Engineering bacterium for producing 3 '-sialyllactose as well as construction method and application of engineering bacterium

Abstract

The invention provides an engineering bacterium for producing 3 '-sialyllactose as well as a construction method and application thereof. The engineering bacterium has the capability of generating 3 '-sialic acid lactose, and an exogenous UDP-N-acetyl glucosamine-2-epimerase gene (neuC), a sialic acid synthase gene (neuB), an N-acetylneuraminic acid cytidyltransferase gene (css) and an alpha-2, 3-sialic acid transferase gene (ST) are introduced into a genome of the engineering bacterium or a recombinant plasmid carried by the engineering bacterium. The engineering bacterium has high capacity of producing 3 '-sialyllactose under the condition that antibiotics are not needed.

Description

Technical Field

[0001] The invention belongs to the field of fermentation, and in particular relates to an engineering bacterium for producing 3'-sialyllactose without the need for antibiotics, a construction method thereof and an application thereof. Background Art

[0002] Human milk oligosaccharides (HMOs) are an important class of oligosaccharides found in breast milk. They are composed of five monomeric sugars plus lactose molecules arranged in various combinations under the action of specific glycosyltransferases. Currently, more than 30 HMOs have been identified. Studies have shown that HMOs can act as prebiotics to affect the intrinsic components of the intestinal microbiota and maintain the balance of the intestinal microbial environment; HMOs and their metabolites (such as sialic acid, etc.) play an important role in brain development, neurotransmission and synapse formation, and have the function of promoting brain development in infants and young children and improving cognition; in addition, HMOs also have antiviral, immune regulation and human recovery promotion effects.

[0003] The main types of human milk oligosaccharides found so far include fucosylated HMOs and sialylated HMOs. Among the sialylated HMOs, sialyllactose has the highest content, including 3'-sialyllactose (3'-Sialyllactose, 3'-SL) and 6'-sialyllactose (6'-Sialyllactose, 6'-SL). 3'-SL and 6'-SL are respectively made of sialic acid and lactose as precursors connected by α-2,3 or α-2,6 glycosidic bonds. The structural formulas of 3'-SL and 6'-SL are shown in Formula I and Formula II respectively:

[0004]

[0005] At present, the production methods of sialyllactose include chemical synthesis, enzyme catalysis synthesis, etc. The chemical synthesis method requires expensive raw materials, harsh reaction conditions, high cost and low yield. Compared with chemical synthesis, the enzyme catalysis synthesis method has a higher yield, but the enzyme catalysis reaction requires the use of expensive enzymes, so the comprehensive production cost is high, and in the enzyme catalysis reaction, the enzyme is more likely to be inactivated, thereby affecting the enzyme catalysis efficiency and the yield of the product, so this method is also difficult to achieve large-scale production. Summary of the invention

[0006] In order to overcome the problem of low yield of 3'-sialyllactose in the prior art during synthesis, the present invention provides an engineering bacterium that does not require antibiotics and has high yield of 3'-sialyllactose, as well as a construction method and application thereof.

[0007] The Escherichia coli EBSL005 strain (also known as SLIS109 strain) in this article has been deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration (CGMCC) on August 24, 2023, with the deposit number CGMCC No.: 28244. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing; it is classified and named Escherichia coli.

[0008] In one aspect, the present invention provides a genetically engineered bacterium having the ability to produce 3'-sialyllactose and having a UDP-N-acetylglucosamine-2-epimerase gene (neuC), a sialic acid synthase gene (neuB), an N-acetylneuraminic acid cytidylyltransferase gene (css) and an α-2,3-sialyltransferase gene (ST), wherein the UDP-N-acetylglucosamine-2-epimerase gene (neuC), the sialic acid synthase gene (neuB), the N-acetylneuraminic acid cytidylyltransferase gene (css) and the α-2,3-sialyltransferase gene (ST) are integrated independently of each other or integrated together in any combination into the genome of the genetically engineered bacterium, or are present in a recombinant plasmid carried by the genetically engineered bacterium.

[0009] In one embodiment, the genetically engineered bacteria have introduced exogenous UDP-N-acetylglucosamine-2-epimerase gene (neuC), exogenous sialic acid synthase gene (neuB), exogenous N-acetylneuraminic acid cytidylyltransferase gene (css) and exogenous α-2,3-sialyltransferase gene (ST). The introduced exogenous UDP-N-acetylglucosamine-2-epimerase gene (neuC), the introduced exogenous sialic acid synthase gene (neuB), the introduced exogenous N-acetylneuraminic acid cytidylyltransferase gene (css) and the introduced exogenous α-2,3-sialyltransferase gene (ST) are integrated independently of each other or integrated together in any combination into the genome of the genetically engineered bacteria, or are present in a recombinant plasmid carried by the genetically engineered bacteria.

[0010] In one embodiment, the UDP-N-acetylglucosamine-2-isomerase gene (neuC), the sialic acid synthase gene (neuB), the N-acetylneuraminic acid cytidylyltransferase gene (css) and the α-2,3-sialyltransferase gene (ST) are integrated into the genome of the genetically engineered bacteria by being respectively connected to a separate first promoter and / or terminator, or are present in a recombinant plasmid carried by the genetically engineered bacteria.

[0011] In one embodiment, any two, three or four of the UDP-N-acetylglucosamine-2-isomerase gene (neuC), sialic acid synthase gene (neuB), N-acetylneuraminic acid cytidylyltransferase gene (css) and α-2,3-sialyltransferase gene (ST) are integrated into the genome of the genetically engineered bacterium by being jointly connected to a first promoter and / or terminator, or exist together in a recombinant plasmid carried by the genetically engineered bacterium.

[0012] In one embodiment, the first promoter is a T2 promoter. In one embodiment, the terminator is a T7 terminator.

[0013] In one embodiment, the recombinant plasmid uses pTU2 as a backbone plasmid.

[0014] In one embodiment, the UDP-N-acetylglucosamine-2-isomerase gene (neuC), the sialic acid synthase gene (neuB), the N-acetylneuraminic acid cytidylyltransferase gene (css) and the α-2,3-sialyltransferase gene (ST) are inserted into one or more insertion sequences 1 (IS1) of the genome of the genetically engineered bacteria as a gene combination in a manner where each is connected to a separate first promoter and / or terminator, or as a gene combination in a manner where they are jointly connected to a first promoter and / or terminator.

[0015] In one embodiment, the copy number of UDP-N-acetylglucosamine-2-isomerase gene (neuC), sialic acid synthase gene (neuB), N-acetylneuraminic acid cytidylyltransferase gene (css) and α-2,3-sialyltransferase gene (ST) as a gene combination at the insertion sequence 1 (IS1) is 10-28, 12-26 or 14-22, for example, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.

[0016] In one embodiment, the UDP-N-acetylglucosamine-2-epimerase comprises: an amino acid sequence as shown in SEQ ID NO:82, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in SEQ ID NO:82.

[0017] In one embodiment, the sialic acid synthase comprises: an amino acid sequence as shown in SEQ ID NO:83, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in SEQ ID NO:83.

[0018] In one embodiment, the N-acetylneuraminic acid cytidylyltransferase comprises: an amino acid sequence as shown in SEQ ID NO:84, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in SEQ ID NO:84.

[0019] In one embodiment, the α-2,3-sialyltransferase comprises: an amino acid sequence as shown in SEQ ID NO:85, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in SEQ ID NO:85.

[0020] In one embodiment, the genetically engineered bacteria are modified in one or more of the following ways:

[0021] (1) Genetically engineered bacteria are modified to increase the production of glucosamine-6-phosphate;

[0022] (2) genetically engineered bacteria are modified to continuously recycle cytidine-5′-triphosphate;

[0023] (3) genetically engineered bacteria modified to inactivate or block the bypass degradation pathway of N-acetylmannosamine (ManNAc);

[0024] (4) genetically engineered bacteria modified to inactivate or block the alternative decomposition pathway of phosphoenolpyruvate; and

[0025] (5) Genetically engineered bacteria are modified to increase the expression or activity of proteins involved in the oligosaccharide transport pathway.

[0026] (6) Genetically engineered bacteria are modified to reduce or eliminate the expression or activity of endogenous β-galactosidase.

[0027] (7) Genetically engineered bacteria are modified to reduce or eliminate the bypass degradation pathway of N-acetylneuraminic acid.

[0028] In one embodiment, the genetically engineered bacteria is a bacterium of the genus Escherichia, preferably Escherichia coli, more preferably Escherichia coli BL21 (DE3) strain.

[0029] In one embodiment, the genetically engineered bacteria are modified in one or more of the following ways:

[0030] (8) Genetically engineered bacteria modified to reduce or eliminate the expression or activity of 6-phosphofructokinase (pfkA);

[0031] (9) Genetically engineered bacteria modified to increase the expression or activity of glucosamine-6-phosphate synthase (glmS);

[0032] (10) The genetically engineered bacteria are modified to reduce or eliminate the expression or activity of N-acetylglucosamine-6-phosphate deacetylase (nagA) and / or glucosamine-6-phosphate deaminase (nagB);

[0033] (11) Genetically engineered bacteria modified to increase the expression or activity of cytidine monophosphate kinase (CMK) and polyphosphate kinase (PPK);

[0034] (12) Genetically engineered bacteria modified to reduce or eliminate the expression or activity of pyruvate kinase (pykA);

[0035] (13) Genetically engineered bacteria modified to increase the expression or activity of oligosaccharide transporters;

[0036] (14) Genetically engineered bacteria modified to reduce or eliminate the expression or activity of N-acetylneuraminic acid aldolase (nanA);

[0037] (15) Genetically engineered bacteria modified to reduce or eliminate the expression or activity of N-acetylmannosamine kinase (nanK);

[0038] (16) The genetically engineered bacteria are modified to reduce or eliminate the expression or activity of N-acetylmannosamine-6-phosphate isomerase (nanE);

[0039] (17) The genetically engineered bacteria are modified to reduce or eliminate the expression or activity of β-galactosidase (lacZ).

[0040] In one embodiment, the genetically engineered bacteria lacks the 6-phosphofructokinase (pfkA) gene.

[0041] In one embodiment, the genetically engineered bacteria overexpresses the glucosamine-6-phosphate synthase (glmS) gene.

[0042] In one embodiment, the genetically engineered bacteria lacks the glucosamine-6-phosphate deaminase (nagB) gene.

[0043] In one embodiment, the genetically engineered bacteria lacks the N-acetylmannosamine kinase (nanK) gene.

[0044] In one embodiment, the genetically engineered bacteria lacks the N-acetylmannosamine-6-phosphate epimerase (nanE) gene.

[0045] In one embodiment, the genetically engineered bacteria lacks the N-acetylglucosamine-6-phosphate deacetylase (nagA) gene.

[0046] In one embodiment, the genetically engineered bacteria overexpresses the cytidine monophosphate kinase (CMK) gene and the polyphosphate kinase (PPK) gene.

[0047] In one embodiment, the genetically engineered bacteria lacks the N-acetylneuraminic acid aldolase (nanA) gene.

[0048] In one embodiment, the genetically engineered bacteria lacks the pyruvate kinase (pykA) gene.

[0049] In one embodiment, the genetically engineered bacteria overexpresses the oligosaccharide transporter setB gene.

[0050] In one embodiment, the genetically engineered bacteria lacks the β-galactosidase (lacZ) gene.

[0051] In one embodiment, the deletion of the 6-phosphofructokinase (pfkA) gene comprises: knocking out a portion of the nucleotide sequence of the 6-phosphofructokinase (pfkA) gene, wherein the knocking out of the portion of the nucleotide sequence comprises: the length of the nucleotide sequence retained at the 5' end accounts for 0%-7% of the total nucleotide sequence length of the 6-phosphofructokinase (pfkA) gene, and the length of the nucleotide sequence retained at the 3' end accounts for 70%-80% of the total nucleotide sequence length of the 6-phosphofructokinase (pfkA) gene.

[0052] In one embodiment, the glucosamine-6-phosphate synthase (glmS) comprises: the amino acid sequence shown in SEQ ID NO:89, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:89.

[0053] In one embodiment, the overexpression of the glucosamine-6-phosphate synthase (glmS) gene is: integrating the glucosamine-6-phosphate synthase (glmS) gene into the genome of the genetically engineered bacteria.

[0054] In one embodiment, the deletion of the pyruvate kinase (pykA) gene comprises: knocking out a portion of the nucleotide sequence of the pyruvate kinase (pykA) gene, wherein the knocking out of the portion of the nucleotide sequence comprises: the length of the nucleotide sequence retained at the 5' end accounts for 4%-5.5% of the total nucleotide sequence length of the pyruvate kinase (pykA) gene, and the length of the nucleotide sequence retained at the 3' end accounts for 10%-20% of the total nucleotide sequence length of the pyruvate kinase (pykA) gene.

[0055] In one embodiment, the cytidine monophosphate kinase (CMK) comprises: the amino acid sequence shown in SEQ ID NO:91, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:91; the polyphosphate kinase (PPK) comprises: the amino acid sequence shown in SEQ ID NO:90, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:90.

[0056] In one embodiment, overexpressing the cytidine monophosphate kinase (CMK) gene and the polyphosphate kinase (PPK) gene comprises integrating the cytidine monophosphate kinase (CMK) gene and the polyphosphate kinase (PPK) gene into the genome of the genetically engineered bacteria.

[0057] In one embodiment, the oligosaccharide transporter setB comprises: the amino acid sequence shown in SEQ ID NO:93 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:93.

[0058] In one embodiment, overexpressing the oligosaccharide transporter setB gene comprises: integrating the oligosaccharide transporter setB gene into the genome of a genetically engineered bacterium.

[0059] In one embodiment, under the condition that part of the nucleotide sequence of the 6-phosphofructokinase (pfkA) gene is knocked out, the glucosamine-6-phosphate synthase (glmS) gene is inserted into the position where the 6-phosphofructokinase (pfkA) gene is knocked out.

[0060] In one embodiment, the genetically engineered bacteria lacks the melibiose permease (melB) gene, and the cytidine monophosphate kinase (CMK) gene and the polyphosphate kinase (PPK) gene are inserted into the position where the melibiose permease (melB) gene is knocked out.

[0061] In one embodiment, under the condition that a part of the nucleotide sequence of the pyruvate kinase gene (pykA) gene is knocked out, the oligosaccharide transporter setB gene is inserted into the knocked-out position of the pyruvate kinase gene (pykA) gene.

[0062] In one embodiment, the cytidine monophosphate kinase (CMK) gene and the polyphosphate kinase (PPK) gene are inserted into the position where the melibiose permease (melB) gene is knocked out, either by being linked to a separate second promoter or by being linked to a second promoter together.

[0063] In one embodiment, the cytidine monophosphate kinase (CMK) gene is located upstream of the polyphosphate kinase (PPK) gene. In one embodiment, the second promoter is a T5 promoter or a tetracycline promoter tet.

[0064] In another aspect, an Escherichia coli strain is provided, whose deposit number is CGMCC No.:28244.

[0065] In another aspect, provided is a use of the genetically engineered bacteria as described herein or the Escherichia coli strain as described herein in producing 3'-sialyllactose using glucose or glycerol as a carbon source.

[0066] In another aspect, a method for constructing a genetically engineered bacterium capable of producing 3'-sialyllactose is provided, comprising: integrating a UDP-N-acetylglucosamine-2-epimerase gene (neuC), a sialic acid synthase gene (neuB), an N-acetylneuraminic acid cytidylyltransferase gene (css) and an α-2,3-sialyltransferase gene (ST) independently or in any combination into the genome of a host bacterium to obtain a genetically engineered bacterium; or,

[0067] The UDP-N-acetylglucosamine-2-epimerase gene (neuC), the sialic acid synthase gene (neuB), the N-acetylneuraminic acid cytidylyltransferase gene (css) and the α-2,3-sialyltransferase gene (ST) are co-introduced into a backbone plasmid to obtain a recombinant plasmid; the recombinant plasmid is introduced into a host bacterial body to obtain a genetically engineered bacterium.

[0068] In one embodiment, the method for constructing a genetically engineered bacterium capable of producing 3'-sialyllactose comprises: independently integrating an introduced exogenous UDP-N-acetylglucosamine-2-epimerase gene (neuC), an introduced exogenous sialic acid synthase gene (neuB), an introduced exogenous N-acetylneuraminic acid cytidylyltransferase gene (css) and an introduced exogenous α-2,3-sialyltransferase gene (ST) into the genome of a host bacterium or integrating them together in any combination to obtain a genetically engineered bacterium; or,

[0069] The introduced exogenous UDP-N-acetylglucosamine-2-epimerase gene (neuC), the introduced exogenous sialic acid synthase gene (neuB), the introduced exogenous N-acetylneuraminic acid cytidylyltransferase gene (css) and the introduced exogenous α-2,3-sialyltransferase gene (ST) are introduced into the backbone plasmid to obtain a recombinant plasmid; the recombinant plasmid is introduced into a host bacterial body to obtain a genetically engineered bacterium.

[0070] In one embodiment, the host bacteria is a bacterium of the genus Escherichia, preferably Escherichia coli, more preferably Escherichia coli BL21 (DE3) strain.

[0071] In one embodiment, the method for constructing a genetically engineered bacterium capable of producing 3'-sialyllactose further comprises: modifying the host bacterium in one or more of the following ways:

[0072] (1) The host bacteria are modified to increase the production of glucosamine-6-phosphate;

[0073] (2) the host cell is modified to continuously recycle cytidine-5′-triphosphate;

[0074] (3) the host bacteria are modified to inactivate or block the bypass degradation pathway of N-acetylmannosamine (ManNAc);

[0075] (4) the host bacteria are modified to inactivate or block the alternative decomposition pathway of phosphoenolpyruvate;

[0076] (5) host bacteria are modified to increase the expression or activity of proteins involved in the oligosaccharide transport pathway;

[0077] (6) modifying the host cell to reduce or eliminate the expression or activity of β-galactosidase; and

[0078] (7) The host bacteria are modified to reduce or eliminate the bypass degradation pathway of N-acetylneuraminic acid.

[0079] In one embodiment, the genetically engineered bacteria are modified in one or more of the following ways:

[0080] (8) the host bacteria are modified to reduce or eliminate the expression or activity of 6-phosphofructokinase (pfkA);

[0081] (9) the host bacteria are modified to increase the expression or activity of glucosamine-6-phosphate synthase (glmS);

[0082] (10) the host bacteria are modified to reduce or eliminate the expression or activity of N-acetylglucosamine-6-phosphate deacetylase (nagA) and / or glucosamine-6-phosphate deaminase (nagB);

[0083] (11) The host bacteria are modified to increase the expression or activity of cytidine monophosphate kinase (CMK) and polyphosphate kinase (PPK);

[0084] (12) The host bacteria are modified to reduce or eliminate the expression or activity of pyruvate kinase (pykA);

[0085] (13) Modification of the host bacteria to increase the expression or activity of oligosaccharide transporters;

[0086] (14) The host bacteria are modified to reduce or eliminate the expression or activity of N-acetylneuraminic acid aldolase (nanA);

[0087] (15) The host bacteria are modified to reduce or eliminate the expression or activity of N-acetylmannosamine kinase (nanK);

[0088] (16) the host cell is modified to reduce or eliminate the expression or activity of N-acetylmannosamine-6-phosphate isomerase (nanE); and

[0089] (17) The host bacteria are modified to reduce or eliminate the expression or activity of β-galactosidase (lacZ).

