Recombinant escherichia coli for producing lactose-N-trisaccharide and double-bacterium coupling fermentation method based on recombinant escherichia coli

Abstract

The invention relates to recombinant escherichia coli for producing lactose-N-trisaccharide and a double-bacterium coupling fermentation method based on the recombinant escherichia coli, and belongs to the technical field of biology. The recombinant escherichia coli for producing the lactose-N-trisaccharide is used for expressing three genes, namely, an N-acetyl hexosamine 1-kinase gene Nahk, a uridine diphosphate-N-acetyl glucosamine pyrophosphorylase gene GlmU and a beta-1, 3-N-acetyl glucosamine aminotransferase gene LgtA, so that the lactose-N-trisaccharide can be obtained; the method for producing the lactose-N-trisaccharide through coupling catalysis of the recombinant escherichia coli and the yeast is a two-stage fermentation process, engineering escherichia coli is cultured in the first stage, the yeast and the recombinant escherichia coli are coupled and fermented in the second stage, and the yield on a 5L fermentation tank is 52.2 g / L. The fermentation production process disclosed by the invention is relatively simple, relatively high in conversion rate and good in industrialization prospect.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a recombinant Escherichia coli for producing lactose-N-triose and a dual-bacteria coupled fermentation method based thereon. Background Art

[0002] Lactose-N-triose (LNT II) is the core sugar in the human milk oligosaccharide group. LNT II (GlcNAcβ1-3Galβ1-4Glc) is generated by GlcNAc and lactose through a β-1,3-glycosidic bond. It is an important backbone precursor for the synthesis of higher-order human milk oligosaccharides such as LNnT and LNT. It can be used as a precursor substance for the production of many linear or branched neutral oligosaccharides in human milk oligosaccharides. In addition to LNT II having a variety of physiological activities, it plays an important role in aspects including immunomodulation, anti-inflammation and activation of natural killer cells, showing the great potential of LNT II as a human milk oligosaccharide component in functional food ingredients. In summary, LNT II has become a research and development hotspot for scientific researchers due to its important role in physiological efficacy, application prospects in functional foods and as a precursor substance for the synthesis of complex HMOs.

[0003] With the rapid development of the infant formula industry in recent years, large-scale and efficient LNT II synthesis methods have become the focus of relevant researchers. Previously, the method of manufacturing this type of compound was to directly separate natural LNT II from breast milk by means of gel chromatography, reverse HPLC, solid phase extraction, etc. However, due to the complex composition of breast milk and the low abundance of natural LNT II, ​​not only is the operation troublesome, but the prepared oligosaccharides are of low purity and low yield, and often exist in the form of mixtures. Most importantly, breast milk is scarce and expensive. Therefore, it is still a difficult challenge to use separation and purification methods to mass produce LNT II. A large number of researchers have focused on direct synthesis methods. Technologies developed in recent years include enzymatic synthesis and microbial cell factory synthesis.

[0004] The core technology of direct synthesis of LNT II is the expression of β-1,3-N-acetylglucosamine transferase. In various domestic and foreign studies, the synthesis of LNT II was achieved by expressing this enzyme. For example, Hu et al. constructed the lgtA gene into Escherichia coli Nissle 1917 and obtained an LNT II yield of 2.04 g / L in a 3L fermenter. In 2023, China's invention patent CN116590325A disclosed the construction and application of an Escherichia coli engineered strain for producing lactose-N-triose, which mainly used gene editing technology to construct a cell factory. The yield on a 5L fermenter with glucose and lactose as substrates was about 20 g / L. Similarly, the current LNT II biosynthesis technology is mainly carried out in the form of constructing a cell factory. Its transformation steps are cumbersome and the reaction path is long, which limits its large-scale application to a certain extent. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a recombinant Escherichia coli for producing lactose-N-triose and a dual-bacteria coupled fermentation method based thereon, which is a simpler lactose-N-triose biosynthesis strategy, using the equally cheap N-acetylglucosamine as a substrate, expressing the three enzymes required for synthesizing lactose-N-triose in the chassis strain Escherichia coli, and then coupling it with yeast cells for fermentation to achieve the bioconversion of lactose-N-triose.

[0006] The present invention selects three enzymes, namely Nahk (N-acetylhexosamine 1-kinase) from Bifidobacterium breve, EcGlmU (fused N-acetylglucosamine-1-phosphateuridyltransferase and glucosamine-1-phosphate acetyltransferase) from Escherichia coli, and LgtA (lacto-N-neotetraose biosynthesis glycosyl tranferase) from Neisseria meningitidis, integrates the genes expressing the three enzymes into Escherichia coli JM109 (DE3) for expression, and produces lactose-N-triose through coupling catalysis of Escherichia coli and yeast, with an output of 52.19 g / L in a 5L fermentation tank. The present invention has a relatively simple synthesis route, a high conversion rate, and good industrialization prospects.

[0007] The present invention is achieved through the following technical solutions:

[0008] The first object of the present invention is to provide a recombinant Escherichia coli for producing lactose-N-triose, wherein the recombinant Escherichia coli uses Escherichia coli JM109 (DE3) as a starting strain and expresses the N-acetylglucosamine 1-kinase gene Nahk, the uridine diphosphate-N-acetylglucosamine pyrophosphorylase gene EcGlmU and the β-1,3-N-acetylglucosamine aminotransferase gene LgtA.

[0009] In one embodiment of the present invention, the host bacteria of the recombinant Escherichia coli can be a common model strain or a modified strain, including Escherichia coli JM109 (DE3), BL21 (DE3) or EcN03 (GDMCC 63722) and the like.

[0010] In one embodiment of the present invention, the recombinant Escherichia coli is any one of the following combinations:

[0011] (1) The recombinant Escherichia coli uses pRSFDuet-1 plasmid or pETDuet-1 plasmid to express N-acetylglucosamine 1-kinase gene Nahk, uridine diphosphate-N-acetylglucosamine pyrophosphorylase gene EcGlmU and β-1,3-N-acetylglucosamine aminotransferase gene LgtA; the N-acetylglucosamine 1-kinase gene Nahk and the β-1,3-N-acetylglucosamine aminotransferase gene LgtA are connected by a flexible protein Linker;

[0012] (2) the recombinant Escherichia coli uses the pRSFDuet-1 plasmid to express the β-1,3-N-acetylglucosamine aminotransferase gene LgtA and the uridine diphosphate-N-acetylglucosamine pyrophosphorylase gene EcGlmU located after LgtA, and uses the pETDuet-1 plasmid to express the N-acetylhexosamine 1-kinase gene Nahk;

