Engineering bacterium of high-yield N-acetylglucosamine as well as construction method and application of engineering bacterium

Abstract

The invention discloses an engineering bacterium for high yield of N-acetylglucosamine as well as a construction method and application of the engineering bacterium, and the construction method comprises the following steps: sequentially knocking out nagA, nagB, nagP, gamA and gamP genes in a bacillus subtilis 168 strain to obtain an MT101 strain; the method comprises the following steps: carrying out series connection on a GNA1 gene in Saccharomyces cerevisiae W303 yeast and a glmS gene in a Bacillus subtilis 168 strain by adopting a chemical synthesis method, so as to obtain a GNA1 and glmS series connection gene; the method comprises the following steps: introducing a GNA1 and glmS tandem gene into a pP43NMK plasmid, and enabling the GNA1 and glmS tandem gene to be positioned between a KpnI restriction enzyme cutting site and a HindIII restriction enzyme cutting site, so as to obtain a pP43NMK-GNA1-glmS plasmid; and transferring the pP43NMK-GNA1-glmS plasmid into an MT101 strain, so as to obtain the engineering bacterium capable of producing the N-acetylglucosamine at high yield. According to the engineering bacterium for producing the N-acetylglucosamine at high yield, high expression of the N-acetylglucosamine can be realized, and the yield of the N-acetylglucosamine is remarkably improved.

Description

Technical Field

[0001] The present invention relates to the technical field of engineering bacteria construction, and in particular to an engineering bacteria with high N-acetylglucosamine production, a construction method and an application thereof. Background Art

[0002] N-acetylglucosamine (NAG) is a fundamental building block of many important polysaccharides within biological cells, and is particularly abundant in the exoskeletons of crustaceans. It is an essential precursor for the synthesis of bifidobacteria and has many important physiological functions within the body. For example, it can serve as a monomer for chitin, forming chitin through β-1,4-glycosidic bonds. It also exhibits anti-inflammatory, anti-tumor, and antioxidant properties, and is clinically used as a drug for the treatment of osteoarthritis and rheumatoid arthritis. It is widely used in the food, pharmaceutical, and cosmetic industries, serving as a food antioxidant, an additive for infant food, and a sweetener for diabetics. It is primarily used clinically to enhance the function of the human immune system, inhibit the excessive growth of cancer cells or fibroblasts, and inhibit and treat cancer and malignant tumors. It is effective in treating various inflammatory conditions, and also has therapeutic effects on osteoarthritis and joint pain.

[0003] As N-acetylglucosamine is increasingly used in the pharmaceutical and food industries, the demand for its production is also increasing. To meet market demand, researchers have proposed a variety of production methods, of which synthesis and fermentation are the two most important. Synthesis refers to the preparation of N-acetylglucosamine through chemical synthesis. Its principle is to react glucosamine with an acetyl compound using a chemical synthesis reaction to obtain the target product. This method has the advantages of strict reaction conditions and high product purity, making it one of the most widely used production methods. When using synthesis to prepare N-acetylglucosamine, the common advantages are easy control of reaction conditions and high product purity, making it suitable for large-scale industrial production. However, synthesis also has disadvantages such as high raw material costs and significant environmental impact, which restrict its application in sustainable development.

[0004] The fermentation method for producing N-acetylglucosamine utilizes microbial biotransformation to produce N-acetylglucosamine. The principle is to ferment suitable raw materials, causing the microbial strain to produce N-acetylglucosamine. This method has advantages such as high raw material utilization and environmental friendliness, and has attracted increasing attention. However, it also has disadvantages such as difficulty in selecting microbial strains and low product yields.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The first purpose of the present invention is to provide a method for constructing an engineered bacterium that can produce high N-acetylglucosamine yield. The engineered bacterium constructed by this construction method can produce high N-acetylglucosamine under anaerobic culture conditions, significantly improving the yield of N-acetylglucosamine and increasing the yield of the target product.

[0007] The second purpose of the present invention is to provide an application of an engineered bacterium with high N-acetylglucosamine production, which can achieve large-scale production of N-acetylglucosamine and improve the production efficiency of N-acetylglucosamine.