[0090] In one embodiment, the host cell is deleted of the 6-phosphofructokinase (pfkA) gene.

[0091] In one embodiment, the host cell overexpresses the glucosamine-6-phosphate synthase (glmS) gene.

[0092] In one embodiment, the host cell is deleted of the glucosamine-6-phosphate deaminase (nagB) gene.

[0093] In one embodiment, the host cell is deleted of the N-acetylmannosamine kinase (nanK) gene.

[0094] In one embodiment, the host cell is deleted of the N-acetylmannosamine-6-phosphate epimerase (nanE) gene.

[0095] In one embodiment, the host cell is deleted of the N-acetylglucosamine-6-phosphate deacetylase (nagA) gene.

[0096] In one embodiment, the host bacteria overexpress cytidine monophosphate kinase (CMK) gene and polyphosphate kinase (PPK) gene.

[0097] In one embodiment, the host cell is deleted of the N-acetylneuraminic acid aldolase (nanA) gene.

[0098] In one embodiment, the host cell is deleted for the pyruvate kinase (pykA) gene.

[0099] In one embodiment, the host bacteria overexpresses the oligosaccharide transporter setB gene.

[0100] In one embodiment, the host cell is deleted for the β-galactosidase (lacZ) gene.

[0101] In one embodiment, the host bacteria is made to lack the 6-phosphofructokinase (pfkA) gene by: knocking out part of the nucleotide sequence of the 6-phosphofructokinase (pfkA) gene, wherein the knocking out of the part of the nucleotide sequence includes: the length of the nucleotide sequence retained at the 5' end accounts for 0%-7% of the total nucleotide sequence length of the 6-phosphofructokinase (pfkA) gene, and the length of the nucleotide sequence retained at the 3' end accounts for 70%-80% of the total nucleotide sequence length of the 6-phosphofructokinase (pfkA) gene.

[0102] In one embodiment, the glucosamine-6-phosphate synthase (glmS) comprises: the amino acid sequence shown in SEQ ID NO:89, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:89.

[0103] In one embodiment, the method of causing the host cell to overexpress the glucosamine-6-phosphate synthase (glmS) gene comprises integrating the glucosamine-6-phosphate synthase (glmS) gene into the genome of the host cell.

[0104] In one embodiment, the host bacteria is made to lack the pyruvate kinase (pykA) gene by: knocking out part of the nucleotide sequence of the pyruvate kinase (pykA) gene, wherein the knocking out of the part of the nucleotide sequence includes: the length of the nucleotide sequence retained at the 5' end accounts for 4%-5.5% of the total nucleotide sequence length of the pyruvate kinase (pykA) gene, and the length of the nucleotide sequence retained at the 3' end accounts for 10%-20% of the total nucleotide sequence length of the pyruvate kinase (pykA) gene.

[0105] In one embodiment, the cytidine monophosphate kinase (CMK) comprises: the amino acid sequence shown in SEQ ID NO:91, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:91; the polyphosphate kinase (PPK) comprises: the amino acid sequence shown in SEQ ID NO:90, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:90.

[0106] In one embodiment, the method of causing the host bacteria to overexpress the cytidine monophosphate kinase (CMK) gene and the polyphosphate kinase (PPK) gene is: integrating the cytidine monophosphate kinase (CMK) gene and the polyphosphate kinase (PPK) gene into the genome of the host bacteria.

[0107] In one embodiment, the oligosaccharide transporter setB comprises: the amino acid sequence shown in SEQ ID NO:93 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:93.

[0108] In one embodiment, causing the host bacteria to overexpress the oligosaccharide transporter setB gene comprises: integrating the oligosaccharide transporter setB gene into the genome of the host bacteria.

[0109] In one embodiment, under the condition that part of the nucleotide sequence of the 6-phosphofructokinase (pfkA) gene is knocked out, the glucosamine-6-phosphate synthase (glmS) gene is also inserted into the position where the 6-phosphofructokinase (pfkA) gene is knocked out.

[0110] In one embodiment, the melibiose permease (melB) gene of the host bacteria is deleted, and the cytidine monophosphate kinase (CMK) gene and the polyphosphate kinase (PPK) gene are inserted into the position where the melibiose permease (melB) gene is knocked out.

[0111] In one embodiment, under the condition that part of the nucleotide sequence of the pyruvate kinase gene (pykA) gene is knocked out, the oligosaccharide transporter setB gene is inserted into the knocked-out position of the pyruvate kinase gene (pykA) gene.

[0112] In one embodiment, the cytidine monophosphate kinase (CMK) gene and the polyphosphate kinase (PPK) gene are inserted into the position where the melibiose permease (melB) gene is knocked out, either by being connected to a separate second promoter or by being connected to a second promoter together.

[0113] In one embodiment, the cytidine monophosphate kinase (CMK) gene is located upstream of the polyphosphate kinase (PPK) gene. In one embodiment, the second promoter is a T5 promoter or a tetracycline promoter tet.

[0114] In one embodiment, the UDP-N-acetylglucosamine-2-isomerase gene (neuC), the sialic acid synthase gene (neuB), the N-acetylneuraminic acid cytidylyltransferase gene (css) and the α-2,3-sialyltransferase gene (ST) are inserted into one or more insertion sequence 1 (IS1) sites of the host bacterial genome as a gene combination in a manner where each is connected to a separate first promoter and / or terminator, or in a manner where they are commonly connected to a first promoter and / or terminator.

[0115] In one embodiment, the introduced exogenous UDP-N-acetylglucosamine-2-isomerase gene (neuC), sialic acid synthase gene (neuB), N-acetylneuraminic acid cytidylyltransferase gene (css) and α-2,3-sialyltransferase gene (ST) are inserted as a gene combination at the insertion sequence 1 (IS1) in a copy number of 10-28, 12-26 or 14-22.

[0116] In one embodiment, pTargetF-nagAB comprising homology arm 1 and homology arm 2 is used to knock out nagA and nagB genes, homology arm 1 is amplified using primers of SEQ ID NO: 3 and SEQ ID NO: 4 with the host bacterial genome as a template, and homology arm 2 is amplified using primers of SEQ ID NO: 5 and SEQ ID NO: 6 with the host bacterial genome as a template.

[0117] In one embodiment, a glmS knock-in Donor-DNA fragment comprising homology arm 3 and homology arm 4 and a pTargetF-pfkA-NT1 recombinant plasmid are used to knock out the pfkA gene and knock the glmS gene into the position in the host bacterial genome where the pfkA gene is knocked out. Homology arm 3 is amplified using primers of SEQ ID NO: 11 and SEQ ID NO: 12 with the host bacterial genome as a template, and homology arm 4 is amplified using primers of SEQ ID NO: 19 and SEQ ID NO: 20 with the host bacterial genome as a template.

[0118] In one embodiment, the ppk gene is knocked into the host bacterial genome using a ppk knock-in Donor-DNA and a pTargetF-melB-NT1 recombinant plasmid containing homology arms 5 and 6, homology arm 5 is amplified using primers of SEQ ID NO:35 and SEQ ID NO:36 with the host bacterial genome as a template, and homology arm 6 is amplified using primers of SEQ ID NO:41 and SEQ ID NO:42 with the host bacterial genome as a template.

[0119] In one embodiment, the cmk gene is knocked into the host bacterial genome using a cmk knock-in Donor-DNA and a pTargetF-melB-NT2 recombinant plasmid containing homology arms 7 and 8, homology arm 7 is amplified using primers of SEQ ID NO:43 and SEQ ID NO:44 with the host bacterial genome as a template, and homology arm 8 is amplified using primers of SEQ ID NO:49 and SEQ ID NO:50 with the host bacterial genome as a template.

[0120] In one embodiment, the pykA gene is knocked out using a setB knock-in pykA site Donor-DNA and a pTargetF-pykA-NT2 recombinant plasmid containing homology arms 9 and 10, and the setB gene is knocked into the position where the pykA gene is knocked out in the host bacterial genome, homology arm 9 is amplified using primers of SEQ ID NO: 58 and SEQ ID NO: 59 with the host bacterial genome as a template, and homology arm 10 is amplified using primers of SEQ ID NO: 64 and SEQ ID NO: 65 with the host bacterial genome as a template. The host bacterial cell is preferably Escherichia coli.

[0121] In another aspect, a method for producing 3'-sialyllactose by fermentation is provided, the method comprising the steps of:

[0122] Cultivating the genetically engineered bacteria described herein or the Escherichia coli strain described herein in a seed culture medium to obtain seeds;

[0123] The seeds are inoculated into a fermentation medium for induction culture, and after the fermentation is completed, a fermentation liquid containing 3'-sialyllactose is obtained.

[0124] In one embodiment, the fermentation medium is free of antibiotics.

[0125] In one embodiment, the seed culture medium is LB medium.

[0126] In one embodiment, the fermentation medium comprises TB medium and a carbon source, and the carbon source comprises glucose and / or glycerol.

[0127] Advantages of the present invention include:

[0128] 1. Provided is an integrated engineered bacterium with high 3'-sialyllactose production, which has a higher 3'-sialyllactose production than the production strains in the prior art (such as the SLIS026 strain);

[0129] 2. The genetically engineered bacteria in this article are integrated strains, there is no problem of plasmid loss, and the genetic stability is higher;

[0130] 3. When knocking out a gene (such as the pfkA gene), the method of the present invention integrates a target gene (such as the glmS gene, the setB gene) at the location of the original gene, thereby achieving overexpression of the target gene in the bacterial cell genome, reducing the steps of gene manipulation, and reducing the impact on bacterial cell growth and fermentation. BRIEF DESCRIPTION OF THE DRAWINGS

[0131] Figure 1 This is the 3'-SL metabolic pathway diagram of the integrated engineered bacteria.

[0132] Figure 2 This is the plasmid map of pTU2-A-DOE13.

[0133] Figure 3 This is the HPLC detection result spectrum of 3'-SL.

[0134] Figure 4 The following are the fermentation results of some selected transformants.

[0135] Figure 5 This is the growth and yield curve of shake flask fermentation of EBSL005 strain.

[0136] Figure 6 This is the plasmid map of pCas-Sac. DETAILED DESCRIPTION

[0137] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0138] As used herein, an engineered bacterium "having the ability to produce 3'-sialyllactose" refers to a bacterium that has the ability to produce 3'-sialyllactose as a target when cultured in a culture medium and accumulate it in the culture medium and / or in the bacterial body to a degree that can be recovered. An engineered bacterium having the ability to produce 3'-sialyllactose may be a strain that can accumulate 3'-sialyllactose in a culture medium and / or in the bacterial body in a greater amount than a non-modified strain. A "non-modified strain" refers to a control strain that has not undergone a specific genetic modification.

[0139] As used herein, the "SLIS026" strain is an Escherichia coli BL21 strain with the LacZ gene, nanA gene, and nanKE gene knocked out. The SLIS026 strain is a chassis strain constructed in Example 1 of the patent application with publication number CN 116200316A applied by Hongmo Biotechnology (Shanghai) Co., Ltd., which is incorporated herein by reference. The integrated engineered bacteria herein are constructed using the SLIS026 strain (see CN 116200316A) as the starting strain.

[0140] The increase in the number of copies of a gene can be achieved by introducing the gene into the host's genome. For example, homologous recombination can be used to introduce a gene into the genome. The gene introduction method using homologous recombination can include methods using linear DNA such as Red drive integration, methods using plasmids containing temperature-sensitive replication origins, methods using plasmids that can be transferred by conjugation, methods using suicide vectors that do not have replication origins that function in the host, and transduction methods using bacteriophages. Specifically, the host can be transformed with a recombinant DNA containing a target gene, and homologous recombination occurs with the target site on the host's genome, and the gene is introduced into the host's genome. As far as the structure of the recombinant DNA used in homologous recombination is concerned, there is no particular restriction as long as homologous recombination can occur in a desired manner. For example, a linear DNA containing a target gene can be used to transform the host, and the linear DNA is a linear DNA having the same base sequence as the upstream and downstream of the target site on the genome at both ends of the gene, and the gene can be replaced to the target site by causing homologous recombination in the upstream and downstream of the target site. The recombinant DNA used in homologous recombination can be equipped with a marker gene for selecting transformants. The gene can be introduced into only one copy, or two copies or more than two copies. For example, by carrying out homologous recombination with a base sequence having multiple copies on the genome as a target, multiple copies of the gene can be introduced into the genome. As base sequences having multiple copies on the genome, repetitive DNA sequences (repetitive DNA) and inverted repeats present at both ends of a transposon can be cited. In addition, homologous recombination can also be carried out with appropriate base sequences on the genome such as genes not needed in the production of the target substance as a target. In addition, the gene can also be randomly introduced into the genome using a transposon or Mini-Mu. It should be noted that, for the modification technique of the genome utilizing homologous recombination in this way, it is not limited to the introduction of the target gene, and can also be used for arbitrary modification of the genome such as the modification of the expression regulatory sequence.

[0141] Confirmation of the introduction of the target gene into the genome can be carried out by Southern hybridization using a probe having a sequence complementary to all or part of the gene, or PCR using primers prepared based on the sequence of the gene.

[0142] In addition, the increase in the copy number of a gene can also be achieved by introducing a vector containing the gene into a host. For example, a DNA fragment containing a target gene is connected to a vector that functions in a host to construct an expression vector of the gene, and by transforming the host with the expression vector, the copy number of the gene can be increased. For example, for a DNA fragment containing a target gene, it can be obtained by PCR using the genomic DNA of a microorganism having the target gene as a template. As a vector, a vector that can replicate autonomously in the cell of the host can be used. The vector is preferably a multicopy vector. In addition, in order to select transformants, it is preferred that the vector has markers such as antibiotic resistance genes. In addition, the vector can have a promoter and a terminator for expressing the inserted gene. The vector can be, for example, a vector derived from a bacterial plasmid, a vector derived from a yeast plasmid, a vector derived from a phage, a clay or a phagemid, etc.

[0143] In the case of introducing a gene, the gene can be maintained in the host in an expressible manner. Specifically, the gene can be maintained in a manner that is controlled and expressed based on a promoter that functions in the host. As far as the promoter is concerned, there is no particular limitation as long as it is a promoter that functions in the host. "Promoter that functions in the host" may refer to a promoter that has promoter activity in the host. The promoter may be a promoter derived from the host or a promoter derived from a heterologous species. The promoter may be an intrinsic promoter of the gene to be introduced or a promoter of other genes. As a promoter, for example, a strong promoter may be used.

[0144] A terminator for terminating transcription can be configured downstream of the gene. As far as the terminator is concerned, there is no particular limitation as long as it is a terminator that functions in the host. The terminator can be a terminator derived from the host or a terminator derived from a heterospecies. The terminator can be a terminator inherent to the gene to be introduced or a terminator of another gene.

[0145] In addition, when two or more genes are introduced, each gene can be maintained in the host in an expressible manner. For example, each gene can be maintained on a single expression vector or on a genome. In addition, each gene can be maintained on a plurality of expression vectors or on a single or multiple expression vectors and on a genome.

[0146] As for the gene introduced, there is no particular limitation as long as it is a gene encoding a protein that functions in the host. The gene introduced may be a gene derived from the host or a gene derived from a heterologous species. As for the gene introduced, for example, a primer designed based on the base sequence of the gene can be used, and the genomic DNA of the organism having the gene or a plasmid carrying the gene can be used as a template to obtain it by PCR. In addition, the introduced gene can also be fully synthesized based on the base sequence of the gene, for example. The obtained gene can be used as is, or it can be used after being appropriately modified. That is, by modifying the gene, its variant can be obtained. The modification of the gene can be carried out by known techniques. For example, by a site-specific mutation method, a target mutation can be introduced into a target site of DNA. That is, for example, by a site-specific mutation method, the coding region of the gene can be modified so that the encoded protein contains substitution, deletion, insertion and / or addition of amino acid residues at a specific site. Or a variant of the gene can be fully synthesized.

[0147] In addition, the increase of gene expression can be achieved by improving the transcription efficiency of the gene. In addition, the increase of gene expression can be achieved by improving the translation efficiency of the gene. The increase of gene transcription efficiency or translation efficiency can be achieved, for example, by modification of expression regulatory sequences.

[0148] The improvement of gene transcription efficiency can be achieved, for example, by replacing the promoter of the gene on the genome with a stronger promoter. A "stronger promoter" may refer to a promoter that improves gene transcription compared to an originally existing wild-type promoter.

[0149] "The activity or expression of a protein or enzyme is reduced" means that the activity of the protein or enzyme is reduced compared to a non-modified strain or a starting strain. It should be noted that "the activity or expression of a protein or enzyme is reduced" also includes the situation where the activity of the protein or enzyme disappears completely. More specifically, "the activity or expression of a protein or enzyme is reduced" may mean that the number of molecules per cell of the protein or enzyme is reduced and / or the function of each molecule of the protein or enzyme is reduced compared to a non-modified strain. As for the degree of reduction in the activity or expression of a protein or enzyme, there is no particular limitation as long as the activity of the protein or enzyme is reduced compared to a non-modified strain or a starting strain. The activity of a protein or enzyme can be reduced to, for example, less than 50%, less than 20%, less than 10%, less than 5% or 0% of that of a non-modified strain.

[0150] The reduction of gene expression can be caused by, for example, the reduction of transcription efficiency, the reduction of translation efficiency, or a combination thereof. For the reduction of gene expression, for example, the expression regulatory sequences such as the promoter, Shine-Dalgarno (SD) sequence (also referred to as ribosome binding site (RBS)), the spacer region between RBS and the start codon of the gene can be modified and achieved. In the case where the expression regulatory sequence is modified, it is preferred that more than one base of the expression regulatory sequence, more preferably more than two bases, and particularly preferably more than three bases are modified. For the reduction of gene transcription efficiency, for example, the promoter of the gene on the genome can be replaced by a weaker promoter. "Weaker promoter" refers to a promoter whose transcription of the gene is weakened compared to the promoter of the wild type that originally existed. As a weaker promoter, for example, an inducible promoter can be cited. That is, an inducible promoter can function as a weaker promoter under non-inducing conditions (for example, under the condition of the absence of an inducing substance). In addition, a part or all of the regions of the expression regulatory sequence can also be deleted (deleted). In addition, with regard to the reduction of gene expression, for example, it is also possible to achieve by operating the factors related to expression control. As factors related to expression control, low molecules (inducing substances, inhibitory substances, etc.), proteins (transcription factors, etc.), nucleic acids (siRNA, etc.) related to transcription and translation control can be enumerated. In addition, with regard to the reduction of gene expression, for example, it is also possible to achieve by introducing a variation that reduces the expression of the gene in the coding region of the gene. For example, by replacing the codons in the coding region of the gene with synonymous codons used with lower frequency in the host, the expression of the gene can be reduced. In addition, for example, it is also possible to reduce the expression of the gene itself by the destruction of the gene as described later.

