A method for synthesis of lacto-n-neotetraose based on protein assembly scaffolds

Abstract

The application discloses a method for synthesizing lacto-N-neotetraose based on a protein assembly scaffold, and belongs to the technical field of metabolic engineering. Based on the problem that the spatial distribution of key enzymes in the synthesis of lacto-N-neotetraose is scattered, intermediate diffusion is caused, and finally the intracellular enzymatic reaction rate is reduced, the plasmid is used to heterogeneously express beta-1, 3-N-acetylglucosamine amino transferase lgtA containing a small peptide SH3 lig and beta-1, 4-galactosyltransferase lgtB containing a small peptide PDZ lig, and the protein assembly scaffold PDZ and SH3 are fused and expressed, so that a recombinant escherichia coli for synthesizing lacto-N-neotetraose based on the protein assembly scaffold is constructed, the key enzyme gene is efficiently catalyzed in the escherichia coli, and finally the yield of lacto-N-neotetraose reaches 1699 mg / L in a flask, which lays a foundation for efficient synthesis and further industrialization of lacto-N-neotetraose.

Description

Technical Field

[0001] This invention relates to a method for synthesizing lactyl-N-neotetrasaccharides based on protein assembly scaffolds, belonging to the field of metabolic engineering technology. Background Technology

[0002] Human milk oligosaccharides (HMOs) are the third largest solid component in breast milk. They promote the growth of gut microbiota and have antibacterial, anti-inflammatory, and brain-development-promoting effects. Lacto-N-neotetraose (LNnT) is a non-fucosylated neutral HMO with the structural formula Gal. β 1-4GlcNAc β 1-3Gal β 1-4Glc, molecular formula C 26 H 45 NO 21 The molecular weight is 707.63. Lactoyl-N-neotetrasaccharide has been approved by the European Food Safety Authority and the U.S. Food and Drug Administration as a novel food additive for use in infant formula.

[0003] Oligosaccharides extracted from breast milk or other animal milk are low in content and mixed; chemical synthesis involves multiple protection and deprotection steps, resulting in low yields; and enzymatic synthesis of lactyl-N-neotetrasaccharides requires expensive substrates. Therefore, environmentally friendly microbial fermentation is a more promising approach.

[0004] *Escherichia coli* is an industrial model strain approved by the EU for the production of lactyl-N-neotetrasaccharide. It is characterized by rapid growth, ease of operation, and suitability for large-scale fermentation, making it suitable for high-density cultivation. Previous studies have directly introduced key enzyme genes into *E. coli* to synthesize lactyl-N-neotetrasaccharide; however, the spatially dispersed distribution of the enzyme leads to intermediate diffusion, ultimately resulting in a reduced intracellular enzymatic reaction rate. Currently, no research focuses on using protein assembly scaffolds to improve the yield of lactyl-N-neotetrasaccharide. Summary of the Invention

[0005] To address the aforementioned issues, this invention provides a recombinant Escherichia coli strain that synthesizes lactyl-N-neotetrasaccharides based on protein assembly scaffolds PDZ / PDZlig and SH3 / SH3lig. By introducing and optimizing the protein assembly scaffold structure, the product yield is significantly increased, laying the foundation for the development of health foods rich in LNnT, especially formula milk powder.

[0006] The first objective of this invention is to provide a method for synthesizing lactyl-N-neotetrasaccharides by introducing a protein assembly scaffold, comprising the following steps:

[0007] Production was carried out by fermentation using recombinant Escherichia coli containing a protein assembly scaffold;

[0008] The recombinant E. coli had the β-galactosidase gene knocked out ( lacZ ), glucosamine-6-phosphate deaminase gene ( nagB ), UDP-acetylglucosamine epiisomerase gene ( wecB UDP-glucose dehydrogenase gene ( ugd ), overexpressed the lactose transport enzyme gene ( lacY ), glucose-6-phosphate isomerase gene ( pgi ), glutamine-fructose-6-phosphate aminotransferase gene ( glmS ) and glucosamine-1-phosphate acetyltransferase gene ( glmU ), and heterologously expressed the β-1,3-N-acetylglucosamine aminotransferase gene fused with at least one SH3lig at the C-terminus ( lgtA ), a β-1,4-galactosyltransferase gene fused to at least one PDZlig at its C-terminus ( lgtB ) as well as protein assembly scaffolds PDZ and SH3.

