Transporter mutant with high efficiency of transporting lnt and application thereof

Abstract

The application discloses a mutant of a transport protein with high-efficiency LNT transport function and application thereof, and belongs to the technical field of biotechnology. The technical problem to be solved is to provide a mutant of a transport protein capable of efficiently transporting lactose-N-tetrasaccharide (LNT) from a bacterial cell to outside the cell, so as to solve the technical problem that LNT intracellular accumulation leads to short cell production cycle, low yield and difficulty in separation and purification of a downstream product in the prior art. The technical solution is as follows: key amino acid residues in a substrate binding pocket of a sugar efflux transport protein JWZ8 from Bacillus are subjected to site-directed mutation, a mutant of the transport protein with enhanced recognition ability and transport efficiency for the substrate LNT is obtained, and the mutant is expressed in recombinant Escherichia coli producing LNT, so that the extracellular yield of LNT can be significantly improved.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically to the fields of genetic engineering and metabolic engineering, and specifically to a transporter protein mutant that efficiently transports LNTs and its applications. Background Technology

[0002] For understanding the technical content of this invention: Human milk oligosaccharides (HMOs) are a group of structurally diverse non-conjugated polysaccharides, ranking as the third most abundant nutrient in breast milk after lactose and lipids. Lactose-N-tetrasaccharide (LNT) is one of the core structures of HMOs and also an important precursor backbone for fucoidylation and sialylation. Therefore, the synthesis of LNTs is crucial for the synthesis of many HMOs.

[0003] Current research indicates that lactobacilli (LNTs) can increase the abundance of beneficial gut bacteria, prevent pathogen adhesion, strengthen the intestinal barrier, regulate immunity, and have antiviral effects. As the biological value of LNTs as a bioactive component of breast milk continues to be confirmed, their commercial value is also gradually increasing.

[0004] Microbial fermentation using metabolically engineered bacteria is currently the main method for large-scale production of lactose (LNT). The LNT synthesis process is as follows: β-1,3-N-acetylglucosamine transferase catalyzes the synthesis of lactose-N-trisaccharide from UDP-GlcNAc and lactose. Subsequently, lactose-N-trisaccharide reacts with UDP-gal under the action of β-1,3-galactosyltransferase to generate LNT.

[0005] Relevant patent documents retrieved: The country of origin for this publication is China, publication number CN121472116A, publication date February 6, 2026. The document, titled "A Recombinant Escherichia coli Synthesizing Sialyllactose-N-Tetrasaccharide c, Its Construction Method and Application," discloses a recombinant Escherichia coli strain that synthesizes sialyllactose-N-tetrasaccharide c, its construction method, and its application. This involves optimizing the supply of its precursor CMP-Neu5Ac by adding a new synthetic pathway; further increasing the yield of sialyllactose-N-tetrasaccharide c and reducing the accumulation of precursors LNnT, LNTⅡ, and the byproduct 6'-SL by screening for highly efficient and specific α2,6-sialyltransferases and integrating them into the E. coli BL21(DE3) genome; and reducing the inhibitory effect caused by the intracellular accumulation of high concentrations of sugar products by adding a sugar efflux transporter gene. By culturing and fermenting recombinant Escherichia coli, a high yield of sialyl lactose-N-tetrasaccharide c can be synthesized, and the proportion of the obtained product sialyl lactose-N-tetrasaccharide c in the total HMO can be further increased.

[0006] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: In actual fermentation processes, lactose-N-tetrasaccharide (LNT) with a tetrasaccharide structure is difficult for bacterial cells to transfer from intracellular to extracellular medium. With prolonged fermentation, intracellular LNT accumulation leads to cell rupture and premature fermentation termination, limiting yield increases and causing difficulties in the separation and purification of downstream products. However, there are currently few reports on LNT efflux, thus requiring suitable methods to promote LNT efflux, which is crucial for large-scale microbial production of lactose-N-tetrasaccharides. While patent document CN121472116A mentions integrating the sugar efflux transporter gene setS to reduce the inhibitory effect of high-concentration sugar product accumulation in cells, it does not address the modification of the transporter itself to enhance its recognition and transport efficiency of the specific substrate LNT, resulting in limited transport efficiency. Current research on LNT efflux is scarce, especially research on improving LNT transport efficiency through site-directed mutagenesis of transporters. Existing technologies lack effective solutions for modifying transporters to improve LNT transport efficiency. Summary of the Invention

[0007] The purpose of this invention is to provide: A highly efficient LNT transporter mutant and its application, along with related technologies, to address technical problems in existing technologies, such as the short cell production cycle and low yield caused by intracellular LNT accumulation, difficulties in the separation and purification of downstream products, and the lack of effective solutions for modifying transporters to improve LNT transport efficiency, or combinations thereof.