[0008] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted:

[0009] The first aspect of the present invention provides a method for constructing an engineered bacterium that produces high N-acetylglucosamine yield, the method comprising the following steps:

[0010] (a) The nagA (N-acetylglucosamine-6-phosphate deacetylase), nagB (glucosamine 6-phosphate deaminase), nagP (N-acetylglucosamine phosphotransferase), gamA (glucosamine 6-phosphate deaminase), and gamP (glucosamine 6-phosphate deaminase) genes in Bacillus subtilis 168 were sequentially knocked out to generate strain MT101;

[0011] (b) The GNA1 gene from the yeast Saccharomyces cerevisiae W303 and the glmS gene from the strain Bacillus subtilis 168 were tandemly synthesized to obtain a tandem GNA1 and glmS gene.

[0012] (c) The tandem genes of GNA1 and glmS were introduced into the pP43NMK plasmid and located between the KpnI and HindIII restriction sites to obtain the pP43NMK-GNA1-glmS plasmid;

[0013] (d) The pP43NMK-GNA1-glmS plasmid is transformed into the MT101 strain to obtain the engineered bacteria that produces high N-acetylglucosamine.

[0014] Preferably, in step (a), the nagA, nagB, nagP, gamA and gamP genes in Bacillus subtilis 168 are knocked out in sequence using the Cre / loxP homologous recombination method.

[0015] Preferably, the primer sequences used in the Cre / loxP homologous recombination method are as follows:

[0016] The sequence of nagP-L-1F is shown in SEQ ID NO.1; the sequence of nagP-L-1R is shown in SEQ ID NO.2

[0017] As shown;

[0018] The sequence of nagP-R-1F is shown in SEQ ID NO. 3; the sequence of nagP-R-1R is shown in SEQ ID NO. 4;

[0019] The sequence of gamPA-L-1F is shown in SEQ ID NO. 5; the sequence of gamPA-L-1R is shown in SEQ ID NO. 6;

[0020] The sequence of gamPA-R-1F is shown in SEQ ID NO. 7; the sequence of gamPA-R-1R is shown in SEQ ID NO. 8;

[0021] The sequence of nagAB-L-1F is shown in SEQ ID NO. 9; the sequence of nagAB-L-1R is shown in SEQ ID NO. 10;

[0022] The sequence of nagAB-R-2F is shown in SEQ ID NO.11; the sequence of nagAB-R-2R is shown in SEQ ID NO.12.

[0023] Preferably, in step (d), the pP43NMK-GNA1-glmS plasmid is transformed into the MT101 strain using electroporation technology.

[0024] The second aspect of the present invention provides a method for constructing an engineered bacterium with high N-acetylglucosamine production, and the obtained engineered bacterium with high N-acetylglucosamine production.

[0025] The third aspect of the present invention provides an application of the engineered bacteria with high N-acetylglucosamine production constructed by the above-mentioned method for constructing an engineered bacteria with high N-acetylglucosamine production in the production of N-acetylglucosamine.

[0026] A third aspect of the present invention provides a method for producing N-acetylglucosamine, comprising:

[0027] The engineered bacteria with high N-acetylglucosamine production are inoculated into a culture medium for anaerobic culture.

[0028] Preferably, the culture medium is LB medium.

[0029] Preferably, the culture temperature is 35-38°C and the culture time is 8-12 hours.

[0030] Preferably, the inoculation concentration of the engineered bacteria with high N-acetylglucosamine production is 10 6 cfu / mL.

[0031] Compared with the prior art, the beneficial effects of the present invention include at least:

[0032] Compared with the starting strain, the engineered bacteria with high N-acetylglucosamine production of the present invention can achieve high expression of the glmS and GNA1 genes, and reduce the expression of the gamA, nagB, nagA, and nagP genes, thereby enabling the engineered bacteria with high N-acetylglucosamine production to accumulate N-acetylglucosamine under anaerobic culture conditions, thereby significantly improving the yield of N-acetylglucosamine.

[0033] In addition, the construction method of the present invention adopts traditional genetic engineering methods, which is relatively reliable and requires skilled operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly describes the drawings required for the specific embodiments or the description of the prior art. Similar elements or parts are generally identified by similar reference numerals throughout the drawings. Elements or parts in the drawings are not necessarily drawn to scale.