[0009] Furthermore, overexpression on the genome lacY , pgi , glmS and glmU Heterologous expression of plasmids fused with SH3lig and PDZlig was performed. lgtAB And protein assembly scaffolds PDZ and SH3.

[0010] Furthermore, SH3lig and PDZlig are respectively fused to the C-terminus of the β-1,3-N-acetylglucosamine aminotransferase gene and the β-1,4-galactosyltransferase gene via a first flexible linker peptide.

[0011] Furthermore, the first flexible linker peptide used in this embodiment of the invention is GGGGSGGGGS. Of course, those skilled in the art can choose (GGGGS)m as the linker peptide as needed, where m is an integer from 1 to 5.

[0012] Furthermore, the protein assembly scaffold PDZ and SH3 are connected via a second flexible linker peptide.

[0013] Furthermore, the second flexible linker peptide used in this embodiment of the invention is GSGSGSGSGSGSGS. Of course, those skilled in the art can choose (GS)n as the linker peptide as needed, where n is an integer from 3 to 10.

[0014] Furthermore, the ratio of PDZlig to SH3lig is 1:1-4, with the optimal ratio being 1:1.

[0015] Furthermore, the β-1,3-N-acetylglucosamine aminotransferase gene fused with SH3lig at the C-terminus and the β-1,4-galactosyltransferase gene fused with PDZlig at the C-terminus were expressed by inducible promoters, while the protein assembly scaffolds PDZ and SH3 were expressed by constitutive promoters.

[0016] Furthermore, the inducible promoters include, but are not limited to, the tac promoter, and the constitutive promoters include, but are not limited to, the J23119 promoter.

[0017] Furthermore, for heterologous expression, pCDFDuet-1 was used as the vector.

[0018] Furthermore, Escherichia coli K-12 MG1655 was used as the host.

[0019] Furthermore, the protein assembly scaffold SH3 is located downstream of PDZ.

[0020] Further, the sequences of β-1,3-N-acetylglucosamine aminotransferase are shown in SEQ ID NO.1-2, the sequences of β-1,4-galactosyltransferase are shown in SEQ ID NO.3-4, the sequences of protein assembly scaffold PDZ are shown in SEQ ID NO.5-6, the sequences of protein assembly scaffold SH3 are shown in SEQ ID NO.7-8, the amino acid sequences of PDZlig are shown in SEQ ID NO.9, and the amino acid sequences of SH3lig are shown in SEQ ID NO.10.

[0021] Furthermore, the IDs of the β-galactosidase gene are 945006, the glucosamine-6-phosphate deaminase gene is 945290, the UDP-acetylglucosamine epiisomerase gene is 944789, the UDP-glucose dehydrogenase gene is 946571, the lactose transport enzyme gene is 949083, the glucose-6-phosphate isomerase gene is 948535, the glutamine-fructose-6-phosphate aminotransferase gene is 948241, and the glucosamine-1-phosphate acetyltransferase gene is 948246.

[0022] Further, the fermentation production includes the following steps: picking a single colony of recombinant Escherichia coli into a seed culture medium, obtaining a seed liquid, and then inoculating it into the fermentation culture medium at an inoculation rate of 3%-10%, waiting for OD... 600 When the concentration reaches 0.5-1.2, production is induced at 25-35℃.

[0023] Furthermore, the seed culture medium comprises: 8-12 g / L tryptone, 3-7 g / L yeast extract, and 8-12 g / L sodium chloride.

[0024] Furthermore, the fermentation medium comprises: peptone 10-14 g / L, yeast extract 22-26 g / L, glycerol 3-5 g / L, dipotassium hydrogen phosphate 2.2-2.5 g / L, dipotassium hydrogen phosphate 12.2-12.5 g / L, and lactose 4-6 g / L.

[0025] A second objective of this invention is to provide a recombinant *E. coli* strain that synthesizes lactyl-N-neotetrasaccharides, wherein the recombinant *E. coli* strain has the β-galactosidase gene knocked out. lacZ ), glucosamine-6-phosphate deaminase gene ( nagB ), UDP-acetylglucosamine epiisomerase gene ( wecB UDP-glucose dehydrogenase gene ( ugd ), overexpressed the lactose transport enzyme gene ( lacY ), glucose-6-phosphate isomerase gene ( pgi ), glutamine-fructose-6-phosphate aminotransferase gene ( glmS ) and glucosamine-1-phosphate acetyltransferase gene ( glmU ), and heterologously expressed the β-1,3-N-acetylglucosamine aminotransferase gene fused with at least one SH3lig at the C-terminus ( lgtA ), a β-1,4-galactosyltransferase gene fused to at least one PDZlig at its C-terminus ( lgtB ) as well as protein assembly scaffolds PDZ and SH3.

