Engineered bacteria producing 3'-sialyllactose and construction method and application thereof

Abstract

Provided are an engineered bacterium producing 3'-sialyllactose, a construction method and application thereof. The engineered bacterium has the ability to produce 3'-sialyllactose, and has an introduced exogenous UDP-N-acetylglucosamine-2-epimerase gene (neuC), a sialic acid synthase gene (neuB), an N-acetylneuraminic acid cytidyltransferase gene (css) and an alpha-2,3-sialyltransferase gene (ST) in its genome or a recombinant plasmid carried thereby. The engineered bacterium has a higher ability to produce 3'-sialyllactose without the need for antibiotics.

Description

Technical Field

[0001] This invention belongs to the field of fermentation, specifically relating to an engineered strain that produces 3'-sialic acid lactose without the need for antibiotics, its construction method, and its application. Background Technology

[0002] Human milk oligosaccharides (HMOs) are an important class of oligosaccharides found in breast milk. They are composed of five monomeric sugars and lactose molecules arranged in various combinations under the action of specific glycosyltransferases. Currently, more than 30 HMOs have had their structures identified. Studies have shown that HMOs can act as prebiotics, influencing the intrinsic components of the gut microbiota and maintaining the balance of the gut microbiome. HMOs and their metabolites (such as sialic acid) play important roles in brain development, neurotransmission, and synapse formation, promoting brain development and improving cognition in infants and young children. Furthermore, HMOs also have antiviral, immunomodulatory, and healing-promoting effects.

[0003] Currently, the main types of human lactose oligosaccharides discovered include fucoidylated HMOs and sialylated HMOs. Among sialylated HMOs, sialyllactose is the most abundant, including 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL). 3'-SL and 6'-SL are formed by sialic acid and lactose as precursors linked by α-2,3 or α-2,6 glycosidic bonds, respectively. The structural formulas of 3'-SL and 6'-SL are shown in Formula I and Formula II, respectively.

[0004]

[0005] Currently, methods for producing sialic acid lactose include chemical synthesis and enzymatic synthesis. Chemical synthesis requires expensive raw materials, stringent reaction conditions, high costs, and low yields. Compared to chemical synthesis, enzymatic synthesis offers higher yields, but the enzyme-catalyzed reaction requires expensive enzymes, resulting in high overall production costs. Furthermore, enzymes are prone to inactivation during the catalytic reaction, affecting enzyme efficiency and product yield, thus making this method difficult to implement on a large scale. Summary of the Invention

[0006] In order to overcome the problem of low yield of 3'-sialic acid lactose in the prior art, this invention provides an engineered bacterium that produces high yield of 3'-sialic acid lactose without the need for antibiotics, as well as its construction method and application.

[0007] The *Escherichia coli* strain EBSL005 (also known as strain SLIS109) described in this article was deposited on August 24, 2023, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No.:28244, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; and classified as *Escherichia coli*.

[0008] In one aspect, the present invention provides a genetically engineered bacterium capable of producing 3'-sialyl lactose, and possessing a UDP-N-acetylglucosamine-2-epomerase gene (neuC), a sialic acid synthase gene (neuB), an N-acetylneuraminic acid cytidine transferase gene (css), and an α-2,3-sialic acid transferase gene (ST), wherein the UDP-N-acetylglucosamine-2-epomerase gene (neuC), the sialic acid synthase gene (neuB), the N-acetylneuraminic acid cytidine transferase gene (css), and the α-2,3-sialic acid transferase gene (ST) are independently integrated into each other or in any combination thereof in 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 bacterium possesses an introduced exogenous UDP-N-acetylglucosamine-2-epomerase gene (neuC), an exogenous sialic acid synthase gene (neuB), an exogenous N-acetylneuraminic acid cytidine transferase gene (css), and an exogenous α-2,3-sialic acid transferase gene (ST). The introduced exogenous UDP-N-acetylglucosamine-2-epomerase gene (neuC), the introduced exogenous sialic acid synthase gene (neuB), the introduced exogenous N-acetylneuraminic acid cytidine transferase gene (css), and the introduced exogenous α-2,3-sialic acid transferase gene (ST) are independently integrated into each other or in any combination integrated into the genome of the genetically engineered bacterium, or are present in a recombinant plasmid carried by the genetically engineered bacterium.

[0010] In one embodiment, the UDP-N-acetylglucosamine-2-epimerase gene (neuC), sialic acid synthase gene (neuB), N-acetylneuraminic acid cytidine transferase gene (css), and α-2,3-sialic acid transferase gene (ST) are integrated into the genome of the genetically engineered bacterium, respectively, with separate first promoters and / or terminators linked to them, or are present in a recombinant plasmid carried by the genetically engineered bacterium.

[0011] In one embodiment, any two, three, or four of the following genes—UDP-N-acetylglucosamine-2-epimerase (neuC), sialic acid synthase (neuB), N-acetylneuraminic acid cytidine transferase (css), and α-2,3-sialic acid transferase (ST)—are integrated into the genome of the genetically engineered bacterium in a manner that is jointly linked to a first promoter and / or terminator, or coexist 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 the backbone plasmid.

[0014] In one embodiment, the UDP-N-acetylglucosamine-2-epimerase gene (neuC), the sialic acid synthase gene (neuB), the N-acetylneuraminic acid cytidine transferase gene (css), and the α-2,3-sialic acid transferase gene (ST) are inserted as a gene combination, either individually linked to a first promoter and / or terminator, or as a gene combination jointly linked to a first promoter and / or terminator, into one or more insert sequences 1 (IS1) in the genome of the genetically engineered bacterium.

[0015] In one embodiment, the copy number of the UDP-N-acetylglucosamine-2-epimerase gene (neuC), sialic acid synthase gene (neuB), N-acetylneuraminic acid cytidine transferase gene (css), and α-2,3-sialic acid transferase 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 with 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 with the amino acid sequence shown in SEQ ID NO:83.

[0018] In one embodiment, the N-acetylneuraminic acid cytyltransferase 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 with the amino acid sequence shown in SEQ ID NO:84.

[0019] In one embodiment, the α-2,3-sialic acid transferase 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.

[0020] In one implementation, 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) The genetically engineered bacteria are modified to enable the continuous recycling and regeneration of cytidine-5'-triphosphate;

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

[0024] (4) Genetically engineered bacteria are modified to inactivate or block the phosphoenolpyruvate bypass pathway; and

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

[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 are Escherichia coli, preferably Escherichia coli, and more preferably Escherichia coli BL21(DE3) strain.

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

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

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

[0032] (10) 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 are modified to increase the expression or activity of cytidine monophosphate kinase (CMK) and polyphosphate kinase (PPK);

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

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

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

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

[0038] (16) Genetically engineered bacteria are modified to reduce or eliminate the expression or activity of N-acetylmnosamine-6-phosphate epimerase (nanE);

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

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

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

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

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

[0044] In one implementation, the genetically engineered bacteria lack the N-acetylmnosamine-6-phosphate epimerase (nanE) gene.

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

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

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

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

[0049] In one implementation, the genetically engineered bacteria overexpressed the oligosaccharide transporter setB gene.

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

[0051] In one embodiment, the deletion of the 6-phosphofructokinase (pfkA) gene is defined as: partial nucleotide sequence knockout of the 6-phosphofructokinase (pfkA) gene, wherein the partial nucleotide sequence knockout includes: the nucleotide sequence length retained at the 5' end accounting 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 accounting 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 with SEQ ID NO:89.

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

[0054] In one embodiment, the deletion of the pyruvate kinase (pykA) gene is defined as: partial nucleotide sequence knockout of the pyruvate kinase (pykA) gene, wherein the partial nucleotide sequence knockout includes: the nucleotide sequence length retained at the 5' end accounting for 4%-5.5% of the total nucleotide sequence length of the pyruvate kinase (pykA) gene, and the nucleotide sequence length retained at the 3' end accounting 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 with 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 with SEQ ID NO:90.

[0056] In one embodiment, overexpression of the cytidine monophosphate kinase (CMK) gene and the polyphosphate kinase (PPK) gene is achieved by integrating the cytidine monophosphate kinase (CMK) gene and the polyphosphate kinase (PPK) gene into the genome of a genetically engineered bacterium.

[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, overexpression of the oligosaccharide transporter setB gene includes: integrating the oligosaccharide transporter setB gene into the genome of a genetically engineered bacterium.

[0059] In one embodiment, the glucosamine-6-phosphate synthase (glmS) gene is inserted at the site where the 6-phosphate fructose kinase (pfkA) gene is knocked out, provided that a portion of the nucleotide sequence of the pfkA gene has been knocked out.

[0060] In one embodiment, the genetically engineered bacteria lack the melibiase (melB) gene, and the cytidine monophosphate kinase (CMK) and polyphosphate kinase (PPK) genes are inserted at the site where the melibiase (melB) gene is knocked out.

[0061] In one embodiment, the oligosaccharide transporter setB gene is inserted into the knocked-out position of the pyruvate kinase gene (pykA) after a portion of the nucleotide sequence of the pyruvate kinase gene (pykA) has been knocked out.

[0062] In one embodiment, the cytidine monophosphate kinase (CMK) gene and the polyphosphate kinase (PPK) gene are inserted at the site where the melibiose permease (melB) gene is knocked out, either by linking their respective second promoters or by linking them together to a second promoter.

[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 the T5 promoter or the tetracycline promoter tet.

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

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

[0066] In another aspect, a method is provided for constructing genetically engineered bacteria capable of producing 3'-sialyl lactose, comprising: independently integrating the UDP-N-acetylglucosamine-2-epomerase gene (neuC), the sialic acid synthase gene (neuB), the N-acetylneuraminic acid cytidine transferase gene (css), and the α-2,3-sialic acid transferase gene (ST) into the genome of a host bacterial cell, or,

[0067] The UDP-N-acetylglucosamine-2-epomerase gene (neuC), sialic acid synthase gene (neuB), N-acetylneuraminic acid cytidine transferase gene (css), and α-2,3-sialic acid transferase gene (ST) were co-introduced into a backbone plasmid to obtain a recombinant plasmid; the recombinant plasmid was then introduced into host bacteria to obtain genetically engineered bacteria.

[0068] In one embodiment, a method for constructing genetically engineered bacteria capable of producing 3'-sialic acid lactose includes: 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 cytidine transferase gene (css), and an introduced exogenous α-2,3-sialic acid transferase gene (ST) into the genome of a host bacterial cell, or in any combination thereof, to obtain genetically engineered bacteria; 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 cytidine transferase gene (css), and the introduced exogenous α-2,3-sialic acid transferase gene (ST) were co-introduced into the backbone plasmid to obtain a recombinant plasmid; the recombinant plasmid was then introduced into the host bacterial cells to obtain genetically engineered bacteria.

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

[0071] In one embodiment, the method for constructing genetically engineered bacteria capable of producing 3'-sialic acid lactose further includes modifying the host bacterial cells in one or more of the following ways:

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

[0073] (2) Host cell modification enables continuous cyclic regeneration of cytidine-5'-triphosphate;

[0074] (3) The host cell is modified to inactivate or block the bypass degradation pathway of N-acetylmnosamine (ManNAc);

[0075] (4) The host cell is modified to inactivate or block the bypass decomposition pathway of phosphoenolpyruvate.

