Method for increasing yield of lactose-N-tetrasaccharide based on dynamic regulation and control system
By constructing a lactose-N-tetrasaccharide synthesis pathway in Escherichia coli and utilizing the interaction of micro-region proteins and RIAD/RIDD tags for spatial regulation, the problem of lack of dynamic regulation in existing technologies has been solved, achieving efficient production of lactose-N-tetrasaccharide using glucose as a substrate and reducing production costs.
Patent Information
- Application Number
- CN202511212696.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-28
- Publication Date
- 2025-12-02
AI Technical Summary
Existing technologies lack dynamic regulation methods for the microbial synthesis of lactose-N-tetrasaccharides, and most cell factories require the use of glycerol as a base material, which increases production costs.
A lactose-N-tetrasaccharide synthesis pathway was constructed in *E. coli*. Spatial regulation of β-1,3-N-acetylglucosamine transferase and β-1,3-N-acetylglucosamine transferase was achieved by utilizing micro-region proteins and RIAD/RIDD tag interactions. A growth-coupled promoter was used to express 6-phosphofructokinase and a degradation tag to achieve uncoupling of cell growth and product synthesis, thus constructing an engineered *E. coli* strain for glucose production.
It achieved a significant increase in lactose-N-tetrasaccharide yield, reaching 2.89 g/L after 72 hours of fermentation, with a production intensity of 0.04 g/L/h, reducing production costs and providing a new tool and dynamic control method for lactose-N-tetrasaccharide production.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of bioengineering technology, and in particular to a method for increasing the yield of lactose-N-tetrasaccharide based on a dynamic regulation system. Background Technology
[0002] Lactose-N-tetrasaccharide is an important component of human milk oligosaccharides, a tetrasaccharide compound composed of galactose, glucose, and N-acetylglucosamine. It has health benefits such as stimulating beneficial intestinal bacteria, regulating gut microbiota, and modulating the immune system. Microbial fermentation synthesis of lactose-N-tetrasaccharide offers advantages such as environmental friendliness and high product yield, and is widely used in infant formula additives, special medical foods, and healthcare industries.
[0003] Currently, green biomanufacturing centers on the reaction processes of microbial cells and their enzymes, utilizing advanced synthetic biology techniques and metabolic engineering strategies to efficiently synthesize lactose-N-tetrasaccharides. Therefore, microbial manufacturing using metabolic engineering and synthetic biology methods has become an important means of constructing 2'-fucosylated lactose cell factories, mainly including: (i) synthetic pathway construction, producing lactose-N-tetrasaccharides by overexpressing key genes glmM, glmU, glmS, lgtA, and wbgO in plasmids and deleting byproduct genes; (ii) cofactor regeneration, enhancing the catalytic efficiency of the key enzyme GlmU for LNT production by regenerating uridine 5'-triphosphate based on optimized metabolic pathways; and (iii) transporter protein engineering, screening endogenous E. coli transporter protein genes (setA, setB, ydeA, and mdfA) and identifying the key gene setA to enhance precursor LNT production. II. This increases the yield of the target LNT; (iv) Genome integration: To reduce the metabolic burden of the lactose-N-tetrasaccharide pathway, key genes lgtA (β-1,3-N-acetylglucosamine transferase), wbdO (β-1,3-galactosyltransferase), and galE (UDP-galactose-4-epimerase) were integrated into the genome, realizing a plasmid-free lactose-N-tetrasaccharide synthesis system. Currently, existing technologies lack methods for the dynamic regulation of enzymes in the microbial colocalization pathway of lactose-N-tetrasaccharide synthesis, and most cell factories require glycerol as a substrate for fermentation, increasing production costs. Summary of the Invention
[0004] To address the aforementioned technical problems, this invention constructs a lactose-N-tetrasaccharide synthesis pathway in *E. coli*. Spatial regulation of β-1,3-N-acetylglucosamine transferase and β-1,3-galactosyltransferase is achieved through micro-region proteins and RIAD / RIDD tag interactions. A growth-coupled promoter is used to express 6-phosphofructokinase and a degradation tag, thereby uncoupling cell growth from product synthesis. This results in an engineered *E. coli* strain with significantly increased lactose-N-tetrasaccharide production, capable of producing lactose using glucose as a substrate.