[0008] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0009] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0010] Definitions of the standard terminology can be found in the references “Molecular Cloning: A Laboratory Manual (Sambrook J, Russell D W. Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press, 2001),” “Microbiology Tutorial (4th Edition), Higher Education Press, author: Zhou Deqing, 2020,” “Modern Molecular Biology (5th Edition), Higher Education Press, authors: Zhu Yuxian, Li Yi, Zheng Xiaofeng, and Guo Hongwei, 2019-06-19,” and “Genetic Engineering, Higher Education Press, 2013-08-01.”

[0011] Unless otherwise stated, conventional methods within the scope of the art, such as gene cloning, PCR amplification, site-directed mutagenesis, seamless cloning, heat shock conversion, electroporation conversion, microbial culture, and high-performance liquid chromatography detection, shall be used.

[0012] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0013] The terms “optional / arbitrary” or “optionally / arbitrarily” mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0014] As used herein, the term "transporter protein" refers to a membrane protein located on the cell membrane that can transport a specific substrate from inside the cell to outside the cell. In this invention, transporter proteins specifically refer to those derived from Bacillus subtilis (Bacillus). Bacillus sp The sugar efflux transporter JWZ8 (Genbank ID: WP_090989553.1) and its mutants of OV322.

[0015] The term “lactose-N-tetrasaccharide” used in this article refers to LNT, a human lactose oligosaccharide core component with a tetrasaccharide structure, namely Gal-β1,3-GlcNAc-β1,3-Gal-β1,4-Glc.

[0016] As used herein, the term "mutant" refers to a protein variant obtained by substituting, deleting, or inserting one or more amino acid residues in the amino acid sequence of a parent protein using site-directed mutagenesis. In this invention, a mutant specifically refers to a variant obtained by substituting alanine or tyrosine residues of a specific amino acid residue in the JWZ8 protein.

[0017] The term "substrate-binding pocket" used in this article refers to the region in the three-dimensional structure of a transporter protein that specifically binds to substrate molecules. It consists of several amino acid residues, the spatial arrangement and chemical properties of which determine the transporter protein's ability to recognize and transport substrates.

[0018] In a first aspect, the present invention provides: a transporter protein mutant for efficient LNT transport, wherein the transporter protein mutant is formed by mutating amino acid residues of a sugar transporter protein with an amino acid sequence as shown in SEQ ID NO:4, and the mutation is any one of the following: (i) The methionine at position 35 was mutated to alanine and named JWZ8-M35A; (ii) The phenylalanine at position 159 was mutated to tyrosine and named JWZ8-F159Y; (iii) The lysine at position 212 is mutated to alanine and named JWZ8-K212A; (iv) Tyrosine at position 237 was mutated to alanine and named JWZ8-Y237A; (v) The phenylalanine at position 266 is mutated to alanine and named JWZ8-F266A; (vi) Glycine at position 332 is mutated to alanine and named JWZ8-G332A; (vii) The isoleucine at position 335 is mutated to alanine and named JWZ8-I335A; (viii) Glutamine at position 339 was mutated to alanine and named JWZ8-Q339A; (ix) Glutamine at position 339 is mutated to alanine and phenylalanine at position 266 is mutated to alanine, named JWZ8-Q339A / F266A.

[0019] Among them, the sugar transporter protein is derived from Bacillus ( Bacillus sp. The gene encoding a sugar efflux transporter (OV322, ncbitaxon:1882764) (Genbank ID:WP_090989553.1), named JWZ8, was optimized according to the codon preference of *E. coli*. The amino acid sequence of this sugar transporter is shown in SEQ ID NO:4.