[0076] (5) Host cell modification to increase the expression or activity of proteins involved in oligosaccharide transport pathway;

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

[0078] (7) Host cell modification is a bypass degradation pathway that reduces or eliminates N-acetylneuraminic acid.

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

[0080] (8) Host cell modification to reduce or eliminate the expression or activity of 6-phosphofructokinase (pfkA);

[0081] (9) Host cell modification to increase the expression or activity of glucosamine-6-phosphate synthase (glmS);

[0082] (10) Host cell modification to reduce or eliminate the expression or activity of N-acetylglucosamine-6-phosphate deacetylase (nagA) and / or glucosamine-6-phosphate deaminase (nagB);

[0083] (11) Host cell modification to increase the expression or activity of cytidine monophosphate kinase (CMK) and polyphosphate kinase (PPK);

[0084] (12) Host cell modification to reduce or eliminate the expression or activity of pyruvate kinase (pykA);

[0085] (13) Host cell modification to increase the expression or activity of oligosaccharide transport proteins;

[0086] (14) Host cell modification reduces or eliminates the expression or activity of N-acetylneuraminic acid aldolase (nanA);

[0087] (15) Host cell modification to reduce or eliminate the expression or activity of N-acetylmnosamine kinase (nanK);

[0088] (16) Host cell modification to reduce or eliminate the expression or activity of N-acetylmnosamine-6-phosphate epimerase (nanE); and

[0089] (17) Host cell modification to reduce or eliminate the expression or activity of β-galactosidase (lacZ).

[0090] In one implementation, the host bacterial cell is deprived of the 6-phosphofructokinase (pfkA) gene.

[0091] In one implementation, the host bacterial cells are overexpressed with the glucosamine-6-phosphate synthase (glmS) gene.

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

[0093] In one implementation, the host bacterial cells are deprived of the N-acetylmannosamine kinase (nanK) gene.

[0094] In one implementation, the host cell is deprived of the N-acetylmnosamine-6-phosphate epimerase (nanE) gene.

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

[0096] In one implementation, the host bacterial cells are overexpressed with the cytidine monophosphate kinase (CMK) gene and the polyphosphate kinase (PPK) gene.

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

[0098] In one implementation, the host bacterial cell is deprived of the pyruvate kinase (pykA) gene.

[0099] In one implementation, the host bacterial cells are overexpressed with the oligosaccharide transporter gene setB.

[0100] In one implementation, the host bacterial cell is deprived of the β-galactosidase (lacZ) gene.

[0101] In one embodiment, the deletion of the 6-phosphofructokinase (pfkA) gene in the host bacteria comprises: partial nucleotide sequence knockout of the 6-phosphofructokinase (pfkA) gene, wherein the partial nucleotide sequence knockout includes: the nucleotide sequence length retained at the 5' end being 0%-7% of the total nucleotide sequence length of the 6-phosphofructokinase (pfkA) gene, and the nucleotide sequence length retained at the 3' end being 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 with SEQ ID NO:89.

[0103] In one embodiment, the overexpression of the glucosamine-6-phosphate synthase (glmS) gene in the host bacterial cells is achieved by integrating the glucosamine-6-phosphate synthase (glmS) gene into the genome of the host bacterial cells.

[0104] In one embodiment, the deletion of the pyruvate kinase (pykA) gene in the host bacteria is achieved by: partially knocking out the pyruvate kinase (pykA) gene, wherein the partial nucleotide sequence knockout includes: retaining a nucleotide sequence at the 5' end that accounts for 4%-5.5% of the total nucleotide sequence length of the pyruvate kinase (pykA) gene, and retaining a nucleotide sequence at the 3' end that 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 with 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 with SEQ ID NO:90.

[0106] In one embodiment, the overexpression of the cytidine monophosphate kinase (CMK) gene and the polyphosphate kinase (PPK) gene in the host bacterial cell is achieved by integrating the cytidine monophosphate kinase (CMK) gene and the polyphosphate kinase (PPK) gene into the genome of the host bacterial cell.

[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, the overexpression of the oligosaccharide transporter setB gene in the host bacterial cells comprises: integrating the oligosaccharide transporter setB gene into the genome of the host bacterial cells.

[0109] In one embodiment, the glucosamine-6-phosphate synthase (glmS) gene is inserted into the site where the 6-phosphate fructose kinase (pfkA) gene was knocked out, provided that a portion of the nucleotide sequence of the pfkA gene has been knocked out.

[0110] In one embodiment, the melbiose permease (melB) gene of the host bacterial cell is deleted, and the cytidine monophosphate kinase (CMK) gene and polyphosphate kinase (PPK) gene are inserted at the site where the melbiose permease (melB) gene is knocked out.

[0111] In one embodiment, the oligosaccharide transporter setB gene is inserted into the knocked-out position of the pyruvate kinase gene (pykA) after a portion of the nucleotide sequence of the pyruvate kinase gene (pykA) has been knocked out.

[0112] In one embodiment, the cytidine monophosphate kinase (CMK) gene and the polyphosphate kinase (PPK) gene are inserted at the site where the melibiose permease (melB) gene is knocked out, either by linking their respective second promoters or by linking them together to a second promoter.

[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 the T5 promoter or the tetracycline promoter tet.

[0114] In one embodiment, the UDP-N-acetylglucosamine-2-epimerase gene (neuC), sialic acid synthase gene (neuB), N-acetylneuraminic acid cytidine transferase gene (css), and α-2,3-sialic acid transferase gene (ST) are inserted as a gene combination, each with its own first promoter and / or terminator, or as a gene combination with the first promoter and / or terminator jointly linked, into one or more insertion sequence 1 (IS1) sites in the host bacterial genome.

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

[0116] In one implementation, the nagA and nagB genes are knocked out using pTargetF-nagAB containing homologous arm 1 and homologous arm 2. Homologous arm 1 is amplified using primers SEQ ID NO:3 and SEQ ID NO:4 with the host bacterial genome as a template, and homologous arm 2 is amplified using primers SEQ ID NO:5 and SEQ ID NO:6 with the host bacterial genome as a template.

[0117] In one embodiment, the pfkA gene is knocked out using a glmS knock-in Donor-DNA fragment containing homologous arms 3 and 4 and a pTargetF-pfkA-NT1 recombinant plasmid, and the glmS gene is knocked into the position where the pfkA gene was knocked out in the host bacterial genome. Homologous 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 homologous 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 plasmid containing homologous arms 5 and 6 and a pTargetF-melB-NT1 recombinant plasmid. Homologous 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 homologous 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 plasmid containing homologous arms 7 and 8 and a pTargetF-melB-NT2 recombinant plasmid. Homologous arm 7 is amplified using the host bacterial genome as a template using primers of SEQ ID NO:43 and SEQ ID NO:44, and homologous arm 8 is amplified using the host bacterial genome as a template using primers of SEQ ID NO:49 and SEQ ID NO:50.

[0120] In one embodiment, the pykA gene is knocked out using the setB knock-in pykA site Donor-DNA and pTargetF-pykA-NT2 recombinant plasmid containing homologous arms 9 and 10, and the setB gene is knocked into the knockout position of the pykA gene in the host bacterial genome. Homologous 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 homologous 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 preferred host bacterial strain is *Escherichia coli*.

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

[0122] Seeds are obtained by culturing the genetically engineered bacteria or the Escherichia coli strain described herein in a seed culture medium.

[0123] The seeds were inoculated into a fermentation medium for induction culture, and after fermentation, a fermentation broth containing 3'-sialic acid lactose was obtained.

[0124] In one embodiment, the fermentation medium does not contain 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, the carbon source including glucose and / or glycerol.

[0127] The advantages of this invention include:

[0128] 1. An integrated engineered strain that produces high levels of 3'-sialic acid lactose is provided, exhibiting a higher yield of 3'-sialic acid lactose compared to existing production strains (e.g., SLIS026 strain);

[0129] 2. The genetically engineered bacteria in this paper are integrative strains, which do not suffer from plasmid loss and have higher genetic stability;

[0130] 3. The method of the present invention integrates a target gene (e.g., glmS gene, setB gene) at the original gene location when knocking out a gene (e.g., pfkA gene), thereby achieving overexpression of the target gene in the bacterial cell genome, reducing gene manipulation steps, and minimizing the impact on bacterial cell growth and fermentation. Attached Figure Description

[0131] Figure 1 This is a 3'-SL metabolic pathway diagram of an integrative engineered bacterium.

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

[0133] Figure 3 The HPLC detection result chromatogram of 3'-SL.

[0134] Figure 4 This is a graph showing the fermentation results of a selected subset of transformants.

[0135] Figure 5 This is a graph showing the growth and yield of strain EBSL005 during shake-flask fermentation.

[0136] Figure 6 This is a plasmid diagram of pCas-Sac. Detailed Implementation

[0137] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0138] As used herein, an engineered bacterium "capable of producing 3'-sialic acid lactose" refers to a bacterium that, when cultured in a culture medium, produces 3'-sialic acid lactose as a target and accumulates it in the culture medium and / or within the bacterial cell to a level that allows for recovery. An engineered bacterium capable of producing 3'-sialic acid lactose can be a strain that accumulates 3'-sialic acid lactose in the culture medium and / or within the bacterial cell in a greater quantity than an unmodified strain. "Unmodified strain" refers to a control strain that has not undergone specific gene modification.

[0139] As used herein, the "SLIS026" strain is an *E. coli* BL21 strain with the LacZ, nanA, and nanKE genes knocked out. The SLIS026 strain is the chassis strain constructed according to Example 1 of patent application CN 116200316A filed by Hongmo Biotechnology (Shanghai) Co., Ltd., which is incorporated herein by reference. The integrative engineered bacteria described herein were constructed using the SLIS026 strain (see CN 116200316A) as the starting strain.

[0140] An increase in gene copy number can be achieved by introducing the gene into the host genome. For example, homologous recombination can be used to introduce a gene into the genome. Gene introduction methods using homologous recombination can include methods using linear DNA, such as Red-driven integration; methods using plasmids containing temperature-sensitive origins of replication; methods using conjugative plasmids; methods using suicide vectors that do not have origins of replication that function within the host; and methods using phage transduction. Specifically, the gene can be introduced into the host genome by transforming the host with recombinant DNA containing the target gene, which then undergoes homologous recombination with the target site on the host genome. There are no particular restrictions on the structure of the recombinant DNA used in homologous recombination, as long as homologous recombination can occur in the desired manner. For example, a linear DNA containing the target gene can be used to transform the host, wherein the linear DNA has the same base sequences at both ends of the gene as those upstream and downstream of the target site on the genome. By inducing homologous recombination upstream and downstream of the target site, the gene can be substituted into the target site. The recombinant DNA used in homologous recombination can contain marker genes for selecting transformants. A single copy of the gene can be introduced, or two or more copies can be introduced. For example, multiple copies of a gene can be introduced into the genome by targeting a base sequence that exists in multiple copies on the genome. Examples of base sequences that exist in multiple copies on the genome include repetitive DNA sequences and inverted repeats located at both ends of transposons. Furthermore, appropriate base sequences on the genome, such as genes not needed in the production of the target substance, can also be used as targets for homologous recombination. Additionally, genes can be randomly introduced into the genome using transposons or Mini-Mu. It should be noted that this method of genome modification utilizing homologous recombination is not limited to the introduction of target genes; it can also be used for any modification of the genome, such as the modification of expression regulatory sequences.