[0005] The first objective of this invention is to provide a recombinant *E. coli* strain that produces lactose-N-tetrasaccharide, said recombinant *E. coli* strain obtained by the following modifications to a chassis strain:
[0006] (1) Knock out the UDP-N-acetylglucosamine-2-epimerase encoding gene wecB, the β-galactosidase encoding gene lacZ, and the 6-phosphofructokinase encoding gene pfkA;
[0007] (2) Express the glucosamine 6-phosphate synthase encoding gene glmS, the N-acetylglucosamine 1-phosphate uridine transferase / glucosamine 1-phosphate acetyltransferase encoding gene glmU and the phosphoglucosamine mutase encoding gene glmM;
[0008] (3) Express the β-1,3-N-acetylglucosamine transferase encoding gene lgtA and the β-1,3-galactosyltransferase encoding gene wbgO, and attach a polypeptide tag or tag ligand to the N-terminus or C-terminus of lgtA and wbgO, respectively; express a micro-region protein using a stationary promoter, and attach a polypeptide tag or tag ligand to the N-terminus or C-terminus of the micro-region protein, wherein the polypeptide tag and tag ligand bind specifically.
[0009] In one embodiment of the present invention, the nucleotide sequence of pfkA is shown in SEQ ID NO.15.
[0010] In one embodiment of the present invention, the nucleotide sequence of glmS is shown in SEQ ID NO.7, the nucleotide sequence of glmU is shown in SEQ ID NO.6, and the nucleotide sequence of glmM is shown in SEQ ID NO.5.
[0011] In one embodiment of the present invention, the nucleotide sequence of lgtA is shown in SEQ ID NO.9, and the nucleotide sequence of wbgO is shown in SEQ ID NO.10.
[0012] Furthermore, the polypeptide tag is RIAD, and the tag ligand is RIDD.
[0013] In one embodiment of the present invention, the nucleotide sequence of the RIAD is shown in SEQ ID NO.11, and the nucleotide sequence of the RIDD is shown in SEQ ID NO.12.
[0014] Furthermore, the nucleotide sequence of the micro-region protein is shown in SEQ ID NO.3.
[0015] Furthermore, the modification also includes expressing the phosphofructokinase-encoding gene pfkA and the degradation tag DAS+8 using a growth-coupled promoter.
[0016] In one embodiment of the present invention, the nucleotide sequence of the degradation tag DAS+8 is shown in SEQ ID NO.2.
[0017] Furthermore, the stable promoter is PbolA.
[0018] In one embodiment of the present invention, the nucleotide sequence of PbolA is shown in SEQ ID NO.13.
[0019] A second objective of the present invention is to provide a microbial agent comprising the above-mentioned recombinant Escherichia coli.
[0020] A third objective of this invention is to provide the application of the above-mentioned recombinant Escherichia coli or the above-mentioned microbial agent in the production of lactose-N-tetrasaccharide.
[0021] The fourth objective of this invention is to provide a method for producing lactose-N-tetrasaccharide by adding the aforementioned recombinant Escherichia coli or the aforementioned microbial agent to a fermentation system and fermenting it with glucose as a substrate.
[0022] The fifth objective of this invention is to provide a method for increasing the production of lactose-N-tetrasaccharide in *Escherichia coli* based on a dynamic regulation system. In an engineered bacterium producing lactose-N-tetrasaccharide, a polypeptide tag or tag ligand is attached to the N-terminus or C-terminus of the β-1,3-N-acetylglucosamine transferase gene *lgtA* and the β-1,3-galactosyltransferase gene *wbgO*, respectively. A micro-region protein is expressed using a stationary promoter, and a polypeptide tag or tag ligand is attached to the N-terminus or C-terminus of the micro-region protein. The polypeptide tag and tag ligand bind specifically.