[0026] A third objective of this invention is to provide a method for constructing the aforementioned recombinant Escherichia coli, comprising the following steps:

[0027] S1. Knock out the β-galactosidase gene, glucosamine-6-phosphate deaminase gene, UDP-acetylglucosamine epimerase gene, and UDP-glucose dehydrogenase gene in the host strain of Escherichia coli, and overexpress the lactose transport enzyme gene, glucose-6-phosphate isomerase gene, glutamine-fructose-6-phosphate aminotransferase gene, and glucosamine-1-phosphate acetyltransferase gene in the genome to obtain the starting strain;

[0028] S2. PDZlig and SH3lig were linked to the C-terminus of β-1,3-N-acetylglucosamine aminotransferase and β-1,4-galactosyltransferase, respectively. The protein assembly scaffold PDZ and SH3 were fused and expressed together, and then linked to the vector pCDFDuet-1 to obtain the recombinant plasmid.

[0029] S3. The recombinant plasmid obtained in S2 is introduced into the starting strain in S1 to obtain the above-mentioned recombinant Escherichia coli.

[0030] A fourth objective of this invention is to provide the application of the above-mentioned recombinant Escherichia coli in the preparation of biological products, chemical products, pharmaceuticals, or food.

[0031] Furthermore, all of the above products contain lactoyl-N-neotetrasaccharides, or use lactoyl-N-neotetrasaccharides as an intermediate product. This is especially true for formula milk powders containing human milk oligosaccharides.

[0032] The fifth objective of this invention is to provide a method for promoting the synthesis of lactyl-N-neotetrasaccharides in Escherichia coli, comprising the following steps:

[0033] SH3lig was linked to the C-terminus of β-1,3-N-acetylglucosamine aminotransferase, and PDZlig was linked to the C-terminus of β-1,4-galactosyltransferase. The protein assembly scaffold PDZ and SH3 were fused and expressed together, and then linked to the vector backbone to obtain a recombinant plasmid. The recombinant plasmid was then introduced into Escherichia coli host bacteria.

[0034] Furthermore, the ratio of PDZlig to SH3lig is 1:1-4, with the optimal ratio being 1:1.

[0035] The beneficial effects of this invention are:

[0036] The invention constructs a recombinant Escherichia coli based on protein assembly scaffolds PDZ / PDZlig and SH3 / SH3lig to synthesize lactyl-N-neotetrasaccharides. The titer of lactyl-N-neotetrasaccharides synthesized in shake flasks reached 1699 mg / L, which is 198% higher than that of recombinant E. coli without protein assembly scaffolds. This invention achieves efficient synthesis of lactyl-N-neotetrasaccharides, provides a feasible method for the optimized expression of key enzymes introduced from the exogenous, and lays the foundation for further metabolic engineering of E. coli to produce lactyl-N-neotetrasaccharides and its industrialization. Attached Figure Description

[0037] Figure 1 This invention describes the metabolic pathway by which recombinant Escherichia coli synthesizes lactyl-N-neotetrasaccharides.

[0038] Figure 2 This is a schematic diagram illustrating the construction of the recombinant expression plasmid in recombinant Escherichia coli according to the present invention.

[0039] Figure 3 This is the standard curve of lactyl-N-neotetrasaccharide in this invention. Detailed Implementation

[0040] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0041] The materials and testing methods involved in the following embodiments are as follows:

[0042] (a) Strains and vectors

[0043] plasmid construction in E. coli The plasmid was constructed in DH5α and then transformed into E. coli host bacteria for fermentation to synthesize lactyl-N-neotetrasaccharide.

[0044] The vector pCDFDuet-1 used is a commercial plasmid.

[0045] (ii) Culture medium

[0046] E. coli Cultured using LB medium (containing 10 g tryptone, 5 g yeast extract and 10 g NaCl per liter).