[0141] In terms of confirming the introduction of a target gene into the genome, confirmation can be achieved through Southern hybridization using probes with sequences that are complementary to all or part of the gene, or through PCR using primers prepared based on the gene sequence.

[0142] Furthermore, an increase in gene copy number can also be achieved by introducing a vector containing the gene into the host. For example, an expression vector for the gene can be constructed by linking a DNA fragment containing the target gene to a vector that functions in the host. By transforming the host with this expression vector, the copy number of the gene can be increased. The DNA fragment containing the target gene can be obtained, for example, by PCR using genomic DNA of a microorganism containing the target gene as a template. As the vector, a vector capable of autonomous replication within the host cell can be used. A multi-copy vector is preferred. Furthermore, for the selection of transformants, it is preferable that the vector contains a marker such as an antibiotic resistance gene. In addition, the vector may 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 bacteriophage, a granule, or a phage particle.

[0143] When introducing a gene, the gene only needs to be maintained in the host in a manner that allows for expression. Specifically, the gene only needs to be maintained in a manner controlled by a promoter that functions in the host. Regarding the promoter, there are no particular restrictions as long as it functions in the host. "A promoter that functions in the host" can refer to a promoter that has promoter activity in the host. The promoter can be a promoter derived from the host or a promoter derived from a different species. The promoter can be an intrinsic promoter of the introduced gene or a promoter of another gene. For example, a strong promoter can be used as a promoter.

[0144] Downstream of the gene, a terminator can be configured to terminate transcription. There are no particular restrictions on the terminator, as long as it functions in the host. The terminator can be derived from the host or from a different species. The terminator can be an inherent terminator of the introduced gene or a terminator of another gene.

[0145] Furthermore, when two or more genes are introduced, each gene only needs to be maintained in the host in a manner that allows for expression. For example, each gene can be maintained on a single expression vector or on the genome. Alternatively, each gene can be maintained on multiple expression vectors, or on one or more expression vectors and on the genome.

[0146] Regarding the introduced gene, there are no particular restrictions as long as it encodes a protein that functions in the host. The introduced gene can be derived from the host or from a different species. For example, the introduced gene can be obtained by PCR using primers designed based on its base sequence, with genomic DNA of an organism possessing the gene or a plasmid carrying the gene as a template. Alternatively, the introduced gene can be fully synthesized based on its base sequence. The obtained gene can be used as is or with appropriate modifications. That is, variants can be obtained by modifying the gene. Gene modification can be performed using known methods. For example, site-specific mutation methods can introduce targeted mutations into target sites on the DNA. That is, for example, site-specific mutation methods can modify the coding region of the gene so that the encoded protein contains substitutions, deletions, insertions, and / or additions of amino acid residues at specific sites. Alternatively, gene variants can be fully synthesized.

[0147] Furthermore, increased gene expression can be achieved by improving gene transcription efficiency. Additionally, increased gene expression can be achieved by improving gene translation efficiency. Improvements in gene transcription efficiency or translation efficiency can be achieved, for example, by modifying expression regulatory sequences.

[0148] In terms of improving gene transcription efficiency, this can be achieved, for example, by replacing the promoter of a gene in the genome with a more robust promoter. A "more robust promoter" can refer to a promoter that improves gene transcription compared to the original wild-type promoter.

[0149] "Decreased activity or expression of a protein or enzyme" means that the activity of the protein or enzyme is reduced compared to the unmodified strain or the originating strain. It should be noted that "decreased activity or expression of a protein or enzyme" also includes cases where the activity of the protein or enzyme is completely lost. More specifically, "decreased activity or expression of a protein or enzyme" can mean that, compared to the unmodified strain, the number of molecules of the protein or enzyme per cell is reduced and / or the function of each molecule of the protein or enzyme is reduced. There are no particular limitations on the degree of reduction in protein or enzyme activity or expression, as long as the activity is reduced compared to the unmodified strain or the originating strain. For example, the activity of the protein or enzyme can be reduced to less than 50%, less than 20%, less than 10%, less than 5%, or 0% of that in the unmodified strain.

[0150] A decrease in gene expression can result from a decrease in transcription efficiency, a decrease in translation efficiency, or a combination thereof. Regarding a decrease in gene expression, for example, it can be achieved by modifying expression regulatory sequences such as the gene promoter, the Shine-Dalgarno (SD) sequence (also known as the ribosome binding site (RBS)), and the spacer region between the RBS and the start codon. When modifying expression regulatory sequences, it is preferable that at least one base, more preferably at least two bases, and particularly preferably at least three bases are modified. Regarding a decrease in gene transcription efficiency, for example, it can be achieved by replacing the promoter of a gene in the genome with a weaker promoter. A "weaker promoter" refers to a promoter in which gene transcription is weakened compared to the originally present wild-type promoter. Examples of weaker promoters include, for example, inducible promoters. That is, inducible promoters can function as weaker promoters under non-inducible conditions (e.g., in the absence of inducing substances). Furthermore, a portion or all of the expression regulatory sequence can be deleted (deficiency). Furthermore, the reduction of gene expression can also be achieved, for example, by manipulating factors involved in expression control. Examples of factors involved in expression control include low-molecular-weight molecules (inducing substances, repressing substances, etc.), proteins (transcription factors, etc.), and nucleic acids (siRNA, etc.) involved in transcription and translation control. Additionally, the reduction of gene expression can also be achieved, for example, by introducing mutations that reduce gene expression into the coding region of the gene. For example, gene expression can be reduced by replacing codons in the coding region of the gene with synonymous codons that are used less frequently in the host. Furthermore, gene expression can also be reduced by gene disruption as described later.

[0151] Furthermore, 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 modifying the gene in a way that prevents the production of a normally functioning protein. "Preventing the production of a normally functioning protein" includes: cases where no protein is produced from the gene at all, and cases where the gene produces a protein whose function (e.g., activity, properties) is reduced or absent per molecule.

[0152] Regarding gene disruption, this can be achieved, for example, by deleting (deficiting) a gene in the genome. "Gene deletion" refers to the deletion of part or all of the coding region of a gene. Furthermore, the entire gene can be deleted, including the sequences preceding and following the coding region. The sequences preceding and following the coding region may, for example, contain gene expression regulatory sequences. The deleted region can be any region, such as the N-terminal region (the region flanking the N-terminus of the protein), the internal region, or the C-terminal region (the region flanking the C-terminus of the protein), as long as it reduces protein activity. Generally, the longer the deleted region, the more reliably the gene is inactivated. The deleted region can be, for example, a length of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the full length of the coding region. Furthermore, regarding the sequences preceding and following the deleted region, inconsistency in the reading frames is preferred. Inconsistency in the reading frames allows for frameshifting downstream of the deleted region.

[0153] Furthermore, regarding gene disruption, it can be achieved, for example, by introducing amino acid substitutions (missense variants), stop codons (speechless variants), or additions or deletions of 1-2 bases (frameshift variants) 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. Furthermore, regarding the sequences before and after the insertion site, inconsistent reading frames are preferred. Inconsistent reading frames can cause a frameshift downstream of the insertion site. As for other base sequences, there are no particular restrictions as long as they reduce or eliminate the activity of the encoded protein. In particular, it can be implemented by deleting (deficient) the amino acid sequence of the encoded protein or enzyme. In other words, regarding modifications that reduce the activity of a protein or enzyme, for example, it can be achieved by deleting part or all of the amino acid sequence of the protein.

[0154] As used in this article, the nanA gene encodes N-acetylneuaminic acid aldolase (EC 4.1.3.3). N-acetylneuaminic acid aldolase catalyzes the cleavage of the C-C bond in N-acetylneuraminic acid (Neu5Ac, also known as sialic acid), producing N-acetyl-D-mannosamine (ManNAc) and pyruvic acid (Pyr). Strains that knock out the nanA gene essentially do not express active N-acetylneuaminic acid aldolase, thus preventing the conversion of Neu5Ac to ManNAc and inhibiting its consumption via this pathway, thereby promoting the accumulation of Neu5Ac within the bacterial cells.

[0155] As used in this article, the nanK gene encodes N-acetyl-D-mannosamine kinase (EC 2.7.1.60). N-acetyl-D-mannosamine kinase catalyzes phosphate group transfer, converting ManNAc to N-acetyl-D-mannosamine-6-phosphate (ManNAc-6-P). Strains that knock out the nanK gene essentially do not express active N-acetyl-D-mannosamine kinase, thus preventing the conversion of ManNAc to ManNAc-6-P and inhibiting the consumption of ManNAc through this pathway. This, in turn, promotes the accumulation of ManNAc in the bacterial cell, which in turn promotes the accumulation of Neu5Ac (using ManNAc as a precursor) in the bacterial cell.

[0156] As used in this paper, the nanE gene encodes N-acetyl-D-mannosamine epimerase (also known as ManNAc-6-P epimerase, EC 5.1.3.9). N-acetyl-D-mannosamine epimerase catalyzes the isomerization of N-acetyl-D-mannosamine-6-phosphate (ManNAc-6-P) to N-acetylglucosamine-6-phosphate (GlcNAc-6-P). Strains that have had the nanE gene knocked out essentially do not express active N-acetyl-mannosamine-6-phosphate epimerase, thus preventing the isomerization reaction from ManNAc-6-P to GlcNAc-6-P and inhibiting the consumption of ManNAc-6-P through this pathway.

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

[0158] Previously, CRISPR / Cas9 technology was used to knock out genes related to the intracellular degradation of N-acetylneuraminic acid (Neu5Ac) in the wild-type strain BL2l(DE3), and the lacZ gene encoding β-galactosidase was also knocked out, resulting in a non-naturally occurring E. coli strain, SLIS026, for the production of Neu5Ac. Furthermore, a Neu5Ac or SL synthetic pathway containing at least one heterologous enzyme was constructed in this strain. The resulting genetically engineered strain can synthesize Neu5Ac or SL through autometabolism using a single, inexpensive exogenous carbon source present in the fermentation broth. See patent application CN116200316A for details.

[0159] Based on the previously obtained genetically engineered bacterium SLIS026, the inventors further modified its genes to obtain an integrative engineered bacterium that produces high levels of 3'-sialic acid lactose without the need for antibiotics. The construction of the integrative engineered bacterium of this invention is further described below. The enzymes involved in the gene modification and their encoding genes are described below.

[0160] like Figure 1As shown, the lacZ gene in *E. coli* encodes β-galactosidase. β-galactosidase cleaves the galactosidic bond of lactose, producing galactose and glucose. Strains that knock out the lacZ gene essentially do not express active β-galactosidase, thus inhibiting lactose consumption through this pathway and promoting lactose accumulation within the bacterial cells. Because CMP-N-acetylneuraminic acid (CMP-Neu5Ac) reacts with lactose to produce 3'-sialyllactose under the catalysis of α-2,3-sialyltransferase encoded by the ST gene, the accumulation of lactose concentration within the bacterial cells is beneficial for 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) in strain SLIS026 was modified.