[0023] Furthermore, the micro-region protein is an I8 protein, and its nucleotide sequence is shown in SEQ ID NO.3.
[0024] Furthermore, the engineered bacteria were used to express the phosphofructokinase-encoding gene pfkA and the degradation tag DAS+8 using a growth-coupled promoter.
[0025] In one embodiment of the present invention, the nucleotide sequence of pfkA is shown in SEQ ID NO.15.
[0026] In one embodiment of the present invention, the nucleotide sequence of the degradation tag DAS+8 is shown in SEQ ID NO.2.
[0027] The beneficial effects of this invention are:
[0028] This invention utilizes micro-region proteins and interacting short peptides to achieve spatial regulation of key enzymes in the lactose-N-tetrasaccharide synthesis pathway. It employs growth-phase promoters and degradation tags to uncouple E. coli growth and lactose-N-tetrasaccharide production. The resulting high-yield lactose-N-tetrasaccharide strain, using glucose as a substrate, achieved a lactose-N-tetrasaccharide yield of 2.89 g / L after 72 hours of fermentation, with a production intensity of 0.04 g / L / h. This demonstrates promising application prospects, provides a novel tool for lactose-N-tetrasaccharide production, and offers new ideas and solutions for the dynamic regulation of lactose-N-tetrasaccharide production. Attached Figure Description
[0029] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein:
[0030] Figure 1 Construction and characterization of the growth-coupled promoter in Example 1 of this invention;
[0031] Figure 2 This document describes the construction and characterization of the stable-phase promoter and micro-region protein I8 assembly in Example 2 of the present invention, where A represents the characterization of the stable promoter and B represents the characterization of the micro-region protein I8 assembly.
[0032] Figure 3 This is a schematic diagram of the lactose-N-tetrasaccharide synthesis pathway in Example 3 of the present invention;
[0033] Figure 4 This is a comparison of the yield of lactose-N-tetrasaccharide produced by the recombinant strain L1-L5 in shake-flask fermentation in Example 3 of the present invention;
[0034] Figure 5 This is a comparison of the yield of lactose-N-tetrasaccharide produced by recombinant strains L1-16 and L1-32 during shake-flask fermentation in Example 3 of the present invention;
[0035] Figure 6 This is a comparison of the yield of lactose-N-tetrasaccharide produced by recombinant strains L1-1 and L1-2 in shake-flask fermentation in Example 3 of the present invention. Detailed Implementation
[0036] 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.
[0037] (I) Culture medium
[0038] Seed culture medium: LB medium, containing 5 g / L yeast extract, 10 g / L peptone, and 10 g / L sodium chloride.
[0039] Fermentation medium: Standard fermentation medium (DM medium, 1L), comprising 4.0 g / L ammonium phosphate, 1.4 g / L magnesium sulfate heptahydrate, 13.5 g / L potassium dihydrogen phosphate, 1.7 g / L citric acid, and 10 mL / L trace metal element solution (10 g / L ferric chloride, 2.25 g / L zinc sulfate heptahydrate, 1.0 g / L copper sulfate pentahydrate, 0.35 g / L manganese sulfate monohydrate, 0.23 g / L sodium borate decahydrate, 0.11 g / L ammonium molybdate, 2.0 g / L calcium chloride dihydrate, pH = 6.8); glucose of appropriate concentration was added (40 g / L glucose for shake flask fermentation). Glucose was autoclaved separately. Before adding to the fermentation medium, the pH was adjusted to 7 with KOH and sterilized through a 0.22 μm membrane.