[0020] SEQ ID NO:4: MMNKIAAFRDKLLYIWRIPGLPVLFLLNIIYGLGASFFAPFSSLFGIDEAGMSNIGFGIFMTIMAVGGVVISSYIAKKSDTTLSRRRILIYSSLAGAAGYILFAYLRNYYALCLTGFFVLGSAAATVPQLWAYARDTMTAAKVPAEETPLIMNIFRMFFALSWIGGPALGAWLVMMLDFKGLFYFVAACYLLGSLTAIFLLKEIARQEVP NKESIKVKKYVSRLHILANLAAAFLLYSASSIHMLNVPQFVTKTLHGTEMQVGIIFSVAPIFEVPLMIIVGMMATKMDNSILIKAGYFLAFVYFLIFGFVSEPQQ IYPLQILSAAQVSITAGIAISYFQDFIPEAPGTATTLYMNVTQIGSTAGYLLFGFISSLISYGQLITIYTFFAGIGFLFITVYGKERQTNSKRNKQASESPPTSFK .

[0021] In a second aspect, the present invention provides: a nucleotide molecule that encodes the transporter protein mutant described in the first aspect.

[0022] According to some embodiments of the present invention, the recombinant nucleic acid may be a nucleic acid sequence optimized by E. coli codon preference.

[0023] Thirdly, the present invention provides a recombinant expression vector comprising the nucleotide molecules described in the second aspect.

[0024] The backbone vector of the recombinant expression vector is selected from pACYCDuet-1, pETDuet-1, pRSFDuet-1, or pCOLADuet. 1. Preferably, pCOLADuet-1. It is understood that the skeletal carrier includes, but is not limited to, the aforementioned carrier skeletal structure.

[0025] The recombinant expression vector further includes a promoter operatively linked to the nucleotide molecule.

[0026] Fourthly, the present invention provides: a recombinant host cell, said recombinant host cell comprising the nucleotide molecule described in the second aspect or the recombinant expression vector described in the third aspect.

[0027] According to some embodiments of the present invention, the recombinant host cell may be Escherichia coli.

[0028] Furthermore, the recombinant host cell is preferably BL21(DE3).

[0029] According to some embodiments of the present invention, the recombinant host cell further comprises a gene encoding β-1,3-N-acetylglucosamine transferase lgtA, a gene encoding β-1,3-galactosyltransferase wcf, and / or a gene encoding uridine diphosphate glucose-4-epimerase galE.

[0030] Fifthly, the present invention provides a method for producing LNTs, the method comprising culturing the recombinant host cells described in the fourth aspect and collecting LNTs.

[0031] According to some embodiments of the present invention, the culture is carried out in a culture medium containing a carbon source and a nitrogen source.

[0032] According to some embodiments of the present invention, the method further includes adding an inducer to induce the expression of the transporter mutant.

[0033] Further, the inducer is selected from at least one of IPTG, arabinose, lactose, rhamnose, and tetracycline. Even further, the inducer is preferably IPTG.

[0034] In a sixth aspect, the present invention provides the use of the transporter protein mutant described in the first aspect in the preparation of products for transporting LNTs.

[0035] The product in question is an engineered bacterium used to produce LNTs.

[0036] Examples 1-2 of this invention at least support the protection scope of "transporter mutants of LNT with high efficiency".

[0037] The term "highly efficient LNT transporter mutant" is derived from the foregoing explanation and / or the corresponding JWZ8-M35A, JWZ8-F159Y, JWZ8-K212A, JWZ8-Y237A, JWZ8-F266A, JWZ8-G332A, JWZ8-I335A, JWZ8-Q339A, and JWZ8-Q339A / F266A in Examples 1-2. Therefore, those skilled in the art can reasonably presume that the term "highly efficient LNT transporter mutant," its subordinate concepts, its substantially equivalent technical means, and technical means that can replace it within the scope of conventional and common knowledge based on the existing level of technology should all fall within the protection scope of "highly efficient LNT transporter mutant."

[0038] The present invention has at least the following beneficial effects: 1. Compared with the prior art, the present invention has better technical effect in terms of LNT extracellular yield.

[0039] According to experimental tests, this invention increases the extracellular LNT yield by 20.75%-202.89% compared to the wild-type JWZ8 recombinant strain. The total LNT yield also increases by 12.46%-62.98% compared to the wild-type JWZ8 recombinant strain. Specifically, the double mutant JWZ8-Q339A / F266A achieves an extracellular LNT yield of 3.20 g / L and a total yield as high as 4.71 g / L.

