Genetically engineered bacteria for producing lacto-n-neotetraose and application thereof

Abstract

The present application relates to a genetically engineered bacterium and a preparation method and application thereof, and belongs to the field of biosynthesis. The present application improves the yield of lactose-N-neotetraose by changing the expression of the source of part of key enzymes, and has important significance for the industrial production of lactose-N-neotetraose.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, and in particular to a genetically engineered bacterium for producing lactose-N-neotetrasaccharide. Background Technology

[0002] Human milk oligosaccharides (HMOs) are the third most abundant solid component in human milk after lactose and lipids. They are considered natural prebiotics for infants, shaping the gut microbiota and promoting gut health. Furthermore, they have other unique effects on infant health, including promoting brain and immune system development, exhibiting anti-adhesion and antibacterial activity, and reducing necrotizing enterocolitis. Therefore, HMOs are becoming a central area of ​​infant nutrition research, and commercial supplementation with HMOs is becoming a trend in the future development of infant formula.

[0003] Lacto-N-neotetraose (LNnT) is one of the most abundant oligosaccharides in human milk. It belongs to the non-fucosylated neutral human milk oligosaccharides and possesses physiological functions such as immunomodulation, acting as a regulator of intestinal cell responses, and promoting cell maturation. Initially, LNnT was obtained from human milk, but human milk contains various solid components, making separation complex and the content low, failing to meet the huge market demand. Therefore, the synthesis of LNnT has attracted widespread attention. Currently reported methods for LNnT synthesis include chemical synthesis, enzymatic synthesis, and microbial production. Chemical synthesis is often accompanied by the formation of chemical waste and byproducts, requiring multiple steps to protect and deprotect functional groups. Biosynthesis offers advantages such as environmental friendliness, high specificity, low cost, and renewable substrates. Furthermore, with the development of advanced multi-enzyme reaction and metabolic engineering strategies, the biosynthesis of LNnT has received considerable attention.

[0004] In microorganisms, the precursors for LNnT synthesis include intracellularly synthesized uridine 5'-bisphosphate-N-acetylglucosamine (UDP-G1cNAc) and uridine 5'-bisphosphate-galactose (UDP-Gal), as well as exogenously added lactose. The synthesis process involves the following steps: first, extracellular lactose is transported into the cell by lactose permease (LacY), and then, under the catalysis of heterologously expressed β-1,3-N-acetylglucosamine transferase (LgtA), it reacts with intracellular UDP-G1cNAc to generate lactose-N-triose II (Lacto-N-triose II, LNTII). LNTII and UDP-Gal are then further catalyzed by heterologously expressed β-1,4-galactosyltransferase (LgtB) to generate LNnT.

[0005] Existing research has yielded several strategies for increasing LNnT production. For example, Zhang et al. improved LNnT yield by optimizing the metabolic flux of Bacillus subtilis 168 (0.09 g / L / h) using CRISPRi; Hu et al. increased LNnT yield to 0.34 g / L / h by optimizing the copy number of key genes to adjust the expression intensity of target genes; and Zhang et al. improved LNnT yield by screening for β-1,4-galactosyltransferase (0.25 g / L / h) in Aggregatibacteractinomycetemcomitans NUM4039. Summary of the Invention

[0006] The problem the invention aims to solve

[0007] The synthesis of LNnT via microbial fermentation requires a key β-1,4-galactosyltransferase that catalyzes the conversion of the precursor lactyl-N-trisaccharide II. This β-1,4-galactosyltransferase transfers the galactose moiety from UDP-Gal to LNTII. To date, only a handful of β-1,4-galactosyltransferases capable of producing LNnT have been identified. Studies have shown that LNnT can be successfully synthesized from LNTII using a β-1,4-galactosyltransferase derived from Neisseria meningitidis in metabolically engineered *Escherichia coli* or *Bacillus subtilis*. However, heterologous expression in *E. coli* presents challenges due to poor soluble expression and easy degradation. Another β-1,4-galactosyltransferase derived from *Histophilus somni* can efficiently produce LNnT from LNTII using enzymatic and metabolic engineering methods, exhibiting good solubility. However, in all tested reactions, its hydrolytic activity was relatively higher than its transglycosylation activity, and the enzyme mainly catalyzed hydrolysis when producing LNnT, thus affecting the final yield of LNnT.