[0035] Figure 1 N-acetylglucosamine production of WT, MT101, and MT102 under hypoxic conditions in Example 2 of the present invention;

[0036] Figure 2 The key gene expression of WT and MT102 in Example 2 of the present invention;

[0037] Figure 3 The expression of key metabolites of WT and MT102 in Example 2 of the present invention. DETAILED DESCRIPTION

[0038] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention and are therefore only used as examples and cannot be used to limit the scope of protection of the present invention.

[0039] It should be noted that, unless otherwise specified, the technical or scientific terms used in this application should have the common meanings understood by those skilled in the art to which the present invention belongs.

[0040] The embodiment of the present invention provides a method for constructing an engineered bacterium that produces high N-acetylglucosamine yield, and the method comprises the following steps:

[0041] (a) The nagA, nagB, nagP, gamA, and gamP genes in Bacillus subtilis 168 were sequentially knocked out to generate strain MT101;

[0042] (b) The GNA1 gene from the yeast Saccharomyces cerevisiae W303 and the glmS gene from the strain Bacillus subtilis 168 were tandemly synthesized to obtain a tandem GNA1 and glmS gene.

[0043] (c) The tandem genes of GNA1 and glmS were introduced into the pP43NMK plasmid and located between the KpnI and HindIII restriction sites to obtain the pP43NMK-GNA1-glmS plasmid;

[0044] (d) The pP43NMK-GNA1-glmS plasmid is transformed into the MT101 strain to obtain the engineered bacteria that produces high N-acetylglucosamine.

[0045] Compared with the starting strain, the engineered bacteria with high N-acetylglucosamine production of the present invention can achieve high expression of the glmS and GNA1 genes, and reduce the expression of the gamA, nagB, nagA, and nagP genes, thereby enabling the engineered bacteria with high N-acetylglucosamine production to accumulate N-acetylglucosamine under anaerobic culture conditions, thereby significantly improving the yield of N-acetylglucosamine.

[0046] In one embodiment, in step (a), the nagA, nagB, nagP, gamA and gamP genes in Bacillus subtilis 168 are knocked out in sequence using Cre / loxP homologous recombination.

[0047] The present invention does not specifically limit the method of Cre / loxP homologous recombination. Those skilled in the art can use conventional methods in the art to knock out the target gene. Preferably, in one embodiment, the primer sequences used in the Cre / loxP homologous recombination method are as follows:

[0048] The sequence of nagP-L-1F is shown in SEQ ID NO. 1, specifically 5'-TAATGAGATGGCTGTGTCGGAAT-3'; the sequence of nagP-L-1R is shown in SEQ ID NO. 2, specifically 5'-CCTGTGTGAAATTGTTATGCGCTCATCCACTCTCCAAACGAGTTGAT-3';

[0049] The sequence of nagP-R-1F is shown in SEQ ID NO. 3, specifically 5'-TACGTCGTGACTGGGAAAACCCTGCCCGCGGTCTTAACCGGGT-3'; the sequence of nagP-R-1R is shown in SEQ ID NO. 4, specifically 5'-AACGACAACGCCCAGCTT-3';

[0050] The sequence of gamPA-L-1F is shown in SEQ ID NO. 5, specifically 5'-TCATTCTCCCCATTGTGTATG-3'; the sequence of gamPA-L-1R is shown in SEQ ID NO. 6, specifically 5'-GGTGTGAAATTGTTATCCGCTCTGATATGCCTTATTAAACATGACA-3';

[0051] The sequence of gamPA-R-1F is shown in SEQ ID NO. 7, specifically 5'-GTCGTGACTGGGAAAACCCTGCCATTCTCATAACACAATAAAAGGAGA CT-3'; the sequence of gamPA-R-1R is shown in SEQ ID NO. 8, specifically 5'-AATGGCGGACATGGAATAAATCA-3';

[0052] The sequence of nagAB-L-1F is shown in SEQ ID NO. 9, specifically 5'-TTGTATCAATGATTTTCATGGTCTCT-3'; the sequence of nagAB-L-1R is shown in SEQ ID NO. 10, specifically 5'-CTGTGTGAAATTGTTATCCGCTCTCAATTTCTGTCACGATCGC-3';

[0053] The sequence of nagAB-R-2F is shown in SEQ ID NO. 11, specifically 5'-GTCGTGACTGGGAAAACCCTGGCATGGAACATGCTGACTTATGAATAT C-3'; the sequence of nagAB-R-2R is shown in SEQ ID NO. 12, specifically 5'-GCAATAAGGTGGAAACCGATTAT-3'.

