Recombinant corynebacterium glutamicum with high yield of N-acetylglucosamine and application of recombinant corynebacterium glutamicum

Abstract

The invention discloses recombinant corynebacterium glutamicum for high yield of N-acetylglucosamine, which is characterized in that corynebacterium glutamicum ATCC13032 is used as an original strain; an N-acetylglucosamine-6-phosphate deacetylase encoding gene nagA, a glucosamine-6-phosphate deaminase encoding gene nagB, a glucose-6-phosphate 1-dehydrogenase encoding gene zwf, a lactic dehydrogenase encoding gene ldhA, an N-acetylmannosamine-6-phosphate 2-epimerase encoding gene nano E, a glucosamine-6-phosphate deacetylase encoding gene nagA, a glucosamine-6-phosphate deacetylase encoding gene nagB, a glucose-6-phosphate 1-dehydrogenase encoding gene zwf, a lactic dehydrogenase encoding gene ldhA, a lactic dehydrogenase encoding gene ldhA, a an N-acetylglucosamine specific phosphotransferase encoding gene cgl2642 and a glucose transporter encoding gene ptsG are used as the specific gene of the N-acetylglucosamine specific phosphotransferase; the invention also discloses a method for preparing the recombinant glucosamine-6-phosphate acetyltransferase gene, and expresses an exogenous glucosamine-6-phosphate acetyltransferase coding gene GNA1, an exogenous glutamine-fructose-6-phosphate transaminase coding gene glmS, an exogenous glucose transporter coding gene galP and an exogenous glucose transporter coding gene glf.

Description

Technical Field

[0001] The present invention belongs to the technical field of synthetic biology, and in particular relates to a recombinant Corynebacterium glutamicum capable of producing high N-acetylglucosamine yield and application thereof. Background Art

[0002] N-acetylglucosamine (NAG), a derivative of glucose, has numerous physiological functions in the human body and is widely used in food, medicine, and cosmetics. NAG possesses anti-inflammatory, anti-tumor, and antioxidant properties in the human body. As a major component of cartilage and joint lubricants, it plays a crucial role in bone and joint health. Clinically, it is used as a treatment for osteoarthritis and rheumatoid arthritis. Exogenous glucosamine and NAG can be supplemented to treat joint diseases in middle-aged and elderly people, alleviating joint pain. Furthermore, NAG is widely used in infant food, cosmetics, and feed additives. It can be used as a food antioxidant, an infant food additive, and a sweetener for diabetics, among other applications.

[0003] Currently, the main methods for producing NAG include acid hydrolysis and enzymatic hydrolysis of shrimp and crab shells. The acid hydrolysis method primarily involves the chemical condensation reaction of glucosamine and acetic anhydride. Its primary raw material, glucosamine, is obtained by hydrolyzing shrimp and crab shells with hydrochloric acid or sulfuric acid. This process generates large amounts of industrial wastewater containing hydrochloric (or sulfuric) and acetic acid, placing significant environmental strain. Enzymatic hydrolysis increases production costs for manufacturers and is unsuitable for industrial production. Both methods suffer from high costs, low efficiency, limited raw material supply, and environmental pollution. With the advancement of metabolic engineering and synthetic biology, the use of microbial fermentation to synthesize NAG has gained widespread application due to its advantages of mild production conditions, high product purity, short production cycles, and independence from regional or seasonal restrictions. Genetically engineered microorganisms synthesizing NAG offer high quality and titer, and hold promise for industrial production. Currently, genetically engineered bacteria used for NAG production in research and development both domestically and internationally primarily include Escherichia coli, Bacillus subtilis, and Corynebacterium glutamicum.

[0004] Escherichia coli is the preferred host for a variety of fermentation products due to its ease of cultivation and mature genetic manipulation. Microbial fermentation for NAG production typically involves modifying strains based on the existing glucosamine metabolic pathways in E. coli through metabolic engineering to enable the synthesis of NAG from simple carbon sources such as glucose or glycerol through multiple enzymatic reactions within the E. coli. However, because E. coli secretes endotoxins during fermentation, these substances are difficult to completely remove from the fermentation product. Therefore, NAG produced by colon fermentation does not meet food-grade requirements.