[0163] The aim of this modification is to increase the intracellular supply of glucosamine-6-phosphate by reducing its consumption via an alternative pathway. Since glucosamine-6-phosphate is a precursor for the synthesis of N-acetylneuraminic acid (Neu5Ac), which in turn is a precursor for the synthesis of 3'-sialyl lactose (3'-SL), increasing the supply of glucosamine-6-phosphate helps to increase the supply of Neu5Ac, thereby contributing to increased production of 3'-SL.

[0164] The integrative engineered bacteria of this invention can utilize at least glucose or glycerol as a carbon source to produce 3'-SL. Specifically, after entering the bacterial cell, glucose is converted to glucose-6-phosphate (G-6-P) by glucose phosphorylation and transport-related enzymes encoded by genes such as ptsG, ptsH, crr, and ptsL. Glucose-6-phosphate is then converted to fructose-6-phosphate (F-6-P) by phosphoglucose isomerase (PGI) encoded by the pgi gene. Glycerol, after entering the bacterial cell, is converted to F-6-P by glycerol metabolism-related enzymes encoded by genes such as glpF, glpK, and glpD. F-6-P is then converted to glucosamine-6-phosphate (GlcN-6-P) by glucosamine-6-phosphate synthetase (glmS, EC 2.6.1.16). The same principle applies to the use of other carbon sources (such as sucrose).

[0165] To increase the supply of glucosamine-6-phosphate (GlcN-6-P), the present invention employs the following method:

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

[0167] The *E. coli* pfkA gene encodes 6-phosphofructokinase (EC2.7.1.11). 6-phosphofructokinase catalyzes the transfer of phosphate groups, thereby converting F-6-P into fructose-1,6-bisphosphate (FBP). Strains that knock out the pfkA gene essentially do not express 6-phosphofructokinase, thus preventing the conversion of most F-6-P to FBP. This reduces the consumption of F-6-P within the bacterial cell, promoting its accumulation and consequently, the accumulation of GlcN-6-P. In this invention, only a portion of the nucleotide fragment of the pfkA gene is knocked out, not the entire nucleotide fragment. The knockout of certain nucleotide segments is performed as follows: 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 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 *E. coli* glmS gene encodes glucosamine-6-phosphate synthase (glmS). Glucosamine-6-phosphate synthase glmS catalyzes the conversion of F-6-P to GlcN-6-P. Overexpression of the glmS gene increases the efficiency of F-6-P to GlcN-6-P conversion, thereby increasing the efficiency of GlcN-6-P to glucosamine-1-phosphate (GlcN-1-P), which is beneficial for 3'-SL formation. This invention integrates the glucosamine-6-phosphate synthase (glmS) gene into the genome of a genetically engineered bacterium by increasing the copy number of the glmS gene, thereby achieving glmS gene overexpression.

[0170] In this invention, when knocking out a portion of the pfkA gene, the glmS gene (denoted as ΔpfkA::glmS) can be integrated at the knockout site, thereby achieving overexpression of the glmS gene in the bacterial cell genome. This reduces the number of gene manipulation steps and minimizes the impact on bacterial cell growth and fermentation.

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

[0172] The *E. coli* *nagB* gene encodes glucosamine-6-phosphate deaminase. Glucosamine-6-phosphate deaminase catalyzes the breakdown of glucosamine-6-phosphate (GlcN-6-P) into fructose-6-phosphate (F-6-P) and ammonia. Strains that knock out the *nagB* gene essentially do not express active glucosamine-6-phosphate deaminase, thus preventing the conversion of GlcN-6-P to F-6-P. This reduces the consumption of GlcN-6-P within the bacterial cell and promotes its accumulation.

[0173] As described above, this invention enhances the GlcN-6-P synthesis pathway by overexpressing the glmS gene. Secondly, this invention inhibits the GlcN-6-P degradation pathway by knocking out the nagB and pfkA genes. Therefore, the modification of the GlcN-6-P metabolic pathway within bacterial cells by this invention helps increase the supply of GlcN-6-P within bacterial cells, thereby contributing to increased Neu5Ac supply and ultimately increasing the yield of 3'-SL.

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

[0175] 2. Overexpression of four key genes involved in 3'-SL synthesis was performed.

[0176] GlcN-6-P can be converted into GlcN-1-P by the phosphoglucosamine isomerase encoded by the glmM gene. GlcN-1-P is then converted into GlcNAc-1-P by the enzymes encoded by the glmU gene (fused N-acetylglucosamine-1-phosphate uridyltransferase and glucosamine-1-phosphate acetyltransferase), which in turn generates UDP-GlcNAc. UDP-GlcNAc, as one of the precursors for 3'-SL synthesis, can generate 3'-SL under the action of four key enzymes. Therefore, overexpression of these four key enzyme genes in bacterial cells will enhance the 3'-SL synthesis pathway and help increase the yield of 3'-SL.

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

[0178] The neuC gene encodes UDP-N-acetylglucosamine 2-epimerase. This enzyme catalyzes the conversion of UDP-GlcNAc to ManNAc. In this invention, the UDP-N-acetylglucosamine 2-epimerase was derived from the Campylobacter jejuni strain, NCBI accession number WP_002874240.1.

[0179] The neuB gene encodes sialic acid synthase (also known as neuraminidase). Sialic acid synthase catalyzes the reaction of ManNAc and phosphoenolpyruvate (PEP) to produce Neu5Ac and phosphate. In this invention, the sialic acid synthase gene is derived from the Campylobacter jejuni strain, GenBank accession number WP_002874241.1.

[0180] The CSS gene encodes N-acetylneuraminic cytidylyl transferase. N-acetylneuraminic cytidylyl transferase can catalyze the production of CMP-Neu5Ac and pyrophosphate from Neu5Ac using the cofactor CTP. In this invention, the N-acetylneuraminic cytidylyl transferase is derived from the strain *Neisseria meningitidis*, NCBI accession number WP_002215295.1.

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

[0182] The four genes mentioned above, when overexpressed, are constructed into different transcription units using strong promoters (e.g., the T2 promoter) and terminators, and expressed separately. For example, the transcription units composed of the 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, RBS represents the ribosome binding site, and T7T represents the T7 terminator sequence. The sequence of the T2 promoter is GGATAATCAGATCACGAAATTAATACCACTCACTATAGGAATAGTGCCCGGGTAACCATTCC, sequence number SEQ ID NO:81. The sequence of pET-RBS is TTTAACTTTAAGAAGGAGATATA, sequence number SEQ ID NO:94. The inventors have experimentally verified 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 is more effective, therefore, the T2 promoter was chosen to be used in this invention.

[0183] 3. Modify the supply channels for cofactors.

[0184] In point 2, the N-acetylneuraminic acid cytyltransferase encoded by the css gene requires the cofactor cytidine-5'-triphosphate (CTP) to catalyze the formation of CMP-Neu5Ac and pyrophosphate from Neu5Ac. 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 also releases CMP when it catalyzes the reaction of CMP-Neu5Ac with lactose to produce 3'-SL. The reaction equation is: CMP-Neu5Ac + lactose → 3'-SL + CMP. This reaction leads to the accumulation of CMP.

[0186] If intracellular CTP is insufficient, enough CMP-Neu5Ac cannot be produced, which affects the yield of 3'-SL. Furthermore, the accumulation of CMP also inhibits the formation of 3'-SL. To address these issues, this invention overexpresses the cmk gene encoding cytidine monophosphate kinase (CMK) and the ppk gene encoding polyphosphokinase (PPK). The cytidine monophosphate kinase encoded by the cmk gene catalyzes the phosphorylation of CMP to generate CDP. The polyphosphokinase (PPK) encoded by the ppk gene catalyzes the phosphorylation of CDP to generate CTP, thereby achieving cofactor recycling and avoiding the inhibitory effect of insufficient cofactors on the formation of 3'-SL.

[0187] The CMK and PPK genes of this invention are integrated into the genome of a genetically engineered bacterium. The endogenous melibiase (melB) gene is knocked out of the genome of the genetically engineered bacterium, and the exogenous cytidine monophosphate kinase (CMK) and exogenous polyphosphate kinase (PPK) genes are inserted at the knockout site of the endogenous melibiase (melB) gene. This reduces the number of gene manipulation steps and minimizes the adverse effects of gene manipulation on the growth and metabolism of the strain.

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

[0189] ManNAc is one of the precursors for 3'-SL production. However, the degradation pathway of ManNAc in bacterial cells can reduce the content of ManNAc in bacterial cells, thereby affecting the production of 3'-SL. The degradation pathway of ManNAc includes: (1) N-acetylmnosamine kinase encoded by the nanK gene catalyzes the conversion of ManNAc to ManNAc-6-P; (2) N-acetylmnosamine-6-phosphate epimerase encoded by the nanE gene isomerizes ManNAc-6-P to GlcNAc-6-P; (3) N-acetylglucosamine-6-phosphate deacetylase encoded by the nagA gene can catalyze the deacetylation of GlcNAc-6-P to produce GlcN-6-P.

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

[0191] The strain of this invention lacks the nanK gene encoding N-acetylmnosamine kinase. Strains lacking the nanK gene can essentially not express active N-acetylmnosamine kinase, thus essentially preventing the conversion of ManNAc to ManNAc-6-P, which is conducive to the accumulation of ManNAc in bacterial cells, and consequently, to the accumulation of Neu5Ac (using ManNAc as a precursor) in bacterial cells.

[0192] Furthermore, the strains of this invention also lack the nanE gene encoding N-acetylmnosamine-6-phosphate epimerase. Strains that knock out the nanE gene can essentially not express active N-acetylmnosamine-6-phosphate epimerase, thus essentially preventing the isomerization reaction from ManNAc-6-P to GlcNAc-6-P.

[0193] Furthermore, the strain of this invention knocks out the nagA gene encoding N-acetylglucosamine-6-phosphate deacetylase. N-acetylglucosamine-6-phosphate deacetylase catalyzes the deacetylation of GlcNAc-6-P, thereby producing GlcN-6-P. Therefore, the strain with the nagA gene knocked out essentially does not express active N-acetylglucosamine-6-phosphate deacetylase, thus virtually eliminating the GlcNAc-6-P to GlcN-6-P reaction. Exemplarily, when knocking out the nagA gene, the nagB gene and the nagA gene can be knocked out together, which reduces the number of gene manipulation steps, improves the efficiency of gene manipulation, and reduces the impact on cell growth and fermentation.

[0194] The strain of this invention has knocked out the nanK gene, the nanE gene, and the nagA gene. Therefore, ManNAc is difficult to be consumed through the above-mentioned degradation pathway, which is conducive to the accumulation of ManNAc in the bacterial cells and thus helps to increase the yield of 3'-SL.