[0008] Solution for solving the problem

[0009] On one hand, the present invention provides a genetically engineered bacterium for producing lactose-N-neotetrasaccharide, characterized in that the genetically engineered bacterium expresses a β-1,4-galactosyltransferase gene in chassis cells; the β-1,4-galactosyltransferase gene is derived from Pasteurella dagmatis ATCC43325.

[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 in the chassis cells.

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

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

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

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

[0015] Preferably, the amino acid sequence of the β-1,4-galactosyltransferase derived from Pasteurella dagmatis ATCC43325 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.

[0016] Preferably, the nucleotide sequence of the β-1,4-galactosyltransferase gene derived from Pasteurella dagmatis ATCC43325 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.

[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.2, and is more preferably SEQ ID NO.2.

[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.3, and is preferably SEQ ID NO.3.

[0019] On the one hand, the present invention provides a biological agent characterized in that it comprises the above-mentioned genetically engineered bacteria.

[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-neotetrasaccharide.

[0021] On the one hand, the present invention provides a method for producing lactose-N-neotetrasaccharide, 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-neotetrasaccharide.

[0022] The effects of the invention

[0023] The genetically engineered bacteria constructed in this invention can achieve a higher raw material utilization rate, and lactose-N-neotetrasaccharide can achieve a higher yield within the same fermentation time. Detailed Implementation

[0024] 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.

[0025] On one hand, the present invention provides a genetically engineered bacterium for producing lactose-N-neotetrasaccharide, characterized in that the genetically engineered bacterium expresses a β-1,4-galactosyltransferase gene in chassis cells; the β-1,4-galactosyltransferase gene is derived from Pasteurella dagmatis ATCC43325.

[0026] In some embodiments, the genetically engineered bacteria knock out the β-galactosidase gene lacZ in the chassis cells.

[0027] In some embodiments, the genetically engineered bacteria knocked out the UDP-N-acetylglucosamine-2-epimerase gene wecB in the chassis cells.

[0028] In some embodiments, the genetically engineered bacteria have the glucosamine-6-phosphate deaminase gene nagB knocked out in the chassis cells.

[0029] In some embodiments, the genetically engineered bacteria express the β-1,3-acetylglucosamine transferase gene lgtA in the chassis cells.

[0030] In some embodiments, the β-1,3-acetylglucosamine transferase gene lgtA is derived from Neisseria meningitidis.

[0031] In some embodiments, the genetically engineered bacteria express the UDP-glucose-4-epimerase gene galE in the chassis cells.

[0032] In some embodiments, the UDP-glucose-4-epimerase gene galE is derived from Escherichia coli.

[0033] In some embodiments, the amino acid sequence of the β-1,4-galactosyltransferase derived from Pasteurella dagmatis ATCC43325 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.

[0034] In some embodiments, the amino acid sequence of the β-1,4-galactosyltransferase derived from Pasteurella dagmatis ATCC43325 is shown in SEQ ID NO.4.

[0035] SEQ ID NO.4

[0036] MNFMSLLKNYVISLSSATDRRKHILEEFYQHNIPFLFFDALSPSLEFNQLVEQLIPNLSRCNLT

[0037] NGEKGCLMSHLSLWHKCVEENLPYIGVFEDDILLGKDAEEFLASDEWIHSRFQHGDNFIIRL

[0038] ETFLQKVLCDATNISPYLDRDFLKLRSTHFGTAGYIISFSAAKYFLNIFANMKEEDEIEPIDQLIF

[0039] DRFLPMDNFQVYQLSPAICIQELQVNQEHSLLSSQLEDERIIARLDRDRKRKQKKIKDLFIHFLKKPQRMIEKSKKKRIQLKNNENSVVIEFK*

[0040] In some embodiments, the nucleotide sequence of the β-1,4-galactosyltransferase gene derived from Pasteurella dagmatis ATCC43325 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.

[0041] In some embodiments, the nucleotide sequence of the β-1,4-galactosyltransferase gene derived from Pasteurella dagmatis ATCC43325 is shown in SEQ ID NO.1.