[0013] (3) the recombinant Escherichia coli uses the pRSFDuet-1 plasmid to express the uridine diphosphate-N-acetylglucosamine pyrophosphorylase gene EcGlmU, and uses the pETDuet-1 plasmid to express the β-1,3-N-acetylglucosamine aminotransferase gene LgtA and the N-acetylhexosamine 1-kinase gene Nahk located after LgtA;

[0014] (4) the recombinant Escherichia coli uses the pRSFDuet-1 plasmid to express the β-1,3-N-acetylglucosamine aminotransferase gene LgtA and the N-acetylhexosamine 1-kinase gene Nahk located after LgtA, and uses the pETDuet-1 plasmid to express the uridine diphosphate-N-acetylglucosamine pyrophosphorylase gene EcGlmU;

[0015] (5) The recombinant Escherichia coli uses the pRSFDuet-1 plasmid to express the N-acetylglucosamine 1-kinase gene Nahk, and uses the pETDuet-1 plasmid to express the β-1,3-N-acetylglucosamine aminotransferase gene LgtA and the uridine diphosphate-N-acetylglucosamine pyrophosphorylase gene EcGlmU located after LgtA;

[0016] (6) The recombinant Escherichia coli uses the pRSFDuet-1 plasmid to express the β-1,3-N-acetylglucosamine aminotransferase gene LgtA, and uses the pETDuet-1 plasmid to express the N-acetylhexosamine 1-kinase gene Nahk and the uridine diphosphate-N-acetylglucosamine pyrophosphorylase gene EcGlmU located after Nahk;

[0017] (7) The recombinant Escherichia coli uses the pRSFDuet-1 plasmid to express the N-acetylglucosamine 1-kinase gene Nahk and the uridine diphosphate-N-acetylglucosamine pyrophosphorylase gene EcGlmU located after Nahk, and uses the pETDuet-1 plasmid to express the β-1,3-N-acetylglucosamine aminotransferase gene LgtA.

[0018] In one embodiment of the present invention, the nucleotide sequence of the gene Nahk is shown as SEQ ID NO.5, the nucleotide sequence of the gene EcGlmU is shown as SEQ ID NO.4, and the nucleotide sequence of the gene LgtA is shown as SEQ ID NO.6.

[0019] In one embodiment of the present invention, the sequence of the flexible protein Linker is GGGGS)3.

[0020] In one embodiment of the present invention, at least one of the promoters of the N-acetylglucosamine 1-kinase gene Nahk, the uridine diphosphate-N-acetylglucosamine pyrophosphorylase gene EcGlmU and the β-1,3-N-acetylglucosamine aminotransferase gene LgtA in the recombinant Escherichia coli is the strong promoter T7.

[0021] In one embodiment of the present invention, the N-acetylhexosamine 1-kinase is derived from Bifidobacterium breve;

[0022] And / or, the UDP-N-acetylglucosamine pyrophosphorylase is derived from Escherichia colistr.K-12substr.MG1655;

[0023] And / or, the β-1,3-N-acetylglucosamine aminotransferase is derived from Neisseria meningitidis.

[0024] The second object of the present invention is to provide a dual-bacteria coupling system, which includes the recombinant Escherichia coli and yeast.

[0025] In one embodiment of the present invention, the yeast includes brewer's yeast and / or baker's yeast.

[0026] In one embodiment of the present invention, the amount of the recombinant Escherichia coli used is 10 g / L-200 g / L;

[0027] And / or, the usage amount of the yeast is 10g / L-200g / L.

[0028] The third object of the present invention is to provide the use of the recombinant Escherichia coli or the dual-bacteria coupling system in synthesizing lactose-N-trisaccharide.

[0029] The fourth object of the present invention is to provide a method for synthesizing lactose-N-trisaccharide, using N-acetylglucosamine and lactose as substrates and using the recombinant Escherichia coli or the dual-bacteria coupling system to catalyze the synthesis of lactose-N-trisaccharide.

[0030] In one embodiment of the present invention, the dual-bacteria coupling system catalyzes the synthesis of lactose-N-trisaccharide, including: a recombinant Escherichia coli fermentation stage, and a recombinant Escherichia coli and yeast co-fermentation stage.

[0031] In one embodiment of the present invention, the catalytic synthesis system also includes one or more of fructose, adenosine triphosphate disodium salt, uridine triphosphate trisodium salt, Triton X-100, inorganic salts and buffer; the reaction system is a phosphate buffer or a tris(hydroxymethyl)aminomethane hydrochloride buffer.

[0032] In one embodiment of the present invention, one or more of the following conditions are met:

[0033] 1) The recombinant Escherichia coli is induced to express using isopropyl-β-D-thiogalactoside;

[0034] 2) The catalytic synthesis temperature is 15°C-42°C and the time is 4h-30h;

[0035] 3) The pH value of the catalytic synthesis system is 6.0-9.0.

[0036] The above technical solution of the present invention has the following advantages compared with the prior art:

[0037] The present invention provides a recombinant Escherichia coli for producing lactose-N-triose and a dual-bacteria coupled fermentation method based thereon. With Escherichia coli as the base strain, a recombinant strain with the ability to synthesize lactose-N-triose is constructed, and the N-acetylglucosamine-1-kinase gene, uridine diphosphate-N-acetylglucosamine pyrophosphorylase gene and β-1,3-N-acetylglucosamine aminotransferase gene required for the lactose-N-triose synthesis pathway are integrated into the recombinant strain, which can synthesize lactose-N-triose with N-acetylglucosamine and lactose as substrates. In order to further increase the yield, a method for synthesizing lactose-N-triose based on the coupled catalysis of Escherichia coli cells and yeast cells is developed, which can effectively solve the problem of energy material regeneration in the catalytic synthesis process. The enzyme selected by the present invention has the advantages of high activity and strong substrate recognition ability. The cell-coupled catalysis method developed by the present invention has the characteristics of high yield, high conversion rate and simple route, and has good industrialization prospects.

