Glucosamine-6 phosphate synthetase mutant and application thereof

Abstract

The invention belongs to the technical field of biological enzyme engineering, and particularly relates to a glucosamine-6-phosphate synthetase mutant and application thereof. A plurality of glucosamine-6-phosphate synthase mutants capable of remarkably improving the yield of N-acetylglucosamine are screened by performing single-point mutation or combined mutation on amino acid residues near a substrate binding pocket of the glucosamine-6-phosphate synthase or on the surface of protein of the glucosamine-6-phosphate synthase. According to the technical scheme, a foundation is laid for producing glucosamine by further transforming escherichia coli through metabolic engineering.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological enzyme engineering, in particular, the present application relates to a glucosamine-6-phosphate synthetase mutant and its application. BACKGROUND

[0002] N-acetylglucosamine (GlcNAc) is an amino sugar, which is widely present in organisms and has multiple physiological functions. Currently, GlcNAc has been widely used in the fields of medicine, food and cosmetics. In the human body, GlcNAc is the synthesis precursor of glycosaminoglycan disaccharide units, which plays an important role in repairing and maintaining the function of cartilage and joint tissue. Therefore, GlcNAc is widely used as a drug and nutritional dietary supplement to treat and repair joint damage. In addition, GlcNAc can enhance the immune function of the human body, can be used as a food additive, an antioxidant, and a sweetener for diabetic patients, and can also promote the metabolic synthesis of hyaluronic acid in the human body, thereby improving the skin moisturizing effect.

[0003] Currently, the industrial production methods of GlcNAc mainly include acid hydrolysis method, enzyme hydrolysis method and microbial fermentation method. The traditional acid hydrolysis method uses chitin extracted from shrimp and crab shells as raw material, which needs to use strong acid and strong base, and has the defects of environmental pollution, limited raw material source and product allergenicity; although the enzyme hydrolysis method has mild reaction conditions, it is limited by high enzyme cost, low efficiency and long reaction period. With the rapid development of synthetic biology technology, microbial fermentation method has become the mainstream due to its environmental friendliness and renewable raw materials. The genetically engineered bacteria used mainly include Escherichia coli, Corynebacterium glutamicum and Bacillus subtilis (B. subtilis). By introducing glucosamine-6-phosphate synthetase (glmS) and glucosamine 6-phosphate N-acetyltransferase (gna1) encoding genes into these strains, GlcNAc can be synthesized from glucose as raw material.

[0004] It is found that in the process of synthesizing GlcNAc in Escherichia coli, glutamine acts as an amino donor to generate glucosamine-6-phosphate by glucosamine-6-phosphate synthetase (glmS) catalysis from 6-phosphofructose, and this reaction is the initial rate-limiting step of the synthesis pathway. As a double-substrate binding type amino transferase, glmS has relatively low catalytic efficiency and is subject to feedback inhibition caused by product accumulation, which limits the yield of N-acetylglucosamine and the conversion rate of raw materials. Therefore, obtaining a high-activity glucosamine-6-phosphate synthetase (glmS) can improve the ability of Escherichia coli to produce N-acetylglucosamine, which has important economic value and social significance. Summary of the Invention

[0005] To address the problem of insufficient enzyme activity and low catalytic efficiency of existing glucose-6-phosphate synthase (glmS), the inventors conducted extensive and long-term research, performing various mutations and screenings on glucose-6-phosphate synthase, and finally obtained a high-activity enzyme mutant, thus completing this invention.

[0006] The technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a glucosamine-6-phosphate synthase mutant, wherein the glucosamine-6-phosphate synthase mutant is a mutation of amino acid residues near the substrate binding pocket or on the surface of the protein of wild-type glucosamine-6-phosphate synthase.

[0008] In one embodiment, the mutation comprises any one or a combination of the following mutations 1-4:

[0009] Mutation 1: Located in the substrate pocket of wild-type glucosamine-6-phosphate synthase Some amino acids within the range are mutated to alanine (Ala), which has less steric hindrance;

[0010] Mutation 2: Located in the substrate pocket of wild-type glucosamine-6-phosphate synthase Some key amino acids within the range are mutated into valine (val), which has less steric hindrance and is more hydrophobic.