[0040] 2. Compared with existing technologies, this invention provides a technical solution with a different technical concept. The difference between this invention and existing technologies (CN121472116A) lies in the following: existing technologies only involve the integrated expression of wild-type glycoefflux transporters, while this invention, for the first time, significantly improves the transporter's recognition ability and transport efficiency for LNTs by rationally designing and site-directedly mutagenesis of key amino acid residues in the substrate binding pocket. This invention provides a new technical solution for the extracellular transport of LNTs and other HMOs through computer-aided prediction and molecular docking simulation screening of key amino acid residues and targeted mutagenesis. Attached Figure Description

[0041] Figure 1 This is a plasmid map of the expression plasmid pAC-wcf-galE-lgtA of this invention.

[0042] Figure 2 This is a plasmid map of the expression plasmid pCO-JWZ8 of this invention.

[0043] Figure 3 The figure shows the yield test results of lactose-N-tetrasaccharide in the mutant strain of this invention. Detailed Implementation

[0044] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from commercial suppliers, and experiments were conducted in accordance with the operating instructions. Unless otherwise specified, all instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus, and experiments were conducted in accordance with the operating instructions and the accompanying reagents.

[0045] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.

[0046] All data processing in this invention was performed using OriginPro 2024 software. Analysis between different groups was conducted using independent samples t-tests or one-way ANOVA tests. P <0.05 indicates that the difference is statistically significant.

[0047] The main reagents used in the following examples: LB liquid medium: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L.

[0048] LB solid medium: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar 20 g / L.

[0049] Glucose medium: glucose 15.07 g / L, citric acid 1.7 g / L, KH2PO4 13.5 g / L, (NH4)2PO4 4 g / L, MgSO4·7H2O 1.4 g / L, trace metal solution 10 mL / L (iron citrate 10 g / L, ZnSO4·7H2O 2.25 g / L, CuSO4·5H2O 1.0 g / L, MnSO4·H2O 0.35 g / L, Na2B4O7·2H2O 0.23 g / L, (NH4)6Mo7O2 40.11 g / L, CaCl2·2H2O 2.0 g / L).

[0050] Example 1: Construction of a chassis strain for efficient synthesis of lactose-N-tetrasaccharides (1) Use the NCBI database to query and obtain information from... Neisseria meningitidis The gene encoding β-1,3-N-acetylglucosamine transferase lgtA (Genbank ID: AAC44084.1) and its source. Citrobacter freundii The encoding β of (Genbank ID:WP_187258760.1) 1,3 The galactosyltransferase genes, optimized according to the codon preference of *E. coli*, were named lgtA and wcf, respectively. The full sequences of lgtA and wcf genes were synthesized by Suzhou Genewiz Company (the amino acid sequence of lgtA is shown in SEQ ID NO:1, and the amino acid sequence of wcf is shown in SEQ ID NO:2). E. coliThe genome of MG1655 (strain purchased from Weidi Biotechnology, catalog number: Bec-2030) was used to obtain the gene galE (amino acid sequence SEQ ID NO:3) of uridine diphosphate glucose-4-epimerase galE. Genomic DNA extraction was performed according to the instructions of the Deep Processed Food DNA Extraction Kit (catalog number: DP326) manufactured by Tiangen Biotech (Beijing) Co., Ltd. The concentration of extracted genomic DNA was determined using a UV spectrophotometer and stored at -20℃.

[0051] (2) Primers pAC-HP-F / R, lgtA-F / R, wcf-F / R, and galE-F / R (specific sequences are shown in Table 1) were designed and used with expression vector pACYCDuet-1 (purchased from Addgene, pACYCDuet-1#128837), synthetic gene lgtA, synthetic gene wcf, etc. E. coli PCR was performed using the MG1655 genome as a template. The pACYCDuet-1 linear vector, wcf gene fragment, galE gene fragment, and lgtA gene fragment were obtained. The sizes of the amplified fragments were determined by gel electrophoresis (the fragment sizes were 2570 bp, 789 bp, 1017 bp, and 1005 bp, respectively). The linear fragments were then recovered for seamless cloning (the DNA fragments and plasmids were ligated using the Shanghai Sangon Biotech Seamless Cloning Kit; the ligation method followed the kit's instructions). The fragments and vector were assembled, and the seamless cloning reaction solution was transformed into [a specific product / process] by heat shock. E. coli DH5α competent cells (Weidi Biotechnology, catalog number: DL1001) were plated on chloramphenicol-resistant LB agar plates and incubated overnight at 37°C. Positive clones were then screened and sequenced. After successful sequencing, the cells were amplified and plasmids were extracted to obtain the expression plasmid pAC-wcf-galE-lgtA. The plasmid map is shown below. Figure 1 As shown. The specific steps for heat shock conversion and cultivation are as follows: Remove the [material] stored at -80℃. E. coli DH5α competent cells were thawed on ice, and then plasmids or PCR ligation products were added to the competent cells and mixed thoroughly using a pipette. The mixture was placed on ice for 30 min, heat-shocked in a 42°C water bath for 90 s, and then placed on ice for 2 min. 1 mL of LB medium was added to the mixture in a clean bench, and the mixture was incubated at 37°C and 220 rpm for 45 min. Finally, the mixture was centrifuged at 2000 rpm for 2 min, and 800 μL of the supernatant was discarded. The remaining bacterial cells and medium were mixed thoroughly and spread onto LB agar plates containing the corresponding antibody. The mixture was then incubated overnight at 37°C. Single colonies were selected the following day for verification of positive transformants.