[0047] The engineered strain was first cultured as a seed culture in LB medium. After 12 h of culture, the recombinant E. coli seed culture was inoculated into the fermentation medium at an inoculation rate of 3%-10%. The culture was allowed to mature until the OD (Organic Demand) was reached. 600 When the pH reaches 0.5-1.2, induction is performed at 25-35℃ with IPTG at a final concentration of 0.05-0.25 mM for 48-72 h. The fermentation medium consists of: peptone 10-14 g / L, yeast extract 22-26 g / L, glycerol 3-5 g / L, dipotassium hydrogen phosphate 2.2-2.5 g / L, dipotassium hydrogen phosphate 12.2-12.5 g / L, and lactose 4-6 g / L.

[0048] (III) Detection method for lactyl-N-neotetrasaccharide

[0049] Lactoyl-N-neotetrasaccharide was measured using a CarboPac PA10 (4×250 mm) column via high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The mobile phase was NaOH (36 mM), the flow rate was set to 1.00 mL / min, the column temperature was 30 °C, and the injection volume was 25 µL. Before sample measurement, standards of different concentrations of lactoyl-N-neotetrasaccharide were prepared, and a standard curve was plotted using chromatographic peak areas to calculate the content of each component in each fermentation sample.

[0050] (4) Sequence

[0051] β-1,3-N-acetylglucosamine aminotransferase lgtA, SEQ ID NO.1-2,

[0052] β-1,4-galactosyltransferase IgTB, SEQ ID NO. 3-4,

[0053] Protein assembly scaffold PDZ, SEQ ID NO.5-6

[0054] Protein assembly scaffold SH3, SEQ ID NO.7-8

[0055] PDZlig, a protein assembly scaffold ligand, SEQ ID NO.9

[0056] Protein assembly scaffold ligand SH3lig, SEQ ID NO.10.

[0057] Since the cellular synthesis pathway of lactyl-N-neotetrasaccharide requires exogenous lactose as a substrate and endogenous precursors uridine diphosphate-N-acetylglucosamine and uridine diphosphate-galactose, in order to reduce precursor metabolism and weaken the synthesis of byproducts, the β-galactosidase gene was knocked out using the CRISPR / Cpf1 gene editing system, with Escherichia coli K-12 MG1655 as the starting strain, in order to reduce precursor metabolism and weaken the synthesis of byproducts. lacZ Glucosamine-6-phosphate deaminase gene nagB UDP-acetylglucosamine epiisomerase gene wecB and UDP-glucose dehydrogenase gene ugd In the genome fliK Site-integrated lactose transport enzyme gene lacY , motA Site integration of glucose-6-phosphate isomerase gene pgi , poxB Site integration of glutamine-fructose-6-phosphate aminotransferase gene glmS And in arsB Site integration of glucosamine-1-phosphoacetyltransferase gene glmU The starting strain was constructed in this way. The specific steps of gene knockout and integration are as follows:

[0058] (1) In fliK Site integration lacY Taking genes as an example, in fliKThe PAM site of TTTV was located within the gene, and its 23 bp sgRNA sequence was identified. An sgRNA primer fliK-crRNA-F / R with BasI restriction endonuclease was designed and annealed. The PCR annealing program was 98℃ for 2 min, 4℃ for 2 min, with a cooling rate of 0.1℃ / s. The pcrEG plasmid was digested with BsaI restriction endonuclease. The digestion system (50 μL) consisted of: 42.5 μL plasmid, 5 μL rapid digestion buffer, and 2.5 μL BsaI rapid digestion enzyme. The digestion conditions were 37℃ for 3 h. After successful digestion, the residue was purified using an agarose gel electrophoresis column and diluted to approximately 50 ng / μL for later use. The linearized plasmid vector and annealed sgRNA, after enzyme digestion, were ligated using T4 ligase at 22°C for 3 h. The T4 ligation system (10 μL) consisted of 1 μL each of T4 ligase, T4 ligase buffer, digested vector, and annealed sgRNA, with 6 μL of deionized water added at the end. The ligation product was transformed into the cloning host DH5α, the plasmid was extracted, and the sgRNA sequence was sequenced.

[0059] (2) The plasmid with correct sequence was further linearized using fliK-AF / AR, and upstream and downstream homologous arms were amplified using fliK-HL-F / R and fliK-HR-F / R. The lacY gene expression cassette for integration was amplified using lacY-F / R, ligated using a seamless cloning enzyme, and transformed into DH5α again. After plasmid extraction and sequencing verification, the final pcrEG-lacY plasmid was obtained.