[0042] SEQ ID NO.1

[0043] ATGAACTTCATGTCCTTATTAAAGAATTACGTCATTAGCCTGAGCTCTGCAACCGATCGTC

[0044] GCAAACATATTCTGGAAGAATTCTATCAGCATAACATTCCGTTTCTGTTCTTTGATGCACT

[0045] GAGCCCGTCCCTGGAATTTAATCAGCTGGTCGAACAGCTGATTCCGAATTTATCGCGCTG

[0046] TAACCTGACGAACGGCGAGAAAGGCTGTCTGATGTCTCACCTTAGCCTGTGGCATAAAT

[0047] GCGTCGAAGAGAACCTGCCGTACATCGGCGTGTTTGAAGATGACATTCTGCTGGGTAAA

[0048] GATGCAGAAGAATTCCTGGCGAGCGACGAATGGATTCACTCGCGCTTTCAGCACGGTGA

[0049] TAACTTCATCATTCGTCTTGAAACGTTCCTGCAGAAAGTTCTGTGCGACGCGACCAACAT

[0050] TTCGCCATATCTCGATCGCGACTTTCTGAAACTGCGCAGCACCCATTTCGGTACGGCCGG

[0051] CTACATTATCTCCTTTAGCGCAGCCAAATACTTCCTGAATATCTTCGCGAATATGAAAGAA

[0052] GATGAAATTGAACCGATTGATCAGCTTATCTTCGACCGTTTCTTACCGATGGATAACTTCC

[0053] AGGTTTACCAGCTCAGCCCAGCAATCTGCATCCAGGAACTGCAGGTTAATCAGGAACAC

[0054] AGCCTGCTGTCCTCGCAGCTGGAAGACGAACGCATCATCGCACGCCTCGATCGTGATCG

[0055] TAAACGCAAACAGAAGAAGATTAAAGATCTGTTCATCCACTTCTTAAAGAAACCGCAGC

[0056] GCATGATCGAGAAAAGCAAGAAGAAACGGATTCAACTGAAGAACAATGAGAACTCCGT

[0057] AGTAATCGAATTCAAGTAA

[0058] 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.2.

[0059] 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.2.

[0060] SEQ ID NO.2

[0061] ATGGGCCAGCCGCTGGTTAGCGTTCTGATCTGCGCGTACAACGTTGAAAAATATTTCGCG

[0062] CAGAGCCTGGCAGCTGTTGTTAACCAGACCTGGCGTAACCTGGACATTCTGATCGTTGA

[0063] TGATGGCTCTACCGATGGCACCCTGGCGATCGCGCAGCGTTTCCAGGAACAGGACGGTC

[0064] GTATCCGTATTCTGGCGCAGCCGCGTAACTCTGGTCTGATTCCAAGCCTGAACATCGGCC

[0065] TGGATGAACTGGCGAAAAGCGGCGGTGGTGGTGAATACATCGCGCGTACCGATGCGGAT

[0066] GATATCGCAGCTCCGGATTGGATTGAAAAAATCGTTGGTGAAATGGAAAAAGATCGTAG

[0067] CATCATCGCAATGGGCGCTTGGCTGGAAGTGCTGTCCGAAGAAAAAGATGGCAACCGT

[0068] CTGGCACGTCACCACGAACACGGTAAAATCTGGAAAAAACCGACCCGTCACGAAGACA

[0069] TCGCGGATTTCTTCCCATTCGGCAACCCGATTCACAACAACACCATGATCATGCGTCGTT

[0070] CCGTGATCGATGGCGGCCTGCGTTACAACACCGAACGTGATTGGGCAGAAGACTATCAG

[0071] TTCTGGTATGATGTTTCTAAACTGGGTCGTCTGGCGTACTACCCGGAAGCGCTGGTTAAA

[0072] TACCGTCTGCACGCTAACCAGGTTAGCTCCAAATATAGCATCCGCCAGCACGAAATCGCT

[0073] CAGGGTATCCAGAAAACCGCACGTAACGATTTCCTGCAGTCTATGGGTTTCAAAACCCG

[0074] TTTCGATAGCCTGGAATACCGTCAGATTAAAGCGGTTGCGTATGAACTGCTGGAAAAAC

[0075] ACCTGCCGGAAGAAGATTTTGAACTGGCGCGTCGTTTTCCTGTACCAGTGCTTCAAACGT

[0076] ACCGATACCCTGCCGGCGGGCGCTTGGCTGGATTTCGCGGCGGATGGCCGTATGCGTCG

[0077] TCTGTTCACCCTGCGTCAGTACTTCGGTATCCTGCACCGTCTGCTGAAAAACCGTTAA

[0078] 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.3.

[0079] In some embodiments, the nucleotide sequence of the UDP-glucose-4-epimerase gene galE derived from Escherichia coli is shown in SEQ ID NO.3.