[0151] In addition, modifications that reduce the activity or expression of a protein or enzyme can be achieved, for example, by disrupting the gene encoding the protein. "Gene disruption" means that the gene is modified in such a way that a protein that functions normally is not produced. "No protein that functions normally is produced" includes: a situation where no protein is produced from the gene at all, and a situation where the function (e.g., activity, property) of each molecule produced from the gene is reduced or lost.

[0152] As for the destruction of genes, for example, it can be achieved by making the gene on the genome deleted (deleted). "Gene deletion" refers to the deletion of a part or all of the coding region of the gene. Further, the entire gene can be deleted together with the sequences before and after the coding region of the gene on the genome. In the sequences before and after the coding region of the gene, for example, the expression regulatory sequence of the gene can be included. As long as the reduction of protein activity can be achieved, the region to be deleted can be any region such as the N-terminal region (the region on the N-terminal side of the encoded protein), the internal region, and the C-terminal region (the region on the C-terminal side of the encoded protein). Generally, the longer the region to be deleted, the more reliably the gene can be inactivated. For example, the region to be deleted can be a region of more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90% or more than 95% of the total length of the coding region of the gene. In addition, with respect to the sequences before and after the region to be deleted, it is preferred that the reading frame is inconsistent. By the inconsistency of the reading frame, a frame shift can occur downstream of the deleted region.

[0153] In addition, for example, the disruption of a gene can be achieved by introducing an amino acid substitution (missense variation), introducing a stop codon (nonsense variation), or introducing the addition or deletion of 1 to 2 bases (frameshift variation) into the coding region of a gene on the genome. Alternatively, it can be achieved by inserting other base sequences into the coding region of a gene on the genome. The insertion site can be any region of the gene, and the longer the inserted base sequence, the more reliably the gene can be inactivated. In addition, for the sequences before and after the insertion site, it is preferred that the reading frame is inconsistent. By inconsistency in the reading frame, a frameshift can occur downstream of the insertion site. As other base sequences, there is no particular restriction as long as the activity of the encoded protein is reduced or eliminated. In particular, it can be implemented in a manner that the amino acid sequence of the encoded protein or enzyme is deleted (defective). In other words, for example, with respect to modifications that reduce the activity of a protein or enzyme, the amino acid sequence of the protein (a part or all of the region of the amino acid sequence) can be deleted.

[0154] As used herein, the nanA gene encodes N-acetylneuraminic acid aldolase (EC 4.1.3.3). N-acetylneuraminic acid aldolase can catalyze the C—C bond cleavage of N-acetylneuraminic acid (N-acetylneuraminic acid, Neu5Ac, also known as sialic acid), thereby producing N-acetylmannosamine (N-acetyl-D-mannosamine or N-Acetylmannosamine, ManNAc) and pyruvic acid (Pyr). The strain with the nanA gene knocked out does not express active N-acetylneuraminic acid aldolase, so that Neu5Ac is not converted into ManNAc, which inhibits Neu5Ac from being consumed through this pathway, thereby facilitating the accumulation of Neu5Ac in bacterial cells.

[0155] As used herein, the nanK gene encodes N-acetylmannosamine kinase (N-acetyl-D-mannosaminekinase, EC 2.7.1.60). N-acetylmannosamine kinase can catalyze the transfer of phosphate groups, thereby converting ManNAc into N-acetylmannosamine-6-phosphate (N-Acetyl-D-mannosamine-6-phosphate, ManNAc-6-P). The strain with the nanK gene knocked out does not express active N-acetylmannosamine kinase, thereby basically not converting ManNAc into ManNAc-6-P, inhibiting ManNAc from being consumed through this pathway, thereby facilitating the accumulation of ManNAc in bacterial cells, and then facilitating the accumulation of Neu5Ac (with ManNAc as a precursor) in bacterial cells.

[0156] As used herein, the nanE gene encodes N-acetylmannosamine-6-phosphate epimerase (N-acetyl-D-mannosamine epimerase; also known as ManNAc-6-P epimerase, EC 5.1.3.9). N-acetylmannosamine-6-phosphate epimerase can catalyze the isomerization of N-acetylmannosamine-6-phosphate (N-Acetyl-D-mannosamine-6-phosphate, ManNAc-6-P) to N-acetylglucosamine-6-phosphate (N-acetylglucosamine-6-phosphate, GlcNAc-6-P). The strain with the nanE gene knocked out does not basically express active N-acetylmannosamine-6-phosphate epimerase, so that the isomerization reaction of ManNAc-6-P to GlcNAc-6-P does not occur, which inhibits ManNAc-6-P from being consumed through this pathway.

[0157] Genetically engineered bacteria and construction method of the present invention

[0158] Previously, the CRISPR / Cas9 technology was used to knock out genes related to the intracellular degradation of N-acetylneuraminic acid (Neu5Ac) in the wild-type strain BL21 (DE3), and the lacZ gene encoding β-galactosidase was knocked out, resulting in a non-naturally occurring Escherichia coli strain SLIS026 for producing Neu5Ac, and a Neu5Ac or SL synthesis pathway containing at least one heterologous enzyme was constructed in the strain, and the resulting genetically engineered bacteria were able to synthesize Neu5Ac or SL by self-metabolism using a single cheap exogenous carbon source present in the fermentation broth. For details, see the record of the patent application with publication number CN116200316A.

[0159] Based on the previously obtained genetically engineered bacteria SLIS026, the inventors further genetically modified it and obtained an integrated engineered bacteria that does not require the addition of antibiotics and produces high 3'-sialyllactose. The construction of the integrated engineered bacteria of the present invention is further described below. The enzymes related to genetic modification and their encoding genes are described below.

[0160] like Figure 1As shown in the figure, the lacZ gene in Escherichia coli encodes β-galactosidase. β-galactosidase can cut the galactosidic bond of lactose to produce galactose and glucose. The strain with the lacZ gene knocked out basically does not express active β-galactosidase, thereby inhibiting the consumption of lactose through this pathway, which is conducive to the accumulation of lactose in bacterial cells. Because CMP-N-acetylneuraminic acid (CMP-Neu5Ac) and lactose generate 3'-sialyllactose under the catalysis of α-2,3-sialyltransferase encoded by the ST gene, the accumulation of lactose concentration in bacterial cells is conducive to increasing the production of 3'-sialyllactose.

[0161] The integrated engineered bacteria of the present invention have undergone any one or more of the following modifications relative to the starting strain (SLIS026 strain):

[0162] 1. The metabolic pathway of glucosamine 6-phosphate (GlcN-6-P) of the SLIS026 strain was modified.

[0163] The purpose of this modification is to increase the supply of glucosamine-6-phosphate in bacterial cells by reducing the consumption of glucosamine-6-phosphate in bacterial cells through the bypass pathway. Because glucosamine-6-phosphate is one of the synthetic precursors of N-acetylneuraminic acid (Neu5Ac), and Neu5Ac is one of the synthetic precursors of 3'-sialyllactose (3'-SL), increasing the supply of glucosamine-6-phosphate helps to increase the supply of Neu5Ac, and thus helps to increase the production of 3'-SL.

[0164] The integrated engineered bacteria of the present invention can at least use glucose (Glucose) or glycerol (Glycerol) as a carbon source to produce 3'-SL. Among them, after entering the bacterial cell, glucose can be converted into glucose-6-phosphate (Glucose-6-phosphate, G-6-P) under the action of glucose phosphorylation and transport-related enzymes encoded by genes such as ptsG, ptsH, crr, ptsL, etc. Glucose-6-phosphate generates fructose-6-phosphate (Fructose-6-phosphate, F-6-P) under the action of phosphoglucose isomerase (PGI) encoded by the pgi gene. After entering the bacterial cell, glycerol generates F-6-P under the catalysis of glycerol metabolism-related enzymes encoded by genes such as glpF, glpK, glpD, etc. F-6-P generates glucosamine-6-phosphate (GlcN-6-P) under the action of glucosamine-6-phosphate synthetase (glmS, EC 2.6.1.16). The same applies to the utilization of other carbon sources (such as sucrose).

[0165] In order to increase the supply of glucosamine-6-phosphate (GlcN-6-P), the present invention adopts the following method:

[0166] (1) Knockout the pfkA gene encoding 6-phosphofructokinase;

[0167] The pfkA gene of Escherichia coli can encode 6-phosphofructokinase (EC2.7.1.11). 6-phosphofructokinase can catalyze the transfer of phosphate groups, thereby generating F-6-P into fructose-1,6-bisphosphate (Fructose 1,6-bisphosphate, FBP). The strain with the pfkA gene knocked out basically does not express 6-phosphofructokinase, thereby avoiding the conversion of most F-6-P into FBP, thereby reducing the consumption of F-6-P in bacterial cells, facilitating the accumulation of F-6-P in bacterial cells, and then facilitating the accumulation of GlcN-6-P in bacterial cells. When the pfkA gene is knocked out, the present invention only knocks out part of its nucleotide fragments, without knocking out all of its nucleotide fragments. The knockout method of the partial nucleotide fragment is: the length of the nucleotide sequence retained at the 5' end accounts for 0%-7% of the total nucleotide sequence length of the endogenous 6-phosphofructokinase (pfkA) gene, and the length of the nucleotide sequence retained at the 3' end accounts for 70%-80% of the total nucleotide sequence length of the endogenous 6-phosphofructokinase (pfkA) gene

[0168] (2) overexpression of the glmS gene encoding glucosamine-6-phosphate synthase;

[0169] The glmS gene of Escherichia coli can encode glucosamine-6-phosphate synthase (glmS). Glucosamine-6-phosphate synthase glmS can catalyze F-6-P to generate GlcN-6-P. When the glmS gene is overexpressed, the efficiency of F-6-P to generate GlcN-6-P will be improved, and then the efficiency of GlcN-6-P conversion to glucosamine-1-phosphate (GlcN-1-P) can be improved, which is beneficial to the generation of 3'-SL. The present invention integrates the glucosamine-6-phosphate synthase gene (glmS) into the genome of the genetically engineered bacteria in a manner of increasing the copy number of the glucosamine-6-phosphate synthase (glmS) gene, thereby achieving overexpression of the glmS gene.

[0170] In the present invention, when a partial fragment of the pfkA gene is knocked out, the glmS gene (denoted as ΔpfkA::glmS) can be integrated at the position where the pfkA gene is knocked out, thereby achieving overexpression of the glmS gene in the bacterial cell genome. This can reduce the steps of gene manipulation and reduce the impact on bacterial cell growth and fermentation.

[0171] (3) knocking out the nagB gene encoding glucosamine-6-phosphate deaminase;

[0172] The nagB gene of Escherichia coli encodes glucosamine-6-phosphate deaminase. Glucosamine-6-phosphate deaminase can catalyze the decomposition of glucosamine-6-phosphate (Glucosamine-6-P, GlcN-6-P) into fructose-6-phosphate (F-6-P) and ammonia. The strain with the nagB gene knocked out can basically not express active glucosamine-6-phosphate deaminase, so that GlcN-6-P is basically not converted into F-6-P, thereby reducing the consumption of GlcN-6-P in bacterial cells, which is conducive to the accumulation of GlcN-6-P in bacterial cells.

[0173] As can be seen from the above, the present invention can enhance the synthesis pathway of GlcN-6-P by overexpressing the glmS gene. Secondly, the present invention can inhibit the decomposition pathway of GlcN-6-P by knocking out the nagB gene and the pfkA gene. Therefore, the modification of the metabolic pathway of GlcN-6-P in bacterial cells by the present invention is conducive to increasing the supply of GlcN-6-P in bacterial cells, thereby helping to increase the supply of Neu5Ac, and ultimately helping to increase the yield of 3'-SL.

[0174] In addition, when knocking out the nagB gene, the nagB gene and the nagA gene can be knocked out together, which can reduce the steps of genetic manipulation, improve the efficiency of genetic manipulation, and reduce the impact on bacterial cell growth and fermentation.

[0175] 2. Four key genes related to 3'-SL synthesis were overexpressed

[0176] GlcN-6-P can generate GlcN-1-P under the action of phosphoglucosamine isomerase encoded by glmM gene. GlcN-1-P generates GlcNAc-1-P under the action of enzyme (fused N-acetylglucosamine-1-phosphateuridyltransferase and glucosamine-1-phosphate acetyltransferase) encoded by glmU gene, and then generates UDP-GlcNAc. UDP-GlcNAc, as one of the precursors of 3'-SL synthesis, can generate 3'-SL under the action of four key enzymes. Therefore, if the genes of these four key enzymes are overexpressed in bacterial cells, the synthesis pathway of 3'-SL will be enhanced, which will help to increase the production of 3'-SL.

[0177] The four key genes related to 3'-SL synthesis are: neuC gene, neuB gene, css gene and ST gene.

[0178] Wherein, the neuC gene encodes UDP-N-acetylglucosamine 2-epimerase. The enzyme can catalyze UDP-GlcNAc to generate ManNAc. The UDP-N-acetylglucosamine 2-epimerase in the present invention is derived from Campylobacter jejuni strain, and the NCBI accession number is WP_002874240.1.

[0179] The neuB gene encodes sialic acid synthase (also known as neuraminidase). Sialic acid synthase can catalyze the reaction of ManNAc and phosphoenolpyruvic acid (PEP) to generate Neu5Ac and phosphate. The gene of sialic acid synthase in the present invention is derived from Campylobacter jejuni strain, and the GenBank accession number is WP_002874241.1.

[0180] The css gene encodes N-acetylneuraminate cytidylyltransferase. N-acetylneuraminate cytidylyltransferase can catalyze Neu5Ac to generate CMP-Neu5Ac and pyrophosphate using the cofactor CTP. In the present invention, N-acetylneuraminate cytidylyltransferase is derived from Neisseria meningitidis strain, and the NCBI accession number is WP_002215295.1.

[0181] The ST gene encodes α-2,3-sialyltransferase. α-2,3-sialyltransferase can transfer sialic acid (Sialicacid, also known as N-acetylneuraminic acid, Neu5Ac) from an activated glycosyl donor (CMP-Neu5Ac) to lactose (Lactose), thereby generating 3'-SL and releasing CMP. The ST of the present invention is derived from the Bibersteinia trehalosi strain, and the NCBI accession number is AGH37861.1.

[0182] The above four genes are respectively constructed into different transcription units using strong promoters (such as T2 promoters) and terminators when overexpressed, and are expressed separately. Exemplarily, the transcription units composed of the above four genes are T2-pET-RBS-css-T7T, T2-pET-RBS-neuB-T7T, T2-pET-RBS-neuC-T7T and T2-pET-RBS-ST-T7T. T2 represents the T2 promoter sequence, and RBS represents the ribosome binding site. T7T represents the T7 terminator sequence. The sequence of the T2 promoter is GGATAATCAGATCACGAAATTAATACCACTCACTATAGGAATAGTGCCCGGGTAACCATTCC, and the sequence number is SEQ ID NO:81. The sequence of pET-RBS is TTTAACTTTAAGAAGGAGATATA, and the sequence number is SEQ ID NO:94. The inventors have experimentally confirmed that, in addition to the T2 promoter, the lactose promoter (lac promoter), tryptophan promoter (trp promoter), and T5 promoter can also be used for the above four genes. However, the T2 promoter has a better effect, so the T2 promoter is selected for use in the present invention.

[0183] 3. Modify the supply pathway of cofactors

[0184] In point 2, the N-acetylneuraminic acid cytidyl transferase encoded by the css gene needs the cofactor cytidine 5'-triphosphate (CTP) to catalyze Neu5Ac to generate CMP-Neu5Ac and pyrophosphate. The reaction equation is: CTP+Neu5Ac→pyrophosphate+CMP-Neu5Ac. This reaction consumes CTP.

[0185] In addition, the α-2,3-sialyltransferase encoded by the ST gene will also release CMP when catalyzing the reaction of CMP-Neu5Ac and lactose to generate 3'-SL. The reaction equation is: CMP-Neu5Ac+lactose→3'-SL+CMP. This reaction will lead to the accumulation of CMP.

[0186] If there is insufficient CTP in the bacterial cells, it is impossible to produce enough CMP-Neu5Ac, which will affect the yield of 3'-SL. In addition, as CMP accumulates, the generation reaction of 3'-SL will also be inhibited. In order to solve the above problems, the present invention can overexpress the cmk gene encoding cytidine monophosphate kinase (CMK) and the ppk gene encoding polyphosphate kinase PPK. The cytidine monophosphate kinase encoded by the cmk gene can catalyze the phosphorylation of CMP to produce CDP. The polyphosphate kinase PPK encoded by the ppk gene can catalyze the phosphorylation of CDP to produce CTP, thereby realizing the recycling of cofactors and avoiding the inhibitory effect of insufficient cofactors on the generation reaction of 3'-SL.

[0187] The cmk gene and the ppk gene of the present invention are integrated into the genome of the genetically engineered bacteria. The endogenous melibiose permease (melB) gene in the genome of the genetically engineered bacteria is knocked out, and the exogenous cytidine monophosphate kinase (CMK) gene and the exogenous polyphosphate kinase (PPK) gene are inserted into the position where the endogenous melibiose permease (melB) gene is knocked out, so that the steps of genetic manipulation can be reduced and the adverse effects of genetic manipulation on strain growth and metabolism can be reduced.

[0188] 4. Modification of the decomposition pathway of ManNAc

[0189] ManNAc is one of the precursors for the production of 3'-SL. However, the decomposition pathway of ManNAc in bacterial cells will cause the content of ManNAc in bacterial cells to decrease, thereby affecting the production of 3'-SL. The decomposition pathway of ManNAc includes: (1) N-acetylmannosamine kinase encoded by nanK gene catalyzes the conversion of ManNAc into ManNAc-6-P; (2) N-acetylmannosamine-6-phosphate isomerase encoded by nanE gene isomerizes ManNAc-6-P into GlcNAc-6-P; (3) N-acetylglucosamine-6-phosphate deacetylase encoded by nagA gene can catalyze the removal of acetyl group of GlcNAc-6-P, thereby producing GlcN-6-P.

[0190] Based on this, the present invention modifies the decomposition pathway of ManNAc to inactivate the decomposition pathway, thereby facilitating the accumulation of ManNAc in bacterial cells.

[0191] The strain of the present invention lacks the nanK gene encoding N-acetylmannosamine kinase. The strain lacking the nanK gene can basically not express active N-acetylmannosamine kinase, so that ManNAc is basically not converted into ManNAc-6-P, which is conducive to the accumulation of ManNAc in bacterial cells, and further conducive to the accumulation of Neu5Ac (with ManNAc as a precursor) in bacterial cells.