[0060] (3) The pCpf1 plasmid was transformed into K-12 MG1655 cells using chemical transformation. The specific operation was as follows: K12MG1655 cells were treated with a commercial kit to become competent cells for chemical transformation and placed on ice for transformation. The transformation steps were as follows: 500-1000 ng of pCpf1 plasmid was added to 100 uL of competent cells. The EP tube was gently tapped to mix it evenly. The cells were placed on ice for 30 min, then heat-shocked in a 42℃ water bath for 60-90 s. The cells were then quickly placed on ice for 2-3 min. 500-900 uL of LB liquid medium was added. The cells were cultured at 37℃ for 1 h. The cells were then spread on LB solid medium containing kanamycin and cultured upside down at 37℃ for 12-16 h.

[0061] (4) Select colonies containing the pCpf1 plasmid and incubate them in a solution containing kanamycin and arabinose at a final concentration of 0.1 mol. When OD... 600 When the concentration reaches 0.3-0.5, stop the culture, immediately place the cells on ice, and prepare them into chemically competent cells using a commercially available kit.

[0062] (5) Add 500-1000 ng of the plasmid obtained in step (2) to 100 uL of competent cells prepared in step (4). The specific transformation steps are the same as described in step (3). Spread the cultured cells on LB solid medium containing both kanamycin and spectinomycin resistance, and culture at 37°C for 12-16 h. Select positive clones for PCR verification and sequencing.

[0063] (6) Elimination of pcrEG and pCpf1 plasmids from verified single colonies. First, the pcrEG plasmid needs to be eliminated. The principle of elimination is that the pCpf1 plasmid carries an sgRNA sequence targeting the pcrEG plasmid backbone, strictly regulated by the rhamnose promoter. The specific procedure is as follows: single colonies are picked and placed in LB liquid medium containing kanamycin and a final concentration of 0.1 mol of rhamnose. After incubation at 37°C for 12-16 h, they are streaked onto LB solid medium containing kanamycin. After further incubation at 37°C for 12-16 h, single colonies picked from the solid plates are copied onto LB solid plates containing kanamycin and LB solid plates containing both kanamycin and spectinomycin. Both solid plates are incubated at 37°C for 12-16 h. Single colonies that can grow on the kanamycin plate but cannot grow on the plate containing both kanamycin and spectinomycin are considered to have eliminated the pcrEG plasmid.

[0064] (7) After eliminating the pcrEG plasmid, the next step is to eliminate the pCpf1 plasmid. The plasmid contains... sacB The gene, which cannot grow in a sucrose environment, was used as a selection marker for plasmid elimination. Single colonies after pcrEG plasmid elimination were picked and placed in LB liquid medium, with glucose added to a final concentration of 5 g / L. After incubation at 37°C for 12–16 h, the colonies were streaked onto LB solid medium containing 5 g / L glucose and 10 g / L sucrose. After further incubation for 12–16 h, single colonies picked from the solid plates were copied onto LB solid plates containing kanamycin and those without antibiotics. Both solid plates were incubated at 37°C for 12–16 h. Single colonies that grew on the antibiotic-free LB solid plate but not on the kanamycin-containing plate were considered to have eliminated the pCpf1 plasmid.

[0065] (8) Targeting the genome fliK Site-integrated lactose transport enzyme gene lacY , motA Site integration of glucose-6-phosphate isomerase gene pgi , poxB Site integration of glutamine-fructose-6-phosphate aminotransferase gene glmS And inarsB Site integration of glucosamine-1-phosphoacetyltransferase gene glmU The gene integration procedure follows the steps described above. β-galactosidase gene. lacZ Glucosamine-6-phosphate deaminase gene nagB UDP-acetylglucosamine epiisomerase gene wecB and UDP-glucose dehydrogenase gene ugd The knockout process follows the steps described above, with the only difference being that there are no integration sequences between homologous arms when constructing the pcrEG plasmid.

[0066] Table 1 Primer Information

[0067]

[0068]