[0011] Mutation 3: Located in the substrate pocket of wild-type glucosamine-6-phosphate synthase Some key amino acids within the range may be mutated to one or more of the charged amino acids aspartic acid (Asp), lysine (Lys), arginine (Arg), and histidine (His); or mutated to one or more of the hydrogen bond donor amino acids serine (Ser), threonine (Thr), tyrosine (Tyr), and cysteine ​​(Cys).

[0012] Mutation 4: The amino acids on the surface of the wild-type glucosamine-6-phosphate synthase protein are mutated.

[0013] In this invention, glucosamine-6-phosphate synthase can be derived from Escherichia coli, Bacillus subtilis, or Corynebacterium glutamicum.

[0014] Preferably, in this invention, the wild-type glucosamine-6-phosphate synthase is derived from Escherichia coli, and its amino acid sequence is shown in SEQ ID NO.1, and its nucleotide sequence is shown in SEQ ID NO.2.

[0015] In a second aspect, the present application provides a glucosamine-6-phosphate synthetase mutant, wherein the mutant is mutated at an amino acid residue near a substrate binding pocket or on the surface of a wild-type glucosamine-6-phosphate synthase, and the amino acid sequence of the wild-type glucosamine-6-phosphate synthase is shown as SEQ ID NO. 1, GenBank: HAY0704369.1.

[0016] Preferably, the mutation of the glucosamine-6-phosphate synthetase mutant satisfies at least one of the following conditions:

[0017] 1) In mutation 1, the amino acid at position 485, 399, 302, 348, 346, 352, 304, 351, 355, 381, 396, 481, 484, 486 in the amino acid sequence of the wild-type glucosamine-6-phosphate synthase is mutated to alanine (A);

[0018] 2) In mutation 2, the amino acid at position 304, 381, 346, 484, 486 in the amino acid sequence of the wild-type glucosamine-6-phosphate synthase is mutated to valine (V);

[0019] 3) In mutation 3, the amino acid at position 352, 355, 399, 481, 485 in the amino acid sequence of the wild-type glucosamine-6-phosphate synthase is mutated to one of aspartic acid (D), lysine (K), arginine (R), histidine (H), serine (S), threonine (T), tyrosine (Y), and cysteine (C);

[0020] 4) In mutation 4, the amino acid at position 38, 249, 14, 386, 499, 524, 42, 150, 257, 433, 551 in the amino acid sequence of the wild-type glucosamine-6-phosphate synthase is mutated.

[0021] Preferably, the glucosamine-6-phosphate synthetase mutant of the present application comprises any combination of mutation 1 and mutation 3.

[0022] Preferably, the glucosamine-6-phosphate synthetase mutant of the present application comprises any combination of mutation 2 and mutation 3.

[0023] Preferably, the glucosamine-6-phosphate synthetase mutant of the present application comprises any combination of mutation 1 and mutation 4.

[0024] Preferably, the glucosamine-6-phosphate synthetase mutant of the present application comprises any combination of mutation 2 and mutation 4.

[0025] Preferably, the glucosamine-6-phosphate synthetase mutant of the application comprises any combination of mutations 3 and 4 described above.

[0026] In one embodiment, the glucosamine-6-phosphate synthetase mutation 1 comprises any one of K485A, V399A, T302A, Q348A, L346A, T352A, Y304A, E351A, T355A, L381A, L396A, E481A, L484A, L486A and combinations thereof.

[0027] In one embodiment, the glucosamine-6-phosphate synthetase mutation 2 comprises any one of Y304V, L381V, L346V, E481V, L484V, L486V and combinations thereof.

[0028] In one embodiment, the glucosamine-6-phosphate synthetase mutation 3 comprises any one of T352D, T352S, T352K, T352C, T355D, T355S, T355K, T355C, K485R, K485H, K485S, K485C, V399S, V399C, V399D, V399K, E481S, E481D, E481K, E481C and combinations thereof.