[0080] SEQ ID NO.3

[0081] ATGAGAGTTCTGGTTACCGGTGGTAGCGGTTACATTGGAAGTCATACCTGTGTGCAATTA

[0082] CTGCAAAACGGTCATGATGTCATCATTCTTGATAACCTCTGTAACAGTAAGCGCAGCGTA

[0083] CTGCCTGTTATCGAGCGTTTAGGCGGCAAACATCCAACGTTTGTTGAAGGCGATATTCGT

[0084] AACGAAGCGTTGATGACCGAGATCCTGCACGATCACGCTATCGACACCGTGATCCACTT

[0085] CGCCGGGCTGAAAGCCGTGGGCGAATCGGTACAAAAACCGCTGGAATATTACGACAAC

[0086] AATGTCAACGGCACTCTGCGCCTGATTAGCGCCATGCGCGCCGCTAACGTCAAAAACTT

[0087] TATTTTTAGCTCCTCCGCCACCGTTTATGGCGATCAGCCCAAAATTCCATACGTTGAAAG

[0088] CTTCCCGACCGGCACACCGCAAAGCCCTTACGGCAAAAGCAAGCTGATGGTGGAACAG

[0089] ATCCTCACCGATCTGCAAAAAGCCCAGCCGGACTGGAGCATTGCCCTGCTGCGCTACTT

[0090] CAACCCGGTTGGCGCGCATCCGTCGGGCGATATGGGCGAAGATCCGCAAGGCATTCCGA

[0091] ATAACCTGATGCCATACATCGCCCAGGTTGCTGTAGGCCGTCGCGACTCGCTGGCGATTT

[0092] TTGGTAACGATTATCCGACCGAAGATGGTACTGGCGTACGCGATTACATCCACGTAATGG

[0093] ATCTGGCGGACGGTCACGTCGTGGCGATGGAAAAACTGGCGAACAAGCCAGGCGTACA

[0094] CATCTACAACCTCGGCGCTGGCGTAGGCAACAGCGTGCTGGACGTGGTTAATGCCTTCA

[0095] GCAAAGCCTGCGGCAAACCGGTTAATTATCATTTTGCACCGCGTCGCGAGGGCGACCTT

[0096] CCGGCCTACTGGGCGGACGCCAGCAAAGCCGACCGTGAACTGAACTGGCGCGTAACGC

[0097] GCACACTCGATGAAATGGCGCAGGACACCTGGCACTGGCAGTCACGCCATCCACAGGG

[0098] ATATCCCGATTAA

[0099] In some implementations, the actual sequence used is the same as or similar to the above sequence, as well as the amino acid sequence translated from the nucleic acid sequence, and the codons can be optimized according to the different chassis cells before use.

[0100] On the one hand, the present invention provides a biological agent characterized in that it comprises the above-mentioned genetically engineered bacteria.

[0101] 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-neotetrasaccharide.

[0102] On the one hand, the present invention provides a method for producing lactose-N-neotetrasaccharide, 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-neotetrasaccharide.

[0103] Example 1 Plasmid Construction

[0104] 1. The required gene lgtA was synthesized from *N. meningitidis* (lgtA) after codon optimization; the required gene Nm-lgtB was synthesized from *Neisseria meningitidis* serogroup B (strain MC58) after codon optimization; the required gene Pd-lgtB was synthesized from *Pasteurella dagmatis* ATCC43325 after codon optimization; the required gene Aa-lgtB was synthesized from *Aggregatibacter actinomycetemcomitans* NUM4039 after codon optimization; and the required gene Aj-lgtB was synthesized from *Acinetobacter johnsonii* SH046 after codon optimization. All gene synthesis was performed by Genewiz. The galE gene was directly cloned from *E. coli* MG1655, with *E. coli* DH5α as the cloning host and *E. coli* BL21 (DE3) as the expression host. All three strains were purchased from Genewiz. The vectors pTargetF, pCas, pCOLADuet-1, and pACYCDuet-1 were all purchased from Tolo Biotech.

[0105] 2. High-fidelity enzymes, common amplification enzymes, markers, and DNA gel recovery kits were purchased from Nanjing Novizan Biotechnology Co., Ltd.; GelRed nucleic acid dye and 50×TAE were purchased from Biosharp; bacterial genomic DNA extraction kits and plasmid rapid extraction kits were purchased from Tiangen Biotech (Beijing) Co., Ltd.; glycerol, spectinomycin, kanamycin, L-arabinose, and IPTG (isopropanol-β-D-thiogalactoside) were purchased from Shanghai Maclean Biotechnology Co., Ltd.; seamless ligase, LB, agar, and agarose were purchased from Shanghai Sangon Biotech Co., Ltd.