[0192] In addition, the strain of the present invention also lacks the nanE gene encoding N-acetylmannosamine-6-phosphate epimerase. The strain with the nanE gene knocked out can basically not express active N-acetylmannosamine-6-phosphate epimerase, so that the isomerization reaction from ManNAc-6-P to GlcNAc-6-P basically does not occur.

[0193] Furthermore, the strain of the present invention has knocked out the nagA gene encoding N-acetylglucosamine-6-phosphate deacetylase. N-acetylglucosamine-6-phosphate deacetylase can catalyze the deacetylation of GlcNAc-6-P, thereby producing GlcN-6-P. Therefore, the strain with the nagA gene knocked out does not basically express active N-acetylglucosamine-6-phosphate deacetylase, so that the reaction of GlcNAc-6-P to GlcN-6-P basically does not occur. Exemplarily, when the nagA gene is knocked out, the nagB gene and the nagA gene can be knocked out together, which can reduce the steps of genetic manipulation, improve the efficiency of genetic manipulation, and reduce the impact on bacterial cell growth and fermentation.

[0194] The strain of the present invention has knocked out the nanK gene, the nanE gene, and the nagA gene, so that ManNAc is difficult to be consumed through the above-mentioned decomposition pathway, which is beneficial to the accumulation of ManNAc in bacterial cells and further helps to increase the yield of 3'-SL.

[0195] 5. Modification of the decomposition pathway of phosphoenolpyruvate

[0196] The sialic acid synthase encoded by the neuB gene catalyzes the reaction of ManNAc and phosphoenolpyruvic acid (PEP) to generate Neu5Ac and phosphoric acid. In order to increase the accumulation of Neu5Ac and thus increase the content of 3'-SL, it is necessary to increase the content of PEP. One way to produce PEP is that bacterial cells use glucose through the glycolysis pathway. However, PEP is easy to generate enolpyruvic acid under the catalysis of pyruvate kinase (PykA) in bacterial cells, and the reaction equation is: PEP+2ADP→enolpyruvic acid+2ATP. Enolpyruvic acid is then converted into pyruvic acid (pyr). Therefore, in order to increase the PEP content in bacterial cells, the present invention knocks out a partial fragment of the pykA gene encoding pyruvate kinase. Part of the nucleotide sequence of the endogenous pyruvate kinase (pykA) gene is knocked out, wherein the length of the nucleotide sequence retained at the 5' end accounts for 4%-5.5% of the total nucleotide sequence length of the endogenous pyruvate kinase (pykA) gene, and the length of the nucleotide sequence retained at the 3' end accounts for 10%-20% of the total nucleotide sequence length of the endogenous pyruvate kinase (pykA) gene.

[0197] 6. Modification of oligosaccharide transport pathway

[0198] The 3'-SL produced in the bacterial cell needs to be pumped out of the cell, otherwise it will inhibit the generation of 3'-SL. The present invention uses overexpression of the oligosaccharide transporter gene setB to increase the ability of 3'-SL to be pumped out of the cell. When knocking out a partial fragment of the pykA gene, the present invention integrates the setB gene (denoted as ΔpykA::setB) at the knocked-out position of the original pykA gene, which can reduce the steps of gene manipulation and reduce the impact on bacterial cell growth and fermentation.

[0199] The genes involved in this application are shown in Table 1 below:

[0200] Table 1 Gene information involved in the examples

[0201]

[0202]

[0203] The metabolic pathway diagram of the integrated engineered bacteria of the present invention is as follows: Figure 1 shown.

[0204] Example

[0205] To further illustrate the technical means and effects of the present invention, the following is a detailed description of the preferred embodiments of the present invention in conjunction with the accompanying drawings. The experimental methods in the following embodiments without specifying specific conditions are selected according to conventional methods and conditions or according to the product instructions.

[0206] The plasmids, PCR reagents, restriction endonucleases, plasmid extraction kits, DNA gel recovery kits, etc. used in the following examples are commercial products. Please refer to the instructions for specific operations.

[0207] Primer synthesis, gene synthesis and sequencing were all completed by Beijing Qingke Biotechnology Co., Ltd. (hereinafter referred to as Qingke Biotechnology).

[0208] The competent cells of Escherichia coli were prepared using the competent cells preparation kit of Sangon Biotech (Shanghai) Co., Ltd.

[0209] The SLIS026 strain is a chassis strain constructed in Example 1 of the patent application with publication number CN 116200316A applied by Hongmo Biotechnology (Shanghai) Co., Ltd.

[0210] The map of the plasmid pCas-Sac (KanR resistance) used in the present invention is as follows Figure 6 Other reagents involved in the following examples are shown in Table 2.

[0211] Table 2 Reagent information table in the embodiment

[0212]

[0213]

[0214] The homemade reagents involved in the following examples are as follows:

[0215] (1) Antibiotic and sugar concentrations: CmR (chloramphenicol) is used at a concentration of 25 μg / mL; KanR (kanamycin) is used at a concentration of 50 μg / mL; SmR (Spec) (spectinomycin) is used at a concentration of 50 μg / mL; Amp (ampicillin) is used at a concentration of 100 μg / mL; aTc (tetracycline) is used at a concentration of 100 ng / mL or 1000 ng / mL; Apr (ampramycin) is used at a concentration of 50 μg / mL, rhamnose is used at a concentration of 10 mM; sucrose is used at a concentration of 10 g / L.

[0216] (2) TB medium: weigh 10 g of trypsin, 18 g of yeast extract, and KH 2 PO 4 (potassium dihydrogen phosphate) 2.31 g and K 2 HPO 4 Dissolve 16.43 g of potassium dihydrogen phosphate in distilled water and mix well. Then add 4 mL of glycerol and dilute to 1000 mL with deionized water. Finally, dispense into conical flasks at 100 mL / bottle and wrap them well. Sterilize at 121°C for 20 min and store at room temperature.

[0217] (3) LB medium: Weigh 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, and 15 g of agar, respectively, add distilled water to dissolve and mix, adjust the pH to 7.2 with 1 mol / L NaOH, and then make up to 1 L with distilled water. Finally, dispense into Erlenmeyer flasks and wrap them well. Sterilize at 121°C for 20 min and store at 4°C. Do not add agar to LB liquid medium.

[0218] (4) 200 g / L MgSO 4 7H 2 O mother solution: weigh 10g MgSO 4 7H 2 O solution into deionized water, dilute to 50 mL after complete dissolution, sterilize at 121°C for 20 min, and store at room temperature.

[0219] (5) Trace element mother solution: prepare 54.4 g / L ammonium ferric citrate and 9.8 g / L MnCl 2 ·4H 2 O, 1.6g / LCoCl 2 6H 2 O, 1g / L CuCl 2 ·2H 2 O, 1.9g / LH 3 BO 3 , 9g / L ZnSO 4 7H 2 O, 1.1 g / L Na 2 MoO 4 ·2H 2 O, 1.5 g / L Na 2 SeO 3 and 1.5 g / L NiSO 4 6H 2 O trace element stock solution, sterilize at 121℃ for 20min and store at 4℃ for later use.

[0220] (6) 500 g / L glucose: Weigh 500 g of glucose and dissolve it in deionized water (heat to dissolve). Adjust the volume to 1 L. Then sterilize it at 115°C for 20 min and store it at room temperature.

[0221] (7) 250 g / L lactose: Weigh 250 g of lactose and dissolve it in deionized water (heat to dissolve). Adjust the volume to 1 L. Sterilize the solution at 121°C for 20 min and store at room temperature.

[0222] The HPLC detection method for the 3'-SL obtained by fermentation in the following examples is as follows:

[0223] (I) Fermentation sample processing:

[0224] Sample processing method: Take 1-2mL of sample (such as fermentation broth), then place the sample in a metal bath (95℃) and boil for 30 minutes to remove enzymes, centrifuge after cooling, and test after filtering through a water system membrane.

[0225] (II) HPLC detection of 3'-SL:

[0226] (1) Instruments and reagents: High performance liquid chromatography with UV-visible absorption detector, acetonitrile (chromatographic grade), purified water, ammonium formate (AR), formic acid (chromatographic grade)

[0227] (2) Chromatographic conditions: Chromatographic column: Sepax HP-Amide 250×4.6 mm, 5 μm (amide column); buffer salt: 10 mM ammonium formate (pH 3.0); mobile phase: acetonitrile: buffer salt = 70:30;

[0228] Flow rate: 1.0 mL / min; concentration: 1 mg / mL; detection wavelength: 210 nm; injection volume: 10 μL; column temperature: 35 °C;

[0229] (3) Detection operation:

[0230] ① The blank loading solution and dilution solvent are deionized water.

[0231] ② Preparation of standard solution: Accurately weigh 25 mg of the standard into a 25 mL volumetric flask, add water to dissolve and dilute, make up to volume, and prepare for injection.

[0232] ③ Preparation of test solution: Take a certain amount of fermentation broth, heat and inactivate it, centrifuge it, take the supernatant and filter it, and prepare for sampling.

[0233] (4) Calculation of 3'-SL content: The content was calculated using the external standard method.

[0234] The sample content calculation formula is as follows:

[0235]

[0236] Note: A 供试品 is the peak area of ​​the test sample (mAU), A 对照品 is the peak area of ​​the reference substance (mAU), C 供试品 is the concentration of the test sample (mg / mL), C 对照品 is the concentration of the reference substance (mg / mL), X 供试品 is the content of the test sample (%), X 对照品 is the content of the reference substance (%).

[0237] Example 1: Increasing the supply of glucosamine-6-phosphate (GlcN-6P)

[0238] Glucosamine-6-phosphate is one of the precursors of 3'-SL synthesis and the amount of glucosamine-6-phosphate supplied is one of the rate-limiting steps of 3'-SL synthesis, so increasing the supply of glucosamine-6-phosphate helps to increase the yield of 3'-SL. This embodiment transforms the metabolic pathway of glucosamine-6-phosphate, increases the supply of glucosamine-6-phosphate, and reduces the decomposition amount of glucosamine-6-phosphate. Specifically, this example uses the SLIS026 strain as the starting strain, and uses the CRISPR / Cas9 gene editing system to knock out the nagA gene (the encoding gene for N-acetylglucosamine-6-phosphate deacetylase) and the nagB gene (the encoding gene for glucosamine-6-phosphate deaminase), as well as a partial fragment of the pfkA gene (the encoding gene for 6-phosphofructokinase), and when knocking out the pfkA gene, the glmS gene (the encoding gene for glucosamine-6-phosphate synthase) is integrated into the knocked-out pfkA fragment, so that the glmS gene is overexpressed (recorded as ΔpfkA::glmS), thereby obtaining the SLIS027 strain.

[0239] The steps of this embodiment are as follows:

[0240] 1.1. Design and synthesis of primers required for gene knockout and gene integration

[0241] According to Table 1, the nucleotide sequences of nagA gene, nagB gene, pfkA gene and glmS gene were obtained respectively. According to these sequences, primers for knocking out nagA gene and nagB gene were designed respectively, and primers for knocking out a part of the pfkA gene fragment and integrating the knocked-out pfkA gene fragment into the glmS gene were designed, as shown in Table 3. The sequence of sgRNA is shown in Table 4. The designed primers were submitted to Beijing Qingke Biotechnology Co., Ltd. (hereinafter referred to as Qingke Biotechnology) for synthesis.

[0242] Table 3 Primers required for gene knockout and gene integration

[0243]

[0244]

[0245] In Table 3, in the column pTargetF-nagAB, PAM represents the protospacer adjacent motif, which is used for the Cas9 protein to recognize PAM and target a specific DNA sequence under the guidance of sgRNA (single guide RNA), and then the Cas9 protein excises the specific DNA sequence. The DNA sequence used by homology arms LF1 and RF1 to generate the incision can be accurately repaired.

[0246] The glmS knock-in Donor-DNA fragment is a donor DNA molecule for glmS knock-in, which includes a homology arm LF2 sequence, a Tet promoter sequence, a glmS gene sequence, a T7 terminator sequence, and a homology arm RF2 sequence, so that the glmS gene can be inserted at the position where the pfkA gene is knocked out. The above sequences are amplified by PCR with respective primers and corresponding templates, and then connected into the glmS knock-in Donor-DNA fragment using NEB (New England Biolabs) multi-fragment recombinase. The same applies to the remaining knock-in genes.

[0247] Table 4 sgRNA sequence list

[0248] Genetic manipulation How it works sgRNA sequence Sequence number ΔnagAB Knockout CGGTCTGCCGAAAGAGCATC SEQ ID NO:21 ΔpfkA::glmS Knock out and replace GTTTCTGACATGATCAACCG SEQ ID NO:22

[0249] In Table 4, ΔnagAB indicates that the nagA gene and the nagB gene are knocked out. ΔpfkA::glmS indicates that a portion of the pfkA gene is knocked out and the glmS gene is integrated into the knocked-out pfkA gene.

[0250] 1.2 Construction of related plasmids:

[0251] The primers designed according to Table 3 were used to specifically amplify the gene fragments related to pTargetF-nagAB and pTargetF-pfkA-NT1, and the high-fidelity enzyme 2× FlashKOD Dye Mix was used for PCR reaction. The PCR amplification reaction system is shown in Table 5:

[0252] Table 5 PCR amplification reaction system

[0253] Reagents PCR reaction system (50 μL) DNA template 1μL Primer F 1μL Primer R 1μL PCR Mix 25μL <![CDATA[ddH 2 The]]> 22μL

[0254] The DNA template in Table 5 refers to the template where each gene fragment to be specifically amplified is located, see the record in the "Template" column in Table 3. Primer F refers to each forward primer in Table 3, and primer R refers to each reverse primer in Table 2.

[0255] The PCR amplification program is shown in Table 6:

[0256] Table 6 PCR reaction program

[0257]

[0258] Take 5 μL of the amplified product and perform 1% agarose gel electrophoresis to detect the PCR amplification results. Then perform the following steps:

[0259] (1) The UE DNA gel recovery kit of Suzhou UE Landi Biotechnology Co., Ltd. was used to perform gel excision and recovery on each DNA fragment with correct PCR amplification results, and the concentration of the recovered DNA fragments was measured using the BioSpec-nano instrument.

[0260] (2) Use NEB (New England Biolabs) multi-fragment recombinase to carry out corresponding ligation and recombination of different DNA fragments, and the amount of each DNA fragment is 200 ng. Then add an equal volume of multi-fragment recombinase, mix well, and place in a 50°C water bath for 30-60 minutes to obtain different ligation products.

[0261] (3) taking out the competent E. coli cells Trans 10 stored in the ultra-low temperature refrigerator and storing them on ice (in this example, the competent E. coli cells Trans 10 are used, but the competent E. coli cells C2566 can also be used);

[0262] (4) Use a pipette to take up each ligation product and add it to 100 μL of E. coli competent cells Trans 10. Mix carefully and let it stand on ice for 30 min.

[0263] (5) Turn on the water bath and heat it to 42° C. in advance, place the centrifuge tube containing the competent E. coli cells in step (4) in the water bath for heat shock for 90 seconds, and then quickly place it in an ice bath for 5 minutes;

[0264] (6) Pipette 800 μL of sterilized LB liquid medium into the centrifuge tube in step (5) and culture at 37°C, 250 rpm, and shake for 45 to 60 min;

[0265] (7) After centrifuging the centrifuge tube at 4000 rpm for 1 min, aspirate part of the supernatant, use a pipette to mix the remaining bacterial solution by aspirating it back and forth, and then spread the mixed bacterial solution on a solid LB plate containing the corresponding antibiotics and culture it upside down at 37°C overnight;

[0266] (8) After a single white colony grows, pick the single white colony and place it in a test tube containing 5 mL of LB liquid medium (containing the corresponding antibiotics) and culture it at 37°C with shaking at 250 rpm for 6 h;

[0267] (9) After the bacterial solution is tested by PCR, 500 μL of the bacterial solution that is verified to be positive is sent to Qingke Company for sequencing, or the bacterial solution that has not been tested by PCR is directly sent for sequencing, and the remaining bacterial solution is stored in 30% glycerol.

[0268] (10) The strains verified by sequencing were expanded and cultured, and plasmids were extracted using the Tianluo Diagnostic Plasmid Miniprep Kit to obtain pTargetF-nagAB plasmid and pTargetF-pfkA-NT1 plasmid, respectively.

[0269] 1.3 Construction of glmS knock-in Donor-DNA fragment

[0270] The glmS knock-in Donor-DNA fragment was constructed by overlapping extension PCR (OE-PCR) using primers designed in Table 3. The program of each round of multiple rounds of PCR in the overlapping extension PCR method is shown in Table 6.

[0271] 1.4 Construction of SLIS027 strain

[0272] The SLIS027 strain of this embodiment was transformed with the SLIS026 strain as the starting strain, and the nagA gene and the nagB gene were knocked out relative to the SLIS026 strain, and a partial fragment of the pfkA gene was knocked out, and the glmS gene was integrated into the knocked-out pfkA fragment, and finally the SLIS027 strain was obtained. Among them, the nagAB gene of the SLIS026 strain was knocked out with the pTargetF-nagAB plasmid. The pTargetF-pfkA-NT1 plasmid and the glmS knock-in Donor-DNA fragment were used to knock out a partial fragment of the pfkA gene and integrate the glmS gene into the knocked-out pfkA site.

[0273] The construction method of the SLIS027 strain includes the following steps:

[0274] ①Prepare SLIS026 competent cells: streak a single colony of SLIS026 strain stored at -80°C. Pick a single colony and inoculate it into 5 mL LB liquid culture medium. Cultivate at 37°C with shaking at 250 rpm until the OD of the bacterial solution reaches 600 The bacterial solution was transferred to a pre-cooled sterile centrifuge tube and centrifuged at 4000 rpm for 10 min at 4°C. The supernatant was discarded and the wet cells were collected. 2 The wet cells were resuspended twice in a solution (containing 15% glycerol), and the resuspended solution was centrifuged at 4000 rpm for 10 min at 4°C, and the supernatant was discarded. 2 The cells were resuspended in 1% glycerol (containing 15% glycerol) solution, and 100 μL per tube was dispensed into 1.5 mL centrifuge tubes, which were quickly placed in liquid nitrogen for quick freezing and then stored at -80°C to obtain SLIS026 competent cells.

[0275] ② Take 3 μL of pCas-Sac plasmid and add it to a centrifuge tube containing 100 μL of SLIS026 competent cells. Place the centrifuge tube on ice for 30 minutes, then heat shock at 42°C for 45 seconds, and immediately place it on ice for 2 to 5 minutes; add 800 μL of LB liquid culture medium and place it on a 30°C shaker for incubation for 45 minutes, spread the plate (Kan resistance, LB solid culture medium), invert it in a 37°C incubator, and culture overnight; after a single colony grows, pick it into LB liquid culture medium (Kan resistance), and after culturing for several hours, maintain the bacteria (final glycerol concentration 30%) to obtain the pCas-Sac / SLIS026 strain (capable of expressing Cas protein).