[0040] (II) Fermentation production of lactose-N-tetrasaccharide
[0041] The recombinant strain was inoculated into 20 mL of seed culture medium and cultured at 37 °C and 200-220 rpm for 12-18 h to obtain seed liquid. The seed liquid was then inoculated into a 250 mL shake flask containing 50 mL of DM culture medium at an inoculation rate of 1% (v / v). The pH was 7, and the pressure and temperature were kept constant at 30 °C or 37 °C and 200-220 rpm for a fermentation period of 72 h.
[0042] (III) Determination of lactose-N-tetrasaccharide content
[0043] Fermentation sample preparation: Take the fermentation broth sample, centrifuge at 12000 rpm for 5 min, take the supernatant or dilute the supernatant, filter it through a 0.22 μm aqueous membrane to remove impurities, and then perform quantitative analysis by HPLC.
[0044] Determination of lactose-N-tetrasaccharide content: The sample was analyzed using a Shimadzu high-performance liquid chromatograph and differential detector with a FinoM Carbohydrate Analysis (Rezex ROA-organic acid H+ (8%)) column. The mobile phase was 0.005M H2SO4, filtered through a 0.22μm filter membrane, degassed by sonication for 30 min, with a flow rate of 0.6 mL / min and a column temperature of 60℃. The injection volume was 10 μL.
[0045] Example 1: Characterization and Construction of Growth-Coupled Promoters
[0046] Using the pACYC engineered plasmid as a template, the original T7 promoter was replaced with the growth-coupled promoter PrrnA (nucleotide sequence shown in SEQ ID NO.1) via whole-plasmid PCR to obtain the pACYC-PrrnA plasmid. Green fluorescent protein (GFP) was fused with the degradation tag DAS+8 (nucleotide sequence shown in SEQ ID NO.2) for expression, and the GFP-DAS+8 fusion fragment was amplified. Then, the pACYC-PrrnA plasmid was digested with NdeI, and the GFP-DAS+8 fusion fragment was inserted into the digested pACYC-PrrnA plasmid via Novovitek-derived recombination to obtain the recombinant plasmid pACYC-PrrnA-GFP-DAS+8. This recombinant plasmid was transformed into *E. coli* BL21 competent cells, and fluorescence changes were measured after inoculation and transformation. The results are as follows: Figure 1 As shown, fluorescence expression gradually increased over time, reaching 7210 a.u. after 9 hours, after which the fluorescent protein was gradually degraded until it was completely degraded after 14 hours. This indicates that plasmid pACYC-PrrnA-GFP-DAS+8 has the function of expressing the gene during the growth phase and gradually degrading the gene expression after reaching the stationary phase.
[0047] Example 2: Construction and characterization of the assembly of the stationary promoter and microregion protein I8
[0048] Using the pACYC engineered plasmid as a template, the linearized vector was digested with NdeI and recovered via gel electrophoresis to amplify the green fluorescent protein (GFP). This GFP fragment was then fused with the I8 protein (nucleotide sequence shown in SEQ ID NO. 3) to obtain the I8-GFP fusion fragment. This I8-GFP fragment was inserted into the pACYC engineered plasmid to obtain the pACYC-I8-GFP plasmid. Further, the original promoter T7 of pACYC-I8-GFP was replaced with the stationary promoter Ps60 (nucleotide sequence shown in SEQ ID NO. 4) using whole-plasmid PCR to obtain the pACYC-Ps60-I8-GFP plasmid.
[0049] The recombinant plasmid pACYC-Ps60-I8-GFP was introduced into competent E. coli BL21 cells to obtain the strain E. coli BL21 / Ps60-I8-GFP containing the pACYC-Ps60-I8-GFP plasmid. The strain E. coli BL21 / Ps60-I8-GFP was inoculated into LB medium and induced at 30℃ for 24 h at 200 rpm. Fluorescence microscopy revealed that E. coli BL21 / Ps60-I8-GFP formed a micro-region system with GFP green fluorescence within the cells, i.e., micro-regions at both ends but not in the middle. This is significantly different from the uniform intracellular distribution of conventional GFP. Figure 2 B). Simultaneous fluorescence data analysis of E. coli BL21 / Ps60-I8-GFP revealed that expression controlled by the stationary phase promoter only gradually began to show fluorescence after 10 hours, indicating the effectiveness of the stationary phase promoter control. Figure 2 A).