[0038] Biomaterial Deposit

[0039] Saccharomyces cerevisiae xpli was deposited in the Guangdong Provincial Microbiological Culture Collection Center on May 12, 2021, with the deposit number GDMCC No: 61663, the taxonomic name: Saccharomyces cerevisiae xpli, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0040] Escherichia coli EcN 03 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 7, 2023, with the deposit number GDMCC No: 63722, the taxonomic name: Escherichia coli, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0042] Figure 1 The LNT II synthesis technology route provided by the present invention;

[0043] Figure 2The protein expression and UDP-N-acetylglucosamine synthesis of each strain detected in Example 1 of the present invention; wherein, (A) is the protein detection result of EJM109A strain; (B) is the protein detection result of EJM109ECG strain; (C) is the protein detection result of EJM109PMG strain; (D) is the protein detection result of EJM109CJG strain; (E) is the yield result of UDP-N-acetylglucosamine pyrophosphorylase catalyzing the synthesis of UDP-N-acetylglucosamine;

[0044] Figure 3 The protein expression and lactose-N-triose synthesis of each strain detected in Example 2 of the present invention; wherein, (A) EJM109BG strain protein detection result; (B) EJM109MG strain protein detection result; (C) EJM109LA strain protein detection result; (D) is a Tlc analysis chart (1: EJM109BG strain catalytic synthesis product; 2: EJM109MG strain catalytic synthesis product; 3: EJM109LA strain catalytic synthesis product; 4: LNTII standard, 5: lactose standard;

[0045] Figure 4 Schematic diagram of plasmid and gene combination of co-expression strains in Example 3 of the present invention and yield diagram of corresponding strains: (A) schematic diagram of plasmids and corresponding gene combinations of recombinant E. coli numbered E01-E08; (B) comparative diagram of lactose-N-triose synthesis of recombinant E. coli E01-E08, wherein: M indicates that the gene is expressed in medium copy number, and H indicates that the gene is expressed in high copy number;

[0046] Figure 5 The present invention provides a comparison of the yield of lactose-N-triose or a graph showing a trend of yield change in the experiment of exploring different reaction conditions such as pH, buffer, surfactant, temperature, and reaction time of the catalytic reaction in Example 4 of the present invention: (A) a graph showing a comparison of the yield of lactose-N-triose under different pH conditions; (B) a graph showing a comparison of the yield of lactose-N-triose under different buffer addition amounts; (C) a graph showing a comparison of the yield of lactose-N-triose under different surfactant addition amounts; (D) a graph showing a comparison of the yield of lactose-N-triose under different temperature conditions; (E) a graph showing a trend of the yield of lactose-N-triose changing with catalytic time;

[0047] Figure 6The diagram is a comparison of the yield of lactose-N-triose in the experiment of exploring different reaction conditions such as the amount of metal ion addition, the amount of cell addition, the induction scheme of recombinant Escherichia coli, and the amount of substrate addition in Example 4 of the present invention: (A) a comparison of the yield of lactose-N-triose under different metal ion addition combinations; (B) a comparison of the yield of lactose-N-triose under different cell addition combinations; (C) a comparison of the yield of lactose-N-triose of recombinant Escherichia coli using different induction schemes; (D) a comparison of the yield of lactose-N-triose under different substrate addition combinations;

[0048] Figure 7 Figure 5 shows the growth of recombinant E. coli E07 in a 5L fermenter and the condition change diagram and the yield change diagram of lactose-N-triose produced by the two-step method in a 5L fermenter in Example 5 of the present invention: (A) the biomass (OD 600 ) Changes over time, glucose feeding rate in each time period and detection of residual glucose in the fermenter at different times (B) Trend chart of the product lactose-N-triose and substrate N-acetylglucosamine changing over time in a 5L fermenter. DETAILED DESCRIPTION

[0049] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0050] The first technical solution of the present invention is to screen the enzymes of the designed lactose-N-triose synthesis pathway.

[0051] In the design of the present invention, N-acetylglucosamine (GlcNAc) is first used as a substrate to synthesize 1-phosphate-N-acetylglucosamine (GlcNAc-1-P), then GlcNAc-1-P is used as a substrate to synthesize uridine diphosphate-N-acetylglucose (UDP-GlcNAc), and finally UDP-GlcNAc and lactose are used as substrates to synthesize lactose-N-triose, and the process requires N-acetyhexose-1-kinase, uridine diphosphate-N-acetylglucosamine pyrophosphorylase, and β-1,3-N-acetylglucosamine aminotransferase, respectively.

[0052] The enzyme catalyzing the production of 1-phospho-N-acetylglucosamine (GlcNAc-1-P) is N-acetylhexosamine 1-kinase derived from Bifidobacterium breve; preferably, the NCBI accession number of the N-acetylhexosamine 1-kinase is WP_003829953.1;

[0053] The enzyme catalyzing the generation of uridine diphosphate-N-acetylglucose (UDP-GlcNAc) is uridine diphosphate-N-acetylglucosamine pyrophosphorylase; the corresponding expression gene is derived from humans (Homo sapiens), Pasteurella multocida, Campylobacter jejuni or Escherichia coli (Escherichia coli) K-12MG1655; the NCBI accession numbers of the enzyme are NP_001311042.1, WP_005758006.1, YP_002344229.1, and NP_418186.1, respectively; preferably, the uridine diphosphate-N-acetylglucosamine pyrophosphorylase is derived from Escherichia coli (Escherichia coli) K-12MG1655, and the NCBI accession number is NP_418186.1.

[0054] The enzyme catalyzing the production of lactose-N-triose (LNT II) is β-1,3-N-acetylglucosamine aminotransferase; it is derived from humans (Homo sapiens), Helicobacter pylori (Helicobacterpylori) or Neisseria meningitidis (Neisseria meningitidis); the NCBI accession numbers of the enzymes are NP_001306004.1, WP_000666599.1, and WP_002233115.1, respectively; preferably, the β-1,3-N-acetylglucosamine aminotransferase is derived from Neisseria meningitidis (Neisseria meningitidis); the NCBI accession number is WP_002233115.1.

[0055] The second technical solution of the present invention is to construct an engineered strain of Escherichia coli capable of synthesizing lactose-N-triose, wherein the recombinant bacteria are obtained by expressing N-acetylglucosamine 1-kinase gene, uridine diphosphate-N-acetylglucosamine pyrophosphorylase gene, and β-1,3-N-acetylglucosamine aminotransferase gene in host cells.

[0056] Preferably, the host cell is Escherichia coli JM109 (DE3), BL21 (DE3) or EcN 03.

[0057] In a specific embodiment, the genes are codon-optimized and then fully synthesized by Wuxi Tianlin Biotechnology Co., Ltd. The three genes are connected to an expression vector, and the obtained recombinant expression vector is then introduced into a host cell to obtain a recombinant bacterium.

[0058] Preferably, the expression vector includes pET28a plasmid, pRSFDuet-1 plasmid and pETDuet-1 plasmid.

[0059] Preferably, two or three of the genes are connected by a flexible protein Linker (GGGGS)3

[0060] The third technical solution of the present invention is the coupled fermentation of recombinant Escherichia coli and yeast cells, which is characterized in that the above-mentioned recombinant Escherichia coli and yeast cell dry powder preparation are used as whole-cell catalysts and added to a reaction system containing N-acetylglucosamine and lactose to produce lactose-N-trisaccharide.

[0061] Preferably, the yeast cell is Saccharomyces cerevisiae and / or baker's yeast.

