122] Table 5

[0123]

[0124] Example 2 Construction of genetically engineered bacteria

[0125] 1. Construction of Escherichia coli L2

[0126] The pCas plasmid was chemically transformed into *E. coli* strain BL21(DE3), using *E. coli* strain L1 carrying the pCas plasmid as the host to create electroporation competent cells. Then, the pTargetF-ΔlacZ plasmid was added to the competent cells at approximately 800 ng. Since the strain used to create the electroporation competent cells carried the temperature-sensitive pCas plasmid, all cultures were incubated at 30°C. Finally, the cells were plated on spec+Kana resistant LB agar and incubated overnight at 30°C. Single colonies were selected the following day for positive transformant verification. The primers lacZ-JD-F / R were identified (see Table 6). The cells were then sent to a sequencing company for sequencing, and the correctly sequenced strains were obtained.

[0127] The pTargetF plasmid in the strain was eliminated and verified. Positive single clones were inoculated into LB liquid medium containing Kan and cultured at 30°C and 220 rpm until the logarithmic growth phase. IPTG was then added to a final concentration of 0.5 mmol / L and cultured overnight to induce the loss of the pTargetF plasmid. The bacterial culture was streaked onto LB plates containing Kan and cultured overnight at 30°C. Single clones were selected for identification using the primer pTargetF-smr-JD-F / R (see Table 6). The final strain was Escherichia coli L2.

[0128] 2. Construction of Escherichia coli L3

[0129] Using the same method as in step 1, the pTargetF-ΔwecB plasmid was transformed into Escherichia coli strain L2 to obtain Escherichia coli strain L3. The primers wecB-JD-F / R for identifying positive transformants are shown in Table 6.

[0130] 3. Construction of Escherichia coli L4

[0131] Using the same method as in step 1, the pTargetF-ΔnagB plasmid was transformed into Escherichia coli strain L3 to obtain Escherichia coli strain L4. The primers ngaB-JD-F / R for identifying positive transformants are shown in Table 6.

[0132] 4. Construction of Escherichia coli L5

[0133] The pCas plasmid in Escherichia coli strain L4 was eliminated and verified. Positive single clones lacking pTargetF were transferred to antibiotic-free LB liquid medium and passaged at 42°C to eliminate the pCas plasmid. The bacterial culture was streaked onto antibiotic-free LB plates and incubated overnight at 30°C. Single clones were selected for identification using the Cas9-IS-F / R primers (see Table 6). Finally, strain L5 was obtained.

[0134] Table 6

[0135]

[0136] 5. Construction of Escherichia coli L6

[0137] Using L5 as the host, L5 electroporation competent cells were prepared. The pAC-lgtA plasmid was added to the competent cells at an amount of approximately 200 ng. The cells were then plated on CMR-resistant LB solid medium and incubated overnight at 37°C. The following day, single colonies were selected and incubated overnight on CMR-resistant LB liquid medium to obtain Escherichia coli L6.

[0138] 6. Construction of Escherichia coli L6-5, L6-6, L6-7, and L6-8

[0139] Using L6 as the host, L6 electroporation competent cells were prepared. The plasmids pCO-ec-wbgO-galE, pCO-pf-wbgO-galE, pCO-se-wbgO-galE, and pCO-cf-wbgO-galE were added to the competent cells at a concentration of approximately 200 ng. The cells were plated on LB solid medium resistant to CMR and KANA and incubated overnight at 37°C. The following day, single colonies were selected and incubated overnight on LB liquid medium resistant to CMR and KANA to obtain *E. coli* L6-5, L6-6, L6-7, and L6-8.

[0140] Example 3: Fermentation culture and detection of high-yield lactose-4-tetrasaccharide

[0141] 1. Strains domestication

[0142] Seed cultures of *E. coli* L6-5, L6-6, L6-7, and L6-8 were inoculated at a rate of 2% into 100 mL of glucose fermentation medium containing CMR+KANA resistance. The cultures were incubated at 37°C with a shaker at 200 rpm until the OD reached [value missing]. 600 When the concentration is 1.2, the domesticated strain is stored at -80℃.