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

[0196] The neuB gene encodes sialic acid synthase, which catalyzes the reaction of ManNAc and phosphoenolpyruvic acid (PEP) to produce Neu5Ac and phosphate. To increase the accumulation of Neu5Ac and consequently the content of 3'-SL, the content of PEP needs to be increased. One pathway for PEP production is through glycolysis of glucose by bacterial cells. However, PEP readily converts to enolpyruvic acid under the catalysis of pyruvate kinase (PykA) within the bacterial cell, with the reaction equation: PEP + 2ADP → enolpyruvic acid + 2ATP. Enolpyruvic acid is then converted back to pyruvic acid (pyr). Therefore, to increase the PEP content within bacterial cells, this invention knocks out a portion of the pykA gene encoding pyruvate kinase. The endogenous pyruvate kinase (pykA) gene is partially knocked out, wherein the knockout is such that 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. Modify oligosaccharide transport pathways

[0198] The 3'-SL produced within bacterial cells needs to be pumped out of the cell; otherwise, its production will be inhibited. This invention uses overexpression of the oligosaccharide transporter gene setB to increase the ability of 3'-SL to be pumped out of the cell. In this invention, when knocking out a portion of the pykA gene, the setB gene (denoted as ΔpykA::setB) is integrated at the knockout site. This reduces the number of gene manipulation steps and minimizes the impact on bacterial cell growth and fermentation.

[0199] The genetic information involved in this application is shown in Table 1 below:

[0200] Table 1 shows the gene information involved in the embodiments.

[0201]

[0202]

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

[0204] Example

[0205] To further illustrate the technical means and effects of this invention, a detailed description is provided below in conjunction with the accompanying drawings and preferred embodiments of the invention. Experimental methods in the following embodiments, unless otherwise specified, are performed according to conventional methods and conditions, or as selected in the product manual.

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

[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 Escherichia coli cells were prepared using a competent cell preparation kit from Sangon Biotech (Shanghai) Co., Ltd.

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

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

[0211] Table 2. Reagent Information Table from the Examples

[0212]

[0213]

[0214] The homemade reagents involved in the following examples are shown below:

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

[0216] (2) TB medium: Weigh 10g of trypsin, 18g of yeast extract, 2.31g of KH2PO4 (potassium dihydrogen phosphate) and 16.43g of K2HPO4 (dipotassium hydrogen phosphate), dissolve and mix them in distilled water, then add 4mL of glycerol, and then make up to 1000mL with deionized water. Finally, dispense 100mL into Erlenmeyer flasks and wrap them tightly. Sterilize at 121℃ for 20min and store at room temperature.

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

[0218] (4) 200g / L MgSO4·7H2O stock solution: Weigh 10g of MgSO4·7H2O solution into deionized water, and after it is completely dissolved, make up to 50mL. Then sterilize at 121℃ for 20min and store at room temperature.

[0219] (5) Trace element stock solutions: Prepare trace element stock solutions of 54.4 g / L ferric ammonium citrate, 9.8 g / L MnCl2·4H2O, 1.6 g / L CoCl2·6H2O, 1 g / L CuCl2·2H2O, 1.9 g / L H3BO3, 9 g / L ZnSO4·7H2O, 1.1 g / L Na2MoO4·2H2O, 1.5 g / L Na2SeO3 and 1.5 g / L NiSO4·6H2O respectively, sterilize at 121℃ for 20 min and store at 4℃ for later use.

[0220] (6) 500g / L glucose: Weigh 500g of glucose and dissolve it in deionized water (heat to dissolve), make up to 1L, then sterilize at 115℃ for 20min and store at room temperature.

[0221] (7) 250g / L lactose: Weigh 250g of lactose and dissolve it in deionized water (heat to dissolve), make up to 1L, then sterilize at 121℃ for 20min and store at room temperature.

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

[0223] (I) Fermentation sample processing:

[0224] Sample preparation method: Take 1-2 mL of sample (such as fermentation broth), then place the sample in a metal bath (95℃) and boil for 30 min to remove enzymes. After cooling, centrifuge, filter through an aqueous membrane, and then perform the test.

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

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

[0227] (2) Chromatographic conditions: Column: Sepax HP-Amide 250×4.6mm, 5μm (amide column); Buffer salt: 10mM 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℃;

[0229] (3) Testing operation:

[0230] ① The blank loading solution and diluent were deionized water.

[0231] ② Preparation of standard solution: Accurately weigh 25 mg of 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 to inactivate it, centrifuge it, filter the supernatant, and prepare for sample injection.

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

[0234] The formula for calculating sample content is as follows:

[0235]

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

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

[0238] Glucosamine-6-phosphate is one of the precursors in the synthesis of 3'-SL, and the amount of glucosamine-6-phosphate supplied is one of the rate-limiting steps in the synthesis of 3'-SL. Therefore, increasing the supply of glucosamine-6-phosphate helps to increase the yield of 3'-SL. In this embodiment, the metabolic pathway of glucosamine-6-phosphate was modified to increase the supply of glucosamine-6-phosphate and reduce the amount of glucosamine-6-phosphate decomposed. Specifically, in this embodiment, SLIS026 strain was used as the starting strain. The CRISPR / Cas9 gene editing system was used to knock out the nagA gene (encoding gene of N-acetylglucosamine-6-phosphate deacetylase) and the nagB gene (encoding gene of glucosamine-6-phosphate deaminase), as well as a partial fragment of the pfkA gene (encoding gene of 6-phosphofructokinase). Furthermore, when knocking out the pfkA gene, the glmS gene (encoding gene of glucosamine-6-phosphate synthase) was integrated into the knocked-out pfkA fragment, resulting in overexpression of the glmS gene (recorded as ΔpfkA::glmS), thereby obtaining strain SLIS027.

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

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

[0241] The nucleotide sequences of the nagA, nagB, pfkA, and glmS genes were obtained according to Table 1. Primers for knocking out the nagA and nagB genes were designed based on these sequences, as well as primers for knocking out a portion of the pfkA gene and integrating the knocked-out pfkA gene fragment into the glmS gene, as shown in Table 3. The sgRNA sequences are 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, under the column pTargetF-nagAB, PAM represents the protospacer-jacent motif. Guided by sgRNA (single guide RNA), the Cas9 protein recognizes the PAM and targets a specific DNA sequence, which is then excised by the Cas9 protein. Homologous arms LF1 and RF1 are used to precisely repair the DNA sequence that generates the nick.

[0246] The glmS knock-in Donor-DNA fragment is the donor DNA molecule used for glmS knock-in. It includes the homologous arm LF2 sequence, the Tet promoter sequence, the glmS gene sequence, the T7 terminator sequence, and the homologous arm RF2 sequence, enabling the insertion of the glmS gene at the site where the pfkA gene is knocked out. These sequences are all obtained by PCR amplification using their respective primers and templates, and then ligated into the glmS knock-in Donor-DNA fragment using NEB (New England Biolabs) multi-fragment recombinase. The same process is used for other knock-in genes.

[0247] Table 4 sgRNA sequence listing

[0248] Gene manipulation Operation method sgRNA sequence Serial Number ΔnagAB Knockout CGGTCTGCCGAAAGAGCATC SEQ ID NO:21 ΔpfkA::glmS Knock out and replace GTTTCTGACATGATCAACCG SEQ ID NO:22

[0249] In Table 4, ΔnagAB represents the knockout of the nagA and nagB genes. ΔpfkA::glmS represents the knockout of a portion of the pfkA gene, resulting in the integration of the glmS gene into the knocked-out pfkA gene.

[0250] 1.2 Construction of related plasmids:

[0251] Based on the primers designed in Table 3, specific amplification of each gene fragment related to pTargetF-nagAB and pTargetF-pfkA-NT1 was performed. High-fidelity enzyme 2× from Tianluo Diagnostics Technology (Jiangsu) Co., Ltd. was used. PCR reaction was performed using FlashKOD Dye Mix. 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[ddH2O]]> 22μL

[0254] The DNA templates in Table 5 refer to the templates containing the gene fragments that need to be specifically amplified, as recorded in the "Template" column of Table 3. Primer F refers to the forward primers in Table 3, and primer R refers to the reverse primers in Table 2.

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

[0256] Table 6 PCR Reaction Procedure

[0257]

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

[0259] (1) The DNA fragments with correct PCR amplification results were extracted and recovered by gel cutting using the UE DNA gel recovery kit of Suzhou Youyilandi Biotechnology Co., Ltd., and the concentration of the recovered DNA fragments was determined by the BioSpec-nano instrument.

[0260] (2) Different DNA fragments were ligated and recombined using NEB (New England Biolabs) multi-fragment recombinase, with each DNA fragment being 200 ng in volume. An equal volume of multi-fragment recombinase was then added, and the mixture was incubated at 50°C for 30-60 min to obtain different ligation products.

[0261] (3) Take out the E. coli competent cells Trans 10 stored in the ultra-low temperature freezer and place them on ice (E. coli competent cells Trans 10 are used in this embodiment, but E. coli competent cells C2566 can also be used).

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

[0263] (5) Preheat the water bath to 42°C, place the centrifuge tube containing the competent E. coli cells from 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) Take 800 μL of sterilized LB liquid culture medium and add it to the centrifuge tube in step (5). Incubate at 37°C and 250 rpm for 45-60 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 back and forth, spread the mixed bacterial solution on LB solid plates containing the corresponding antibiotic, and incubate overnight at 37°C with the plates upside down.

[0266] (8) After the white single colony grows, pick the white single colony into a test tube containing 5 mL of LB liquid medium (containing the corresponding antibiotic), and incubate at 37℃ and 250 rpm for 6 h with shaking.

[0267] (9) After performing PCR testing on the bacterial culture, take 500 μL of the positive bacterial culture and send it to Qingke Company for sequencing or directly send the untested bacterial culture for sequencing, and store the remaining bacterial culture in 30% glycerol.

[0268] (10) The bacterial strains that were verified to be correct by sequencing were expanded and cultured, and plasmids were extracted using the Tianluo Diagnostic Plasmid Mini-Extraction Kit. The pTargetF-nagAB plasmid and the pTargetF-pfkA-NT1 plasmid were obtained respectively.

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

[0270] Based on the primers designed in Table 3, the glmS knock-in Donor-DNA fragment was constructed using overlap extension PCR (OE-PCR). The procedure for each round of the multiple PCR rounds in the overlap extension PCR method is shown in Table 6.

[0271] 1.4 Construction of Strain SLIS027

[0272] In this embodiment, strain SLIS027 was modified from strain SLIS026. Compared to strain SLIS026, strain SLIS027 had the nagA and nagB genes knocked out, a portion of the pfkA gene knocked out, and the glmS gene integrated into the knocked-out pfkA site, ultimately obtaining strain SLIS027. Specifically, the nagAB gene of strain SLIS026 was knocked out using the pTargetF-nagAB plasmid. A portion of the pfkA gene was knocked out using the pTargetF-pfkA-NT1 plasmid and the glmS knock-in Donor-DNA fragment, and the glmS gene was integrated into the knocked-out pfkA site.