[0062] Preferably, the recombinant bacteria need to be induced to culture, and the induction culture process is: inoculate the recombinant bacteria into LB medium containing 20 μg / mL-60 μg / mL kanamycin and / or 20 μg / mL-60 μg / mL ampicillin, obtain seed liquid after culture, and then inoculate the seed liquid into LB medium containing the same antibiotics until the bacterial concentration OD 600 When nm reaches 0.6-1.0, inducer is added, and wet bacteria are obtained by separation and washing after induction.

[0063] Preferably, the culture conditions of the above-mentioned recombinant bacteria are: temperature 30°C-38°C, rotation speed 150rpm-250rpm, and culture time 10h-16h.

[0064] Preferably, the induction conditions are as follows: inducer 0.2 mM-0.6 mM isopropyl-β-D-thiogalactoside (IPTG), temperature 16° C.-37° C., time 4 h-24 h.

[0065] Preferably, the above separation and washing conditions are: rotation speed of 6000rpm-9000rpm, centrifugation time of 5min-10min; washing the wet cells with tris(hydroxymethyl)aminomethane hydrochloride buffer, pH of which is 7.0-8.4 and concentration of which is 100mM-1000mM.

[0066] Preferably, the catalytic reaction system includes: recombinant bacteria wet weight 10g / -200g / L, yeast cells 10g / -200g / L, N-acetylglucosamine 10g / -50g / L, lactose 10g / -50g / L, fructose 10g / -50g / L, adenosine triphosphate disodium salt 1g / -20g / L, uridine triphosphate trisodium salt 1g / -20g / L, Triton X-1000 0.1%-1%, inorganic salt 0.01g / -1g / L, buffer 100mM-1000mM.

[0067] Preferably, the inorganic salt is magnesium chloride and / or manganese chloride.

[0068] Preferably, the buffer is Tris hydrochloride buffer or phosphate buffer.

[0069] Preferably, the conditions of the catalytic reaction are: pH=6.0-9.0, temperature of 15°C-42°C, rotation speed of 150rpm-300rpm, and reaction time of 4h-30h.

[0070] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0071] The plasmids and strains involved in the following examples are:

[0072] pET28a(+) plasmid, pRSFDuet-1 plasmid, and pETDuet-1 plasmid are all commercial plasmids;

[0073] E. coli cloning strain: E. coli DH5α, E. coli expression strain: E. coli JM109 (ED3) are both commercially purchased strains.

[0074] The culture medium involved in the following examples:

[0075] LB medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, pH 7.0;

[0076] SOC medium: tryptone 20 g / L, yeast extract 5 g / L, sodium chloride 0.5 g / L, potassium chloride 2.5 mM, magnesium chloride 10 mM, glucose 20 mM;

[0077] Fermentation medium: enzymatic hydrolyzed casein 10.0 g / L, yeast extract 5.0 g / L, ammonium chloride 2.674 g / L, sodium dihydrogen phosphate 3.549 g / L, dipotassium hydrogen phosphate 3.402 g / L, sodium sulfate 0.7102 g / L, magnesium sulfate 0.3244 g / L, glycerol 5.0 g / L, glucose 10 g / L.

[0078] Example 1: Screening of UDP-N-acetylglucosamine pyrophosphorylase from different species

[0079] (1) Gene screening: Four UDP-N-acetylglucosamine pyrophosphorylases from different species were found. Their Genebank numbers are NP_001311042.1, WP_005758006.1, YP_002344229.1, and NP_418186.1, respectively, as listed in Table 1 below. The gene sequences encoding the corresponding enzymes are shown in SEQ ID NO: 1 to SEQ ID NO: 4.

[0080] Table 1 Four screened UDP-N-acetylglucosamine pyrophosphorylases

[0081] name Genebank ID Optimized nucleic acid sequence AGX1 NP_001311042.1 SEQ ID NO:1 PcqI WP_005758006.1 SEQ ID NO:2 QUR YP_002344229.1 SEQ ID NO:3 EqV NP_418186.1 SEQ ID NO:4

[0082] (2) Construction of gene expression vector: The optimized gene sequence was synthesized by Wuxi Tianlin Biotechnology Co., Ltd. and inserted into plasmid pET28a(+). The recombinant plasmids carrying the optimized gene sequence were transformed into competent Escherichia coli JM109(DE3) cells, and positive transformants were picked on LB agar medium containing kanamycin to form four recombinant E. coli strains, namely EJM109A, EJM109PMG, EJM109CJG and EJM109ECG.

[0083] (3) Protein expression of recombinant strains: According to the classic recombinant E. coli culture and induced expression protocol, single colonies of recombinant E. coli were picked and inoculated into 10 mL of LB medium containing 50 μg / mL kanamycin, and cultured at 37°C, 200 rpm overnight as seed solution; 2-5 mL of seed solution was transferred to 100 mL of LB medium containing 50 μg / mL kanamycin, and cultured at 37°C, 200 rpm until OD 600 When the p-value reaches 0.6-0.8, add isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.3-1mM, and induce expression at 16°C, 200rpm for 24h; after the induction of expression, centrifuge at 8000rpm for 8min, wash the bacteria with Tris-HCl buffer, repeat the centrifugation once, and collect the cells.

[0084] (4) Comparison of the synthetic ability of the four recombinant strains: The catalytic system contained 1-phospho-N-acetylglucosamine 50 mM, uridine triphosphate disodium salt 40 mM, MgCl2 10 mM, Tris-HCl 100 mM (pH = 7.4), a total of 1 mL. 50 g / L of recombinant Escherichia coli was resuspended in the catalytic solution and catalyzed at 37°C, 200 rpm for 16 h.

[0085] (5) Detection of uridine diphosphate-N-acetylglucosamine in the catalytic solution: After the catalytic solution sample was centrifuged at 12000rpm for 2min, the supernatant was taken, diluted with distilled water to a certain multiple, and filtered with a 0.22μm water system microporous filter membrane; the product was preliminarily detected by thin layer chromatography (Tlc), the developing solvent was n-butanol: acetic acid: water = 2:1:1, and the dye was anisaldehyde dye. Further, a Shimadzu LC-2010A high performance liquid chromatograph was used for detection with a UV detector; the chromatographic column was Hanbang Lichrospher-5-C 18 The chromatographic column was (250 mm × 4.6 mm id, 5 μm), the mobile phase was methanol-6‰ (volume fraction) phosphoric acid aqueous solution (the pH was adjusted to 6.6 with triethylamine) (volume ratio of 3:97), the flow rate was 0.6 mL / min, the column temperature was 30 °C, the sample volume was 10 μL, the detection wavelength was 261 nm, and the detection time was 30 min.