[0106] 3. Synthesis of plasmid pAC-lgtA

[0107] 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.

[0108] 4. Synthesis of plasmids pCO-Nm-lgtB, pCO-Pd-lgtB, pCO-Aj-lgtB, and pCO-Aa-lgtB

[0109] (1) The lgtB gene from Neisseria meningitidisserogroupB (strain MC58) (Genbank ID: U25839.1) was obtained using NCBI and optimized according to the codon preference of E. coli, and named the gene Nm-lgtB. The Nm-lgtB gene was synthesized by Suzhou Genewiz and the synthesized gene was ligated into the expression vector pCOLADuet-1 to obtain the plasmid pCO-Nm-lgtB.

[0110] (2) The lgtB gene from Pasteurella dagmatis ATCC43325 (Genbank ID: ACZR01000001.1) was obtained using NCBI and optimized according to the codon preference of E. coli, and named the gene Pd-lgtB. The Pd-lgtB gene was synthesized by Suzhou Genewiz and the synthesized gene was ligated into the expression vector pCOLADuet-1 to obtain the plasmid pCO-Pd-lgtB.

[0111] (3) The lgtB gene from Aggregatibacteractinomycetemcomitans NUM4039 (Genbank ID: BAS48030.1) was obtained using NCBI and optimized according to the codon preference of E. coli, and named the gene Aa-lgtB. The Aa-lgtB gene was synthesized by Suzhou Genewiz Company and the synthesized gene was ligated into the expression vector pCOLADuet-1 to obtain the plasmid pCO-Aa-lgtB.

[0112] (4) The gene HMPREF0016_02855 from AcinetobacterjohnsoniiSH046 (Genbank ID: GG704971.1) was obtained using NCBI and optimized according to the codon preference of E. coli, and named the gene Aj-lgtB. The Aj-lgtB gene was synthesized by Suzhou Genewise Co., Ltd. and the synthesized gene was ligated into the expression vector pCOLADuet-1 to obtain the plasmid pCO-Aj-lgtB.

[0113] 5. Construction of plasmids pCO-Nm-lgtB-galE, pCO-Pd-lgtB-galE, pCO-Aa-lgtB-galE, and pCO-Aj-lgtB-galE

[0114] (1) Extraction of E. coilMG1655 genome. Primers pCO-HP-F / R and galE-F / R were designed. PCR reaction was performed using plasmids pCO-Nm-lgtB, pCO-Pd-lgtB, pCO-Aa-lgtB, pCO-Aj-lgtB, and the E. coilMG1655 genome as templates. Linear vectors pCO-Nm-lgtB, pCO-Pd-lgtB, pCO-Aa-lgtB, and pCO-Aj-lgtB, as well as the galE gene fragment, were obtained. The size of the amplified fragment was determined by gel electrophoresis. The linear fragment was then recovered, seamlessly cloned, and assembled with the vector.

[0115] (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, and sequence-positive transformants were further identified by sequencing, and plasmids were extracted. The successfully constructed plasmids pCO-Nm-lgtB-galE, pCO-Pd-lgtB-galE, pCO-Aa-lgtB-galE, and pCO-Aj-lgtB-galE were obtained.

[0116] Table 1. Primers used for constructing pCO-Nm-lgtB-galE, pCO-Pd-lgtB-galE, pCO-Aa-lgtB-galE, and pCO-Aj-lgtB-galE.

[0117]

[0118] 6. Construction of plasmid pTargetF-ΔlacZ

[0119] (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 were designed, and pTargetF was used as a vector to obtain lacZ.pTargetF1 and lacZ.pTargetF2 plasmids that have been replaced with gRNA1 and gRNA2, respectively.

[0120] (2) Extraction of Escherichia coli BL21(DE3) genome. Primers PF1-F / R, PF2-F / R, lacZ-HL-F / R, and lacZ-HR-F / R were designed. PCR reaction was performed using plasmids lacZ.pTargetF1, lacZ.pTargetF2, and Escherichia coli BL21(DE3) genome as templates to obtain the pTargetF1 linear vector, sgRNA2, upstream homologous arm, and downstream homologous arm. 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.