[0054] In the primer naming of the present invention, the front portion corresponds to the knocked-out gene, and the back portion L / R, 1F / 2F and 1R / 2R are used to distinguish different primers.

[0055] In one embodiment, in step (d), the pP43NMK-GNA1-glmS plasmid is transformed into the MT101 strain using electroporation technology.

[0056] Another embodiment of the present invention provides a method for constructing an engineered bacterium with high N-acetylglucosamine production, and the obtained engineered bacterium with high N-acetylglucosamine production.

[0057] Yet another embodiment of the present invention provides a use of the engineered bacteria with high N-acetylglucosamine production constructed by the above-mentioned method for constructing an engineered bacteria with high N-acetylglucosamine production in the production of N-acetylglucosamine.

[0058] Another embodiment of the present invention provides a method for producing N-acetylglucosamine, the method comprising:

[0059] The engineered bacteria with high N-acetylglucosamine production are inoculated into a culture medium for anaerobic culture.

[0060] In one embodiment, the culture medium is LB medium.

[0061] In one embodiment, the culture temperature is 35-38° C. and the culture time is 8-12 h.

[0062] In one embodiment, the inoculation concentration of the engineered bacteria with high N-acetylglucosamine production is 10 6 cfu / ml.

[0063] The technical solution of the present invention is further described in detail below through specific embodiments.

[0064] The primer sequences used in the Cre / loxP homologous recombination method are as follows:

[0065] The sequence of nagP-L-1F is shown in SEQ ID NO. 1, specifically 5'-TAATGAGATGGCTGTGTCGGAAT-3'; the sequence of nagP-L-1R is shown in SEQ ID NO. 2, specifically 5'-CCTGTGTGAAATTGTTATGCGCTCATCCACTCTCCAAACGAGTTGAT-3';

[0066] The sequence of nagP-R-1F is shown in SEQ ID NO. 3, specifically 5'-TACGTCGTGACTGGGAAAACCCTGCCCGCGGTCTTAACCGGGT-3'; the sequence of nagP-R-1R is shown in SEQ ID NO. 4, specifically 5'-AACGACAACGCCCAGCTT-3';

[0067] The sequence of gamPA-L-1F is shown in SEQ ID NO. 5, specifically 5'-TCATTCTCCCCATTGTGTATG-3'; the sequence of gamPA-L-1R is shown in SEQ ID NO. 6, specifically 5'-GGTGTGAAATTGTTATCCGCTCTGATATGCCTTATTAAACATGACA-3';

[0068] The sequence of gamPA-R-1F is shown in SEQ ID NO. 7, specifically 5'-GTCGTGACTGGGAAAACCCTGCCATTCTCATAACACAATAAAAGGAGA CT-3'; the sequence of gamPA-R-1R is shown in SEQ ID NO. 8, specifically 5'-AATGGCGGACATGGAATAAATCA-3';

[0069] The sequence of nagAB-L-1F is shown in SEQ ID NO. 9, specifically 5'-TTGTATCAATGATTTTCATGGTCTCT-3'; the sequence of nagAB-L-1R is shown in SEQ ID NO. 10, specifically 5'-CTGTGTGAAATTGTTATCCGCTCTCAATTTCTGTCACGATCGC-3';

[0070] The sequence of nagAB-R-2F is shown in SEQ ID NO. 11, specifically 5'-GTCGTGACTGGGAAAACCCTGGCATGGAACATGCTGACTTATGAATAT C-3'; the sequence of nagAB-R-2R is shown in SEQ ID NO. 12, specifically 5'-GCAATAAGGTGGAAACCGATTAT-3'.

[0071] Example 1

[0072] This embodiment is a method for constructing an engineered bacterium that produces high-yield N-acetylglucosamine, and the method comprises the following steps:

[0073] (a) The nagA, nagB, nagP, gamA, and gamP genes in Bacillus subtilis 168 were sequentially knocked out using Cre / loxP homologous recombination to generate strain MT101.