[0069] Example 2: Screening of protein assembly scaffolds

[0070] Currently, there are many types of commonly used protein scaffolds. In this embodiment, three different protein assembly scaffolds were first tested: RIAD / RIDD, CC-Di-A / B, and PDZ / PDZlig-SH3 / SH3lig. Since different protein assembly scaffolds and their ligands have different effects on the enzymes themselves at the C-terminus and N-terminus, ligation was attempted at the N-terminus and C-terminus for these three different protein scaffolds. For example, for the RIAD / RIDD protein assembly peptide pair, a total of four plasmids were constructed as shown below: pCDFDuet-RIAD-lgtA-RIDD-lgtB, pCDFDuet-RIAD-lgtA-lgtB-RIDD, pCDFDuet-lgtA-RIAD-RIDD-lgtB, and pCDFDuet-lgtA-RIAD-lgtB-RIDD. Ligation was performed at the C-terminus and N-terminus of the two glycosyltransferases, respectively. Similarly, four plasmids were also constructed for CC-Di-A / B and PDZ / PDZlig-SH3 / SH3lig. The primers required for plasmid construction are shown in Table 2. Taking the construction of a plasmid with both linker peptides at the C-terminus of PDZ / PDZlig-SH3 / SH3lig as an example, the specific method of plasmid construction is as follows: Figure 2 The method shown constructs the recombinant plasmid pCDFDuet-lgtA-SH3lig-lgtB-PDZlig, which mainly includes P tac -lgtAB and P J23119Two expression cassettes, PDZ-SH3 and β-1,3-N-acetylglucosamine aminotransferase (lgtA) and β-1,4-galactosyltransferase (lgtB), as well as the protein assembly scaffolds PDZ and SH3, were codon-optimized. Their amino acid and nucleotide sequences are shown in SEQ ID NO. 1-8. Using the original pCDFDuet-lgtAB as a template, the β-1,3-N-acetylglucosamine aminotransferase and β-1,4-galactosyltransferase genes were linearized using lgtA-F / R and lgtB-F / R, respectively. The fused protein assembly scaffold PDZ-SH3 was linearized using SH3PDZ / FR. The commercial vector pCDFDuet-1 was linearized into two fragments using pCDFDuet-AF / R and SF / R, where PDZlig and SH3lig are already included in the primers. The high-fidelity enzyme used was Takara PrimeSTAR Max. PCR conditions were: 98℃ for 3 minutes, 98℃ for 15 seconds, 55℃ for 15 seconds, and 72℃ for 10 seconds / kbp, for 35 cycles. After PCR, the purified fragments were used in equimolar amounts for plasmid construction using Beyotime one-step cloning. The formulation was: 5 μL premixed enzyme, 1 μL equimolar fragment, 1 μL ddH2O, incubated at 50℃ for 1 hour. The entire 10 μL system was then transformed into... E. coli In DH5α, plasmids were extracted and sequenced. The correctly sequenced pCDFDuet-lgtA-SH3lig-lgtB-PDZlig plasmid was reserved for later use. Then, using the constructed pCDFDuet-lgtA-SH3lig-lgtB-PDZlig plasmid as a template, the remaining three plasmids linked to different CN ends were constructed.

[0071] The remaining plasmids are processed according to the above steps. Unlike PDZ / PDZlig-SH3 / SH3lig, the other two protein assembly scaffolds only require the ligands to be linked to the two glycosyltransferases, without the need to link them to separate protein assembly scaffolds.

[0072] Table 2 Primers required for protein assembly

[0073]

[0074]

[0075] Example 3: Efficient synthesis of lactyl-N-neotetrasaccharides by Escherichia coli based on protein assembly scaffolds

[0076] The competent strain constructed in Example 1 was prepared into competent cells using the Takara competent cell preparation kit. The original plasmid pCDFDuet-lgtAB and 12 plasmids containing a protein assembly scaffold were transformed into the host bacteria, respectively. The transformed cells were plated on plates containing 100 μg / mL spectinomycin to obtain the synthetic strains EL01-EL13. First, the seed culture was cultured in LB medium. After 12 h of culture, the EL01-EL13 seed culture was inoculated into the fermentation medium at a 6% inoculum. The culture was allowed to develop OD... 600 When the concentration reached 0.8, induction was performed at 30°C with IPTG at a final concentration of 0.2 mM for 60 h. The fermentation medium was formulated as follows: peptone 12 g / L, yeast extract 24 g / L, glycerol 5 g / L, dipotassium hydrogen phosphate 2.31 g / L, dipotassium hydrogen phosphate 12.43 g / L, and lactose 5 g / L.

[0077] After fermentation, the supernatant from centrifugation of the fermentation broth was diluted and passed through a 0.22 μm membrane. The yield was detected by high-performance anion exchange chromatography with pulsed amperometry. The results are shown in the table below. The yield of lactoyl-N-neotetrasaccharide in the control strain without using the protein assembly scaffold was 573 mg / L. Using other protein assembly scaffolds did not have a significant effect on increasing the yield of lactoyl-N-neotetrasaccharide. However, the yield of lactoyl-N-neotetrasaccharide in the experimental strain reached 1699 mg / L after using PDZ / PDZlig and SH3 / SH3lig scaffolds, an increase of 198%.