[0276] ③Pick the pCas-Sac transformant (i.e., pCas-Sac / SLIS026 strain) and inoculate it into the LB screen tube (kan resistance). Cultivate the bacterial solution at 37°C until the OD value is 0.2, and then add arabinose with a final concentration of 2 g / L for induction (in addition, arabinose can also be added when a single colony is cultured). OD 600 When the concentration was 0.4, competent cells were prepared in the same way as in operation ① to obtain pCas-Sac / SLIS026 competent cells;

[0277] ④The correctly constructed pTargetF-nagAB plasmid was transformed into pCas-Sac / SLIS026 competent cells by heat shock method, and after recovery, it was spread on LB plate (kan + 、spe + ), place the plate in a 37°C incubator and invert it for overnight culture until a single colony grows on the plate;

[0278] ⑤ Perform PCR verification on the single colony on the resistance plate according to the PCR verification primers in Table 7, and send the PCR product that verifies the successful knockout of the nagAB gene for sequencing. The sequencing verification primers are shown in Table 7;

[0279] ⑥ According to the sequencing results of ⑤, the strains with successful knockout / knockin were selected for shaking, and rhamnose with a final concentration of 10mM was added (the amount of rhamnose can be appropriately increased to improve the efficiency of plasmid elimination) and induced at 37℃ for 5-7h to lose the plasmid pTargetF-nagAB;

[0280] ⑦ Take the bacterial solution obtained in ⑥ and streak the LB plate (kan + , rhamnose) to obtain a single colony, and perform PCR according to the verification primers in Table 7 to verify whether the pTargetF-nagAB plasmid is lost (or streak it on an LB plate with Spec added and culture overnight to verify the elimination of the pTargetF-nagAB plasmid);

[0281] ⑧ Streak the strain that has completed the pTargetF-nagAB plasmid loss treatment onto a 10 g / L sucrose plate (the amount of sucrose can be appropriately increased to improve the efficiency of plasmid elimination), invert and culture overnight at 37°C, and verify whether the pCas-Sac plasmid is lost by PCR using the verification primers in Table 8 (or streak onto an LB plate with Kan added and culture overnight to verify the elimination of the pCas-Sac plasmid).

[0282] ⑨ When knocking in and knocking out ΔpfkA::glmS, the corresponding pTargetF-pfkA-NT1 and the glmS knock-in Donor-DNA fragment (donor DNA, containing the gene to be inserted) were transformed into the corresponding competent cells together. The subsequent operations were the same as the gene knockout operations, and the SLIS027 strain was finally obtained.

[0283] Table 7 SLIS027 strain knockout and knock-in gene verification primers

[0284]

[0285] Table 8 pCas-Sac and pTargetF elimination verification primers

[0286]

[0287] 1.5 Acquisition and fermentation of fermentation strains EBSL001 and EBSL002

[0288] (1) Design the pTU2-A-DOE13 plasmid. The plasmid map of pTU2-A-DOE13 is as follows: Figure 2As shown. The pTU2-A-DOE13 plasmid carries the transcription units of four genes, namely T2-pET-RBS-css-T7T, T2-pET-RBS-neuB-T7T, T2-pET-RBS-neuC-T7T and T2-pET-RBS-ST-T7T. The designed pTU2-A-DOE13 plasmid was handed over to Qingke Biotechnology for synthesis. Among them, T2 represents the T2 promoter, and these four genes are all expressed using the T2 promoter. RBS represents the ribosome binding site. css, neuB, neuC, and ST represent the names of four genes related to 3'-SL synthesis. T7T represents the T7 terminator.

[0289] (2) The pTU2-A-DOE13 plasmid was transformed into the SLIS026 strain to obtain the EBSL001 strain. The pTU2-A-DOE13 plasmid was transformed into the SLIS027 strain to obtain the EBSL002 strain.

[0290] (3) Protein induction expression and fermentation culture: Single colonies of EBSL001 and EBSL002 were picked and inoculated into 5 mL LB resistance test tubes (containing 25 μg / mL chloramphenicol), cultured at 37°C and 250 rpm for 6-7 h, and then inoculated into 100 mL TB resistance shake flasks (containing 25 μg / mL chloramphenicol) at an inoculum size of 2.5-5% (the specific inoculum size was determined according to the cell density in the seed culture medium so that the number of cells inoculated each time was basically the same), and cultured at 37°C and 250 rpm until OD 600 About 1-1.2 (about 4-5h), then add IPTG (final concentration 0.1mM), 1mL 200g / L MgSO 4 7H 2 0 (final concentration of 2g / L), 0.1mL trace element mother solution (final concentration of 0.1%), 2mL 500g / L glucose (final concentration of 10g / L) and 2mL 250g / L lactose (final concentration of 5g / L) were used to ferment the strain producing 3'-SL. The culture conditions were 30°C and 250rpm. The fermentation start time was 0h. 2mL 500g / L glucose (final concentration of 10g / L) was added every 12h to ensure that the glucose concentration added within 24h was at least 20g / L. The total fermentation time was 48h. HPLC was then used to detect the content of 3'-SL in the fermentation broth. The final concentration in this embodiment refers to the initial concentration of each component after being added to the culture broth, i.e., the concentration at the beginning of the fermentation culture.

[0291] The fermentation broth of strain EBSL001 was analyzed by HPLC, and the test result graph is as follows: Figure 3 As shown. Figure 3 It can be seen that the peak time of the 3'-SL standard sample detected by HPLC is 16.397min, and the peak time of 3'-SL in the fermentation broth of the EBSL001 strain is 16.438min, indicating that 3'-SL is synthesized by fermentation.

[0292] According to the shake flask fermentation results of EBSL001 and EBSL002, the 3'-SL production of EBSL001 was 3.1 g / L, and the 3'-SL production of EBSL002 was 3.8 g / L, which was 22.58% higher than that of EBSL001. This indicates that increasing the supply of glucosamine-6-phosphate (GlcN-6P) precursors by knocking out nagAB and pfkA and integrating the glmS gene is beneficial to 3'-SL synthesis.

[0293] Test Example 1

[0294] When knocking out the pfkA gene, the present embodiment 1 only knocks out a partial fragment of the pfkA gene, rather than the knockout of the full gene fragment. This is because the inventor knocked out the full gene sequence (963bp) of the pfkA gene during the experiment, and fermented the genetically engineered bacteria obtained after the knockout. It was found that the growth state of the strain was very poor, and the passage efficiency was not high, which could not meet the basic requirements as chassis cells. The inventor believes that this may be because the fieF gene, pfkA gene and sbp gene in the prokaryotic genome are closely arranged in series, and the knockout of the 3' segment region of the pfkA gene will affect the transcription and expression of other structural genes adjacent to the pfkA gene, thereby affecting the growth and proliferation of the genetically engineered bacteria.

[0295] In this example, the inventors only knocked out the upstream sequence of the pfkA gene, but retained the 3' end sequence equivalent to 76% of the full-length sequence of the pfkA gene (732bp), and fermented the gene-functional bacteria obtained after the knockout. The inventors surprisingly found that compared with the engineered bacteria with the whole gene fragment knocked out previously, the partial knockout of the pfkA gene did not affect the growth state and passage efficiency of the engineered bacteria.

[0296] The inventors also found in the study that if the middle sequence of the pfkA gene is knocked out, if the sequence retained at the 5' end of the pfkA gene is too long and the sequence retained at the 3' end is too short, then it is difficult to select primers with high knockout efficiency and the workload is large. Therefore, based on comprehensive considerations, when knocking out a partial fragment of the pfkA gene, the length of the sequence retained at the 5' end is preferably in the range of 0% to 7%, and the length of the sequence retained at the 3' end is preferably in the range of 70% to 80%.

[0297] Example 2: Optimizing the cofactor circulation system

[0298] N-acetylneuraminic acid (Neu5Ac), also known as sialic acid (SA), is one of the precursors for the synthesis of 3'-SL. Neu5Ac requires the participation of the cofactor CTP in the process of generating CMP-Neu5Ac. UTP produces CTP under the catalysis of CTP synthetase, and the encoding gene of CTP synthetase is the pyrG gene (accession number AAC75822.1). If the supply of cofactor CTP is insufficient, it will affect the yield of 3'-SL. Therefore, this embodiment uses the SLIS027 strain as the starting strain to overexpress the cmk gene encoding cytidine monophosphate kinase (CMK) and the ppk gene encoding polyphosphate kinase PPK, so that the cofactor CTP is continuously recycled to solve the problem of insufficient supply of cofactor CTP. Cofactor cyclic regeneration refers to the cyclic regeneration from CTP→CMP→CDP→CTP. Among them, cytidine triphosphate is abbreviated as CTP; also known as cytidine triphosphate. Cytidine monophosphate is abbreviated as CMP, also known as cytidine monophosphate. Cytidine diphosphate is abbreviated as CDP, also known as cytidine diphosphate.

[0299] The steps of this embodiment are as follows:

[0300] 2.1. Design and synthesis of primers required for gene knockout and integration

[0301] In this example, the gene knockout and integration primers are shown in Table 9. The sequences of sgRNAs used for gene knockout and integration are shown in Table 10.

[0302] Table 9 Gene knockout and integration primers

[0303]

[0304]

[0305] Table 10 sgRNA sequence list

[0306] Genetic manipulation How it works sgRNA sequence Sequence number ΔmelB::T5-ppk replace CAAAGCATTACATTAAGACG SEQ ID NO:51 ΔmelB::tet-cmk replace TGGACAATAGCTAACGAAAA SEQ ID NO:52

[0307] 2.2 Construction of recombinant plasmid and Donor-DNA fragment:

[0308] The pTargetF-melB-NT1 plasmid and the pTargetF-melB-NT2 plasmid were constructed respectively by referring to the method in Section 1.2 of Example 1.

[0309] Referring to the method in Section 1.3 of Example 1, ppk knock-in Donor-DNA and cmk knock-in Donor-DNA were constructed respectively.

[0310] 2.3 Construction of SLIS028 strain

[0311] The SLIS028 strain of this example was transformed with the SLIS027 strain as the starting strain. Compared with the SLIS027 strain, the cmk gene encoding cytidine monophosphate kinase and the ppk gene encoding polyphosphate kinase were integrated and overexpressed, that is, part of the sequence of the melB site was knocked out and the ppk gene and the cmk gene were knocked into the knocked-out position of the melB gene in the genome of the SLIS027 strain by a double knock-in method.

[0312] The double knock-in method refers to (1) using pTargetF-melB-NT1 and the ppk gene to knock into the linearized fragment of Donor-DNA in the form of co-transformation to knock in the ppk gene, and the ppk gene uses the T5 promoter (denoted as ΔmelB::T5-ppk); (2) using pTargetF-melB-NT2 and cmk to knock into the linearized fragment of Donor-DNA in the form of co-transformation to knock in the cmk gene, and the cmk gene uses the tetracycline promoter tet (ΔmelB::tet-cmk) and the cmk gene is inserted in the upstream position of the pkk gene. For specific steps, see Section 1.4 of Example 1. The same applies to the following examples.

[0313] The inventor has found through a large number of experimental verifications that if the knocked-in ppk gene and cmk gene all use the same T2 promoter as the four key genes, then the expression of the ppk gene and cmk gene will interact with the expression of the four key genes, which is not conducive to the improvement of the cofactor circulation efficiency and the 2'-SL synthesis efficiency. Therefore, the knocked-in ppk gene and cmk gene need to use a promoter different from the T2 promoter. In addition, if the knocked-in ppk gene and cmk gene use a promoter stronger than the T2 promoter, then the expression of the ppk gene and cmk gene will also increase. But in fact, since the ppk gene and cmk gene belong to genes related to the cofactor CTP cycle, they are not genes directly related to the synthesis of 3'-SL. There is no linear relationship between the expression of these two genes and the improvement of the synthesis efficiency of 3'-SL. Therefore, the expression of these two genes does not need a strong promoter to regulate, and a promoter weaker than the T2 promoter is used to maintain a basic transcription level so as to promote the cofactor cycle. Furthermore, if the ppk gene and the cmk gene use the same weak promoter to regulate gene expression, the same weak promoter will competitively bind to the same type of RNA polymerase, which will also have a certain effect on the transcription of the ppk gene and the cmk gene. Therefore, the inventors selected different types of weak promoters to regulate the transcription of the ppk gene and the cmk gene respectively. In this application, the ppk gene uses the T5 promoter to regulate expression, and the cmk gene uses the tetracycline promoter tet to regulate expression. But in fact, the ppk gene can also use the tetracycline promoter tet to regulate expression, and the cmk gene can also use the T5 promoter to regulate expression.

[0314] The strong promoter and weak promoter in this application are relative concepts. A strong promoter refers to a promoter sequence that has a stronger recognition and binding ability with RNA polymerase than a weak promoter, thereby making the transcription efficiency or transcription level of the downstream structural gene connected thereto higher than that of a weak promoter. Specifically, the T2 promoter has a stronger ability to regulate gene transcription than the T5 promoter and the tet promoter.

[0315] The melB gene is a gene located in the Escherichia coli genome, which encodes a transporter protein related to the transport of melibiose, namely, melibiose permease (MelB). Experiments have shown that the knockout of the melB gene in the Escherichia coli genome does not affect the normal growth, metabolism, and proliferation of Escherichia coli. Therefore, this embodiment selects to knock in the ppk gene and the cmk gene to the position where the melB gene is knocked out.

[0316] PCR detection and sequencing showed that the ppk gene and cmk gene were successfully knocked into SLIS027 to obtain the SLIS028 strain. The validation primer table is shown in Table 11.

[0317] Table 11 Verification primers

[0318]

[0319] 2.4 Acquisition and fermentation of fermentation strain EBSL003

[0320] The pTU2-A-DOE13 plasmid was transformed into the SLIS028 strain according to the method in Section 1.5 of Example 1 to obtain the EBSL003 strain, and shake flask fermentation was performed on EBSL001, EBSL002 and EBSL003 respectively. The total fermentation time was 48 h. The content of 3'-SL in the fermentation broth was then detected by HPLC.

[0321] According to the shake flask fermentation results, the 3'-SL production of EBSL001 strain was 3.0g / L, the 3'-SL production of EBSL002 strain was 3.8g / L, and the 3'-SL production of EBSL003 strain was 4.5g / L, which was 18.42% higher than that of EBSL002 strain. Therefore, knocking ΔmelB::T5-ppk and ΔmelB::tet-cmk into the genome of EBSL002 strain increased the production of 3'-SL, indicating that optimizing the cofactor circulation system is beneficial to increasing the supply of CTP, thereby increasing the production of 3'-SL.

[0322] Example 3: Optimization of PEP supply and oligosaccharide transport system

[0323] The sialic acid synthase encoded by the neuB gene catalyzes the reaction of ManNAc and phosphoenolpyruvic acid (PEP) to generate Neu5Ac and phosphate, and Neu5Ac is one of the precursors for the synthesis of 3'-SL. Therefore, increasing the supply of PEP is conducive to increasing the yield of 3'-SL. In addition, if the 3'-SL produced in the cell can be pumped out of the bacterial cell in time, it is conducive to reducing the degree of inhibition of the synthesis reaction of 3'-SL by the excessive accumulation of 3'-SL in the bacterial cell. Therefore, in this embodiment, the EBSL003 strain is used as the starting strain to perform the knockout treatment of the pyruvate kinase gene pykA and the enhanced expression treatment of the oligosaccharide transporter gene setB. After knocking out the pyruvate kinase gene pykA, the catabolic pathway of PEP is inhibited, which is conducive to the accumulation of PEP in the bacterial cell. After the enhanced expression of the oligosaccharide transporter gene setB, the 3'-SL in the bacterial cell can be pumped out of the cell in time after production, which helps to increase the cumulative concentration of 3'-SL outside the cell.

[0324] The steps of this embodiment are as follows:

[0325] 3.1. Design and synthesis of primers required for gene knockout and integration

[0326] In this example, the gene knockout and integration primers are shown in Table 12. The sequences of sgRNAs used for gene knockout and integration are shown in Table 13.

[0327] Table 12 Gene knockout and integration primers

[0328]

[0329] Table 13 Gene knockout and integration sgRNA sequence list

[0330] Genetic manipulation How it works sgRNA sequence Sequence number ΔpykA::tet-setB Deletion and Replacement TCGTGAGATTGCCGCAAAAC SEQ ID NO:66

[0331] In Table 13, tet indicates that the promoter of the setB gene is the tet promoter.

[0332] 3.2 Construction of plasmid:

[0333] The pTargetF-pykA-NT2 plasmid was constructed by referring to the method in Section 1.2 of Example 1. Overlap PCR was used to construct Donor-DNA to knock the setB gene into the pykA locus.

[0334] 3.3 Construction of SLIS029 strain

[0335] The SLIS029 strain of this embodiment is modified with the SLIS028 strain as the starting strain. On the basis of the SLIS028 strain, the pTargetF-pykA-NT2 plasmid and the setB gene knock-in pykA site Donor-DNA are used to knock out a partial fragment of the pyruvate kinase gene pykA and enhance the expression of the oligosaccharide transporter gene setB to increase the supply of phosphoenolpyruvate PEP in SL synthesis, and the SLIS029 strain is obtained.

[0336] PCR detection and sequencing showed that part of the pykA gene in the SLIS028 strain had been successfully knocked out as designed, and the oligosaccharide transporter gene setB had been enhanced to obtain the SLIS029 strain. The validation primer table is shown in Table 13.

[0337] Table 14 SLIS029 strain knock-in gene verification primer list

[0338]

[0339] 3.4 Acquisition and fermentation of fermentation strain EBSL004

[0340] The pTU2-A-DOE13 plasmid was transformed into the SLIS029 strain according to the method in Section 1.5 of Example 1 to obtain the EBSL004 strain, and shake flask fermentation was performed on EBSL001, EBSL002, EBSL003 and EBSL004, respectively. The total fermentation time was 48 h. The content of 3'-SL in the fermentation broth was then detected by HPLC.

[0341] From the shake flask fermentation results, we can see that the 3'-SL production of EBSL001 strain is 3.0g / L, the 3'-SL production of EBSL002 strain is 3.7g / L, the 3'-SL production of EBSL003 strain is 4.5g / L, and the 3'-SL production of EBSL004 strain is 5.2g / L. It can be seen that compared with the production of 4.5g / L of EBSL003 strain, the production of EBSL004 strain increased by 15.56%. This shows that the partial deletion of the pykA gene and the knock-in of tet-setB (ΔpykA::tet-setB) are beneficial to 3'-SL synthesis.

[0342] The strains and their characteristics involved in Examples 1 to 3 are shown in Table 15.

[0343] Table 15 Strains and their characteristics involved in Examples 1 to 3

[0344]

[0345] Test Example 2

[0346] In this Example 2, when knocking out the pykA gene, the middle region fragment of the pykA gene is knocked out, but the two end regions of the pykA gene are retained. Therefore, this Example only knocks out a partial fragment of the pykA gene, rather than knocking out the entire gene fragment.