[0050] Example 3: Detection of lactose-N-tetrasaccharide content in shake flasks
[0051] The de novo synthetic pathway of lactose-N-tetrasaccharide is as follows: Figure 3 As shown, using the E. coli K12 genome as a template, three fragments were amplified: the glmM gene encoding phosphoglucosamine mutase (nucleotide sequence shown in SEQ ID NO. 5), the glmU gene encoding N-acetylglucosamine 1-phosphouridine transferase / glucosamine 1-phosphoacetyltransferase (nucleotide sequence shown in SEQ ID NO. 6), and the glmS gene encoding glucosamine 6-phosphate synthase (nucleotide sequence shown in SEQ ID NO. 7). glmM was inserted into a pCDF plasmid digested with EcoRI and Hind III via homologous recombination to obtain the recombinant plasmid pCDF-glmM. glmU and glmS were fused using an RBS sequence (nucleotide sequence shown in SEQ ID NO. 8) to obtain the glmU-glmS fragment, which was then ligated to the pCDF-glmM plasmid via homologous recombination to obtain the pCDF-glmM-glmU-glmS plasmid.
[0052] The β-1,3-N-acetylglucosamine transferase encoding gene lgtA (nucleotide sequence shown in SEQ ID NO. 9) and the β-1,3-galactosyltransferase encoding gene wbgO (nucleotide sequence shown in SEQ ID NO. 10) were derived from Neisseria meningitidis and E. coli O55:H7, respectively. Through homologous recombination, lgtA was ligated into the Nde I-digested pRSF plasmid to obtain the pRSF-lgtA plasmid. wbgO was then ligated into the Bam H-digested pRSF-lgtA plasmid via homologous recombination to obtain the recombinant plasmid pRSF-lgtA-wbgO. Similarly, RIAD tags (nucleotide sequences shown in SEQ ID NO. 11) were ligated to the C-terminus of both lgtA and wbgO, respectively. The pRSF-lgtA-RIAD-wbgO and pRSF-lgtA-RIAD-wbgO-RIAD plasmids were obtained by whole-plasmid PCR.
[0053] The pACYC-Ps60-I8-GFP plasmid from Example 2 was modified by whole-plasmid PCR to replace the GFP protein with the tag RIDD (nucleotide sequence shown in SEQ ID NO. 12) to obtain the pACYC-Ps60-I8-RIDD plasmid. The pACYC-PrrnA-pfkA-DAS+8 plasmid was digested with Xho I to obtain a linearized vector. Then, the amplified Ps60-I8-RIDD fragment was ligated into the PrrnA-pfkA-DAS+8 plasmid via homologous recombination to obtain the pACYC-PrrnA-pfkA-DAS+8-Ps60-I8-RIDD recombinant plasmid. The Ps60 promoter in the recombinant plasmid pACYC-PrrnA-pfkA-DAS+8-Ps60-I8-RIDD was replaced with the PbolA (nucleotide sequence shown in SEQ ID NO.13) and Pfic (nucleotide sequence shown in SEQ ID NO.14) promoters by whole plasmid PCR, resulting in the recombinant plasmids pACYC-PrrnA-pfkA-DAS+8-PbolA-I8-RIDD and pACYC-PrrnA-pfkA-DAS+8-Pfic-I8-RIDD, respectively.