[0121] (3) A suitable amount of 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.

[0122] Table 2 Primers used to construct pTargetF-ΔlacZ

[0123]

[0124]

[0125] 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.

[0126] 7. Construction of plasmid pTargetF-ΔwecB

[0127] The plasmid pTargetF-ΔwecB was constructed using the same method described in 6.(1). The selected gRNA1 and gRNA2 were GACTGTATTTGGTACGCGCC and AGCCGCGCCCATAACCCGTA, respectively. The primers used are as follows:

[0128] Table 3 Primers used to construct pTargetF-ΔwecB

[0129]

[0130] 8. Construction of plasmid pTargetF-ΔnagB

[0131] The plasmid pTargetF-ΔnagB was constructed using the same method described in 6.(1). The selected gRNA1 and gRNA2 were CGCTGAACAGGTCGGCAAAT and TTAGAAGCAGAAAATATCAA, respectively, and the primers used were as follows:

[0132] Table 4 Primers used to construct pTargetF-ΔnagB

[0133]

[0134] Example 2: Strain Construction

[0135] 1. Construction of Escherichia coli L2

[0136] 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 electrotransformation competent cells. Then, the pTargetF-ΔlacZ plasmid was added to the competent cells at approximately 800 ng. Since the strain used to create the electrotransformation competent cells carried the temperature-sensitive pCas plasmid, all cultures were incubated at 30°C. Finally, the cells were plated on spectinomycin and kanamycin-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, and the cells were sent to a sequencing company for sequencing, yielding correctly sequenced strains.

[0137] The pTargetF plasmid in the strain was eliminated and verified. Positive single clones were inoculated into LB liquid medium containing kanamycin 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 agar plates containing kanamycin and cultured overnight at 30°C. Single clones were selected for identification using the primer pTargetF-smr-JD-F / R. The final strain was *Escherichia coli* L2.

[0138] 2. Construction of Escherichia coli L3

[0139] Using the same method as in Part 1 of Example 2, the pTargetF-ΔwecB plasmid was transformed into Escherichia coli strain L2 to obtain Escherichia coli strain L3.

[0140] 3. Construction of Escherichia coli L4

[0141] Using the same method as in Part 1 of Example 2, the pTargetF-ΔnagB plasmid was transformed into Escherichia coli strain L3 to obtain Escherichia coli strain L4.

[0142] 4. Construction of Escherichia coli L5

[0143] The pCas plasmid in Escherichia coli strain L4 was eliminated and verified. Positive single clones lacking pTargetF were transferred to antibiotic-free LB broth 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 primer. Finally, strain L5 was obtained.

[0144] 5. Construction of Escherichia coli L6

[0145] 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 chloramphenicol-resistant LB agar and incubated overnight at 37°C. The following day, single colonies were selected and incubated overnight on chloramphenicol-containing LB liquid agar to obtain Escherichia coli L6.

[0146] 5. Construction of Escherichia coli L6-1, L6-2, L6-3, and L6-4

[0147] Using L6 as the host, L6 electroporation competent cells were prepared. The plasmids pCO-Nm-lgtB-galE, pCO-Pd-lgtB-galE, pCO-Aa-lgtB-galE, and pCO-Aj-lgtB-galE were added to the competent cells at a concentration of approximately 200 ng. The cells were plated on LB solid medium resistant to chloramphenicol and kanamycin and incubated overnight at 37°C. The following day, single colonies were selected and incubated overnight on LB liquid medium resistant to chloramphenicol and kanamycin to obtain *Escherichia coli* L6-1, L6-2, L6-3, and L6-4.

[0148] Table 5 Primers used to construct the strain

[0149]

[0150]

[0151] Example 3 Fermentation Test

[0152] 1. Main reagents

[0153] Glucose, citric acid, KH₂PO₄, (NH₄)₂PO₄, MgSO₄·7H₂O, ferric citrate, ZnSO₄·7H₂O, CuSO₄·5H₂O, MnSO₄·H₂O, Na₂B₄O₇·2H₂O, (NH₄)₆Mo₇O 24CaCl2·2H2O, lactose, and IPTG (isopropanol-β-D-thiogalactoside) were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; LB was purchased from Shanghai Sangon Biotech Co., Ltd.; H2SO4 was purchased from Sinopharm Reagent; and lactyl-N-neotetrasaccharide (LNnT) was purchased from the National Institute of Metrology, China.