[0029] In one embodiment, the glucosamine-6-phosphate synthetase mutation 4 comprises any one of A38T, R249C, E14K, D386V, S499P, E524G, M42K, P150T, Y257S, I433T, A551G and combinations thereof.

[0030] In one embodiment, the glucosamine-6-phosphate synthetase mutant comprises one or more of mutations L346A, L381A, Y304V, T352S, V399S.

[0031] In one embodiment, the glucosamine-6-phosphate synthetase mutant comprises one or more of mutations Y304V / V399S, Y304V / T352S, L381A / V399S, L381A / T352S, L346A / V399S or L346A / T352S, preferably L381A / V399S.

[0032] In one embodiment, the glucosamine-6-phosphate synthetase mutant comprises one or more of mutations I433T, Y257S, D386V.

[0033] In one embodiment, the mutations in the glucosamine-6-phosphate synthase mutant are one or more of L381A / V399S, I433T, Y257S, D386V. Preferably, the mutations are L381A / V399S / I433T, L381A / V399S / Y257S, L381A / V399S / D386V, L381A / V399S / I433T / Y257S, L381A / V399S / I433T / D386V, L381A / V399S / Y257S / D386V, or L381A / V399S / I433T / Y257S / D386V.

[0034] In a second aspect of the present application, the present application provides a nucleic acid molecule comprising a nucleotide sequence encoding the glucosamine-6-phosphate synthase mutant of the first aspect of the present application.

[0035] In a third aspect of the present application, the present application provides an expression vector comprising the nucleic acid molecule of the second aspect of the present application. In one embodiment, the expression vector is a plasmid vector.

[0036] In a fourth aspect of the present application, the present application provides a genetically engineered bacterium comprising the expression vector of the third aspect of the present application. The chassis bacterium of the genetically engineered bacterium is one of Escherichia coli, Corynebacterium glutamicum, and Bacillus subtilis.

[0037] In one embodiment, the chassis bacterium is Escherichia coli, and the Escherichia coli strain comprises BL21, C41, OrigamB, Rosetta, Turner, JM109, HMS174, NovaBlue, AD494, Origami, and Nissle 1917.

[0038] Preferably, in the present application, the chassis bacterium used is Escherichia coli Nissle 1917, which does not contain pathogenic factors such as enterotoxin, hemolytic toxin, and cytotoxin, and has no safety risk to the host.

[0039] In one embodiment, the Escherichia coli Nissle 1917 is a genetically engineered Escherichia coli Nissle 1917, and the engineering includes integrating a T7 RNA polymerase expression frame of a specific sequence at a specific site of the Nissle 1917 strain, deleting a cryptic plasmid, and knocking out two additional interfering genes, an endA gene and an ompT gene, on the genome. For details of the specific engineering method, see CN202310516515.6.

[0040] In a fifth aspect, the present application provides a method for producing N-acetylglucosamine, wherein the method is catalyzed by the aforementioned glucosamine-6-phosphate synthetase mutant of the present application to catalyze the production of N-acetylglucosamine from glucose.

[0041] Advantages

[0042] The present application obtains a plurality of glucosamine-6-phosphate synthetase mutants by mutation and screening, in particular, the genetically engineered bacteria containing the genes of five mutants of L381A, L381A / V399S, L381A / V399S / Y257S, L381A / V399S / I433T / Y257S, and L381A / V399S / I433T / Y257S / D368V, and the yield of N-acetylglucosamine in the fermentation process is significantly improved. The technical scheme of the present application lays a foundation for further metabolic engineering of Escherichia coli to produce glucosamine. The recombinant Escherichia coli provided by the present application has a simple construction method, is easy to use, and has a good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 is the overall three-dimensional structure of the glucosamine-6-phosphate synthetase from Escherichia coli.

[0044] Figure 2 is a local spatial position diagram of the mutation site of the present application based on the spatial conformation of the glucosamine-6-phosphate synthetase from Escherichia coli, wherein (a) represents the key site of the substrate binding pocket mutation, and (b) represents the key site of the protein surface mutation.