[0086] (6) Results

[0087] The conversion rates of UDP-N-acetylglucosamine pyrophosphorylase from different sources are shown in Table 2. The conversion rate of EcGlmU is as high as 74%. Figure 2 As shown, up to 22.5 g / l. Therefore, EcGlmU was used to synthesize LNTII.

[0088] Table 2 Comparison of the yield of strains expressing UDP-N-acetylglucosamine pyrophosphorylase from different sources

[0089]

[0090]

[0091] Example 2: Screening of β-1,3-N-acetylglucosamine aminotransferase from different species

[0092] (1) Gene screening: Three β-1,3-N-acetylglucosamine aminotransferases from different species were found. Their Genebank numbers are WP_002233115.1, NP_001306004.1, and WP_000666599.1, respectively, as listed in Table 3 below. The gene sequences encoding the corresponding enzymes are shown in SEQ ID NO: 6 to SEQ ID NO: 8.

[0093] Table 3 Three β-1,3-N-acetylglucosamine aminotransferases

[0094] name Genebank ID Optimized nucleic acid sequence LqCy WP_002233115.1 SEQ ID NO:6 B3GNT2 NP_001306004.1 SEQ ID NO:7 murG WP_000666599.1 SEQ ID NO:8

[0095] (2) Construction of gene expression vector: The optimized gene sequence was synthesized by Wuxi Tianlin Biotechnology Co., Ltd. and inserted into the plasmid pET28a(+). The recombinant plasmids carrying the optimized gene sequence were transformed into competent Escherichia coli JM109(DE3) cells, and the positive transformants were picked on LB agar medium containing kanamycin to form three recombinant Escherichia coli strains, namely EJM109LA, EJM109BG, and EJM109MG.

[0096] (3) Protein expression of recombinant strains: According to the classic recombinant E. coli culture and induced expression protocol, single colonies of recombinant E. coli were picked and inoculated into 10 mL of LB medium containing 50 μg / mL kanamycin, and cultured at 37°C, 200 rpm overnight as seed solution; 2-5 mL of seed solution was transferred to 100 mL of LB medium containing 50 μg / mL kanamycin, and cultured at 37°C, 200 rpm until OD 600 When the p-value reaches 0.6-0.8, add isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.3-1mM, and induce expression at 16°C, 200rpm for 24h; after the induction of expression, centrifuge at 8000rpm for 8min, wash the bacteria with Tris-HCl buffer, repeat the centrifugation once, and collect the cells.

[0097] (4) Comparison of the synthetic ability of the three recombinant strains: The catalytic system contained 40 mM UDP-N-acetylglucosamine disodium salt, 40 mM lactose, 10 mM MgCl2, and 100 mM Tris-HCl (pH = 7.4), a total of 1 mL. 50 g / L of recombinant Escherichia coli was resuspended in the catalytic solution and catalyzed at 37°C and 200 rpm for 16 h.

[0098] (5) Detection of lactose-N-triose in the catalytic solution: The catalytic solution sample was centrifuged at 12000 rpm for 2 min, the supernatant was taken, and diluted with distilled water to a certain multiple; the product was preliminarily detected by thin layer chromatography (Tlc), the developing solvent was n-butanol: acetic acid: water = 2:1:1, and the dye was anisaldehyde dye.

[0099] (6) The results are as follows Figure 3 As shown, only the β-1,3-N-acetylglucosamine aminotransferase derived from Neisseria meningitidis has the ability to synthesize lactose-N-triose.

[0100] Example 3: Lactose-N-triose production using co-expression strategy

[0101] Table 4 Primers involved in co-expression plasmid construction

[0102]

[0103] (1) Plasmid construction: Use the primers in Table 4 to amplify the Nahk (SEQ ID NO.5), EcGlmU (SEQ ID NO.4), and LgtA (SEQ ID NO.6) genes, and use the Suzhou Novogene ClonExpress Ultra One Step Cloning Kit V3 homologous recombination enzyme to insert the amplified genes into the expression frame 1 or expression frame 2 of pRSFDuet-1 and pETDuet-1 in the form of homologous recombination to achieve the integrated expression of the three genes. The constructed plasmid was transformed into Escherichia coli JM109 (DE3) competent cells by chemical transformation, and positive transformants were picked from the solid LB plate containing 50 μg / mL kanamycin and 100 μg / mL ampicillin, forming a total of 6 recombinant Escherichia coli, numbered E01-E06. Specifically, the Nahk and EcGlmU genes were linked by (GGGGS)3 flexible protein linker using overlap PCR to form the Nahk-(GGGGS)3-EcGlmU gene; the gene and LgtA gene were inserted into expression cassette 1 or expression cassette 2 of pRSFDuet-1 and pETDuet-1, respectively, and positive transformants were picked from solid LB plates containing 50 μg / mL kanamycin or 100 μg / mL ampicillin to form two additional recombinant Escherichia coli, numbered E07 and E08 (see Figure 4 A).

[0104] (2) Synthesis of lactose-N-triose by coupling catalysis of two bacteria: Referring to the protein induction and expression scheme in Example 1, the recombinant Escherichia coli was cultured to obtain recombinant Escherichia coli cells. The catalytic system contained 40 mM N-acetylglucosamine, 40 mM lactose, 50 mM fructose, 40 mM adenosine triphosphate disodium salt, 40 mM uridine triphosphate trisodium salt, 0.5% Triton X-100, 100 mM Tris-HCl buffer (pH=7.4), 10 mM MgCl2, 10 mM MnCl2, 50 g / L recombinant Escherichia coli cells, and 50 g / L cerevisiae (GDMCC61663) cells. The reaction was carried out at 30°C and 200 rpm for 16 h. After the reaction was completed, the reaction was centrifuged at 12000 rpm for 2 min. The supernatant was collected, diluted with distilled water to a certain multiple, and filtered with a 0.22 μm water system microporous filter membrane.

[0105] (3) Detection of lactose-N-triose in the catalytic solution: Thin layer chromatography (Tlc) was used to preliminarily detect the product, the developing solvent was n-butanol: acetic acid: water = 2:1:1, and the dye was anisaldehyde dye. Further, a Shimadzu LC-2010A high performance liquid chromatograph was used for detection with a UV detector; the chromatographic column was Bio-Rad Aminex HPX-87H (4.6mm×250mm), the mobile phase was 5mM H2SO4, the flow rate was 0.6mL / min, the column temperature was 60℃, the sample volume was 10μL, the detection wavelength was 210nm, and the detection time was 30min.

[0106] (4) Comparison of lactose-N-triose production of different E. coli recombinant strains (Table 5 and Figure 4 As shown in B. The best combination is E07: E. coli JM109 (DE3) / pRSFDuet-Nahk (GGGGS) 3GlmU-LgtA strain. The lactose-N-triose yield is 6.88 g / L and the conversion rate is 0.32.