[0078] Table 3. LNnT yield using scaffolds assembled with different proteins

[0079] Therefore, assembling scaffolds using PDZ / PDZlig and SH3 / SH3lig proteins and attaching their ligands to the C-terminus of the two glycosyltransferases significantly promotes the production of lactyl-N-neotetrasaccharide.

[0080] After screening out the only protein scaffold that can be used to promote the assembly of lactyl-N-neotetrasaccharide, studying the stoichiometry between the scaffold and ligand may further optimize the ratio between enzymes, and has the potential to further improve the yield of lactyl-N-neotetrasaccharide.

[0081] Plasmids PDZSH3-2, PDZSH3-3, and PDZSH3-4 were constructed using ratios of 1:2, 1:3, and 1:4, respectively. The construction method for the plasmids is as follows: using the original pCDFDuet-lgtAB as a template, and employing the same GS flexible linker peptide as described above, ligands of different lengths were introduced using primers. Specific fermentation and detection steps were performed as described in Example 3. The results (Table 4) showed that the original 1:1 stoichiometric ratio was most favorable for the synthesis of lactyl-N-neotetrasaccharide.

[0082] Table 4 Yield Results for Different Mixture Ratios

[0083] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for synthesizing lactyl-N-neotetrasaccharide by introducing a protein assembly scaffold, characterized in that, Includes the following steps: The product was produced by fermentation using recombinant Escherichia coli containing a protein assembly scaffold. The recombinant *E. coli* strain had the β-galactosidase gene, glucosamine-6-phosphate deaminase gene, UDP-acetylglucosamine epimerase gene, and UDP-glucose dehydrogenase gene knocked out. It overexpressed the lactose transport enzyme gene, glucose-6-phosphate isomerase gene, glutamine-fructose-6-phosphate aminotransferase gene, and glucosamine-1-phosphate acetyltransferase gene. It also heterologously expressed the β-1,3-N-acetylglucosamine aminotransferase gene fused with at least one SH3lig at the C-terminus, the β-1,4-galactosyltransferase gene fused with at least one PDZlig at the C-terminus, and the protein assembly scaffolds PDZ and SH3. The amino acid sequences of the β-1,3-N-acetylglucosamine aminotransferase are shown in SEQ ID NO.1, the β-1,4-galactosyltransferase gene in SEQ ID NO.3, the PDZ protein assembly scaffold in SEQ ID NO.5, the SH3 protein assembly scaffold in SEQ ID NO.7, and the PDZlig protein assembly scaffold in SEQ ID NO.

8. As shown in NO.9, the amino acid sequence of SH3lig is shown in SEQ ID NO.10; the IDs of the β-galactosidase gene are 945006, glucosamine-6-phosphate deaminase gene, UDP-acetylglucosamine epiisomerase gene, UDP-glucose dehydrogenase gene, lactose transport enzyme gene, glucose-6-phosphate isomerase gene, glutamine-fructose-6-phosphate aminotransferase gene, glutamine-fructose-6-phosphate aminotransferase gene, and glucosamine-1-phosphate acetyltransferase gene are 948246. The SH3lig and PDZlig are respectively fused to the C-terminus of the β-1,3-N-acetylglucosamine aminotransferase gene and the β-1,4-galactosyltransferase gene via a first flexible linker peptide, wherein the first flexible linker peptide is (GGGGS)m, and m is an integer from 1 to 5. The protein assembly scaffold PDZ and SH3 are connected by a second flexible linker peptide, which is (GS)n, where n is an integer from 3 to 10.

2. The method according to claim 1, characterized in that, The ratio of PDZlig to SH3lig is 1:1-4.

3. The method according to claim 1, characterized in that, The β-1,3-N-acetylglucosamine aminotransferase gene fused with SH3lig at the C-terminus and the β-1,4-galactosyltransferase gene fused with PDZlig at the C-terminus are expressed by inducible promoters, while the protein assembly scaffolds PDZ and SH3 are expressed by constitutive promoters.

4. The method according to claim 3, characterized in that, The inducible promoters include the tac promoter, and the constitutive promoters include the J23119 promoter.