[0347] The inventor previously knocked out the full gene sequence (1443 bp) of the pykA gene and cultured the gene-functional bacteria obtained after the knockout. The inventor found that the strain with the full gene sequence of the pykA gene knocked out also had a poor growth state and low passage efficiency, which could not meet the basic requirements as a chassis cell.

[0348] In this embodiment, the inventors only knocked out the middle sequence of the pykA gene, but retained the 4.7% length sequence (68bp) of the full-length sequence of the pykA gene at the 5' end, and retained the 12.1% length sequence (175bp) of the full-length sequence of the pykA gene at the 3' end, and cultured the gene functional bacteria obtained after the knockout. Surprisingly, the inventors found that the engineered bacteria with the partial knockout of the pykA gene prepared in the embodiment had a good growth state and high passage efficiency, meeting the basic requirements as chassis cells.

[0349] The inventors also found in the study that if the middle sequence of the pykA gene is knocked out, if the sequence retained at the 5' end of the pykA gene is too long and the sequence retained at the 3' end is too short, then it is difficult to select primers with high knockout efficiency and the workload is large. Therefore, for comprehensive consideration, when a partial fragment of the pykA gene is knocked out, the length of the sequence retained at the 5' end is preferably in the range of 4% to 5.5%, and the length of the sequence retained at the 3' end is preferably in the range of 10% to 20%.

[0350] Example 4: Insertion of css, neuB, neuC and ST genes into the genome of engineered bacteria

[0351] The pTU2-A-DOE13 plasmid exists in EBSL001, EBSL002, EBSL003 and EBSL004. The plasmid is easily lost during the subsequent passage, resulting in poor stability of the strain passage. Therefore, this example uses the multi-copy chromosomal integration by CRISPR-associated transposase (MUCICAT) technology to insert the synthetic 3'-SL key genes css, neuB, neuC and ST genes into the genome of the engineered bacteria.

[0352] The steps of this embodiment are as follows:

[0353] 4.1 Construction of pRE57-DOE13 plasmid

[0354] The primers required for constructing the pRE57-DOE13 plasmid were designed and submitted to Beijing Qingke Biotechnology Co., Ltd. for synthesis. The required primers are shown in Table 16. The crRNA sequence is shown in Table 17.

[0355] Table 16 Primers for constructing pRE57-DOE13 plasmid and sequencing primers

[0356]

[0357]

[0358] Table 17 crRNA sequence information

[0359]

[0360] 4.2 Using MUCICAT to obtain antibiotic-free multi-copy integrated EBSL005 strain:

[0361] In order to obtain an integrated strain that does not require a plasmid and has high genetic stability, the gene combination (css, neuB, neuC, ST gene combination) on the pTU2-A-DOE13 plasmid was integrated into the IS1 site of the SLIS029 strain through the multi-copy genome integration technology (MUCICAT) of CRISPR-associated transposons. There are 28 IS1 sites in the E. coli genome, and each IS1 site has a chance to insert one or several css, neuB, neuC, and ST gene combinations. With each round of induction, different IS1 sites have a chance to insert css, neuB, neuC, and ST gene combinations. With the increase in the number of induction rounds, the copy number of these gene combinations at different IS1 sites will gradually increase. The inventors found in the study that when the copy number of these gene combinations is within a certain range, the content of 3'-SL increases with the increase of the copy number of the gene combination, but when the copy number of these gene combinations increases to a certain value, even if the copy number of these gene combinations continues to increase, the content of 3'-SL no longer increases with the increase of the copy number of the gene combination, but decreases to a certain extent. The number of other IS sequences (such as IS186, IS3, and IS4) in the Escherichia coli cell is small and cannot meet the copy number requirements after the insertion of the structural gene. Therefore, IS1 is selected as the insertion site of the structural gene.

[0362] The MUCICAT technology requires multiple rounds of induction of the SLIS029 strain, and after each round of induction, multiple transformants are randomly selected for fermentation screening (primary screening) on ​​a 96-well plate to screen out transformants with a higher content of 3'-SL. When selecting transformants, each selected transformant only picks a part of the colony, and the amount picked is based on the ability to perform preliminary screening. After each round, the remaining transformants after picking are mixed with the unpicked transformants for the next round of induction, and then the multiple transformants obtained after the next round of induction repeat the above process. The transformants picked after each round of induction need to be treated for plasmid loss before shake flask fermentation, while the unpicked transformants do not need to be treated for plasmid loss.

[0363] The steps of the initial screening are: the transformants selected after multiple rounds of induction are added to a 96-well plate containing TB liquid medium (600 μL) for IPTG induction fermentation screening, the fermentation time is 48 hours, and then the supernatant of the fermentation liquid is taken, and the content of 3'-SL in the supernatant is determined by HPLC. Finally, 36 transformants with high 3'-SL content are screened out, and then shake flask fermentation (rescreening) is carried out.

[0364] The steps of rescreening (shake flask fermentation) are: using rhamnose to lose pTet-tns, pQCascade-IS1 and pRE57-DOE13 plasmids, using sucrose to lose pCutamp plasmid, and finally obtaining multiple plasmid-free multi-copy strains. These plasmid-free multi-copy strains were transferred to shake flasks containing TB liquid medium, IPTG induced fermentation for 48 hours, and then the fermentation broth was taken, the supernatant was taken after centrifugation, and the content of 3'-SL in the supernatant was determined by HPLC.

[0365] In this example, some strains with different yields were selected from 36 transformants to measure the copy number (refer to Section 4.8 of CN113249400 A), as shown in Table 18 below.

[0366] Table 18 Yield and copy number of some transformants

[0367] Strain number 3'-SL yield (g / L) Copy number 2 3.92 5 4 2.61 3 5 2.30 3 6 0.35 1 12 4.78 10 14 4.35 6 20 4.61 8 24 5.19 14 32 3.31 4 34 6.15 18 35 6.50 16 36 5.92 20

[0368] From the results in Table 18 and Figure 4 It can be seen that as the copy number increases, the production of 3'-SL also increases. When the copy number reaches 16, the 3'-SL production is the highest. After that, as the copy number continues to increase, the 3'-SL production no longer increases. We named the strain No. 35 with a copy number of 16 as the EBSL005 strain (also known as the SLIS109 strain).

[0369] Table 19 Gene information of EBSL005 strain (SLIS109 strain)

[0370]

[0371] The specific steps of MUCICAT in this embodiment are as follows:

[0372] (1) Using the ClonExpress II One Step Cloning Kit (Nanjing Novogene Biotech Co., Ltd., Catalog No.: C112-01), the crRNA sequence corresponding to the target gene was seamlessly cloned into the pRE57-Ter plasmid to prepare different pRE57-DOE13 (subsequently used with pQCascade-IS1). The crRNA sequences used in the gene site-directed insertion process are shown in Table 17.

[0373] (2) 300 ng of pDonor (pRE57-Ter or other variants), 300 ng of pQCascade and 300 ng of pTnsABC (pTet-tns or other variants) were co-transformed into E. coli BL21 (DE3) cells by electroporation. After recovering in fresh LB liquid medium at 37°C for 2 hours, the E. coli BL21 (DE3) cells were inoculated on triple antibiotic LB agar plates containing 100 μg / mL ampicillin (Amp), 50 μg / mL kanamycin (Kan) and 50 μg / mL spectinomycin (Spec).

[0374] (3) After growing at 37°C for 16 hours, scrape dozens of colonies from the culture dish and resuspend some of them in fresh LB liquid medium. Then transfer the resuspension containing these colony cells to LB agar plates containing three antibiotics and 100 ng / mL to induce the expression of transposition-related proteins and continue to culture at 30°C for another 16 hours. After the biofilm is formed, scrape the cells and resuspend them in LB liquid medium. Re-dilute the cells appropriately, place them on triple antibiotic LB agar plates (i.e., induction plates) containing 100 ng / mL tetracycline (aTc), and culture them overnight at 37°C.

[0375] (4) To improve clonal growth and integration rate, colonies were transferred by re-splitting as follows. The colonies grown on the above induction plate were defined as Transfer 1, and a portion of the colonies were streaked onto a new triple antibiotic LB agar plate containing aTc and grown overnight at 30°C to generate a new transfer, Transfer 2. Continuous transfers will generate Transfer n (n = 3, 4...).

[0376] (5) Colony PCR is then performed to identify whether the combination of the four genes has been correctly inserted into the genome of the cell, so as to obtain cells into which the four genes have been correctly inserted.

[0377] (6) The pCutamp plasmid (used to eliminate the three plasmids) was electroporated into cells (still carrying the above three plasmids) into which the four genes had been correctly inserted, and recovered in 1 mL LB medium at 37°C for 1 hour and recovered. In order to improve the elimination efficiency of the plasmid, the recovered cells were transferred to 4 mL LB medium containing 50 μg / mL apramycin and 10 mM rhamnose for culture and shaken at 37°C for 7-10 hours (in order to further improve the elimination efficiency of the above three plasmids, the induction time can be appropriately extended). Then 100 μL of the culture was placed on an LB agar plate containing 50 μg / mL apramycin (Apr) and 10 mM rhamnose and cultured overnight at 37°C.

[0378] (7) The pCutamp plasmid also contains the SacB gene, which encodes a levulosidase that catalyzes sucrose hydrolysis and fructan polymerization. Therefore, E. coli carrying the pCutamp plasmid cannot grow on LB agar plates containing 10 g / L sucrose. Therefore, colonies that grow on LB agar plates containing apramycin and rhamnose are selected and replaced on four LB agar plates containing 100 μg / mL ampicillin, 50 μg / mL kanamycin, 50 μg / mL spectinomycin, and 10 g / L sucrose, respectively. Colonies that cannot grow on LB agar plates containing ampicillin, kanamycin, and streptomycin are considered to have eliminated the three plasmids pDonor, pTnsABC, and pQCascade. The corresponding colonies grown on LB agar plates containing sucrose were then re-plated on non-selective LB agar plates and LB agar plates containing apramycin to verify the digestion of the pCutamp plasmid. Colonies that fail to grow on LB agar plates containing apramycin are strains without plasmid multi-copy integration.

[0379] According to the records in Table 19, the four gene combinations of the SLIS109 strain all carry their own promoters and terminators, thus forming four separate transcription units.

[0380] 4.3 Fermentation of the antibiotic-free multi-copy integrated EBSL005 strain

[0381] The EBSL005 strain (see Table 18) was fermented in a shake flask. After 48 hours of shake flask fermentation, the 3'-SL yield reached 6.60 g / L. After 52 hours of fermentation, the 3'-SL yield reached 6.90 g / L, which was significantly higher than that of the EBSL004 strain. Figure 5 This is the growth and yield curve of shake flask fermentation of EBSL005 strain.

[0382] Furthermore, the EBSL005 strain was subjected to a 5L fermentation tank for amplification fermentation. The conditions for the 5L fermentation tank for amplification fermentation are as follows:

[0383] A single colony of the EBSL005 strain was inoculated into 5 mL of LB medium, cultured at 37°C and 250 rpm for 6-7 h, inoculated into a 5 L fermenter at an inoculum size of 2.5-5% (the specific inoculum size was determined according to the bacterial density in the seed culture medium so that the number of bacteria inoculated each time was basically the same), and cultured at 37°C and 250 rpm until the OD 600 About 1-1.2, then add IPTG (final concentration 0.1mM), 200g / L MgSO 4 7H 2O (final concentration of 2g / L), trace element mother solution (final concentration of 0.1%), 500g / L glucose (final concentration of 10g / L) and 250g / L lactose (final concentration of 5g / L) were used for fermentation culture of the 3'-SL producing strain. The culture conditions were 30°C and 250rpm. The fermentation culture started at 0h, and 500g / L glucose (final concentration of 10g / L) was added every 12h. The total fermentation time was 52h. Then HPLC was used to detect the content of 3'-SL in the fermentation broth. After the fermentation, the 3'-SL production of the EBSL005 strain reached 79g / L.

[0384] The results of this example show that the EBSL005 strain was obtained by integrating the css-neuB-neuC-ST gene combination into the genome of the strain multiple times using the multi-copy genome integration technology (MUCICAT) of CRISPR-associated transposons. Compared with the strains carrying the plasmid of the css-neuB-neuC-ST gene combination (strains EBSL001 to EBSL004), the EBSL005 strain has a higher yield of 3'-SL. Because EBSL005 is an integrated strain, there is no problem of loss of the plasmid carrying the css-neuB-neuC-ST gene combination during the fermentation process, and the genetic stability is higher.

[0385] In summary, the EBSL005 strain was able to achieve a high level of 3'-SL synthesis in both shake flask fermentation and 5L fermenter fermentation.

[0386] Although the present invention has been described with reference to its specific embodiments, it will be appreciated by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the spirit and scope of the present invention. In addition, many modifications may be made to adapt specific circumstances, materials, compositions, methods, one or more method steps to the purpose, spirit and scope of the present invention. All such modifications are intended to be within the scope of the appended claims.

[0387] sequence

[0388] >WP_002874240.1UDP-N-acetylglucosamine 2-epimerase[Campylobacterjejuni]

[0389] SEQ ID NO:82

[0390] MKKILFITGSRADYSKIKSLMYRVQNSSEFELYIFATGMHLSKNFGYTVKELYKNGFKNIYEFINYDKYYQTDKALATTIDGFSRYANELKPDLIVVHGDRIEPLAAAIVGALNNILVAHIEGGEISGTIDDSLRHAISKLAHIHLVNDEFAKRRLMQLGEDEKSIFIIGSPDLELLNDNKISLSEAKKYYDINYENYALLMFHPVTTEITSIKNQADNLVKALIQSNKNYIVIYPNNDLGFELILQSYEEFKNNPRFKLFPSLRFEYFITLLKNADFIIGNSSCILKEALYLKTAGILVGSRQNGRLGNENTLKVNANSDEILKAINTIHKKQDLFSAKLEILDSSKLFFEYLQSGDFFKLSTQKVFKDIK

[0391] >WP_002874241.1sialic acid synthase[Campylobacter jejuni subsp.jejuni81-176]

[0392] SEQ ID NO:83

[0393] MKEIKIQNIIISEEKAPLVVPEIGINHNGSLELAKIMVDAAFSAGAKIIKHQTHIVEDEMSKAAKKVIPGNAKISIYEIMQKCALDYKDELALKEYTEKLGLVYLSTPFSRAGANRLEDMGVSAFKIGSGECNNYPLIKHIAAFKKPMIVSTGMNSIESIKPTVKILLDNEIPFVLMHTTNLYPTPHNLVRLNAMLELKKEFSCMVGLSDHTTDNLACLGAVVLGACVLERHFTDSMHRSGPDIVCSMDTKALKELIIQSEQMAIIRGNNESKKAAKQEQVTIDFAFASVVSIKDIKKGEVLSMDNIWVKRPGLGGISAAEFENILGKKALRDIENDAQLSYEDFA

[0394] >WP_002215295.1 N-acylneuraminate cytidylyltransferase[Neisseriameningitidis]

[0395] SEQ ID NO:84

[0396] MEKQNIAVILARQNSKGLPLKNLRKMNGISLLGHTINAAISSKCFDRIIVSTDGGLIAEEAKNFGVEVVLRPAELASDTASSISGVIHALETIGSNSGTVTLLQPTSPLRTGAHIREAFSLFDEKIKGSVVSACPMEHHPLKTLLQINNGEYAPMRHLSDLEQPRQQLPQAFRPNGAIYINDTASLIANNCFFIAPTKLYIMSHQDSIDIDTELDLQQAENILNHKES

[0397] AGH37861.1 Bibersteinia trehalosi

[0398] SEQ ID NO:85

[0399] MEFCKMATTQKICVYLDYATIPSLNYILHFAQHFEDQETIRLFGLSRFHIPESVIQRYPKGVVQFYPNQEKDFSALLLALKNILIEVKQQQRKCEIELHLNLFHYQLLLLPFLSLYLDTQDYCHLTLKFYDDGSEAISALQELALAPDLAAQIQFEKQQFDELVVKKSFKLSLLSRYFWGKLFESEYIWFNQAILQKAELQILKQEISSSRQMDFAIYQQMSDEQKQLVLEILNIDLNKVAYLKQLMENQPSFLFLGTTLFNITQETKTWLMQMHVDLIQQYCLPSGQFFNNKAGYLCFYKGHPNEKEMNQMILSQFKNLIALPDDIPLEILLLLGVIPSKVGGFASSALFNFTPAQIENIIFFTPRYFEKDNRLHATQYRLMQGLIELGYLDAEKSVTHFEIMQLLTKE

[0400] >ACT42510.1 N-acetylglucosamine-6-phosphate deacetylase[EscherichiacoliBL21(DE3)]

[0401] SEQ ID NO:86

[0402] MYALTQGRIFTGHEFLDDHAVVIADGLIKSVCPVAELPPEIEQRSLNGAILSPGFIDVQLNGCGGVQFNDTAEAVSVETLEIMQKANEKSGCTNYLPTLITTSDELMKQGVRVMREYLAKHPNQALGLHLEGPWLNLVKKGTHNPNFVRKPDAALVDFLCENADVITKVTLAPEMVPAEVISKLANAGIVVSAGHSNATLKEAKAGFRAGITFATHLYNAMPYITGREPGLAGAILDEADIYCGIIADGLHVDYANIRNAKRLKGDKLCLVTDATAPAGANIEQFIFAGKTIYYRNGLCVDENGTLSGSSLTMIEGVRNLVEHCGIALDEVLRMATLYPARAIGVEKRLGTLAAGKVANLTAFTPDFKITRTIVNGNEVVTQ

[0403] >ACT42511.1 glucosamine-6-phosphate deaminase[Escherichia coli BL21(DE3)]

[0404] SEQ ID NO:87

[0405] MRLIPLTTAEQVGKWAARHIVNRINAFKPTADRPFVLGLPTGGTPMTTYKALVEMHKAGQVSFKHVVTFNMDEYVGLPKEHPESYYSFMHRNFFDHVDIPAENINLLNGNAPDIDAECRQYEEKIRSYGKIHLFMGGVGNDGHIAFNEPASSLASRTRIKTLTHDTRVANSRFFDNDVNQVPKYALTVGVGTLLDAEEVMILVLGSQKALALQAAVEGCVNHMWTISCLQLHPKAIMVCDEPSTMELKVKTLRYFNELEAENIKGL

[0406] >ACT45594.1 6-phosphofructokinase I[Escherichia coli BL21(DE3)]