[0107] Table 5 Comparison of lactose-N-triose production of different recombinant E. coli strains

[0108]

[0109] Example 4: Investigation of the ability to synthesize lactose-N-triose under different reaction conditions

[0110] In this example, the strain E. coli JM109 (DE3) /

[0111] pRSFDuet-Nahk(GGGGS)3GlmU-LgtA (named E07) was used to investigate the ability of lactose-N-triose synthesis under different reaction conditions (see Figure 5 ).

[0112] (1) Investigation of lactose-N-triose production under different pH conditions: Referring to the protein induction and expression scheme in Example 1, the recombinant Escherichia coli was cultured to obtain recombinant Escherichia coli cells. The basic catalytic system contains 40 mM N-acetylglucosamine, 40 mM lactose, 50 mM fructose, 40 mM adenosine triphosphate disodium salt, 40 mM uridine triphosphate trisodium salt, 0.5% Triton X-100, 10 mM MgCl2, 10 mM MnCl2, 50 g / L recombinant Escherichia coli cells, and recombinant Escherichia coli cells and Saccharomyces cerevisiae (GDMCC61663) are added in a 1:1 weight ratio. 100 mM Tris-Hcl buffer with pH values ​​of 6, 6.5, 7, 7.5, 8, 8.5, and 9 is added to the basic catalytic system, respectively, and catalyzed at 200 rpm for 16 h at 30 ° C. After the catalysis is completed, centrifuge at 12000 rpm for 2 min, collect the supernatant, dilute it with distilled water for a certain multiple, and filter it with a 0.22 μm water system microporous filter membrane to form a total of 7 experimental groups. The yield and conversion rate are both the highest when the pH is 9 ( Figure 5 A).

[0113] (2) Investigation of lactose-N-triose production under different buffer conditions: Referring to the protein induction and expression scheme in Example 1, the recombinant Escherichia coli was cultured to obtain recombinant Escherichia coli cells. The basic catalytic system contains 40mM N-acetylglucosamine, 40mM lactose, 50mM fructose, 40mM adenosine triphosphate disodium salt, 40mM uridine triphosphate trisodium salt, 0.5% Triton X-100, 10mM MgCl2, 10mM MnCl2, 50g / L recombinant Escherichia coli cells, and recombinant Escherichia coli cells and Saccharomyces cerevisiae (GDMCC61663) are added in a 1:1 weight ratio. Phosphate buffer or Tris-HCl buffer with a pH of 7.4 is added to the basic catalytic system at concentrations of 100, 300, 600, and 1000mM, respectively. The catalysis is carried out at 200rpm for 16h at 30°C. After the catalysis is completed, the supernatant is centrifuged at 12000rpm for 2min, the supernatant is collected, diluted with distilled water for a certain multiple, and filtered with a 0.22μm water system microporous filter membrane to form a total of 8 experimental groups. Figure 5 As can be seen from B, the optimal buffer is 100 mM Tris-HCl.

[0114] (3) Investigation of lactose-N-triose production under different surfactant addition conditions: Referring to the protein induction and expression scheme in Example 1, the recombinant E. coli was cultured to obtain recombinant E. coli cells. The basic catalytic system contained 40 mM N-acetylglucosamine, 40 mM lactose, 50 mM fructose, 40 mM adenosine triphosphate disodium salt, 40 mM uridine triphosphate trisodium salt, 100 mM Tris-HCl buffer (pH = 7.4), 10 mM MgCl2, 10 mM MnCl2 mM, recombinant Escherichia coli cells 50g / L, recombinant Escherichia coli cells and Saccharomyces cerevisiae (GDMCC61663) were added at a weight ratio of 1:1, octadecylamine polyoxyethylene ether solution was added to the basic catalytic system to make the final concentration of 1%, 5%, 10%, and 20%; or Triton X-100 solution was added to make the final concentration of 0.1%, 0.5%, 1%, and 2%, and catalyzed at 200rpm for 16h at 30°C. After the catalysis, it was centrifuged at 12000rpm for 2min, the supernatant was collected, diluted with distilled water for a certain multiple, and filtered with a 0.22μm water system microporous filter membrane to form a total of 8 experimental groups. Figure 5 It can be seen from C that the best final concentration of octadecylamine polyoxyethylene ether solution is 1%, or the concentration of Triton X-100 is 0.1%.

[0115] (4) Investigation of lactose-N-triose production under different temperature conditions: Referring to the protein induction and expression scheme in Example 1, the recombinant Escherichia coli was cultured to obtain recombinant Escherichia coli cells. The basic catalytic system contains 40 mM N-acetylglucosamine, 40 mM lactose, 50 mM fructose, 40 mM adenosine triphosphate disodium salt, 40 mM uridine triphosphate trisodium salt, 0.5% Triton X-100, 100 mM Tris-HCl buffer (pH = 7.4), 10 mM MgCl2, 10 mM MnCl2, 50 g / L recombinant Escherichia coli cells, and recombinant Escherichia coli cells and Saccharomyces cerevisiae (GDMCC61663) are added in a 1:1 weight ratio, and catalyzed at 200 rpm for 16 h at temperatures of 42 ° C, 37 ° C, 30 ° C, 25 ° C, and 20 ° C, respectively. After the catalysis is completed, centrifuge at 12000 rpm for 2 min, collect the supernatant, dilute it with distilled water to a certain multiple, and filter it with a 0.22 μm water system microporous filter membrane to form a total of 5 experimental groups. Figure 5 It can be seen from D that 20℃ is the optimal temperature.

[0116] (5) Investigation of lactose-N-triose production under different time conditions: Referring to the protein induction and expression scheme in Example 1, the recombinant Escherichia coli was cultured to obtain recombinant Escherichia coli cells. The basic catalytic system contains 40 mM N-acetylglucosamine, 40 mM lactose, 50 mM fructose, 40 mM adenosine triphosphate disodium salt, 40 mM uridine triphosphate trisodium salt, 0.5% Triton X-100, 100 mM Tris-HCl buffer (pH = 7.4), 10 mM MgCl2, 10 mM MnCl2, 50 g / L recombinant Escherichia coli cells, and recombinant Escherichia coli cells and Saccharomyces cerevisiae (GDMCC61663) are added in a 1:1 weight ratio, catalyzed at 200 rpm at 30 ° C, the total catalytic time is 40 h, and sampling is performed every 2 h. The pH is readjusted to 7.4 at 20 h of catalytic time, forming a total of 20 samples, each sample is centrifuged at 12000 rpm for 2 min, the supernatant is collected, diluted a certain multiple with distilled water, and filtered with a 0.22 μm water system microporous filter membrane. The results are as follows Figure 5 As shown in E.