[0045] Figure 3 is the HPLC chromatogram of GlcNAc, wherein (a) represents the HPLC result of the GlcNAc standard, and (b) represents the HPLC result of the GlcNAc sample.

[0046] Figure 4 is the yield result of the wild type and the mutant-containing engineered strain for the production of GlcNAc by shake flask fermentation. DETAILED DESCRIPTION

[0047] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified, specific conditions in the examples are performed under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the operating methods involved in this invention are based on textbooks or reference books in the field, including but not limited to "Molecular Biology," "Genetic Engineering," "Enzyme Engineering," "Molecular Cloning: A Laboratory Manual," and "A Concise Guide to Molecular Biology Experiments." Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0048] Example 1: Determination of mutation sites in glucosamine-6-phosphate synthase (glmS)

[0049] The catalytic mechanism of glmS follows an ordered reaction mechanism. Fructose-6-phosphate initially binds to glutamine. Following the hydrolysis of glutamine, amino groups are transferred and the amino sugar isomerizes, leading to the sequential release of glutamate and GlcN-6-P from glmS. The glmS of *E. coli* is a homodimer composed of two domains. The isomerase domains on the two different monomers are located inside the dimer, while the two glutamine-binding domains are located on the outside of the dimer, as shown in the image. Figure 1 As shown. Based on PDB crystal structure analysis (PDB ID: 4amv) of glmS derived from E. coli and previous preliminary experimental analysis, the region near the binding site with the substrate Fru-6P was selected. The residues within the defined range are key residues at the substrate-binding pocket, which may affect glmS enzyme activity. Specifically, these include the following 14 sites: K485, V399, T302, Q348, L346, T352, Y304, E351, T355, L381, L396, E481, L484, and L486. Furthermore, 11 sites—A38, R249, E14, D386, S499, E524, M42, P150, Y257, I433, and A551—were identified as potentially influencing product inhibition; these 11 sites are defined as key residues on the protein surface. The specific spatial locations of the aforementioned sites are as follows: Figure 2 As shown.

[0050] Example 2: Screening of genetically engineered bacteria with glmS substrate-binding pocket single mutants

[0051] 2.1 Construction of glmS substrate binding pocket single mutant plasmid

[0052] The recombinant plasmid containing the glmS mutant gene was constructed by whole plasmid amplification method: see Table 1, 39 different point mutation sites were designed, among which, mutants M1-M19 were mainly for key sites of substrate binding pocket for alanine or valine scanning, in order to expand the volume of substrate binding pocket and enhance the hydrophobic interaction; M20-M39 were for introducing strong ionic bond and hydrogen bond at specific sites. According to the desired mutant site design, the upstream and downstream primers were designed, and the plasmid pRSF-glmS-gna1 expressing wild-type glmS gene (nucleotide sequence as shown in SEQ ID NO. 2, corresponding amino acid sequence as shown in SEQ ID NO. 1) was used as template, and PrimeSTAR Max (Takara) was used for whole plasmid amplification. The glmS gene mutant plasmid was amplified by PCR, transformed into E. coli, and the colonies of recombinant plasmid were screened by resistance. The colonies were cultured in LB liquid medium overnight, and then the plasmid was extracted and sequenced for verification.

[0053] Table 1: glmS substrate binding pocket single mutant

[0054]

[0055] 2.2 Construction of genetically engineered strains of glmS substrate binding pocket single mutant

[0056] (1) Preparation of probiotic E. coli Nissle 1917-G7

[0057] In this embodiment, Nissle 1917 is used as the host strain, and the following modifications are made: a T7 RNA polymerase expression frame is inserted into the attB site of the genome of Nissle 1917, the endA gene and ompT gene on the genome are knocked out, and the deaminase gene nagB, the deacetylase gene nagA, the mannose phosphate transporter encoding gene manX, the acetylglucosamine transporter encoding gene nagE and the acetylglucosamine kinase nagK are further knocked out (the specific strain construction method is recorded in patent CN202311431661.5), and the strain is named Nissle 1917-G7.