5. The method according to claim 1, characterized in that, The host was Escherichia coli K-12 MG1655.

6. The method according to claim 1, characterized in that, The fermentation production includes the following steps: single colonies of recombinant *E. coli* are picked and placed in a seed culture medium to obtain a seed solution, which is then inoculated into the fermentation medium at an inoculation rate of 3%-10%. The fermentation process is then carried out until the OD (Organic Demand) is reached. 600 When the concentration reaches 0.5-1.2, production is induced at 25-35℃.

7. The method according to claim 6, characterized in that, The fermentation medium comprises: peptone 10-14 g / L, yeast extract 22-26 g / L, glycerol 3-5 g / L, dipotassium hydrogen phosphate 2.2-2.5 g / L, dipotassium hydrogen phosphate 12.2-12.5 g / L, and lactose 4-6 g / L.

8. A recombinant *Escherichia coli* strain that synthesizes lactyl-N-neotetrasaccharide, characterized in that, The recombinant *E. coli* strain had the following genes knocked out: β-galactosidase, glucosamine-6-phosphate deaminase, UDP-acetylglucosamine epimerase, and UDP-glucose dehydrogenase. It overexpressed the following genes: lactose transport enzyme, glucose-6-phosphate isomerase, glutamine-fructose-6-phosphate aminotransferase, and glucosamine-1-phosphate acetyltransferase. It also heterologously expressed the following genes: β-1,3-N-acetylglucosamine aminotransferase fused to at least one SH3lig at the C-terminus; β-1,4-galactosyltransferase fused to at least one PDZlig at the C-terminus; and the protein assembly scaffolds PDZ and SH3. The amino acid sequences of the β-1,3-N-acetylglucosamine aminotransferase are shown in SEQ ID NO.1, β-1,4-galactosyltransferase in SEQ ID NO.3, PDZ in SEQ ID NO.5, SH3 in SEQ ID NO.7, and PDZlig in SEQ ID NO.

8. As shown in NO.9, the amino acid sequence of SH3lig is shown in SEQ ID NO.10; the IDs of the β-galactosidase gene are 945006, glucosamine-6-phosphate deaminase gene, UDP-acetylglucosamine epiisomerase gene, UDP-glucose dehydrogenase gene, lactose transport enzyme gene, glucose-6-phosphate isomerase gene, glutamine-fructose-6-phosphate aminotransferase gene, glutamine-fructose-6-phosphate aminotransferase gene, and glucosamine-1-phosphate acetyltransferase gene are 948246. The SH3lig and PDZlig are respectively fused to the C-terminus of the β-1,3-N-acetylglucosamine aminotransferase gene and the β-1,4-galactosyltransferase gene via a first flexible linker peptide, wherein the first flexible linker peptide is (GGGGS)m, and m is an integer from 1 to 5. The protein assembly scaffold PDZ and SH3 are connected by a second flexible linker peptide, which is (GS)n, where n is an integer from 3 to 10.

9. The recombinant Escherichia coli according to claim 8, characterized in that, The ratio of PDZlig to SH3lig is 1:1-4.

10. The method for constructing recombinant Escherichia coli according to claim 8 or 9, characterized in that, Includes the following steps: S1. Knock out the β-galactosidase gene, glucosamine-6-phosphate deaminase gene, UDP-acetylglucosamine epimerase gene, and UDP-glucose dehydrogenase gene in the host strain of Escherichia coli, and overexpress the lactose transport enzyme gene, glucose-6-phosphate isomerase gene, glutamine-fructose-6-phosphate aminotransferase gene, and glucosamine-1-phosphate acetyltransferase gene in the genome to obtain the starting strain; S2. PDZlig and SH3lig were linked to the C-terminus of β-1,3-N-acetylglucosamine aminotransferase and β-1,4-galactosyltransferase, respectively. The protein assembly scaffold PDZ and SH3 were fused and expressed together, and then linked to the vector pCDFDuet-1 to obtain the recombinant plasmid. S3. The recombinant plasmid obtained in S2 is introduced into the starting strain in S1 to obtain the above-mentioned recombinant Escherichia coli.

11. The use of the recombinant Escherichia coli according to claim 8 or 9 in the preparation of lactoyl-N-neotetrasaccharide.

12. A method for promoting the synthesis of lactyl-N-neotetrasaccharide in Escherichia coli, characterized in that, The method involves synthesizing lactoyl-N-neotetrasaccharide using the recombinant Escherichia coli as described in claim 8 or 9.

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