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

[0274] ① Preparation of SLIS026 competent cells: SLIS026 bacterial strain stored at -80℃ was streaked to form single colonies. A single colony was picked and inoculated into 5 mL of LB broth, and cultured at 37℃ with shaking at 250 rpm until the OD of the bacterial culture reached its maximum. 600 The bacterial culture was incubated for approximately 3 hours (approximately 0.5 μL) on ice for 30 minutes. The culture was then transferred to pre-chilled sterile centrifuge tubes and centrifuged at 4000 rpm for 10 minutes at 4°C. The supernatant was discarded, and the wet cells were collected. The wet cells were resuspended twice in pre-chilled 0.1M CaCl2 (containing 15% glycerol) solution. The resuspended cells were centrifuged at 4000 rpm for 10 minutes at 4°C, and the supernatant was discarded. Finally, the cells were resuspended in pre-chilled 0.1M CaCl2 (containing 15% glycerol) solution, aliquoted into 100 μL portions into 1.5 mL centrifuge tubes, rapidly frozen in liquid nitrogen, and 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 min, then heat shock it at 42℃ for 45 s, and immediately place it on ice for 2–5 min. Add 800 μL of LB liquid medium and incubate it on a shaker at 30℃ for 45 min. Spread the mixture onto a plate (Kan resistant, LB solid medium), invert it in a 37℃ incubator, and incubate overnight. After single bacteria have grown, pick them and transfer them to LB liquid medium (Kan resistant). After incubating for several hours, preserve the bacteria (final glycerol concentration 30%) to obtain the pCas-Sac / SLIS026 strain (capable of expressing Cas protein).

[0276] ③ Pick pCas-Sac transformants (i.e., pCas-Sac / SLIS026 strain) and inoculate them into LB sieve tubes (Kan resistant). Incubate the bacterial culture at 37°C until the OD value reaches 0.2. Then add arabinose to a final concentration of 2 g / L for induction (alternatively, arabinose can be added during single-colony culture). OD... 600 Competent cells were prepared at a concentration of 0.4, using the same method as operation ①, to obtain pCas-Sac / SLIS026 competent cells;

[0277] ④ The correctly constructed pTargetF-nagAB plasmid was transformed into pCas-Sac / SLIS026 competent cells using the heat shock method. After recovery, the cells were plated on LB agar plates. + spe + Place the plate in a 37°C incubator and invert it overnight until single colonies grow on the plate;

[0278] ⑤ Perform PCR verification on single colonies on resistant plates using the PCR verification primers in Table 7. Send the PCR products that verify the successful knockout of the nagAB gene for sequencing. See Table 7 for the sequencing verification primers.

[0279] ⑥ Based on the sequencing results in ⑤, select the strains that have successfully undergone knockout / knockin and culture them by shaking. Add rhamnose to a final concentration of 10mM (the amount of rhamnose can be increased appropriately to improve the plasmid elimination efficiency) and induce culture at 37℃ for 5-7h to lose the plasmid pTargetF-nagAB.

[0280] ⑦ Take the bacterial suspension obtained in ⑥ and streak it onto LB agar plates (kan). + Single colonies were obtained by rhamnose. PCR was performed using the verification primers in Table 7 to verify whether the pTargetF-nagAB plasmid was lost (or by streaking the plasmid onto an LB plate with Spec and incubating overnight to verify the elimination of the pTargetF-nagAB plasmid).

[0281] ⑧ Streak the strain that has undergone pTargetF-nagAB plasmid loss treatment onto a 10 g / L sucrose plate (the amount of sucrose can be increased appropriately to improve the plasmid elimination efficiency), incubate overnight at 37°C upside down, and verify whether the pCas-Sac plasmid has been lost by PCR according to the verification primers in Table 8 (or streak onto an LB plate with added Kan and incubate 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 glmS knock-in Donor-DNA fragments (donor DNA containing the gene to be inserted) are transformed together into the corresponding competent cells. The subsequent operations are the same as the gene knockout operation, and finally the SLIS027 strain is obtained.

[0283] Table 7 Primer list for gene knockout and knock-in verification in strain SLIS027

[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 of pTU2-A-DOE13 plasmid. The pTU2-A-DOE13 plasmid map is shown below. Figure 2 As shown, the pTU2-A-DOE13 plasmid carries transcription units for four genes: 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 given to Qingke Biotechnology for synthesis. Here, T2 represents the T2 promoter, and all four genes are 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 strains EBSL001 and EBSL002 were picked and inoculated into 5 mL LB resistant test tubes (containing 25 μg / mL chloramphenicol). After incubation at 37℃ and 250 rpm for 6-7 h, 2.5-5% of the colonies were inoculated into 100 mL TB resistant shake flasks (containing 25 μg / mL chloramphenicol) at an inoculation rate (determined based on the cell density in the seed culture medium to ensure a consistent number of cells per inoculation). The flasks were then incubated at 37℃ and 250 rpm until OD500. 600 The fermentation time was approximately 1-1.2 hours (about 4-5 hours). Then, IPTG (final concentration 0.1 mM), 1 mL of 200 g / L MgSO4·7H2O (final concentration 2 g / L), 0.1 mL of trace element stock solution (final concentration 0.1%), 2 mL of 500 g / L glucose (final concentration 10 g / L), and 2 mL of 250 g / L lactose (final concentration 5 g / L) were added to the 3'-SL-producing strain for fermentation culture. The culture conditions were 30°C and 250 rpm. Starting at fermentation time 0 hours, 2 mL of 500 g / L glucose (final concentration 10 g / L) was added every 12 hours to ensure a glucose concentration of at least 20 g / L within 24 hours. The total fermentation time was 48 hours. The 3'-SL content in the fermentation broth was then detected by HPLC. In this example, the final concentration refers to the initial concentration of each component after addition to the culture medium, i.e., the concentration at the beginning of fermentation.

[0291] The fermentation broth of strain EBSL001 was analyzed by HPLC, and the chromatogram of the results is shown below. Figure 3 As shown. By Figure 3 It can be seen that the peak position of 3'-SL standard detected by HPLC was 16.397 min, and the peak position of 3'-SL in the fermentation broth of EBSL001 strain was 16.438 min, indicating that 3'-SL was synthesized by fermentation.

[0292] The shake-flask fermentation results of strains EBSL001 and EBSL002 showed that the 3'-SL yield of strain EBSL001 was 3.1 g / L, while that of strain EBSL002 was 3.8 g / L, representing a 22.58% increase compared to strain EBSL001. This indicates that increasing the supply of glucosamine-6-phosphate (GlcN-6P) precursors by knocking out the nagAB and pfkA genes and integrating the glmS gene is beneficial for 3'-SL synthesis.

[0293] Experimental Example 1

[0294] In Example 1, only a partial segment of the pfkA gene was knocked out, not the entire gene. This is because the inventors knocked out the entire pfkA gene sequence (963 bp) during the experiment and fermented the resulting genetically engineered bacteria. The results showed that the strain exhibited poor growth and low passage efficiency, failing to meet the basic requirements for use as a chassis cell. The inventors believe this may be because the fieF gene, pfkA gene, and sbp gene are tightly tandemly arranged in the prokaryotic genome. Knocking out the 3' region of the pfkA gene affects the transcription and expression of other adjacent structural genes, thus impacting the growth and proliferation of the genetically engineered bacteria.

[0295] In this embodiment, the inventors knocked out only the upstream sequence of the pfkA gene, but retained a 76% (732 bp) 3' end sequence equivalent to the full-length pfkA gene. The resulting gene-functional bacteria were then fermented. Surprisingly, the inventors found that, compared to previously engineered bacteria with the entire gene segment knocked out, partial knockout of the pfkA gene did not affect the growth status or passage efficiency of the engineered bacteria.

[0296] The inventors also found in their research that if the 5' end of the pfkA gene is too long and the 3' end is too short when knocking out the middle sequence, it is difficult to select primers with high knockout efficiency and the workload is large. Therefore, taking all factors into consideration, when knocking out a partial fragment of the pfkA gene, the length of the 5' end sequence should be in the range of 0% to 7%, and the length of the 3' end sequence should be 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. The production of CMP-Neu5Ac from Neu5Ac requires the cofactor CTP. UTP is converted to CTP by CTP synthase, whose encoding gene is pyrG (accession number AAC75822.1). Insufficient CTP supply will affect the yield of 3'-SL. Therefore, this embodiment uses strain SLIS027 as the starting strain and overexpresses the cmk gene encoding cytidine monophosphate kinase (CMK) and the ppk gene encoding polyphosphokinase (PPK), thereby continuously regenerating CTP to solve the problem of insufficient CTP supply. Cofactor regeneration refers to the cycle from CTP→CMP→CDP→CTP. Cytidine triphosphate (CTP) is also known as cytidine triphosphate. Cytidine monophosphate (CMP) is also known as cytidine monophosphate. Cytidine diphosphate (CDP) is also known as cytidine diphosphate.

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

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

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

[0302] Table 9 Primer list for gene knockout and integration

[0303]

[0304]

[0305] Table 10 sgRNA sequence listing

[0306] Gene manipulation Operation method sgRNA sequence Serial 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 plasmids and Donor-DNA fragments:

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

[0309] Following the method described 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 Strain SLIS028

[0311] In this embodiment, the SLIS028 strain was modified from the SLIS027 strain. Compared with the SLIS027 strain, it integrates and overexpresses the cmk gene encoding cytidine monophosphate kinase and the pppk gene encoding polyphosphate kinase. That is, a part of the melB site sequence was knocked out and the pppk gene and cmk gene were knocked into the knocked-out position of the melB gene in the SLIS027 strain genome using a two-step knock-in method.

[0312] The two-step knock-in method refers to (1) using pTargetF-melB-NT1 and the ppk gene to knock in a linearized fragment of Donor-DNA via co-transformation to knock in the ppk gene, with the ppk gene using the T5 promoter (denoted as ΔmelB::T5-ppk); and (2) using pTargetF-melB-NT2 and cmk to knock in a linearized fragment of Donor-DNA via co-transformation to knock in the cmk gene, with the cmk gene using the tetracycline promoter tet (ΔmelB::tet-cmk) and the cmk gene inserted upstream of the ppk gene. See Section 1.4 of Example 1 for specific steps. The following examples are similar.

[0313] Through extensive experimental verification, the inventors discovered that if the knock-in ppk and cmk genes use the same T2 promoter as the four key genes, their expression will interact with the expression of the four key genes, which is detrimental to improving cofactor cycling efficiency and 2'-SL synthesis efficiency. Therefore, the knock-in ppk and cmk genes need to use promoters different from the T2 promoter. Furthermore, if the knock-in ppk and cmk genes use promoters stronger than the T2 promoter, their expression levels will also increase. However, in reality, since the ppk and cmk genes are related to the cofactor CTP cycle and not directly related to 3'-SL synthesis, a significant increase in their expression levels does not have a linear relationship with improving 3'-SL synthesis efficiency. Therefore, the expression of these two genes does not require regulation by strong promoters; using promoters weaker than the T2 promoter to maintain a basic transcriptional level is sufficient to drive the cofactor cycle. Furthermore, if the ppk and cmk genes use the same weak promoter to regulate their expression, the same weak promoter will competitively bind to the same type of RNA polymerase. This will also have a certain impact on the transcription of the ppk and cmk genes respectively. Therefore, the inventors chose different types of weak promoters to regulate the transcription of the ppk and cmk genes respectively. In this application, the ppk gene uses the T5 promoter to regulate its expression, and the cmk gene uses the tetracycline promoter tet to regulate its expression. However, in reality, the ppk gene can also be regulated using the tetracycline promoter tet, and the cmk gene can also be regulated using the T5 promoter.