[0058] (2) Construction of genetically engineered strains containing mutants

[0059] The single mutant plasmid obtained in section 2.1 above was transformed into Nissle 1917-G7 by electroporation to obtain genetically engineered strains containing mutants. At the same time, the plasmid containing the wild-type sequence of glmS was also transformed into Nissle 1917-G7 as a control (WT).

[0060] 2.3 Expression of glmS substrate binding pocket single mutant and determination of GlcNAc yield

[0061] The genetically engineered bacteria containing glmS and its mutants in step (2) in section 2.2 were inoculated into LB liquid medium containing kanamycin, and seed liquid was obtained by overnight culture at 37℃, 220 rpm. 25 mL fermentation medium (fermentation medium composition: tryptone 4 g / L, yeast powder 2 g / L, potassium dihydrogen phosphate 13.5 g / L, dihydrogen phosphate 4 g / L, citric acid 1.7 g / L, magnesium sulfate 1.4 g / L, 1% trace elements, pH adjusted to 7.0) was inoculated at 2% (v / v) inoculation amount. Glucose was added to a final concentration of 40 g / L, and the culture was incubated at 37℃, 220 rpm for 2-3 h. When the OD 600 When the OD reached 0.6-0.8, IPTG was added to a final concentration of 0.2 mM to induce the expression of glmS and its mutants, and the fermentation was continued at 25℃, 250 rpm for 72 h to produce N-acetylglucosamine from glucose by the recombinant bacteria. 1 mL of fermentation broth was centrifuged at 12000 rpm for 5 min, filtered through a 0.22 μm water phase filter, and then the N-acetylglucosamine content was determined.

[0062] High performance liquid chromatography (HPLC) detection method: Agilent 1260, RID detector, HPX-87H column (Bio-Rad Hercules, CA), mobile phase: 5 mM H2SO4, flow rate 0.5 mL / min, column temperature 40℃, injection volume 10 μL. The HPLC results of GlcNAc standard and sample are shown in Figure 2. Figure 3 The peak time of GlcNAc standard is 13.103 min, and the peak time of product in GlcNAc sample is consistent with that of GlcNAc standard

[0063] The results are shown in Table 2. The production of GlcNAc by shake flask fermentation of the engineered bacterial strains of glmS substrate binding pocket single mutant M1-M39 was detected.

[0064] Table 2: GlcNAc production of glmS substrate binding pocket single mutant

[0065]

[0066]

[0067] The present embodiment obtains five positive mutations M5, M11, M17, M30 and M34 for significantly improving the yield of GlcNAc, which are specifically: mutating the leucine at position 346 to alanine (L346A), or mutating the leucine at position 381 to alanine (L381A), or mutating the tyrosine at position 304 to valine (Y304V); or mutating the threonine at position 352 to serine (T352S); or mutating the valine at position 399 to serine (V399S). After 72 hours of fermentation, the yield of M5 (L346A) reaches 9.25 g / L, which is 8.56% higher than that of the wild-type strain; the yield of M11 (L381A) reaches 10.33 g / L, which is 21.24% higher than that of the wild-type strain; the yield of M17 (Y304V) reaches 9.52 g / L, which is 11.17% higher than that of the wild-type strain; the yield of M30 (T352S) reaches 9.7 g / L, which is 11.38% higher than that of the wild-type strain; and the yield of M34 (V399S) reaches 9.30 g / L, which is 9.15% higher than that of the wild-type strain.

[0068] Example 3 Screening of genetically engineered bacteria of double mutants in the glmS substrate binding pocket

[0069] 3.1 Construction of plasmids of double mutants in the glmS substrate binding pocket

[0070] The double mutants in the glmS substrate binding pocket are obtained by multiple rounds of site-directed mutagenesis. After obtaining single point mutants, the upstream and downstream primers of the mutation site are designed, and the whole plasmid is amplified by PCR. The recombinant plasmid containing the mutated sequence of glucosamine-6-phosphate synthase gene is obtained by PCR amplification, transformation screening and plasmid extraction. Specifically, the five positive mutants obtained in Example 2 are combined from two dimensions of expanding the size of the substrate binding pocket and enhancing hydrophobicity, and introducing strong interaction force. The relevant double mutation sites are shown in Table 3.