[0074] (b) The GNA1 gene from the yeast Saccharomyces cerevisiae W303 and the glmS gene from the strain Bacillus subtilis 168 were tandemly synthesized to obtain a tandem GNA1 and glmS gene.

[0075] (c) The tandem genes of GNA1 and glmS were introduced into the pP43NMK plasmid and located between the KpnI and HindIII restriction sites to obtain the pP43NMK-GNA1-glmS plasmid;

[0076] (d) The pP43NMK-GNA1-glmS plasmid was transformed into the MT101 strain using electroporation technology to obtain the engineered bacteria with high N-acetylglucosamine production.

[0077] Example 2

[0078] This example is the determination of N-acetylglucosamine production of the engineered bacteria with high N-acetylglucosamine production.

[0079] The production of N-acetylglucosamine in wild type (WT), intermediate mutant (MT101), and engineered strain with high N-acetylglucosamine production (MT102) under hypoxic conditions was determined by targeted metabolomics methods.

[0080] The high-yield mechanism of the engineered strain that produces high N-acetylglucosamine production was revealed by comparing the transcriptome and metabolome of the engineered strain with that of the wild type. Intermediates in the engineered strain are rapidly converted to N-acetylglucosamine, while the metabolic flux to other products is reduced.

[0081] The specific experimental measurement and data analysis methods are as follows: the internal calibration standards were purchased from Sigma-Aldrich Shanghai Co., Ltd.;

[0082] The wild type WT Bacillus subtilis 168 wild type (WT), intermediate mutant (MT101) and engineered strain with high N-acetylglucosamine production (MT102) were cultured in LB medium under anaerobic conditions. The culture conditions were as follows: the inoculum size was 10 6 cfu / mL, culture conditions were 37°C, rotation speed was 150 rpm and culture time was 10 h; samples were taken for targeted metabolomics and transcriptomics determination.

[0083] The targeted metabolome assay is as follows:

[0084] The disrupted cells were extracted with a solvent of methanol / methyl cyanide / water (2:2:1, volume ratio) for 20 minutes. The sample was then shaken and mixed thoroughly before centrifugation at 14,000 g for 20 minutes. The extracted sample was then vacuum-dried at low temperature and used for later use. The dry powder was then redissolved in methanol / methyl cyanide / water (1:1, volume ratio). After separation on a UPLC chromatograph, the compounds were fragmented and polarized. The resulting positive and negative compounds were then determined by mass spectrometry. Data processing was performed using MultiQuant software.

[0085] Transcriptome determination and analysis methods are as follows:

[0086] Total RNA was extracted using a commercial extraction kit (Qiagen RNeasy Micro Kit); after removing ribosomal RNA (rRNA), mRNA was fragmented and then reverse transcribed to obtain cDNA with random primers for library construction; sequencing was performed on the Illumina Hiseq 2500 platform; sequence quality control was performed using FASTQC software and Trimmomatic; gene expression levels were determined using RSEM software, and differential expression was determined using DESeq2 software; the functions of differentially expressed genes were determined using the website DAVID.

[0087] The experimental results are as follows:

[0088] The N-acetylglucosamine production of the wild type (WT), the intermediate mutant (MT101), and the engineered strain with high N-acetylglucosamine production (MT102) under hypoxic conditions is shown in Figure 2. Figure 1 As shown;

[0089] Depend on Figure 1 It can be seen that compared with the wild type, the engineered bacteria with high N-acetylglucosamine production in the present application can produce high N-acetylglucosamine.

[0090] The key gene expression of wild type (WT) and high-yield N-acetylglucosamine engineered bacteria (MT102) is shown in Figure 2. Figure 2 As shown;

[0091] Depend on Figure 2 It can be seen that the engineered bacteria with high N-acetylglucosamine production in the present application can achieve high expression of glmS and GNA1 genes, and reduce the expression of gamA, nagB, nagA, and nagP genes compared to the wild-type strain.