[0314] In this application, "strong promoter" and "weak promoter" are relative concepts. A strong promoter refers to a promoter sequence that has a stronger ability to recognize and bind to RNA polymerase than a weak promoter, thereby resulting in higher transcription efficiency or transcription level of downstream structural genes linked to it. 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 *E. coli* genome that encodes a transport protein involved in the transport of melibiose, namely melibiose permease (MelB). Experiments have shown that knocking out the melB gene in the *E. coli* genome does not affect the normal growth, metabolism, or proliferation of *E. coli*. Therefore, in this embodiment, the ppk and cmk genes were knocked into the location where the melB gene was knocked out.

[0316] PCR detection and sequencing confirmed that the ppk and cmk genes were successfully knocked into SLIS027, resulting in strain SLIS028. The validation primers are shown in Table 11.

[0317] Table 11 Validation Primer List

[0318]

[0319] 2.4 Obtaining and fermenting fermentation strain EBSL003

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

[0321] The shake-flask fermentation results showed that the 3'-SL yield of strain EBSL001 was 3.0 g / L, that of strain EBSL002 was 3.8 g / L, and that of strain EBSL003 was 4.5 g / L, representing an 18.42% increase compared to strain EBSL002. Therefore, knocking ΔmelB::T5-ppk and ΔmelB::tet-cmk into the genome of strain EBSL002 increased the 3'-SL yield, indicating that optimizing the cofactor cycling system is beneficial for increasing CTP supply, thereby improving 3'-SL yield.

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

[0323] The neuB gene encodes sialic acid synthase, which catalyzes the reaction of ManNAc and phosphoenolpyruvic acid (PEP) to produce Neu5Ac and phosphate. Neu5Ac is one of the precursors for 3'-SL synthesis; therefore, increasing PEP supply is beneficial for increasing 3'-SL production. Furthermore, timely pumping of intracellularly produced 3'-SL out of the cell helps reduce the inhibitory effect of excessive intracellular 3'-SL accumulation on 3'-SL synthesis. Therefore, this example uses strain EBSL003 as the starting strain for pyruvate kinase gene pykA knockout treatment and oligosaccharide transporter gene setB enhanced expression treatment. After knocking out pyruvate kinase gene pykA, the PEP catabolism pathway is inhibited, promoting PEP accumulation within the cell. After enhanced expression of oligosaccharide transporter gene setB, intracellular 3'-SL can be promptly pumped out of the cell after production, helping to increase the extracellular concentration of 3'-SL.

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

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

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

[0327] Table 12 Primer list for gene knockout and integration

[0328]

[0329] Table 13 Sequence List of Gene Knockout and Integration sgRNAs

[0330] Gene manipulation Operation method sgRNA sequence Serial Number ΔpykA::tet-setB Delete and Replace 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 Plasmid Construction:

[0333] The pTargetF-pykA-NT2 plasmid was constructed according to the method described in Section 1.2 of Example 1. Overlap extension PCR was used to construct the Donor-DNA that knocks the setB gene into the pykA site.

[0334] 3.3 Construction of Strain SLIS029

[0335] In this embodiment, the SLIS029 strain was modified from the SLIS028 strain. Based on the SLIS028 strain, the pTargetF-pykA-NT2 plasmid and Donor-DNA knock-in of the pykA site of the setB gene were used to knock out a partial fragment of the pykA gene of pyruvate kinase and enhance the expression of the oligosaccharide transporter gene setB, so as to increase the supply of phosphoenolpyruvate (PEP) in SL synthesis and obtain the SLIS029 strain.

[0336] PCR detection and sequencing showed that a partial fragment of the pykA gene in strain SLIS028 had been successfully knocked out as designed, and the oligosaccharide transporter gene setB had been enhanced, resulting in strain SLIS029. The validation primers are shown in Table 13.

[0337] Table 14 Primer list for gene knock-in validation in strain SLIS029

[0338]

[0339] 3.4 Obtaining and fermenting fermentation strain EBSL004

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

[0341] The shake-flask fermentation results showed that the 3'-SL yield of strain EBSL001 was 3.0 g / L, strain EBSL002 was 3.7 g / L, strain EBSL003 was 4.5 g / L, and strain EBSL004 was 5.2 g / L. This indicates that the yield of strain EBSL004 was 15.56% higher than that of strain EBSL003 (4.5 g / L). This suggests that the partial knockout of the pykA gene and the knock-in of tet-setB (ΔpykA::tet-setB) are beneficial for 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] Experimental Example 2

[0346] In this embodiment 2, when knocking out the pykA gene, the middle region of the pykA gene is knocked out, but the two ends of the pykA gene are preserved. Therefore, this embodiment only knocks out a partial segment of the pykA gene, and not the entire gene segment.

[0347] The inventors previously knocked out the entire pykA gene sequence (1443 bp) and cultured the resulting functional bacteria. They found that the pykA gene knockout strains also exhibited poor growth and low passage efficiency, failing to meet the basic requirements for use as chassis cells.

[0348] In this embodiment, the inventors only knocked out the middle sequence of the pykA gene, but retained a 5' end sequence equivalent to 4.7% (68 bp) of the full-length pykA gene and a 3' end sequence equivalent to 12.1% (175 bp) of the full-length pykA gene. The resulting gene-functional bacteria were then cultured. Surprisingly, the inventors found that the partially knocked-out pykA gene engineered bacteria prepared in this embodiment exhibited good growth and high passage efficiency, meeting the basic requirements for use as chassis cells.

[0349] The inventors also found in their research that if the 5' end of the pykA gene is too long and the 3' end is too short when knocking out the middle sequence, it is difficult to select primers with high knockout efficiency and the workload is large. Therefore, taking all factors into consideration, when knocking out a partial fragment of the pykA gene, the length of the 5' end sequence should be in the range of 4% to 5.5%, and the length of the 3' end sequence should be 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 was present in EBSL001, EBSL002, EBSL003, and EBSL004. This plasmid is easily lost during subsequent passaging, leading to poor strain stability. Therefore, this embodiment employs CRISPR-associated transposase (MUCICAT) technology to insert the synthesized 3'-SL key genes css, neuB, neuC, and ST into the engineered bacterial genome.

[0352] The steps in 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 sequences are shown in Table 17.

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

[0356]

[0357]

[0358] Table 17 crRNA sequence information

[0359]

[0360] 4.2 Obtaining antibiotic-free, multi-copy, integrated EBSL005 strain using MUCICAT:

[0361] To obtain a plasmid-free and genetically stable integrative strain, gene combinations (css, neuB, neuC, and ST gene combinations) from the pTU2-A-DOE13 plasmid were integrated into the IS1 site of the SLIS029 strain using CRISPR-associated transposon multicopy genome integration technology (MUCICAT). The *E. coli* genome has 28 IS1 sites, each with a probability of inserting one or more css, neuB, neuC, and ST gene combinations. With each round of induction, different IS1 sites have a chance of inserting css, neuB, neuC, and ST gene combinations. As the number of induction rounds increases, the copy number of these gene combinations at different IS1 sites gradually increases. The inventors discovered in their research 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. However, 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; instead, it decreases to a certain extent. The number of other IS sequences (such as IS186, IS3, and IS4) in E. coli cells is relatively small and cannot meet the copy number requirements after structural gene insertion. Therefore, IS1 was chosen as the insertion site for the structural gene.

[0362] The MUCICAT technique requires multiple rounds of induction of strain SLIS029. After each round of induction, several transformants are randomly selected for fermentation screening (primary screening) in 96-well plates to identify transformants with high 3'-SL content. During transformant selection, only a portion of the colony is picked from each selected transformant, with the amount sufficient for primary screening. After each round, the remaining transformed transformants are mixed with the unselected transformants for the next round of induction. This process is repeated for the multiple transformants obtained after the next round of induction. Transformants selected after each round of induction undergo plasmid loss treatment before shake-flask fermentation, while unselected transformants do not require plasmid loss treatment.

[0363] The initial screening steps were as follows: transformants selected after multiple rounds of induction were added to 96-well plates containing TB liquid medium (600 μL) for IPTG-induced fermentation screening for 48 h. The supernatant of the fermentation broth was then collected, and the 3'-SL content in the supernatant was determined by HPLC. Finally, 36 transformants with high 3'-SL content were selected, and then subjected to shake-flask fermentation (secondary screening).

[0364] The secondary screening (shake-flask fermentation) steps were as follows: rhamnose was used to lose the pTet-tns, pQCascade-IS1, and pRE57-DOE13 plasmids, and sucrose was used to lose the pCutamp plasmid, ultimately obtaining multiple plasmid-free multi-copy strains. These plasmid-free multi-copy strains were transferred to shake flasks containing TB liquid medium, and IPTG was used to induce fermentation for 48 h. The fermentation broth was then collected, centrifuged, and the supernatant was taken. The 3'-SL content in the supernatant was determined by HPLC.

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

[0366] Table 18 lists the 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 the yield of 3'-SL increases with the increase of copy number, reaching its highest level when the copy number reaches 16. After that, the yield of 3'-SL no longer increases with further increases in copy number. We named strain 35, with a copy number of 16, strain EBSL005 (also known as strain SLIS109).

[0369] Table 19 Genetic 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 Novizan Biotechnology Co., Ltd., catalog number: C112-01), the crRNA sequence corresponding to the target gene was seamlessly cloned into the pRE57-Ter plasmid to prepare different pRE57-DOE13 (which were subsequently used with pQCascade-IS1). The crRNA sequences used in the gene insertion process are shown in Table 17.

[0373] (2) 300 ng pDonor (pRE57-Ter or other variants), 300 ng pQCascade, and 300 ng pTnsABC (pTet-tns or other variants) were co-transformed into Escherichia coli BL21(DE3) cells by electroporation. After recovering in fresh LB liquid medium at 37°C for 2 hours, E. coli BL21(DE3) cells were seeded on LB agar plates containing triple antibiotics: 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, several dozen colonies were scraped from the culture dish, and some were resuspended in fresh LB liquid medium. The resuspension containing these colony cells was then transferred to LB agar plates containing three antibiotics and 100 ng / mL to induce transposon-related protein expression, and cultured at 30°C for another 16 hours. After biofilm formation, the cells were scraped off and resuspended in LB liquid medium. The cells were appropriately redissolved and placed on triple antibiotic LB agar plates (i.e., induction plates) containing 100 ng / mL tetracycline (aTc), and cultured overnight at 37°C.

[0375] (4) To improve clonal growth and integration rate, colonies are transferred by re-division as follows. Colonies grown on the induction plate are defined as Transfer1, a portion of which are transferred to LB agar plates containing a novel triple antibiotic containing aTc and grown overnight at 30°C to generate a new transfer, i.e., Transfer2. Continuous transfers will generate transfers n (n = 3, 4...).

[0376] (5) Then colony PCR was performed to identify whether the combination of the four genes was correctly inserted into the cell genome, so as to obtain cells in which the four genes were correctly inserted.