[0071] Table 3: Double mutants in the glmS substrate binding pocket

[0072] Number Mutant Template M40 Y304V / V399S Y304V M41 Y304V / T352S Y304V M42 L381A / V399S L381A M43 L381A / T352S L381A M44 L346A / V399S L346A M45 L346A / T352S L346A

[0073] 3.2 Construction of genetically engineered bacteria of double mutants in the glmS substrate binding pocket

[0074] As shown in the detailed steps in section 2.2 of Example 2, the double mutant plasmid with correct sequencing is electroporated into probiotic Escherichia coli Nissle 1917-G7 to obtain genetically engineered strains containing double mutants in the glmS substrate binding pocket.

[0075] 3.3 Expression of double mutants in the glmS substrate binding pocket and determination of GlcNAc yield

[0076] The genetically engineered bacteria obtained above were subjected to shake flask fermentation test and the GlcNAc production was detected according to the detailed steps shown in section 2.3 of Example 2.

[0077] Results: Table 4 shows the GlcNAc production of the genetically engineered bacteria containing double mutants M40-M45 in the substrate binding pocket of glmS after shake flask fermentation.

[0078] Table 4: GlcNAc production of double mutants in the substrate binding pocket of glmS

[0079]

[0080] The present embodiment obtains a double mutant M42 (L381A / V399S) that significantly improves the production of GlcNAc, wherein the mutation is that the 381st leucine is mutated to alanine and the 399th valine is mutated to serine; the GlcNAc production of the genetically engineered bacteria M42 (L381A / V399S) after 72h fermentation is 15.13g / L, which is increased by 80.98% compared with the wild type strain, increased by 46.46% compared with L381A, and increased by 62.68% compared with V399S.

[0081] Example 4: Screening of genetically engineered bacteria of glmS protein surface mutants

[0082] 4.1 Construction of plasmid of glmS protein surface mutants

[0083] Based on the previous research that A38, R249, E14, D386, S499, E524, M42, P150, Y257, I433, and A551 may have an impact on product inhibition, see Table 5, mutations were designed at the corresponding sites (M46-M56) for these 11 sites. Specifically, the plasmid pRSF-glmS-gna1 expressing the wild type glmS gene was used as a template, and the whole plasmid was amplified using PrimeSTAR Max (Takara). The mutant sequence containing the glmS gene was amplified by PCR, transformed into E. coli, and the colonies containing the recombinant plasmid with mutations were screened by resistance. After overnight culture in LB liquid medium, the plasmid was extracted and sequenced to verify.

[0084] Table 5: Design of glmS protein surface mutants

[0085]

[0086]

[0087] 4.2 Construction of genetically engineered bacteria of glmS protein surface mutants

[0088] The plasmid containing the mutant of the surface of the glmS protein was transformed into the probiotic Escherichia coli Nissle 1917-G7 by electroporation, to obtain a genetically engineered strain containing the mutant of the surface of the glmS protein.

[0089] 4.3 Expression of the mutant of the surface of the glmS protein and determination of GlcNAc production

[0090] The genetically engineered strain obtained above was subjected to a shake flask fermentation experiment and the production of GlcNAc was detected, as shown in the detailed steps in Section 2.3 of Example 2.

[0091] As a result, referring to Table 6, the production of GlcNAc produced by the shake flask fermentation of the engineered strain containing the mutant M46-M56 of the surface of the glmS protein was detected.

[0092] Table 6

[0093] Number GlcNAc production (g / L) Number GlcNAc production (g / L) WT 8.23 M51 9.47 M46 5.35 M52 6.70 M47 9.81 M53 7.55 M48 10.21 M54 6.44 M49 4.93 M55 6.16 M50 5.58 M56 6.44

[0094] Three positive mutations M47, M48 and M51 that can improve the production of GlcNAc were obtained in this example, and the mutations are as follows: the isoleucine at position 433 is mutated to threonine (I433T); or the tyrosine at position 257 is mutated to serine (Y257S), or the aspartic acid at position 386 is mutated to valine (D386V). After 72 hours of fermentation, the production of GlcNAc of M47 (I433T), M48 (Y257S) and M51 (D386V) reached 9.81 g / L, 10.21 g / L and 9.47 g / L, respectively, which was 19.20%, 24.06% and 15.07% higher than the wild-type strain, respectively.