[0052] (3) with E. coliUsing BL21(DE3) (Weidi Biotechnology, catalog number: EE1002) as the starting strain, plasmid pAC-wcf-galE-lgtA was transformed into... E. coli BL21(DE3) electrotransfer competent cells were plated on chloramphenicol-resistant LB agar plates and incubated overnight at 37°C. Positive clones were then screened, and the correctly identified positive transformants were identified as the chassis strain LNT-01, which efficiently synthesizes lactose-N-tetrasaccharide. E. coli BL21(DE3)-pAC-wcf-galE-lgtA)).

[0053] SEQ ID NO:1: MGQPLVSVLICAYNVEKYFAQSLAAVVNQTWRNLDILIVDDGSTDGTLAIAQRFQEQDGRIRILAQPRNSGLIPSLNIGLDELAKSGGGGEYIARTDADDIAAPDWIEKIVGEMEKDRSIIAMGAWLEVLSEEKDGNRLARHHEHGKIWKKPTRHEDIADFFPFGNP IHNNNTMIMRRSVIDGGLRYNTERDWAEDYQFWYDVSKLGRLAYYPEALVKYRLHANQVSSKYSIRQHEIAQGIQKTARNDFLQSMGFKTRFDSLEYRQIKAVAYELLEKHLPEEDFELARRFLYQCFKRTDTLPAGAWLDFAADGRMRRLFTLRQYFGILHRLLKNR.

[0054] SEQ ID NO:2: MIIDETESAQSSYPVVSVILPVNKKNPFFDEAINSILSQTFSLFEIIIVANCCTDDFFNELKCKVNDKIKLVRTDIAYLPYSLNKAIDLSRGEFIARMDSDDISHPDRFTKQVSFLKNNPHVDVVGTNAIFID EKGLEISKTKLPEENLDIVKTLPYKCCIVHPSVMFRKEVIASMGGYMFSNYSEDYELWNRLSLAKIRFQNLPEYLFYYRLHEGQSTDKKNLYIVMVNDLAIKMKCFFVTGNIHYIFGGIRTIASVIYCKYIK.

[0055] SEQ ID NO:3: MRVLVTGGSGYIGSHTCVQLLQNGHDVIILDNLCNSKRSVLPVIERLGGKHPTFVEGDIRNEALMTEILHDHAIDTVIHFAGLKAVGESVQKPLEYYDNNVNGTLRLISAMRAANVKNFIFSSSATVYGDQPKIPYVESFPTGTPQSPYGKSKLMVEQILTDLQKAQPD WSIALLRYFNPVGAHPSGDMGEDPQGIPNNLMPYIAQVAVGRRDSLAIFGNDYPTEDGTGVRDYIHVMDLADGHVVAMEKLANKPGVHIYNLGAGVGNSVLDVVNAFSKACGKPVNYHFAPRREGDLPAYWADASKADRELNWRVTRTLDEMAQDTWHWQSRHPQGYPD.

[0056] Table 1 Primer sequences used for constructing the pAC-wcf-galE-lgtA plasmid

[0057] Example 2: Construction of a high-efficiency LNT transporter mutant and a recombinant strain of the transporter protein JWZ8. (1) Use the NCBI database to query and obtain information from... Bacillus sp The gene encoding a sugar efflux transporter derived from OV322 (Genbank ID: WP_090989553.1), named gene JWZ8, was optimized according to the codon preference of E. coli and synthesized in its entirety by Suzhou Genewiz Company (amino acid sequence is SEQ ID NO:4).