[0092] The expression of key metabolites of wild type (WT) and high-yield N-acetylglucosamine engineered bacteria (MT102) are shown in Figure 2. Figure 3 As shown; N-acetylglucosamine is the extracellular concentration, and the other three compounds are the intracellular concentrations. Figure 3In the formula, Fructose 6-P is fructose 6-phosphate; Glucosamine 6-P is glucosamine 6-phosphate; N-acetylglucosamine 6-P is N-acetylglucosamine 6-phosphate; N-acetylglucosamine is N-acetylglucosamine;

[0093] Depend on Figure 3 It can be seen that compared with the wild type, the engineered bacteria with high N-acetylglucosamine production in the present application can highly express N-acetylglucosamine extracellularly.

[0094] The above experiments are only part of the experiments conducted by the inventors of this application, but they are sufficient to prove that the engineered bacteria with high N-acetylglucosamine production in this application can produce high N-acetylglucosamine, realize the fermentation production of N-acetylglucosamine and improve the yield.

[0095] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or make equivalent replacements for some or all of the technical features therein. These modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and description of the present invention.

Claims

1. A method for constructing an engineered bacterium that produces high-yield N-acetylglucosamine, characterized in that: The steps include: (a) The nagA, nagB, nagP, gamA, and gamP genes in Bacillus subtilis 168 were sequentially knocked out to generate strain MT101; (b) The GNA1 gene from the yeast Saccharomyces cerevisiae W303 and the glmS gene from the strain Bacillus subtilis 168 were tandemly synthesized to obtain a tandem GNA1 and glmS gene. (c) The tandem genes of GNA1 and glmS were introduced into the pP43NMK plasmid and located between the KpnI and HindIII restriction sites to obtain the pP43NMK-GNA1-glmS plasmid; (d) The pP43NMK-GNA1-glmS plasmid is transformed into the MT101 strain to obtain the engineered bacteria that produces high N-acetylglucosamine.

2. The method for constructing an engineered bacterium for high-yield N-acetylglucosamine production according to claim 1, wherein: In the step (a), the nagA, nagB, nagP, gamA and gamP genes in the Bacillus subtilis 168 strain are knocked out in sequence using the Cre / loxP homologous recombination method.

3. The method for constructing an engineered bacterium for high-yield N-acetylglucosamine production according to claim 2, wherein: The primer sequences used in the Cre / loxP homologous recombination method are as follows: The sequence of nagP-L-1F is shown in SEQ ID NO. 1; the sequence of nagP-L-1R is shown in SEQ ID NO. 2; The sequence of nagP-R-1F is shown in SEQ ID NO. 3; the sequence of nagP-R-1R is shown in SEQ ID NO. 4; The sequence of gamPA-L-1F is shown in SEQ ID NO. 5; the sequence of gamPA-L-1R is shown in SEQ ID NO. 6; The sequence of gamPA-R-1F is shown in SEQ ID NO. 7; the sequence of gamPA-R-1R is shown in SEQ ID NO. 8; The sequence of nagAB-L-1F is shown in SEQ ID NO. 9; the sequence of nagAB-L-1R is shown in SEQ ID NO. 10; The sequence of nagAB-R-2F is shown in SEQ ID NO.11; the sequence of nagAB-R-2R is shown in SEQ ID NO.

12.

4. The method for constructing an engineered bacterium for high-yield N-acetylglucosamine production according to claim 1, wherein: In the step (d), the pP43NMK-GNA1-glmS plasmid is transformed into the MT101 strain using electroporation technology. 5 . The engineered bacteria with high N-acetylglucosamine production obtained by the method for constructing an engineered bacteria with high N-acetylglucosamine production as claimed in claim 1 .

6. Use of the engineered bacteria with high N-acetylglucosamine production constructed by the method for constructing the engineered bacteria with high N-acetylglucosamine production according to any one of claims 1 to 4 in the production of N-acetylglucosamine.

7. A method for producing N-acetylglucosamine, characterized in that: The production method of the N-acetylglucosamine comprises: The engineered bacteria with high N-acetylglucosamine production according to any one of claim 5 is inoculated into a culture medium for anaerobic culture.

8. The method for producing N-acetylglucosamine according to claim 7, wherein The culture medium is LB culture medium.

9. The method for producing N-acetylglucosamine according to claim 1, wherein The culture temperature is 35-38° C., and the culture time is 8-12 hours.

10. The method for producing N-acetylglucosamine according to claim 1, wherein The inoculation concentration of the high-yield N-acetylglucosamine engineered bacteria is 10 6 cfu / mL.

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