[0095] Example 5: Screening of genetically engineered strains of glmS substrate binding pocket and protein surface combined mutants

[0096] 5.1 Construction of glmS combined mutant plasmid

[0097] The optimal double mutant M42 (L381A / V399S) at the substrate binding pocket obtained in Example 3 was combined with the positive mutants M47 (I433T), M48 (Y257S) and M51 (D386V) at the surface of the protein, in order to improve the final production of GlcNAc from the aspects of improving catalytic activity and relieving product inhibition. The specific combination of different mutants and sites is shown in Table 7.

[0098] Table 7: glmS combined mutants and their sites

[0099]

[0100] 5.2 Construction of genetically engineered bacteria containing glmS combinatorial mutants

[0101] Following the detailed procedures described in Section 2.2 of Example 2, the correctly sequenced glmS combinatorial mutant plasmids were electroporated into probiotic E. coli Nissle 1917-G7 to obtain genetically engineered strains containing glmS combinatorial mutants of the substrate binding pocket and the protein surface.

[0102] 5.3 Expression of glmS combinatorial mutants and determination of GlcNAc production

[0103] Following the detailed procedures described in Section 2.3 of Example 2, the genetically engineered bacteria obtained above were subjected to shake flask fermentation experiments and the production of GlcNAc was determined.

[0104] Results: Table 8 shows the production of GlcNAc by the genetically engineered strains containing glmS combinatorial mutants M57-M63 in shake flask fermentation.

[0105] Table 8 - GlcNAc production by glmS combinatorial mutants

[0106]

[0107] In the present embodiment, the GlcNAc production of M57-M63 is significantly improved compared with the wild type (WT), and the production is increased by more than 50%. The three combination mutants of the substrate binding pocket optimal mutant M42 (L381A / V399S) further improving the production are M58, M60 and M63. The M58 mutant is specifically: the 381st leucine is mutated to alanine, the 399th valine is mutated to serine, and the 257th tyrosine is mutated to serine (L381A / V399S / Y257S), and the GlcNAc production after 72h fermentation is 15.82g / L, which is increased by 83.52% compared with the WT production, and increased by 4.01% compared with the M42 production. The M60 mutant is specifically: the 381st leucine is mutated to alanine, the 399th valine is mutated to serine, the 433rd isoleucine is mutated to threonine, and the 257th tyrosine is mutated to serine (L381A / V399S / I433T / Y257S), and the GlcNAc production after 72h fermentation is 16.71g / L, which is increased by 93.85% compared with the WT production, and increased by 9.86% compared with the M42 production. The M63 mutant is specifically: the 381st leucine is mutated to alanine, the 399th valine is mutated to serine, the 434th isoleucine is mutated to threonine, the 258th tyrosine is mutated to serine, and the 386th aspartic acid is mutated to valine (L381A / V399S / I433T / Y257S / D368V), and the GlcNAc production after 72h fermentation is 18.50g / L, which is increased by 114.61% compared with the WT production, and increased by 21.63% compared with the M42 production.

[0108] The above embodiments only express several embodiments of the present application, and the description is more specific and detailed, but it cannot be understood as limiting the scope of the patent. It should be noted that for ordinary skilled in the art, without departing from the concept of the present application, several modifications and improvements can be made, which are within the scope of the present application. Therefore, the scope of protection of the present application should be subject to the appended claims.

Claims

1. A glucosamine-6-phosphate synthetase mutant, characterized in that, The amino acid residue in the vicinity of the substrate binding pocket or the surface of the protein of the wild-type glucosamine-6-phosphate synthetase is mutated.