[0143] 2-3L tank fermentation

[0144] Seed cultures of *E. coli* L6-5, L6-6, L6-7, and L6-8 were inoculated at a rate of 10% into a 3L fermentation medium. The fermentation tank was maintained at 37°C, with a stirring speed of 800 rpm, an aeration rate of 1 vvm, and a pH of 7.0 (ammonia replenishment was automatically controlled). Fermentation time was 12 hours (OD). 600 Approximately 30 μL of lactose was added to a final concentration of 20 g / L and IPTG to a final concentration of 0.4 mM, and the mixture was incubated at 25 °C. During this period, glucose and lactose were manually added to maintain cell growth and the synthesis of lactose-N-tetrasaccharides. After 70 hours of cultivation, the cell OD... 600 It reached around 200.

[0145] 3. Methods for detecting LNT production

[0146] Sample preparation: Take 2 mL of fermentation broth, centrifuge at 8000 r / min for 10 min, take the supernatant, dilute the supernatant by 2 times, and wait for HPLC detection; all samples need to be filtered through a 0.22 μm aqueous filter membrane.

[0147] HPLC detection conditions: Samples were analyzed using a high-performance liquid chromatography (HPLC) system (Shimadzu LC-20AT) and a CarbohydrateAnalysis (Rezex ROA-organic acid H+ (8%)) column. Mobile phase: 5 mmol / L H2SO4; flow rate: 1 mL / min; column temperature: 50℃; injection volume: 10 μL.

[0148] 4. Experimental Results

[0149] The HPLC results of Escherichia coli L6-5, L6-6, L6-7, and L6-8 after fermentation in a 3L reactor for 48 hours are shown in Table 7.

[0150] Table 7

[0151]

[0152] The results in the table show that the LNT yields of *Escherichia coli* L6-5 (β-1,3-galactosyltransferase derived from *Escherichiacoli* O55:H7), L6-6 (β-1,3-galactosyltransferase derived from *Pseudogulbenkiania ferrooxidans*), L6-7 (β-1,3-galactosyltransferase derived from *Salmonella enterica*), and L6-8 (β-1,3-galactosyltransferase derived from *Citrobacter freundii*) after fermentation in a 3L fermenter for 48 hours were 18.4 g / L, 53.8 g / L, 86.2 g / L, and 107.8 g / L, respectively. L6-8 (β-1,3-galactosyltransferase derived from Citrobacter freundii) is a newly screened β-1,3-galactosyltransferase in this invention, with the highest yield of 107.8 g / L, which is much higher than the 57.5 g / L reported in the literature (the highest reported yield of β-1,3-galactosyltransferase is derived from Pseudogulbenkiania ferrooxidans).

[0153] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations included in the claims. Various modifications and changes can be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments can be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only illustrate several implementations of the present invention and do not limit the scope of protection of this patent.

Claims

1. A genetically engineered bacterium that produces lactose-N-tetrasaccharide, characterized in that, The genetically engineered bacteria expresses the gene for β-1,3-galactosyltransferase, which is derived from *Citrobacter freundii*. The amino acid sequence of the β-1,3-galactosyltransferase derived from *Citrobacter freundii* is shown in SEQ ID NO.

1. The genetically engineered bacteria expressed the β-1,3-acetylglucosamine transferase gene lgtA derived from Neisseria meningitidis and the UDP-glucose-4-epimerase gene galE derived from Escherichia coli. The genetically engineered bacteria had the β-galactosidase gene lacZ, the UDP-N-acetylglucosamine-2-epimerase gene wecB, and the glucosamine-6-phosphate deaminase gene nagB knocked out.

2. The genetically engineered bacterium according to claim 1, characterized in that, The nucleotide sequence of the β-1,3-galactosyltransferase derived from Citrobacter freundii is shown in SEQ ID NO.

2.

3. The genetically engineered bacterium according to claim 1, characterized in that, The nucleotide sequence of the β-1,3-acetylglucosamine transferase gene lgtA derived from Neisseria meningitidis is shown in SEQ ID NO.

3.

4. The genetically engineered bacterium according to claim 1, characterized in that, The nucleotide sequence of the UDP-glucose-4-epimerase gene galE derived from Escherichia coli is shown in SEQ ID NO.

4.

5. A biological agent, characterized in that, Includes the genetically engineered bacteria as described in any one of claims 1-4.

6. The use of a genetically engineered bacterium according to any one of claims 1-4 or the biological agent according to claim 5 in the production of lactose-N-tetrasaccharide.

7. A method for producing lactose-N-tetrasaccharide, characterized in that, Lactose-N-tetrasaccharide is produced by fermentation using glucose as a carbon source and lactose as a substrate, with the genetically engineered bacteria described in any one of claims 1-4 or the biological agent described in claim 5.

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