[0377] (6) Electroporate the pCutamp plasmid (used to eliminate the three plasmids) into cells that have been correctly inserted with the four genes (still carrying the three plasmids mentioned above), and incubate them in 1 mL LB medium at 37°C for 1 hour before recovery. To improve the plasmid elimination efficiency, transfer the recovered cells to 4 mL LB medium containing 50 μg / mL apramycin and 10 mM rhamnose and culture them at 37°C with shaking for 7-10 hours (the induction time can be appropriately extended to further improve the elimination efficiency of the three plasmids). Then, place 100 μL of culture on LB agar plates containing 50 μg / mL apramycin (Apr) and 10 mM rhamnose and culture overnight at 37°C.

[0378] (7) The pCutamp plasmid also contains the SacB gene, which encodes L-glucosidase, an enzyme 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 grew on LB agar plates containing apramycin and rhamnose were selected and replaced on four different LB agar plates containing 100 μg / mL ampicillin, 50 μg / mL kanamycin, 50 μg / mL spectinomycin, and 10 g / L sucrose, respectively. Colonies that could not grow on LB agar plates containing ampicillin, kanamycin, and streptomycin were considered to have eliminated the pDonor, pTnsABC, and pQCascade plasmids. The corresponding colonies that grew on sucrose-containing LB agar plates were then re-placed on non-selective LB agar plates and LB agar plates containing apramycin to verify the digestion of the pCutamp plasmid. Colonies that cannot grow on LB agar plates containing apramycin are plasmid-free multicopy integrated strains.

[0379] As shown in Table 19, the four gene combinations of strain SLIS109 each have their own promoters and terminators, thus forming four separate transcription units.

[0380] 4.3 Fermentation of antibiotic-free multicopy integrated EBSL005 strain

[0381] The EBSL005 strain (see Table 18) was subjected to shake-flask fermentation. 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 a significant increase compared to the 3'-SL yield of the EBSL004 strain. Figure 5 This is a graph showing the growth and yield of strain EBSL005 during shake-flask fermentation.

[0382] Furthermore, the EBSL005 strain was scaled up for fermentation in a 5L fermenter. The scaled-up fermentation conditions in the 5L fermenter are as follows:

[0383] Single colonies of strain EBSL005 were picked and inoculated into 5 mL of LB medium. After incubation at 37°C and 250 rpm for 6-7 hours, 2.5-5% of the culture medium (the specific inoculation amount was determined based on the cell density in the seed culture medium to ensure a consistent number of cells per inoculation) were transferred to a 5 L fermenter and incubated at 37°C and 250 rpm until OD500 was reached. 600The concentration was approximately 1-1.2. Then, IPTG (final concentration 0.1 mM), 200 g / L MgSO4·7H2O (final concentration 2 g / L), trace element stock solution (final concentration 0.1%), 500 g / L glucose (final concentration 10 g / L), and 250 g / L lactose (final concentration 5 g / L) were added to the 3'-SL-producing strain for fermentation culture. The culture conditions were 30℃ and 250 rpm. Fermentation was started at 0 h, and 500 g / L glucose (final concentration 10 g / L) was added every 12 h. The total fermentation time was 52 h. The 3'-SL content in the fermentation broth was then detected by HPLC. After fermentation, the 3'-SL yield of strain EBSL005 reached 79 g / L.

[0384] The results of this embodiment show that strain EBSL005 was obtained by repeatedly integrating the css-neuB-neuC-ST gene combination into the genome of the strain using CRISPR-associated transposon multiple copy genome integration technology (MUCICAT). Compared with strains carrying plasmids containing the css-neuB-neuC-ST gene combination (strains EBSL001 to EBSL004), strain EBSL005 has a higher 3'-SL yield. Because EBSL005 is an integrative strain, there is no loss of plasmids carrying the css-neuB-neuC-ST gene combination during fermentation, resulting in higher genetic stability.

[0385] In summary, strain EBSL005 achieved very high levels of synthesis of 3'-SL in both shake-flask fermentation and 5L fermenter fermentation.

[0386] While the invention has been described with reference to specific embodiments thereof, those skilled in the art will understand that various changes can be made and equivalents can be substituted without departing from the spirit and scope of the invention. Furthermore, numerous modifications can be made to adapt the specific circumstances, materials, compositions, methods, or one or more method steps to the purpose, spirit, and scope of the invention. All such modifications are intended 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 capable of producing 3'-sialic acid lactose and possessing the UDP-N-acetylglucosamine-2-epomerase gene (neuC), sialic acid synthase gene (neuB), N-acetylneuraminic acid cytidine transferase gene (css) and α-2,3-sialic acid transferase gene (ST). in, The UDP-N-acetylglucosamine-2-epomerase gene (neuC), sialic acid synthase gene (neuB), N-acetylneuraminic acid cytidine transferase gene (css), and α-2,3-sialic acid transferase gene (ST) are integrated into the genome of the genetically engineered bacteria as a gene combination, each linked to a separate first promoter and terminator. The copy number of the UDP-N-acetylglucosamine-2-epomerase gene (neuC), sialic acid synthase gene (neuB), N-acetylneuraminic acid cytidine transferase gene (css) and α-2,3-sialic acid transferase gene (ST) as a gene combination at the insertion site is 10-20. The amino acid sequence of UDP-N-acetylglucosamine-2-epomerase is shown in SEQ ID NO: 82, the amino acid sequence of sialic acid synthase is shown in SEQ ID NO: 83, the amino acid sequence of N-acetylneuraminic acid cytidine transferase is shown in SEQ ID NO: 84, and the amino acid sequence of α-2,3-sialic acid transferase is shown in SEQ ID NO: 85; and the genetically engineered bacterium is Escherichia coli. Among them, the genetically engineered bacteria lacked the following genes: β-galactosidase (lacZ), N-acetylneuraminic acid aldolase (nanA), N-acetylmannosamine kinase (nanK), N-acetylmannosamine-6-phosphate epimerase (nanE), N-acetylglucosamine-6-phosphate deacetylase (nagA), and glucosamine-6-phosphate deaminase (nagB). Specifically, a partial nucleotide sequence knockout of the 6-phosphofructokinase (pfkA) gene was performed; under the condition of partial nucleotide sequence knockout of the 6-phosphofructokinase (pfkA) gene, the glucosamine-6-phosphate synthase (glmS) gene was inserted at the knockout site of the 6-phosphofructokinase (pfkA) gene; the amino acid sequence of glucosamine-6-phosphate synthase (glmS) is the amino acid sequence shown in SEQ ID NO: 89; and In this study, the genetically engineered bacteria lacked the melbiose permease (melB) gene, and the cytidine monophosphate kinase (CMK) and polyphosphate kinase (PPK) genes were inserted at the knockout site of the melbiose permease (melB) gene. The amino acid sequence of the cytidine monophosphate kinase (CMK) is shown in SEQ ID NO: 91; the amino acid sequence of the polyphosphate kinase (PPK) is shown in SEQ ID NO: 90; the cytidine monophosphate kinase (CMK) and polyphosphate kinase (PPK) genes were inserted at the knockout site of the melbiose permease (melB) gene, each linked to a separate second promoter. In this case, the genetically engineered bacteria lacked the pyruvate kinase (pykA) gene. Under the condition of knocking out part of the nucleotide sequence of the pyruvate kinase (pykA) gene, the oligosaccharide transporter setB gene was inserted into the knocked-out position of the pyruvate kinase (pykA) gene. The amino acid sequence of the oligosaccharide transporter setB is the amino acid sequence shown in SEQ ID NO:

93. Partial nucleotide sequence knockout of the pyruvate kinase (pykA) gene includes: retaining 4%-5.5% of the total nucleotide sequence length at the 5' end and 10%-20% of the total nucleotide sequence length at the 3' end. The partial nucleotide sequence knockout of the 6-phosphofructokinase (pfkA) gene includes: retaining 0%-7% of the total nucleotide sequence length of the 6-phosphofructokinase (pfkA) gene at the 5' end, and retaining 70%-80% of the total nucleotide sequence length of the 6-phosphofructokinase (pfkA) gene at the 3' end.

2. The genetically engineered bacteria according to claim 1, wherein, The genetically engineered bacteria is Escherichia coli BL21(DE3) strain.

3. The genetically engineered bacteria according to claim 1 or 2, wherein, The first promoter is the T2 promoter; the terminator is the T7 terminator.

4. The genetically engineered bacteria according to claim 1 or 2, wherein, The second promoter is either the T5 promoter or the tetracycline promoter tet.

5. A strain of Escherichia coli, with accession number CGMCC No.:28244.

6. The use of a genetically engineered bacterium as described in any one of claims 1 to 4 or an Escherichia coli strain as described in claim 5 in the production of 3'-sialic acid lactose using glucose or glycerol as a carbon source.

7. A method for constructing a genetically engineered bacterium with the ability to produce 3'-sialic acid lactose according to any one of claims 1-4, comprising: The UDP-N-acetylglucosamine-2-epimerase gene (neuC), sialic acid synthase gene (neuB), N-acetylneuraminic acid cytidine transferase gene (css), and α-2,3-sialic acid transferase gene (ST) were integrated into the genome of the host bacteria to obtain genetically engineered bacteria.

8. The method according to claim 7, wherein, The nagA and nagB genes were knocked out using pTargetF-nagAB containing homologous arms 1 and 2. Homologous arm 1 was amplified using primers SEQ ID NO: 3 and SEQ ID NO: 4 with the host bacterial genome as a template, and homologous arm 2 was amplified using primers 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 homologous arms 3 and 4 and a pTargetF-pfkA-NT1 recombinant plasmid, and the glmS gene was knocked into the host bacterial genome at the knockout position of the pfkA gene. Homologous 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 homologous 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 was knocked into the host bacterial genome using the ppk knock-in Donor-DNA and pTargetF-melB-NT1 recombinant plasmids containing homologous arms 5 and 6. Homologous arm 5 was amplified using primers SEQ ID NO: 35 and SEQ ID NO: 36 with the host bacterial genome as a template, and homologous arm 6 was amplified using primers 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 the cmk knock-in Donor-DNA and pTargetF-melB-NT2 recombinant plasmid containing homologous arms 7 and 8. Homologous arm 7 was amplified using the host bacterial genome as a template using primers SEQ ID NO: 43 and SEQ ID NO: 44, and homologous arm 8 was amplified using the host bacterial genome as a template using primers SEQ ID NO: 49 and SEQ ID NO:

50. and / or The pykA gene was knocked out using the setB knock-in pykA site Donor-DNA and pTargetF-pykA-NT2 recombinant plasmids containing homologous arms 9 and 10, and the setB gene was knocked into the pykA gene knocked out at the knocked-out position in the host bacterial genome. Homologous 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 homologous arm 10 was amplified using primers of SEQ ID NO: 64 and SEQ ID NO: 65 with the host bacterial genome as a template.

9. A method for producing 3'-sialic acid lactose by fermentation, the method comprising the following steps: Seeds are obtained by culturing the genetically engineered bacteria of any one of claims 1-4 or the Escherichia coli strain of claim 5 in a seed culture medium; The seeds were inoculated into a fermentation medium for induction culture, and after fermentation, a fermentation broth containing 3'-sialic acid lactose was obtained.

10. The method according to claim 9, wherein, The fermentation medium does not contain antibiotics; and / or The seed culture medium is LB medium; and / or The fermentation medium comprises TB medium and a carbon source, wherein the carbon source includes glucose and / or glycerol.

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