[0054] Based on the previously constructed *E. coli* BL21ΔwecBΔlacZ strain (derived from *Combinatorial Engineering of Microbial Cell Factory for 2'-fucosyllactose Biosynthesis*, *Process Biochemistry*, 2025, 156, 202-208.), the pfkA gene encoding phosphofructokinase PfkA (nucleotide sequence shown in SEQ ID NO. 15) was knocked out using CRISPR / CAS9 technology. Nucleic acid gel imaging confirmed successful pfkA gene knockout, yielding the *E. coli* BL21ΔwecBΔlacZΔpfkA strain. The plasmid pACYC-PrrnA-GFP-DAS+8 from Example 1 was then replaced with pfkA via whole-plasmid PCR to obtain the recombinant plasmid pACYC-PrrnA-pfkA-DAS+8.
[0055] The effectiveness of the regulatory system was verified by transforming different plasmids based on the experimental strain combinations.
[0056] (1) The recombinant plasmids pACYC-pCDF-glmM-glmU-glmS, pRSF-lgtA-RIAD-wbgO-RIAD and pACYC-PrrnA-pfkA-DAS+8-Ps60-I8-RIDD were introduced into Escherichia coli BL21ΔwecBΔlacZΔpfkA strain to construct strain L1;
[0057] (2) The recombinant plasmid pACYC-pCDF-glmM-glmU-glmS+pRSF-lgtA-wbgO+PrrnA-pfkA-DAS+8-Ps60-I8-RIDD was introduced into Escherichia coli BL21ΔwecBΔlacZΔpfkA strain to construct strain L2.
[0058] (3) The recombinant plasmid pACYC-pCDF-glmM-glmU-glmS+pRSF-lgtA-RIAD-wb gO-RIAD+Ps60-I8-RIDD was introduced into Escherichia coli BL21ΔwecBΔlacZΔpfkA strain to construct strain L3.
[0059] (4) The recombinant plasmid pACYC-pCDF-glmM-glmU-glmS+pRSF-lgtA-RIAD-wb gO-RIDD was introduced into Escherichia coli BL21ΔwecBΔlacZΔpfkA strain to construct strain L4.
[0060] (5) The recombinant plasmid pACYC-pCDF-glmM-glmU-glmS+pRSF-lgtA-wbgO was introduced into Escherichia coli BL21ΔwecBΔlacZΔpfkA strain to construct strain L5.
[0061] The above-mentioned lactose-N-tetrasaccharide-producing strain was cultured in shake flasks in DM medium containing glucose, and the lactose-N-tetrasaccharide content in the fermentation broth was identified. The results are as follows: Figure 5 As shown, with the extension of culture time, the lactose-N-tetrasaccharide yield of recombinant strain L1 was 2.35 g / L, while the lactose-N-tetrasaccharide yields of the control group (unregulated micro-region strain L2, unregulated growth strain L3, regulated short peptide interaction strain L4, and strain L5 without a regulatory system) reached 2.06 g / L, 2.15 g / L, 1.95 g / L, and 1.85 g / L, respectively. The yield of recombinant strain L1 was increased by 14.08%, 9.3%, 20.51%, and 27.03% compared to the control group, respectively. The results indicate that this dynamic regulation system can effectively increase the lactose-N-tetrasaccharide yield. Figure 4 ).
[0062] Furthermore, based on strain L1, the I8 protein in pACYC-PrrnA-pfkA-DAS+8-Ps60-I8-RIDD was replaced with I16 (nucleotide sequence as shown in SEQ ID NO.16) and I32 (nucleotide sequence as shown in SEQ ID NO.17) using whole-plasmid PCR, thus constructing the plasmids pACYC-PrrnA-pfkA-DAS+8-Ps60-I16-RIDD and pACYC-PrrnA-pfkA-DAS+8-Ps60-I32-RIDD. By replacing the pACYC-PrrnA-pfkA-DAS+8-Ps60-I8-RIDD plasmid of strain L1, strains L1-16 and L1-32 were obtained. The lactose-N-tetrasaccharide yields of strains L1-16 and L1-32 reached 2.3 g / L and 2.2 g / L, respectively, indicating that the repeated fusion micro-region protein has a certain influence on the recruitment of pathway enzymes. Therefore, subsequent experiments will continue to optimize based on strain L1. Figure 5 ).