[0407] SEQ ID NO:88

[0408] MIKKIGVLTSGGDAPGMNAAIRGVVRSALTEGLEVMGIYDGYLGLYEDRMVQLDRYSVSDMINRGGTFLGSARFPEPFRDENIRAVAIENLKKRGIDALVVIGGDGSYMGAMRLTEMGFPCIGLPGTIDNDIKGTDYTIGFFTALSTVVEAIDRLRDTSSSHQRISVVEVMGRYCGDLTLAAAIAGGCEFVVVPEVEFSREDLVNEIKAGIAKGKKHAIVAITEHMCDVDELAHFIEKETGRETRATVLGHIQRGSPVPYDRILASRMGAYAIDLLLAGYGGRCVGIQNEQLVHHDIIDAIENMKRPFKGDWLDCAKKLY

[0409] SEQ ID NO:89

[0410] MCGIVGAIAQ RDVAKILLEG LRRLEYRGYD SAGLAVVDAE GHMTRLRRLG KVQMLAQAAEEHPLHGGTGIAHTRWATHGE PSEVNAHPHV SEHIVVVHNG IIENHEPLRE ELKARGYTFVSETDTEVIAHLVNWELKQGGTLREAVLRAI PQLRGAYGTVIMDSRHPDTL LAARSGSPLVIGLGMGENFIASDQLALLPVTRRFIFLEEG DIAEITRRSVNIFDKTGAEV KRQDIESNLQYDAGDKGIYRHYMQKEIYEQPNAIKNTLTGRISHGQVDLSELGPNADELL SKVEHIQILACGTSYNSGMVSRYWFESLAGIPCDVEIASEFRYRKSAVRRNSLMITLSQS GETADTLAGLRLSKELGYLGSLAICNVPGSSLVRESVLALMTNAGTEIGVASTKAFTTQL TVLLMLVAKLSRLKGLDASIEHDIVHGLQALPSRIEQMLPQDKRIEALAEDFSDKHHALF LGRGDQYPIALEGALKLKEISYIHAEAYAAGELKHGPLALIDADMPVIVVAPNNGLLEKL KSNIEEVRARGGQLYVFADQDAGFVSSDNMHIIEMPHVEEVIAPIFYTVPLQLLAYHVAL IKGTDVDQPRNLAKSVTVE

[0411] >ACT44213.1 polyphosphate kinase,component of RNA degradosome[Escherichiacoli BL21(DE3)]

[0412] SEQ ID NO:90

[0413] MGQEKLYIEKELSWLSFNERVLQEAADKSNPLIERMRFLGIYSNNLDEFYKVRFAELKRRIIISEEQGSNSHSRHLLGKIQSRVLKADQEFDGLYNELLLEMARNQIFLINERQLSVNQQNWLRHYFKQYLRQHITPILINPDTDLVQFLKDDYTYLAVEIIRGDTIRYALLEIPSDKVPRFVNLPPEAPRRRKPMILLDNILRYCLDDIFKGFFDYDALNAYSMKMTRDAEYDLVHEMEASLMELMSSSLKQRLTAEPVRFVYQRDMPNALVEVLREKLTISRYDSIVPGGRYHNFKDFINFPNVGKANLVNKPLPRLRHIWFDKAQFRNGFDAIRERDVLLYYPYHTFEHVLELLRQASFDPSVLAIKINIYRVAKDSRIIDSMIHAAHNGKKVTVVVELQARFDEEANIHWAKRLTEAGVHVIFSAPGLKIHAKLFLISRKENGEVVRYAHIGTGNFNEKTARLYTDYSLLTADARITNEVRRVFNFIENPYRPVTFDYLMVSPQNSRRLLYEMVDREIANAQQGLPSGITLKLNNLVDKGLVDRLYAASSSGVPVNLLVRGMCSLIPNLEGISDNIRAISIVDRYLEHDRVYIFENGGDKKVYLSSADWMTRNIDYRIEVATPLLDPRLKQRVLDIIDILFSDTVKARYIDKELSNRYVPRGNRRKVRAQLAIYDYIKSLEQPE

[0414] SEQ ID NO:91

[0415] MTAIAPVITIDGPSGAGKGTLCKAMAEALQWHLLDSGAIYRVLALAALHHHVDVASEDALVPLASHLDVRFVSTNGNLEVILEGEDVSGEIRTQEVANAASQVAAFPRVREALLRRQRAFRELPGLIADGRDMGTVVFPDAPVKIFLDASSEERAHRRMLQLQVKGFSVNFERLLAEIKERDDRDRNRAVAPLVPAADALVLDSTTLSIEQVIEKALQYARQKLALA

[0416] >ACT43679.1 pyruvate kinase II[Escherichia coli BL21(DE3)]

[0417] SEQ ID NO:92

[0418] MSRRLRRTKIVTTLGPATDRDNNLEKVIAAGANVVRMNFSHGSPEDHKMRADKVREIAAKLGRHVAILGDLQGPKIRVSTFKEGKVFLNIGDKFLLDANLGKGEGDKEKVGIDYKGLPADVVPGDILLLDDGRVQLKVLEVQGMKVFTEVTVGGPLSNNKGINKLGGGLSAEALTEKDKADIKTAALIGVDYLAVSFPRCGEDLNYARRLARDAGCDAKIVAKVERAEAVCSQDAMDDIILASDVVMVARGDLGVEIGDPELVGIQKALIRRARQLNRAVITATQMMESMITNPMPTRAEVMDVANAVLDGTDAVMLSAETAAGQYPSETVAAMARVCLGAEKIPSINVSKHRLDVQFDNVEEAIAMSAMYAANHLKGVTAIITMTESGRTALMTSRISSGLPIFAMSRHERTLNLTALYRGVTPVHFDSANDGVAAASEAVNLLRDKGYLMSGDLVIVTQGDVMSTVGSTNTTRILTVE

[0419] >ACT43922.1 lactose / glucose efflux system[Escherichia coli BL21(DE3)]

[0420] SEQ ID NO:93

[0421] MHNSPAVSSAKSFDLTSTAFLIVAFLTGIAGALQTPTLSIFLTDEVHARPAMVGFFFTGSAVIGILVSQFLAGRSDKRGDRKSLIVFCCLLGVLACTLFAWNRNYFVLLFVGVFLSSFGSTANPQMFALAREHADKTGREAVMFSSFLRAQVSLAWVIGPPLAYALAMGFSFTVMYLSAAVAFIVCGVMVWLFLPSMQKELPLATGTIEAPRRNRRDTLLLFVICTLMWGSNSLYIINMPLFIINELHLPEKLAGVMMGTAAGLEIPTMLIAGYFAKRLGKRFLMRVAAVGGVCFYAGMLMAHSPVILLGLQLLNAIFIGILGGIGMLYFQDLMPGQAGSATTLYTNTSRVGWIIAGSVAGIVAEIWNYHAVFWFAMVMIIATLFCLLRIKDV

[0422] SEQ ID NO:95

[0423]

[0424] SEQ ID NO:96

[0425]

[0426] SEQ ID NO:97

[0427] ATGGAAAAACAGAACATCGCGGTTATCCTGGCGCGTCAGAACAGCAAAGGCCTGCCGCTGAAAAACCTGCGTAAAATGAACGGTATCAGCCTGCTGGGTCACACCATCAACGCGGCGATCAGCAGCAAATGCTTCGATCGTATCATCGTTTCCACCGATGGCGGCCTGATCGCGGAAGAAGCGAAAAACTTCGGCGTTGAAGTTGTTCTGCGTCCGGCGGAACTGGCGTCTGATACCGCATCTAGCATCAGCGGCGTTATCCACGCGCTGGAAACCATCGGCTCTAACAGCGGCACCGTTACCCTGCTGCAGCCGACCAGCCCGCTGCGTACCGGCGCGCACATCCGTGAAGCGTTCAGCCTGTTCGATGAAAAAATCAAAGGTAGCGTTGTTAGCGCGTGCCCGATGGAACACCACCCGCTGAAAACCCTGCTGCAGATCAA

[0428] CAACGGCGAATACGCGCCGATGCGTCACCTGAGCGATCTGGAACAGCCGCGTCAG

[0429] CAGCTGCCGCAGGCGTTCCGTCCGAACGGCGCGATCTACATCAACGATACCGCGA

[0430] GCCTGATCGCGAACAACTGCTTCTTCATCGCGCCGACCAAACTGTACATCATGAGC

[0431] CACCAGGATTCTATCGATATCGATACCGAACTGGATCTGCAGCAGGCGGAAAACA

[0432] TCCTGAACCACAAAGAATCT

[0433] ST SEQ ID NO:98

[0434] ATGGAGTTCTGCAAAATGGCGACCACCCAGAAAATCTGCGTGTACCTGGATTATG

[0435] CGACCATCCCGAGCCTGAACTACATCCTGCACTTCGCGCAGCACTTCGAAGATCAG

[0436] GAAACCATCCGCCTGTTCGGTCTGAGCCGTTTCCACATCCCGGAATCCGTGATCCA

[0437] GCGTTACCCGAAAGGTGTGGTGCAGTTCTACCCGAACCAGGAAAAGGACTTCAGC

[0438] GCTCTGCTGCTGGCGCTGAAAAACATTCTGATCGAAGTTAAACAGCAGCAGCGTA

[0439] AATGCGAAATCGAACTGCACCTGAACCTGTTCCACTACCAGCTGCTGCTGCTGCCG

[0440] TTCCTGTCCCTGTACCTGGACACCCAGGACTACTGCCACCTGACCCTGAAATTCTA

[0441] CGATGACGGCTCTGAAGCGATCAGCGCGCTCCAGGAACTGGCTCTGGCTCCGGAT

[0442] CTGGCGGCGCAGATCCAGTTTGAAAAACAGCAGTTCGACGAACTGGTTGTGAAAA

[0443] AGAGCTTCAAACTGAGCCTGCTGTCCCGTTACTTCTGGGGTAAACTGTTCGAAAGC

[0444] GAATACATCTGGTTCAACCAGGCGATCTTACAGAAAGCTGAATTACAGATCCTGA

[0445] AACAGGAAATCTCCAGCAGCCGTCAGATGGATTTCGCGATCTACCAGCAGATGAG

[0446] CGACGAACAGAAACAGCTGGTTCTGGAAATCCTGAACATCGATCTGAACAAAGTT

[0447] GCGTACCTGAAACAGCTGATGGAAAACCAGCCGAGCTTCCTGTTCCTGGGCACCA

[0448] CCCTGTTCAACATTACCCAGGAAACCAAAACCTGGCTGATGCAGATGCACGTTGAT

[0449] CTGATCCAGCAGTACTGCCTGCCGTCCGGCCAGTTCTTCAACAACAAAGCGGGTTA

[0450] CCTGTGCTTCTACAAAGGCCACCCGAACGAAAAAGAAATGAACCAGATGATCCTG

[0451] AGCCAGTTTAAAAACCTGATCGCGCTGCCGGATGACATCCCGCTGGAAATCTTACT

[0452] GCTGCTGGGCGTTATCCCGTCTAAAGTTGGCGGCTTCGCAAGCAGCGCTCTGTTCA

[0453] ACTTCACCCCGGCGCAGATCGAAAACATCATCTTCTTCACCCCGCGTTACTTCGAA

[0454] AAAGATAACCGCCTGCACGCAACCCAGTATCGTCTGATGCAGGGTCTGATCGAAC

[0455] TGGGCTACCTGGATGCGGAAAAAAGCGTTACCCACTTCGAAATCATGCAGCTGCT

[0456] GACCAAAGAA

[0457] SEQ ID NO:99

[0458] Atgtgcggtatcgttggtgctatcgcacagcgtgatgtagcgaaaatcctcctggaaggtctgcgtcgtctcgaataccgtggttacgact

[0459] ctgccggtctggcagtagtggatgcagaaggtcacatgactcgtctgcgtcgtctgggtaaagtgcagatgctcgcgcaggcggcgga

[0460] agaacacccactccacggtggtacgggtatcgcacacactcgttgggcaacccacggtgaaccgtctgaggtcaacgcacacccgcat

[0461] gttagcgagcacatcgtagtcgttcacaacggtatcatcgagaaccacgaaccactccgtgaggaactcaaagcccgtggttacaccttc

[0462] gtaagcgaaaccgacacggaagttatcgcccacctcgttaactgggaactcaaacagggtggtactctgcgtgaagcagttctgcgtgc

[0463] cattccacagctgcgtggtgcatacggtaccgtgatcatggactctcgtcatccggataccctgctcgccgcacgttctggttctccactcg

[0464] ttatcggtctgggtatgggtgagaacttcatcgcctctgatcagctggccctgctcccagttacccgtcgcttcatcttcctggaagagggtg

[0465] acatcgccgaaatcacccgtcgttccgttaacatcttcgacaaaacgggtgcggaagttaaacgtcaggacatcgagtctaacctgcagt

[0466] atgacgctggtgacaaaggcatctaccgtcactacatgcagaaagagatctacgaacagccgaacgcgatcaaaaacaccctgaccgg

[0467] tcgtatctctcacggtcaggttgacctgtctgagctgggtccaaacgcggacgaactcctgtccaaagtcgagcacatccagatcctggct

[0468] tgtggtacctcttacaactccggtatggtttctcgttactggttcgaatctctggcaggtatcccatgcgacgttgaaatcgcctccgaattcc

[0469] gttatcgtaaatctgcggtacgtcgtaactccctcatgatcaccctgtctcagtctggtgaaaccgctgatactctggcaggtctgcgtctca

[0470] gcaaagaactgggttacctgggttctctggccatctgcaacgttccgggttctagcctggttcgtgagtctgtgctggctctgatgaccaac

[0471] gcgggtacggagatcggtgttgcctctaccaaagcgttcactacccagctcactgtcctgctgatgctggttgccaaactgtctcgtctcaa

[0472] aggcctcgacgctagcatcgaacacgacatcgtacacggtctgcaggccctcccatctcgtatcgagcagatgctgccgcaggacaaa

[0473] cgtatcgaagcactggcagaagacttcagcgacaaacaccacgcgctgtttctgggtcgtggtgaccagtacccaattgcgctggaagg

[0474] tgccctgaaactgaaagagatcagctacatccatgcagaggcatacgcagcgggtgagctgaaacatggtccactggccctgatcgac

[0475] gcagatatgccggttattgtggttgctccgaacaacggcctgctggagaaactgaaatccaacatcgaggaagtacgtgcgcgtggtggt

[0476] cagctgtacgtgtttgctgaccaggacgcgggtttcgtttccagcgacaacatgcacatcatcgaaatgccgcatgttgaagaggtaatcg

[0477] cgccaatcttctacaccgtaccgctgcagctgctggcgtaccatgtagccctgatcaaaggtacggacgttgaccagccgcgtaacctggcgaaatccgtgaccgtggaa

[0478] SEQ ID NO:100

[0479]

[0480] SEQ ID NO:101

[0481] 1 atgacggcaa ttgccccggt tattaccatt gatggcccaa gcggtgcagg gaaaggcacc

[0482] 61 ttgtgtaagg ctatggcgga agcgttgcaa tggcatctgc tggactcggg tgcaatttat

[0483] 121 cgcgtactgg cattggcggc attacatcac catgttgatg ttgcgtcgga agatgcgctg

[0484] 181 gtaccgctgg catcccatct ggatgtacgt tttgtgtcga ccaatggcaa tctggaagtg

[0485] 241 atcctcgaag gggaagatgt cagcggcgaa attcgtactc aggaagtggc gaatgcagct

[0486] 301 tcacaagtcg cggcattccc acgcgttcgt gaagcattat tgcgtcgcca acgcgcgttt

[0487] 361 cgcgaattac caggtctgat tgccgatggc cgcgacatgg gaacggtggt attccctgat

[0488] 421 gcaccagtga aaattttcct tgacgcctcc tcggaagaac gtgcgcatcg ccgcatgcta

[0489] 481 cagttgcagg tgaagggctt tagtgttaac tttgagcgcc ttttggccga gatcaaagaa

[0490] 541 cgcgacgacc gcgatcgtaa ccgagcggta gcgccactgg ttccggcagc cgatgcttta

[0491] 601 gtgttggatt ccaccacctt aagcattgag caagtgattg aaaaagcgct acaatacgcg

[0492] 661 cgccagaaat tggctctcgc a

[0493] setB SEQ ID NO:102

[0494]

Claims

1. A genetically engineered bacterium having the ability to produce 3'-sialyllactose and having a UDP-N-acetylglucosamine-2-epimerase gene (neuC), a sialic acid synthase gene (neuB), an N-acetylneuraminic acid cytidylyltransferase gene (css) and an α-2,3-sialyltransferase gene (ST); in, The UDP-N-acetylglucosamine-2-epimerase gene (neuC), the sialic acid synthase gene (neuB), the N-acetylneuraminic acid cytidylyltransferase gene (css) and the α-2,3-sialyltransferase gene (ST) are integrated independently of each other or integrated together in any combination into the genome of the genetically engineered bacteria, or exist in a recombinant plasmid carried by the genetically engineered bacteria.

2. The genetically engineered bacterium according to claim 1, wherein UDP-N-acetylglucosamine-2-epimerase gene (neuC), sialic acid synthase gene (neuB), N-acetylneuraminic acid cytidylyltransferase gene (css) and α-2,3-sialyltransferase gene (ST) are integrated into the genome of the genetically engineered bacteria in a manner of being connected to a separate first promoter and / or terminator, or exist in a recombinant plasmid carried by the genetically engineered bacteria; or, Any two, three or four genes of UDP-N-acetylglucosamine-2-epimerase gene (neuC), sialic acid synthase gene (neuB), N-acetylneuraminic acid cytidylyltransferase gene (css) and α-2,3-sialyltransferase gene (ST) are integrated into the genome of the genetically engineered bacteria in a manner of being jointly connected to a first promoter and / or terminator, or exist together in a recombinant plasmid carried by the genetically engineered bacteria; and / or The genetically engineered bacteria are bacteria of the genus Escherichia, preferably Escherichia coli, more preferably Escherichia coli BL21 (DE3) strain; Preferably, the first promoter is a T2 promoter; the terminator is a T7 terminator; and / or The recombinant plasmid uses pTU2 as a backbone plasmid.

3. The genetically engineered bacteria according to claim 2, wherein UDP-N-acetylglucosamine-2-epimerase gene (neuC), sialic acid synthase gene (neuB), N-acetylneuraminic acid cytidylyltransferase gene (css) and α-2,3-sialyltransferase gene (ST) are respectively connected to a separate first promoter and / or terminator as a gene combination, or are commonly connected to a first promoter and / or terminator as a gene combination and inserted into one or more insertion sequences 1 (IS1) of the genome of the genetically engineered bacteria; More preferably, the copy number of UDP-N-acetylglucosamine-2-isomerase gene (neuC), sialic acid synthase gene (neuB), N-acetylneuraminic acid cytidylyltransferase gene (css) and α-2,3-sialyltransferase gene (ST) as a gene combination at the insertion sequence 1 (IS1) is 10-28, 12-26 or 14-22.