[0154] 2. Preparation of culture medium

[0155] LB liquid medium: LB 20.0 g / L, autoclaved.

[0156] LB solid medium: Add 1.5% agar to LB liquid medium and autoclave.

[0157] 0.4 MIPTG: Accurately weigh 9.532 g IPTG and dissolve it in deionized water to 400 mL. Filter through a 0.22 μm filter membrane and dispense into sterile 15 mL centrifuge tubes and 1.5 mL EP tubes. Store at -80 °C for later use.

[0158] 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, CaCl2·2H2O2.0g / L).

[0159] 3. Experimental Methods

[0160] (1) Strain domestication

[0161] Seed cultures of *Escherichia coli* L6-1, L6-2, L6-3, and L6-4 were inoculated at a rate of 2% into 100 mL of glucose fermentation medium containing chloramphenicol and kanamycin resistance. The cultures were then 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℃.

[0162] (2) 3L tank fermentation

[0163] Seed cultures of *E. coli* L6-1, L6-2, L6-3, and L6-4 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). After 12 hours of fermentation (OD600 approximately 30), lactose to a final concentration of 20 g / L and IPTG to a final concentration of 0.4 mM were added, and the culture was incubated at 25°C. During this period, glucose and lactose were manually added to maintain bacterial growth and the synthesis of lactyl-N-neotetrasaccharides. After 70 hours of cultivation, the bacterial OD600 reached approximately 200.

[0164] (3) The detection method for LNnT production is as follows:

[0165] a. 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.

[0166] b. HPLC detection conditions: The sample was analyzed using a high-performance liquid chromatography (HPLC) system (Shimadzu LC-20AT) and a Carbohydrate Analysis (Rezex ROA-organic acid H+ (8%)) column. Mobile phase: 5 mmol H2SO4; flow rate: 1 mL / min; column temperature: 50℃; injection volume: 10 μL.

[0167] 4. Experimental Results

[0168] The HPLC results of Escherichia coli L6-1, L6-2, L6-3, and L6-4 after fermentation in a 3L reactor for 48 hours are shown in the table below:

[0169] Table 6 Results of Escherichia coli fermentation for 48 hours

[0170]

[0171] The results in the table show that *E. coli* L6-2 (β-1,4-galactosyltransferase derived from *Pasteurella dagmatis* ATCC43325) achieved a lactoyl-N-neotetrasaccharide yield of 21.8 g / L in a 3L fermenter. *E. coli* L6-1 (β-1,4-galactosyltransferase derived from *Neisseria meningitidis*) and L6-3 (β-1,4-galactosyltransferase derived from *Aggregatibacter actinomycetemcomitans* NUM4039) produced LNnT yields of 8.4 g / L and 10.6 g / L, respectively, after 48 h of fermentation in a 3L fermenter. L6-4 (β-1,4-galactosyltransferase derived from *Acinetobacter johnsonii* SH046) had a lower yield of 3.3 g / L. The yields of *E. coli* L6-1, L6-3, and L6-4 in the 3L fermenter were all lower than that of *E. coli* L6-2. Furthermore, the lactose content (1.3 g / L) of *E. coli* L6-2 after 48 hours of fermentation in the 3L fermenter was lower than that of L6-1, L6-3, and L6-4, indicating that *E. coli* L6-2 had the highest lactose utilization rate.

[0172] 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-neotetrasaccharide, characterized in that, The genetically engineered bacteria expressed the β-1,4-galactosyltransferase gene in the chassis cells; the β-1,4-galactosyltransferase gene was derived from Pasteurella dagmatis ATCC43325; the amino acid sequence of the β-1,4-galactosyltransferase is shown in SEQ ID NO.

4. The genetically engineered bacteria had 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 in the chassis cells. The genetically engineered bacteria expressed the β-1,3-acetylglucosamine transferase gene lgtA in the chassis cells; the β-1,3-acetylglucosamine transferase gene lgtA was derived from Neisseria meningitidis. The genetically engineered bacteria expressed the UDP-glucose-4-epimerase gene galE in the chassis cells; the UDP-glucose-4-epimerase gene galE was derived from Escherichia coli.

2. The genetically engineered bacterium according to claim 1, characterized in that, The nucleotide sequence of the β-1,4-galactosyltransferase gene derived from Pasteurella dagmatis ATCC43325 is shown in SEQ ID NO.

1.

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.

2.

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.

3.

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-neotetrasaccharide.

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

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