[0058] (2) Primers pCO-HP-F / R and JWZ8-F / R were designed and used as templates for PCR reactions with expression vector pCOLADuet-1 (purchased from Addgene, pCOLADuet-1#254010) and synthetic gene JWZ8, respectively. The linear vector pCOLADuet-1 and the JWZ8 gene fragment were obtained. The amplified fragment sizes were determined by gel electrophoresis (fragment sizes were 3442 bp and 1266 bp, respectively). The linear fragment was then recovered for seamless cloning. The fragment and vector were assembled, and the seamless cloning reaction solution was transformed into [a specific product / process] by heat shock. E. coli DH5α competent cells were plated on ampicillin-resistant LB agar plates and incubated overnight at 37°C. Positive clones were then screened and sequenced. After successful sequencing, the cells were amplified and plasmids were extracted to obtain the expression plasmid pCO-JWZ8. The plasmid map is shown below. Figure 2As shown. The seamless cloning and transformation steps are the same as those in Example 1.

[0059] (3) Based on the designed mutation sites, including M35A, F159Y, K212A, Y237A, F266A, G332A, I335A, and Q339A, design the mutation primers shown in Table 2, and construct the mutant recombinant plasmid using pCO-JWZ8 as a template. Taking the mutant M35A as an example, PCR amplification of the template pCO-JWZ8 was performed using the forward primer M35A-F and the reverse primer M35A-R. The size of the amplified fragment (4728bp) was determined by gel electrophoresis. Subsequently, the linear fragment was recovered for seamless cloning. The seamless cloning reaction solution was transformed into a heat shock solution. E. coli In DH5α competent cells, the cells were plated on ampicillin-resistant LB agar plates and incubated overnight at 37°C. Positive clones were then screened and sequenced. After successful sequencing, the cells were expanded and the plasmid was extracted. This plasmid was the mutant recombinant plasmid pCO-JWZ8 / M35A. Seamless cloning and transformation procedures were performed as described in Example 1. The mutant recombinant plasmids shown in Table 3 were constructed using the same method described above.

[0060] (4) Using engineered strain LNT-01 as the starting strain, a single colony was taken and seed culture was prepared by shaking, and the culture was expanded to OD in a proportional manner. 600 After cooling to 0.4-0.6°C for the entire process, the bacterial cells were collected by low-temperature centrifugation. They were gently resuspended and washed twice with pre-cooled 0.1 M CaCl2 solution, then resuspended in calcium chloride preservation solution containing glycerol, aliquoted, flash-frozen in liquid nitrogen, and stored at -80°C to obtain chemically competent cells. During transformation, the control plasmid pCO-JWZ8 and the constructed mutant recombinant plasmid were mixed with the competent cells and placed in an ice bath. After a short heat shock at 42°C, the cells were rapidly cooled in an ice bath, and then thawed by constant temperature shaking in antibiotic-free LB medium. Finally, the cells were plated on ampicillin and chloramphenicol resistant LB agar plates and cultured overnight at 37°C. Positive clones were then screened. The verified positive transformants were the engineered strains (recombinant strains LNT-02 to LNT-11) that can efficiently transport lactose-N-tetrasaccharides, as shown in Table 4.

[0061] (5) Recombinant strains LNT-02 to LNT-11 were inoculated into glucose fermentation medium containing ampicillin and chloramphenicol resistance for shake-flask fermentation tests. The culture temperature was 37℃, the rotation speed was 220 rpm, and when the OD600 reached about 0.6, the culture temperature was reduced to 30℃. 0.2 mM IPTG was added to induce the expression of the target gene, and 5 g / L of lactose substrate was added simultaneously. After 48 h of induction culture, samples were taken, and the content of lactose-N-tetrasaccharide was determined by high performance liquid chromatography-differential refractive index detector. The results are as follows: Figure 3As shown, compared with the wild-type recombinant strain LNT-02, the mutant strains LNT-03 to LNT-11 increased the extracellular yield of the target product LNT by 20.75%, 62.26%, 47.17%, 63.21%, 99.06%, 32.08%, 44.34%, 128.30%, and 202.89%, respectively, and the total yield increased by 12.46%, 30.10%, 21.80%, 31.83%, 38.06%, 15.57%, 21.45%, 43.94%, and 62.98%, respectively. Among them, the LNT strain with the combination of Q339A and F266A sites was able to produce the largest amount of LNT extracellularly at 3.20 g / L, with a total yield as high as 4.71 g / L. This mutant laid an important foundation for the biosynthesis of LNT.