2. The mutant according to claim 1, wherein The mutation comprises any one or a combination of mutations 1-4 as follows, Mutant 1 : Located in the substrate binding pocket of the wild-type glucosamine-6-phosphate synthase Some of the amino acid mutations within the range were replaced by alanine (Ala) which has less steric hindrance. Mutant 2: Located in the substrate binding pocket of the wild-type glucosamine-6-phosphate synthase Key amino acids in this region were mutated to valine (val), which has less steric hindrance and is more hydrophobic. Mutant 3: Located in the substrate binding pocket of the wild-type glucosamine-6-phosphate synthase The key amino acid mutations in the range are one or more of the charged amino acids aspartate (Asp), lysine (Lys), arginine (Arg), and histidine (His). Or the mutation is one or more of the hydrogen bond donor amino acids serine (Ser), threonine (Thr), tyrosine (Tyr), and cysteine (Cys); Mutation 4: the amino acid on the surface of the wild-type glucosamine-6-phosphate synthetase protein is mutated.

3. A glucosamine-6-phosphate synthetase mutant, characterized in that, The mutation of the glucosamine-6-phosphate synthetase mutant satisfies at least one of the following conditions: 1) In mutation 1, the amino acid at position 485, 399, 302, 348, 346, 352, 304, 351, 355, 381, 396, 481, 484, 486 in the amino acid sequence of the wild-type glucosamine-6-phosphate synthetase is mutated, and any one of the above positions is mutated to alanine; 2) In mutation 2, the amino acid at position 304, 381, 346, 484, 486 in the amino acid sequence of the wild-type glucosamine-6-phosphate synthetase is mutated, and any one of the above positions is mutated to valine; 3) In mutation 3, the amino acid at position 352, 355, 399, 481, 485 in the amino acid sequence of the wild-type glucosamine-6-phosphate synthetase is mutated, and any one of the above positions is mutated to one of aspartic acid, lysine, arginine, histidine, serine, threonine, tyrosine, and cysteine; 4) In mutation 4, the amino acid at position 38, 249, 14, 386, 499, 524, 42, 150, 257, 433, 551 in the amino acid sequence of the wild-type glucosamine-6-phosphate synthetase is mutated; The amino acid sequence of the wild-type glucosamine-6-phosphate synthetase is shown in SEQ ID NO.

1.

4. The mutant according to claim 3, wherein The mutation of the glucosamine-6-phosphate synthetase mutant is any combination between mutation 1, mutation 2, mutation 3, and mutation 4.

5. A glucosamine-6-phosphate synthetase mutant, characterized in that, The mutation of the glucosamine-6-phosphate synthetase mutant is any one of the following ①-④: ①: one or more of L346A, L381A, Y304V, T352S, V399S; ②: Y304V / V399S, Y304V / T352S, L381A / V399S, L381A / T352S, L346A / V399S, or L346A / T352S; ③: one or more of I433T, Y257S, D386V; and ④: one or more of I433T, Y257S, D386V. IV: a combination of one or more of L381A / V399S, I433T, Y257S, D386V; preferably L381A / V399S / I433T, L381A / V399S / Y257S, L381A / V399S / D386V, L381A / V399S / I433T / Y257S, L381A / V399S / I433T / D386V, L381A / V399S / Y257S / D386V, or L381A / V399S / I433T / Y257S / D386V.

6. A nucleic acid molecule, characterized in that, The nucleic acid molecule comprises a nucleotide sequence encoding the glucosamine-6-phosphate synthase mutant according to any one of claims 1-5.

7. An expression vector, characterized by, The expression vector comprises the nucleic acid molecule according to claim 6; preferably, the expression vector is a plasmid vector.

8. A genetically engineered bacterium, characterized by, The genetically engineered bacterium comprises the expression vector according to claim 7.

9. The genetically engineered bacteria of claim 8, wherein, The chassis bacterium of the genetically engineered bacterium is one of Escherichia coli, Corynebacterium glutamicum, and Bacillus subtilis.

10. A method for producing N-acetylglucosamine, characterized by, The method is catalyzing the production of N-acetylglucosamine from glucose by using the glucosamine-6-phosphate synthase mutant according to any one of claims 1-5 as a catalyst.

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