[0063] To improve the yield of lactose-N-tetrasaccharide, the biosynthesis of lactose-N-tetrasaccharide was regulated using promoters of different strengths during the stationary phase. The plasmids pACYC-PrrnA-pfkA-DAS+8-PbolA-I8-RIDD and pACYC-PrrnA-pfkA-DAS+8-Pfic-I8-RIDD in recombinant strain L1 were replaced, respectively, to obtain recombinant strains L1-1 and L1-2. Fermentation was then performed, and the results showed that the PbolA promoter in recombinant strain L1-1 resulted in a lactose-N-tetrasaccharide yield of 2.89 g / L, which was 65.14% higher than that of the control group recombinant strain L5, indicating the feasibility of the optimized dynamic regulation system. Figure 6 ).
[0064] 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 recombinant *Escherichia coli* strain that produces lactose-N-tetrasaccharide, characterized in that, The recombinant Escherichia coli was obtained by modifying the chassis strain as follows: (1) Knock out the UDP-N-acetylglucosamine-2-epimerase encoding gene wecB, the β-galactosidase encoding gene lacZ, and the 6-phosphofructokinase encoding gene pfkA; (2) Express the glucosamine 6-phosphate synthase encoding gene glmS, the N-acetylglucosamine 1-phosphate uridine transferase / glucosamine 1-phosphate acetyltransferase encoding gene glmU and the phosphoglucosamine mutase encoding gene glmM. (3) Express the β-1,3-N-acetylglucosamine transferase encoding gene lgtA and the β-1,3-galactosyltransferase encoding gene wbgO, and attach a polypeptide tag or tag ligand to the N-terminus or C-terminus of lgtA and wbgO, respectively; express a micro-region protein using a stationary promoter, and attach a polypeptide tag or tag ligand to the N-terminus or C-terminus of the micro-region protein, wherein the polypeptide tag and tag ligand bind specifically.
2. The recombinant Escherichia coli according to claim 1, characterized in that: The nucleotide sequence of the microregion protein is shown in SEQ ID NO.
3.
3. The recombinant Escherichia coli according to claim 1, characterized in that: The modification also includes using a growth-coupled promoter to express the phosphofructokinase-encoding gene pfkA and the degradation tag DAS+8.
4. The recombinant Escherichia coli according to claim 1, characterized in that: The stable promoter is PbolA.
5. A microbial agent comprising any one of the recombinant Escherichia coli according to claims 1-4.
6. The use of the recombinant Escherichia coli according to any one of claims 1-4 or the microbial agent according to claim 5 in the production of lactose-N-tetrasaccharide.
7. A method for producing lactose-N-tetrasaccharide, characterized in that: The recombinant Escherichia coli of any one of claims 1-4 or the microbial agent of claim 5 is added to the fermentation system, and glucose is used as the substrate for fermentation production.
8. A method for increasing the yield of lactose-N-tetrasaccharide in Escherichia coli based on a dynamic control system, characterized in that: In engineered bacteria that produce lactose-N-tetrasaccharides, polypeptide tags or tag ligands are linked to the N-terminus or C-terminus of the β-1,3-N-acetylglucosamine transferase gene lgtA and the β-1,3-galactosyltransferase gene wbgO, respectively. Microregion proteins are expressed using a stationary promoter, and polypeptide tags or tag ligands are linked to the N-terminus or C-terminus of the microregion proteins. The polypeptide tags and tag ligands bind specifically.
9. The method according to claim 8, characterized in that: The micro-region protein is the I8 protein, and its nucleotide sequence is shown in SEQ ID NO.
3.
10. The method according to claim 8, characterized in that: The engineered bacteria were used to express the phosphofructokinase-encoding gene pfkA and the degradation tag DAS+8 using a growth-coupled promoter.