[0117] (6) Investigation of lactose-N-triose production under the condition of adding different concentrations of metal ions: Referring to the protein induction and expression scheme in Example 1, the recombinant Escherichia coli was cultured to obtain recombinant Escherichia coli cells. The basic catalytic system contains 40mM N-acetylglucosamine, 40mM lactose, 50mM fructose, 40mM adenosine triphosphate disodium salt, 40mM uridine triphosphate trisodium salt, 100mM Tris-HCl buffer (pH=7.4), 0.5% Triton X-100, 50g / L recombinant Escherichia coli cells, and the recombinant Escherichia coli cells and Saccharomyces cerevisiae (GDMCC61663) are added in a weight ratio of 1:1. On the basis of the addition of 10mM MnCl2, 5mM, 10mM, 20mM, 30mM, 40mM, and 50mM MgCl2 are added, or on the basis of the addition of 10mM MnCl2, 10mM MgCl2 is added. On the basis of MgCl2, MnCl2 with concentrations of 5mM, 10mM, 20mM, 30mM, 40mM, and 50mM were added respectively. The experimental groups were catalyzed at 200rpm for 16h at 30℃. After the catalysis, the supernatant was collected and diluted with distilled water for a certain multiple, and filtered with a 0.22μm water-based microporous filter membrane to form a total of 9 experimental groups. Figure 6 As can be seen from A, MnCl250 mM and MgCl210 mM are the optimal concentrations.

[0118] (7) Investigation of lactose-N-triose production under different cell addition conditions: Referring to the protein induction and expression scheme in Example 1, the recombinant Escherichia coli was cultured to obtain recombinant Escherichia coli cells. The basic catalytic system contains 40mM N-acetylglucosamine, 40mM lactose, 50mM fructose, 40mM adenosine triphosphate disodium salt, 40mM uridine triphosphate trisodium salt, 0.5% Triton X-100, 100mM Tris-Hcl buffer (pH=7.4), 10mM MgCl2, 10mM MnCl2. Different proportions of recombinant Escherichia coli and yeast cells were added to the basic catalytic system to form a total of 7 experimental groups. The experimental group catalyzed at 200rpm for 16h at 30°C. After the catalysis was completed, it was centrifuged at 12000rpm for 2min, the supernatant was collected, diluted a certain number of times with distilled water, and filtered with a 0.22μm water system microporous filter membrane. From Figure 6 As can be seen from B, the optimal ratio of adding recombinant Escherichia coli and Saccharomyces cerevisiae (GDMCC61663) in a weight ratio of 1:1 is optimal.

[0119] (8) Investigation of the lactose-N-triose production of recombinant Escherichia coli under different induction conditions: Based on the recombinant Escherichia coli culture scheme of Example 1, different induction schemes were adopted, namely: 37°C induction for 4 h, 30°C induction for 8 h, 25°C induction for 12 h, and 20°C induction for 24 h. The four induction schemes were cross-combined with different IPTG addition amounts: 0.1 mM, 0.3 mM, 0.6 mM, and 1 mM, forming a total of 16 experimental groups. Recombinant Escherichia coli with 16 induction conditions were used to conduct lactose-N-triose synthesis experiments. The catalytic system contained 40mM N-acetylglucosamine, 40mM lactose, 50mM fructose, 40mM adenosine triphosphate disodium salt, 40mM uridine triphosphate trisodium salt, 0.5% Triton X-100, 100mM Tris-HCl buffer (pH=7.4), 10mM MgCl2, 10mM MnCl2, 50g / L recombinant Escherichia coli cells, and recombinant Escherichia coli cells and Saccharomyces cerevisiae (GDMCC61663) were added in a 1:1 ratio. The reaction was carried out at 200rpm for 16h at 30°C. After the reaction was completed, the reaction was centrifuged at 12000rpm for 2min, and the supernatant was collected, diluted with distilled water to a certain multiple, and filtered with a 0.22μm water system microporous filter membrane. Figure 6 As can be seen from C, the optimal induction conditions are 20°C and 0.3 mM IPTG concentration.

[0120] (9) Investigation of lactose-N-triose production under different substrate addition conditions: Referring to the protein induction and expression scheme in Example 1, the recombinant Escherichia coli was cultured to obtain recombinant Escherichia coli cells. The basic catalytic system contained 50 mM fructose, 40 mM adenosine triphosphate disodium salt, 40 mM uridine triphosphate trisodium salt, 0.5% Triton X-100, 100 mM Tris-HCl buffer (pH = 7.4), 10 mM MgCl2, 10 mM MnCl2, 50 g / L recombinant Escherichia coli cells, and recombinant Escherichia coli cells and Saccharomyces cerevisiae (GDMCC61663) were added in a 1:1 weight ratio. Different proportions of N-acetylglucosamine and lactose were added to the basic catalytic system to form a total of 13 experimental groups. Each experimental group was catalyzed at 200 rpm for 16 h at 30 °C. After the catalysis, the mixture was centrifuged at 12,000 rpm for 2 min. The supernatant was collected, diluted with distilled water to a certain multiple, and filtered with a 0.22 μm water-based microporous membrane. Figure 6 As can be seen from D, the optimal concentrations of N-acetylglucosamine and lactose are both 200 mM.

[0121] (10) Detection of lactose-N-triose in the catalytic solution: Shimadzu LC-2010A high performance liquid chromatograph was used with a UV detector; the chromatographic column was Bio-Rad Aminex HPX-87H (4.6 mm × 250 mm), the mobile phase was 5 mM H2SO4, the flow rate was 0.6 mL / min, the column temperature was 60 °C, the sample volume was 10 μL, the detection wavelength was 210 nm, and the detection time was 30 min.

[0122] Example 5: Production of lactose-N-triose by dual-bacteria coupled two-stage fermentation

[0123] In this example, the strain E. coli JM109 (DE3) / pRSFDuet-Nahk (GGGGS) 3GlmU-LgtA (named E07) was used in a 5L fermenter for a two-step fermentation experiment (see Figure 7 ).

[0124] (1) In the first stage, the 5-liter fermentation tank culture process of the engineered strain E07 was as follows: the recombinant E. coli strain E07 was grown overnight on a solid LB plate containing 50 μg / mL kanamycin, and a single colony was picked and inoculated into 20 mL LB medium, and grown at 37°C, 200 rpm for 16 h as the first-level seed liquid; 10 mL of the first-level seed liquid was inoculated into 300 mL LB medium, and grown at 37°C, 200 rpm for 12-16 h as the second-level seed liquid. Then, the prepared second-level seed liquid was inoculated into a 5-liter fermentation tank containing 3 L fermentation medium at a 10% inoculation amount, the temperature was 37°C, the stirring speed was linked to the DO mode, the DO was controlled at 30%, and the pH value was controlled at 6.98 with a 20% ammonia solution. When the glucose concentration was lower than 1 g / L, 300 g / L of glucose was added; when the OD reached 10, 0.3 mM of isopropyl-β-D-thiogalactoside (IPTG) was added, and the temperature was adjusted to 20°C. The induction time was 22 h (see Figure 7 A).