4. The genetically engineered bacterium according to any one of claims 1 to 3, wherein the UDP-N-acetylglucosamine-2-epimerase comprises: an amino acid sequence as shown in SEQ ID NO: 82, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO: 82; and / or, The sialic acid synthase comprises: an amino acid sequence as shown in SEQ ID NO:83, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as shown in SEQ ID NO:83; and / or, The N-acetylneuraminic acid cytidylyltransferase comprises: an amino acid sequence as shown in SEQ ID NO:84, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as shown in SEQ ID NO:84; and / or, The α-2,3-sialyltransferase comprises: an amino acid sequence as shown in SEQ ID NO:85, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:

85.

5. The genetically engineered bacterium according to any one of claims 1 to 4, wherein: Genetically engineered bacteria have been modified in one or more of the following ways: (1) Genetically engineered bacteria are modified to increase the production of glucosamine-6-phosphate; (2) genetically engineered bacteria are modified to continuously recycle cytidine-5'-triphosphate; (3) genetically engineered bacteria modified to inactivate or block the bypass degradation pathway of N-acetylmannosamine (ManNAc); (4) genetically engineered bacteria modified to inactivate or block the alternative decomposition pathway of phosphoenolpyruvate; and (5) genetically engineered bacteria modified to increase the expression or activity of proteins involved in the oligosaccharide transport pathway; (6) Genetically engineered bacteria modified to reduce or eliminate the expression or activity of β-galactosidase; (7) Genetically engineered bacteria are modified to reduce or eliminate the bypass degradation pathway of N-acetylneuraminic acid.

6. The genetically engineered bacterium according to any one of claims 1 to 5, wherein: Genetically engineered bacteria have been modified in one or more of the following ways: (8) Genetically engineered bacteria modified to reduce or eliminate the expression or activity of 6-phosphofructokinase (pfkA); (9) Genetically engineered bacteria modified to increase the expression or activity of glucosamine-6-phosphate synthase (glmS); (10) The genetically engineered bacteria are modified to reduce or eliminate the expression or activity of N-acetylglucosamine-6-phosphate deacetylase (nagA) and / or glucosamine-6-phosphate deaminase (nagB); (11) Genetically engineered bacteria modified to increase the expression or activity of cytidine monophosphate kinase (CMK) and polyphosphate kinase (PPK); (12) genetically engineered bacteria modified to reduce or eliminate the expression or activity of pyruvate kinase (pykA); and (13) Genetically engineered bacteria modified to increase the expression or activity of oligosaccharide transporters; (14) Genetically engineered bacteria modified to reduce or eliminate the expression or activity of N-acetylneuraminic acid aldolase (nanA); (15) Genetically engineered bacteria modified to reduce or eliminate the expression or activity of N-acetylmannosamine kinase (nanK); (16) The genetically engineered bacteria are modified to reduce or eliminate the expression or activity of N-acetylmannosamine-6-phosphate isomerase (nanE); (17) The genetically engineered bacteria are modified to reduce or eliminate the expression or activity of β-galactosidase (lacZ).

7. The genetically engineered bacterium according to any one of claims 1 to 6, wherein: The genetically engineered bacteria lack the 6-phosphofructokinase (pfkA) gene; and / or The genetically engineered bacteria overexpress the glucosamine-6-phosphate synthase (glmS) gene; and / or The genetically engineered bacteria lack the glucosamine-6-phosphate deaminase (nagB) gene; and / or The genetically engineered bacteria lack the N-acetylmannosamine kinase (nanK) gene; and / or The genetically engineered bacteria lacks the N-acetylmannosamine-6-phosphate epimerase (nanE) gene; and / or The genetically engineered bacteria lacks the N-acetylglucosamine-6-phosphate deacetylase (nagA) gene; and / or The genetically engineered bacteria overexpress cytidine monophosphate kinase (CMK) gene and polyphosphate kinase (PPK) gene; and / or The genetically engineered bacteria lack the N-acetylneuraminic acid aldolase (nanA) gene; and / or The genetically engineered bacteria lack the pyruvate kinase (pykA) gene; and / or The genetically engineered bacteria overexpress the oligosaccharide transporter setB gene; and / or The genetically engineered bacteria lacked the β-galactosidase (lacZ) gene.

8. The genetically engineered bacterium according to claim 7, wherein The deletion of the 6-phosphofructokinase (pfkA) gene is: a partial nucleotide sequence of the 6-phosphofructokinase (pfkA) gene is knocked out, wherein the partial nucleotide sequence knockout includes: the nucleotide sequence length retained at the 5' end accounts for 0%-7% of the total nucleotide sequence length of the 6-phosphofructokinase (pfkA) gene, and the nucleotide sequence length retained at the 3' end accounts for 70%-80% of the total nucleotide sequence length of the 6-phosphofructokinase (pfkA) gene; and / or Glucosamine-6-phosphate synthase (glmS) comprises: the amino acid sequence of SEQ ID NO:89, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:89; and / or The overexpression of the glucosamine-6-phosphate synthase (glmS) gene is: integrating the glucosamine-6-phosphate synthase (glmS) gene into the genome of the genetically engineered bacteria; and / or The deletion of the pyruvate kinase (pykA) gene comprises: knocking out a portion of the nucleotide sequence of the pyruvate kinase (pykA) gene, wherein the knocking out of the portion of the nucleotide sequence comprises: the length of the nucleotide sequence retained at the 5' end accounts for 4%-5.5% of the total nucleotide sequence length of the pyruvate kinase (pykA) gene, and the length of the nucleotide sequence retained at the 3' end accounts for 10%-20% of the total nucleotide sequence length of the pyruvate kinase (pykA) gene; and / or The cytidine monophosphate kinase (CMK) comprises: the amino acid sequence set forth in SEQ ID NO:91, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:91; the polyphosphate kinase (PPK) comprises: the amino acid sequence set forth in SEQ ID NO:90, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:90; and / or The overexpression of the cytidine monophosphate kinase (CMK) gene and the polyphosphate kinase (PPK) gene is: integrating the cytidine monophosphate kinase (CMK) gene and the polyphosphate kinase (PPK) gene into the genome of the genetically engineered bacteria; and / or The oligosaccharide transporter setB comprises: an amino acid sequence as shown in SEQ ID NO:93, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:93; and / or The overexpression of the oligosaccharide transporter setB gene comprises: integrating the oligosaccharide transporter setB gene into the genome of the genetically engineered bacteria.

9. The genetically engineered bacterium according to claim 8, wherein Under the condition that part of the nucleotide sequence of the 6-phosphofructokinase (pfkA) gene is knocked out, the glucosamine-6-phosphate synthase (glmS) gene is inserted into the position where the 6-phosphofructokinase (pfkA) gene is knocked out; and / or The genetically engineered bacteria lacks the melibiose permease (melB) gene, and the cytidine monophosphate kinase (CMK) gene and the polyphosphate kinase (PPK) gene are inserted into the position where the melibiose permease (melB) gene is knocked out; and / or Under the condition that a part of the nucleotide sequence of the pyruvate kinase gene (pykA) is knocked out, the oligosaccharide transporter setB gene is inserted into the knocked-out position of the pyruvate kinase gene (pykA); More preferably, the cytidine monophosphate kinase (CMK) gene and the polyphosphate kinase (PPK) gene are inserted into the position where the melibiose permease (melB) gene is knocked out in a manner where each is connected to a separate second promoter or in a manner where both are connected to a second promoter; Further preferably, the cytidine monophosphate kinase (CMK) gene is located upstream of the polyphosphate kinase (PPK) gene; and / or, the second promoter is a T5 promoter or a tetracycline promoter tet.

10. An Escherichia coli strain, whose preservation number is CGMCC No.: 28244.

11. Use of the genetically engineered bacteria according to any one of claims 1 to 9 or the Escherichia coli strain according to claim 10 in producing 3'-sialyllactose using glucose or glycerol as a carbon source.

12. A method for constructing a genetically engineered bacterium capable of producing 3'-sialyllactose, comprising: Integrating UDP-N-acetylglucosamine-2-epimerase gene (neuC), sialic acid synthase gene (neuB), N-acetylneuraminic acid cytidylyltransferase gene (css) and α-2,3-sialyltransferase gene (ST) independently or in any combination into the genome of the host bacteria to obtain a genetically engineered bacterium; or, The UDP-N-acetylglucosamine-2-epimerase gene (neuC), the sialic acid synthase gene (neuB), the N-acetylneuraminic acid cytidylyltransferase gene (css) and the α-2,3-sialyltransferase gene (ST) are co-introduced into a backbone plasmid to obtain a recombinant plasmid; the recombinant plasmid is introduced into a host bacterial cell to obtain a genetically engineered bacterium; Preferably, the host bacteria is a bacterium of the genus Escherichia, preferably Escherichia coli, more preferably Escherichia coli BL21 (DE3) strain.

13. The method according to claim 12, further comprising: The host cell is modified in one or more of the following ways: (1) The host bacteria are modified to increase the production of glucosamine-6-phosphate; (2) the host bacteria are modified to continuously recycle cytidine-5′-triphosphate; (3) the host bacteria are modified to inactivate or block the bypass degradation pathway of N-acetylmannosamine (ManNAc); (4) the host bacteria are modified to inactivate or block the alternative decomposition pathway of phosphoenolpyruvate; and (5) host bacteria are modified to increase the expression or activity of proteins involved in the oligosaccharide transport pathway; (6) The host bacteria are modified to reduce or eliminate the expression or activity of β-galactosidase; (7) The host bacteria are modified to reduce or eliminate the bypass degradation pathway of N-acetylneuraminic acid.

14. The method according to claim 12 or 13, wherein: The genetically engineered bacteria are modified in one or more of the following ways: (8) the host bacteria are modified to reduce or eliminate the expression or activity of 6-phosphofructokinase (pfkA); (9) the host bacteria are modified to increase the expression or activity of glucosamine-6-phosphate synthase (glmS); (10) the host bacteria are modified to reduce or eliminate the expression or activity of N-acetylglucosamine-6-phosphate deacetylase (nagA) and / or glucosamine-6-phosphate deaminase (nagB); (11) The host bacteria are modified to increase the expression or activity of cytidine monophosphate kinase (CMK) and polyphosphate kinase (PPK); (12) Modifying the host cell to reduce or eliminate the expression or activity of pyruvate kinase (pykA); and (13) Modification of the host bacteria to increase the expression or activity of oligosaccharide transporters; (14) The host bacteria are modified to reduce or eliminate the expression or activity of N-acetylneuraminic acid aldolase (nanA); (15) The host bacteria are modified to reduce or eliminate the expression or activity of N-acetylmannosamine kinase (nanK); (16) The host bacteria are modified to reduce or eliminate the expression or activity of N-acetylmannosamine-6-phosphate isomerase (nanE); (17) The host bacteria are modified to reduce or eliminate the expression or activity of β-galactosidase (lacZ).

15. The method according to any one of claims 12 to 14, wherein: The host bacterial 6-phosphofructokinase (pfkA) gene is deleted; and / or Overexpression of the glucosamine-6-phosphate synthase (glmS) gene in the host bacteria; and / or The host bacteria's glucosamine-6-phosphate deaminase (nagB) gene is deleted; and / or The host bacteria's N-acetylmannosamine kinase (nanK) gene is deleted; and / or The N-acetylmannosamine-6-phosphate isomerase (nanE) gene of the host bacteria is deleted; and / or The N-acetylglucosamine-6-phosphate deacetylase (nagA) gene of the host bacteria is deleted; and / or Overexpression of cytidine monophosphate kinase (CMK) gene and polyphosphate kinase (PPK) gene in the host bacteria; and / or The host bacteria's N-acetylneuraminic acid aldolase (nanA) gene is deleted; and / or The host bacteria's pyruvate kinase (pykA) gene is deleted; and / or Overexpression of the oligosaccharide transporter setB gene in the host bacteria; and / or The host bacterium's β-galactosidase (lacZ) gene was deleted.

16. The method according to claim 15, wherein: The deletion of the 6-phosphofructokinase (pfkA) gene of the host bacteria is as follows: a partial nucleotide sequence of the 6-phosphofructokinase (pfkA) gene is knocked out, wherein the partial nucleotide sequence knockout includes: the nucleotide sequence length retained at the 5' end accounts for 0%-7% of the total nucleotide sequence length of the 6-phosphofructokinase (pfkA) gene, and the nucleotide sequence length retained at the 3' end accounts for 70%-80% of the total nucleotide sequence length of the 6-phosphofructokinase (pfkA) gene; and / or Glucosamine-6-phosphate synthase (glmS) comprises: the amino acid sequence of SEQ ID NO:89, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:89; and / or The overexpression of the glucosamine-6-phosphate synthase (glmS) gene in the host bacteria is: integrating the glucosamine-6-phosphate synthase (glmS) gene into the genome of the host bacteria; and / or The deletion of the pyruvate kinase (pykA) gene of the host bacteria comprises: knocking out a portion of the nucleotide sequence of the pyruvate kinase (pykA) gene, wherein the knocking out of the portion of the nucleotide sequence comprises: the length of the nucleotide sequence retained at the 5' end accounts for 4%-5.5% of the total nucleotide sequence length of the pyruvate kinase (pykA) gene, and the length of the nucleotide sequence retained at the 3' end accounts for 10%-20% of the total nucleotide sequence length of the pyruvate kinase (pykA) gene; and / or The cytidine monophosphate kinase (CMK) comprises: the amino acid sequence set forth in SEQ ID NO:91, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:91; the polyphosphate kinase (PPK) comprises: the amino acid sequence set forth in SEQ ID NO:90, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:90; and / or The overexpression of the cytidine monophosphate kinase (CMK) gene and the polyphosphate kinase (PPK) gene in the host bacteria is: integrating the cytidine monophosphate kinase (CMK) gene and the polyphosphate kinase (PPK) gene into the genome of the host bacteria; and / or The oligosaccharide transporter setB comprises: an amino acid sequence as shown in SEQ ID NO:93, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:93; and / or The overexpression of the oligosaccharide transporter setB gene in the host bacteria includes: integrating the oligosaccharide transporter setB gene into the genome of the host bacteria.

17. The method according to claim 16, wherein: Under the condition that part of the nucleotide sequence of the 6-phosphofructokinase (pfkA) gene is knocked out, the glucosamine-6-phosphate synthase (glmS) gene is also inserted into the position where the 6-phosphofructokinase (pfkA) gene is knocked out; and / or The melibiose permease (melB) gene of the host bacteria is deleted, and the cytidine monophosphate kinase (CMK) gene and the polyphosphate kinase (PPK) gene are inserted into the position where the melibiose permease (melB) gene is knocked out; and / or Under the condition that a part of the nucleotide sequence of the pyruvate kinase gene (pykA) is knocked out, inserting the oligosaccharide transporter setB gene into the knocked-out position of the pyruvate kinase gene (pykA); More preferably, the cytidine monophosphate kinase (CMK) gene and the polyphosphate kinase (PPK) gene are inserted into the position where the melibiose permease (melB) gene is knocked out in a manner where each is connected to a separate second promoter or in a manner where both are connected to a second promoter; Further preferably, the cytidine monophosphate kinase (CMK) gene is located upstream of the polyphosphate kinase (PPK) gene; and / or, the second promoter is a T5 promoter or a tetracycline promoter tet.

18. The method according to any one of claims 12 to 17, wherein: UDP-N-acetylglucosamine-2-epimerase gene (neuC), sialic acid synthase gene (neuB), N-acetylneuraminic acid cytidylyltransferase gene (css) and α-2,3-sialyltransferase gene (ST) are respectively connected to a separate first promoter and / or terminator as a gene combination, or are commonly connected to a first promoter and / or terminator as a gene combination and inserted into one or more insertion sequence 1 (IS1) sites of the genome of the host bacteria; More preferably, the introduced exogenous UDP-N-acetylglucosamine-2-isomerase gene (neuC), sialic acid synthase gene (neuB), N-acetylneuraminic acid cytidylyltransferase gene (css) and α-2,3-sialyltransferase gene (ST) are combined as a gene in a copy number of 10-28, 12-26 or 14-22 at the insertion sequence 1 (IS1) in a manner that each is respectively connected to a separate first promoter and / or terminator.

19. The method according to any one of claims 12 to 18, wherein: pTargetF-nagAB containing homology arm 1 and homology arm 2 was used to knock out nagA and nagB genes. Homology arm 1 was amplified using primers of SEQ ID NO: 3 and SEQ ID NO: 4 with the host bacterial genome as a template, and homology arm 2 was amplified using primers of SEQ ID NO: 5 and SEQ ID NO: 6 with the host bacterial genome as a template; and / or The pfkA gene was knocked out using a glmS knock-in Donor-DNA fragment containing homology arms 3 and 4 and a pTargetF-pfkA-NT1 recombinant plasmid, and the glmS gene was knocked into the position of the host bacterial genome where the pfkA gene was knocked out, homology arm 3 was amplified using primers of SEQ ID NO: 11 and SEQ ID NO: 12 with the host bacterial genome as a template, and homology arm 4 was amplified using primers of SEQ ID NO: 19 and SEQ ID NO: 20 with the host bacterial genome as a template; and / or The ppk gene is knocked into the host bacterial genome using a ppk knock-in Donor-DNA and a pTargetF-melB-NT1 recombinant plasmid containing homology arms 5 and 6, wherein the homology arm 5 is amplified using primers of SEQ ID NO:35 and SEQ ID NO:36 with the host bacterial genome as a template, and the homology arm 6 is amplified using primers of SEQ ID NO:41 and SEQ ID NO:42 with the host bacterial genome as a template; and / or The cmk gene was knocked into the host bacterial genome using a cmk knock-in Donor-DNA containing homology arms 7 and 8 and a pTargetF-melB-NT2 recombinant plasmid. Homology arm 7 was amplified using primers of SEQ ID NO: 43 and SEQ ID NO: 44 with the host bacterial genome as a template. Homology arm 8 was amplified using primers of SEQ ID NO: 49 and SEQ ID NO: 50 with the host bacterial genome as a template. and / or The pykA gene was knocked out using the setB knock-in pykA site Donor-DNA and pTargetF-pykA-NT2 recombinant plasmid containing homology arms 9 and 10, and the setB gene was knocked into the position where the pykA gene was knocked out in the host bacterial genome. Homology arm 9 was amplified using primers of SEQ ID NO:58 and SEQ ID NO:59 with the host bacterial genome as a template, and homology arm 10 was amplified using primers of SEQ ID NO:64 and SEQ ID NO:65 with the host bacterial genome as a template.

20. A method for producing 3'-sialyllactose by fermentation, the method comprising the following steps: Cultivating the genetically engineered bacteria according to any one of claims 1 to 9 or the Escherichia coli strain according to claim 10 in a seed culture medium to obtain seeds; The seeds are inoculated into a fermentation medium for induction culture, and after the fermentation is completed, a fermentation liquid containing 3'-sialyllactose is obtained.

21. The method according to claim 20, wherein: The fermentation medium does not contain antibiotics; and / or The seed culture medium is LB culture medium; and / or The fermentation medium comprises TB medium and a carbon source, and the carbon source comprises glucose and / or glycerol.

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