[0062] Among them, the method for detecting lactose-N-tetrasaccharide using high performance liquid chromatography-differential refractive index detector: The content of lactose-N-tetrasaccharide was determined by high-performance liquid chromatography (HPLC) (Agilent 1260 series, USA) using a refractive index detector (RID), a Rezex™ ROA-Organic Acid H+ column (8%, 300 mm x 7.8 mm), a mobile phase of 10 mM dilute sulfuric acid, a detection temperature of 82°C, and a flow rate of 0.6 mL / min. A quantitative method was established using commercially available standards (CAS No.: 14116-68-8). Sample preparation: 1 mL of fermentation broth was heated at 100°C for 20 min, centrifuged at 12000 rpm for 5 min at room temperature, and the supernatant was collected. The supernatant was then filtered through a 0.22 μm aqueous membrane, and the total concentration of the product was determined by HPLC.

[0063] SEQ ID NO:4: MMNKIAAFRDKLLYIWRIPGLPVLFLLNIIYGLGASFFAPFSSLFGIDEAGMSNIGFGIFMTIMAVGGVVISSYIAKKSDTTLSRRRILIYSSLAGAAGYILFAYLRNYYALCLTGFFVLGSAAATVPQLWAYARDTMTAAKVPAEETPLIMNIFRMFFALSWIGGPALGAWLVMMLDFKGLFYFVAACYLLGSLTAIFLLKEIARQEVP NKESIKVKKYVSRLHILANLAAAFLLYSASSIHMLNVPQFVTKTLHGTEMQVGIIFSVAPIFEVPLMIIVGMMATKMDNSILIKAGYFLAFVYFLIFGFVSEPQQ IYPLQILSAAQVSITAGIAISYFQDFIPEAPGTATTLYMNVTQIGSTAGYLLFGFISSLISYGQLITIYTFFAGIGFLFITVYGKERQTNSKRNKQASESPPTSFK .

[0064] Table 2 Primer sequences used for plasmid construction

[0065] Table 3. Recombinant plasmids of mutants used in this invention

[0066] Table 4. Mutant recombinant strains used in this invention

[0067] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A mutant transporter protein for efficiently transporting LNTs, characterized in that, The transporter protein mutant is a sugar transporter protein with an amino acid sequence as shown in SEQ ID NO:4, wherein the mutation is any one of the following: (i) The methionine at position 35 is mutated to alanine; (ii) The phenylalanine at position 159 is mutated to tyrosine; (iii) The lysine at position 212 is mutated to alanine; (iv) Tyrosine at position 237 is mutated to alanine; (v) The phenylalanine at position 266 is mutated to alanine; (vi) Glycine at position 332 is mutated to alanine; (vii) The isoleucine at position 335 is mutated to alanine; (viii) Glutamine at position 339 is mutated to alanine; (ix) Glutamine at position 339 is mutated to alanine and phenylalanine at position 266 is mutated to alanine.

2. A nucleotide molecule, characterized in that, The nucleotide molecule encodes the transporter protein mutant of claim 1.

3. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleotide molecule of claim 2.

4. A recombinant host cell, characterized in that, The recombinant host cell comprises the nucleotide molecule of claim 2, or the recombinant expression vector of claim 3.

5. The recombinant host cell according to claim 4, characterized in that, The recombinant host cell is *Escherichia coli*; and / or The recombinant host cell also contains encoding β 1,3 N The gene encoding acetylglucosamine transferase IgTA, which encodes β 1,3 The gene encoding galactosyltransferase wcf and uridine diphosphate glucose. 4 At least one of the genes for the epimerase galE.

6. A method for producing lactose N The method of tetrasaccharides is characterized by... The method includes culturing the recombinant host cells as described in claim 4 or 5 and collecting lactose. N Four sugars.

7. The method according to claim 6, characterized in that, The method further includes adding an inducer to induce the expression of the transporter mutant.

8. The method according to claim 6 or 7, characterized in that, The method also includes adding the substrate lactose.

9. The transporter mutant of claim 1 in the preparation of a protein for transporting lactose N Applications of tetrasaccharides in products.

10. The application according to claim 9, characterized in that, The product is used in the production of lactose. N Engineered bacteria with tetrasaccharides.

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