[0125] (2) The second stage, coupled fermentation production of lactose-N-triose synthesis process: After the induction, the rotation speed was adjusted to be constant at 300 rpm, and the following were added to the fermenter: N-acetylglucosamine 100 mM, lactose 100 mM, fructose 50 mM, adenosine triphosphate disodium salt 40 mM, uridine triphosphate trisodium salt 40 mM, Triton X-1000 0.1%, tris(hydroxymethyl)aminomethane 100 mM, MgCl2 10 mM, MnCl2 10 mM, NaOH 4 M was used to adjust the pH to 7.4, 50 g / L of cerevisiae (GDMCC61663) was added, and the coupled fermentation time was 28 h. The amount of lactose-N-triose produced was 52.20 g / L, and the conversion rate of the substrate lactose was 95.95% (see Figure 7 B).

[0126] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A recombinant Escherichia coli for producing lactose-N-triose, characterized in that: The recombinant Escherichia coli expresses an N-acetylglucosamine 1-kinase gene Nahk, a uridine diphosphate-N-acetylglucosamine pyrophosphorylase gene EcGlmU and a β-1,3-N-acetylglucosamine aminotransferase gene LgtA.

2. The recombinant Escherichia coli according to claim 1, characterized in that: The recombinant Escherichia coli is any one of the following combinations: (1) The recombinant Escherichia coli uses pRSFDuet-1 plasmid or pETDuet-1 plasmid to express N-acetylglucosamine 1-kinase gene Nahk, uridine diphosphate-N-acetylglucosamine pyrophosphorylase gene EcGlmU and β-1,3-N-acetylglucosamine aminotransferase gene LgtA; the N-acetylglucosamine 1-kinase gene Nahk and the β-1,3-N-acetylglucosamine aminotransferase gene LgtA are connected by a flexible protein Linker; (2) the recombinant Escherichia coli uses the pRSFDuet-1 plasmid to express the β-1,3-N-acetylglucosamine aminotransferase gene LgtA and the uridine diphosphate-N-acetylglucosamine pyrophosphorylase gene EcGlmU located after LgtA, and uses the pETDuet-1 plasmid to express the N-acetylhexosamine 1-kinase gene Nahk; (3) the recombinant Escherichia coli uses the pRSFDuet-1 plasmid to express the uridine diphosphate-N-acetylglucosamine pyrophosphorylase gene EcGlmU, and uses the pETDuet-1 plasmid to express the β-1,3-N-acetylglucosamine aminotransferase gene LgtA and the N-acetylhexosamine 1-kinase gene Nahk located after LgtA; (4) the recombinant Escherichia coli uses the pRSFDuet-1 plasmid to express the β-1,3-N-acetylglucosamine aminotransferase gene LgtA and the N-acetylhexosamine 1-kinase gene Nahk located after LgtA, and uses the pETDuet-1 plasmid to express the uridine diphosphate-N-acetylglucosamine pyrophosphorylase gene EcGlmU; (5) The recombinant Escherichia coli uses the pRSFDuet-1 plasmid to express the N-acetylglucosamine 1-kinase gene Nahk, and uses the pETDuet-1 plasmid to express the β-1,3-N-acetylglucosamine aminotransferase gene LgtA and the uridine diphosphate-N-acetylglucosamine pyrophosphorylase gene EcGlmU located after LgtA; (6) The recombinant Escherichia coli uses the pRSFDuet-1 plasmid to express the β-1,3-N-acetylglucosamine aminotransferase gene LgtA, and uses the pETDuet-1 plasmid to express the N-acetylhexosamine 1-kinase gene Nahk and the uridine diphosphate-N-acetylglucosamine pyrophosphorylase gene EcGlmU located after Nahk; (7) The recombinant Escherichia coli uses the pRSFDuet-1 plasmid to express the N-acetylglucosamine 1-kinase gene Nahk and the uridine diphosphate-N-acetylglucosamine pyrophosphorylase gene EcGlmU located after Nahk, and uses the pETDuet-1 plasmid to express the β-1,3-N-acetylglucosamine aminotransferase gene LgtA.

3. The recombinant Escherichia coli according to claim 1, characterized in that The N-acetylhexosamine 1-kinase is derived from Bifidobacterium breve; And / or, the UDP-N-acetylglucosamine pyrophosphorylase is derived from Escherichia colistr.K-12substr.MG1655; And / or, the β-1,3-N-acetylglucosamine aminotransferase is derived from Neisseria meningitidis.

4. A dual-bacteria coupling system, characterized in that: The dual-bacteria coupling system comprises the recombinant Escherichia coli and yeast described in any one of claims 1 to 3.

5. The dual bacteria coupling system according to claim 4, characterized in that: The yeast includes brewer's yeast and / or baker's yeast.

6. The dual bacteria coupling system according to claim 4, characterized in that: The usage amount of the recombinant Escherichia coli is 10g / L-200g / L; And / or, the usage amount of the yeast is 10g / L-200g / L.

7. Use of the recombinant Escherichia coli according to any one of claims 1 to 3 or the dual-bacteria coupling system according to any one of claims 4 to 6 in synthesizing lactose-N-trisaccharide.

8. A method for synthesizing lactose-N-triose, characterized in that: With N-acetylglucosamine and lactose as substrates, the recombinant Escherichia coli described in any one of claims 1 to 3 or the dual-bacteria coupling system described in any one of claims 4 to 6 is used to catalyze the synthesis of lactose-N-trisaccharide.

9. The method according to claim 8, characterized in that The dual-bacteria coupling system catalyzes the synthesis of lactose-N-trisaccharide, including: a recombinant Escherichia coli fermentation stage, and a recombinant Escherichia coli and yeast co-fermentation stage.

10. The method according to claim 8, characterized in that The catalytic synthesis system also includes one or more of fructose, adenosine triphosphate disodium salt, uridine triphosphate trisodium salt, Triton X-100, inorganic salts and buffer; the reaction system is a phosphate buffer or a tris(hydroxymethyl)aminomethane hydrochloride buffer.

11. The method according to claim 8, characterized in that One or more of the following conditions are met: 1) The recombinant Escherichia coli is induced to express using isopropyl-β-D-thiogalactoside; 2) The catalytic synthesis temperature is 15°C-42°C and the time is 4h-30h; 3) The pH value of the catalytic synthesis system is 6.0-9.0.

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