[0005] Corynebacterium glutamicum is a Gram-positive bacterium that, as a food-grade microorganism, has been used in the industrial fermentation production of numerous amino acids, such as glutamic acid, valine, and lysine. Compared to Escherichia coli, Corynebacterium glutamicum offers advantages such as high safety, low pathogenicity, high stress resistance, and a low probability of phage contamination, thus playing an important role in the field of genetic engineering. Prior art has disclosed the use of Corynebacterium glutamicum for the production of NAG. For example, patent CN114250188B discloses a genetically engineered bacterium for synthesizing N-acetylglucosamine and its application. This genetically engineered bacterium is a recombinant Corynebacterium glutamicum containing the glucosamine synthase gene glmS, the glucosamine transacetylase gene gna1, and the 6β-acetylglucosamine-specific phosphatase gene yqaB. Furthermore, the bacterium has undergone a chassis microbial modification to remove genes associated with the GlcNAc retrotransport pathway, catabolic pathway, and competitive metabolic pathway. However, the NAG production of this recombinant Corynebacterium glutamicum still fails to meet market demand. Therefore, those skilled in the art need to further modify Corynebacterium glutamicum to improve its NAG production capacity. Summary of the Invention

[0006] The present invention selects Corynebacterium glutamicum which is food safety grade (GARS) and not easily contaminated as a starting strain, and performs modification of relevant genes to achieve the purpose of high production of N-acetylglucosamine.

[0007] Specifically, the present invention includes the following technical solutions.

[0008] In the first aspect, the present invention provides a recombinant Corynebacterium glutamicum with high N-acetylglucosamine production, wherein the recombinant strain is based on Corynebacterium glutamicum ATCC13032 as a starting strain, and the N-acetylglucosamine-6-phosphate deacetylase encoding gene nagA, the glucosamine-6-phosphate deaminase encoding gene nagB, the glucose-6-phosphate 1-dehydrogenase encoding gene zwf, the lactate dehydrogenase encoding gene ldhA, the N-acetylmanosamine-6-phosphate 2-epimerase encoding gene nanE, the N-acetylglucosamine-specific phosphotransferase encoding gene cgl2642 and the glucose transporter encoding gene ptsG are knocked out; and the glucosamine-6-phosphate acetyltransferase encoding gene GNA1 from Saccharomyces cerevisiae BY4742, the glutamine-fructose-6-phosphate transaminase encoding gene glmS from Escherichia coli, the glucose transporter encoding gene galP from Escherichia coli and the glucose transporter encoding gene glf from Zymomonas mobilis are expressed.

[0009] In one or more embodiments, a promoter as shown in sequence SEQ ID NO.2 or SEQ ID NO.3 is inserted before the gene galP; a promoter as shown in sequence SEQ ID NO.1 or SEQ ID NO.4 is inserted before the gene glf; a promoter as shown in sequence SEQ ID NO.1 is inserted before the gene GNA1; and a promoter as shown in sequence SEQ ID NO.3 or SEQ ID NO.5 is inserted before the gene glmS.

[0010] In a second aspect, the present invention provides a method for constructing the recombinant Corynebacterium glutamicum, comprising the following steps:

[0011] (1) Knockout of genes that have a negative impact on the synthesis of N-acetylglucosamine: knockout of genes nagA, nagB, zwf, ldhA, nanE, and cgl2642 in the starting strain;

[0012] (2) adding a sugar transporter system: integrating the glucose transporter encoding genes galP and glf at the ptsG knockout site;

[0013] (3) Expression of genes related to the synthesis of N-acetylglucosamine: Integrating the genes glmS and GNA1 into the strain described in step (2) to obtain the recombinant Corynebacterium glutamicum.

[0014] In one or more embodiments, in step (2), a recombinant plasmid containing genes galP and glf is constructed based on plasmid pK18mobsacB, and is transferred into the strain described in step (1);

[0015] In one or more embodiments, in step (3), a recombinant plasmid containing genes glmS and GNA1 is constructed based on plasmid pEKEx2, and is transferred into the strain described in step (2).

[0016] In a third aspect, the present invention provides the use of the recombinant Corynebacterium glutamicum in the production of N-acetylglucosamine.

[0017] In a fourth aspect, the present invention provides a method for efficiently producing N-acetylglucosamine, which uses the recombinant Corynebacterium glutamicum described in the present invention for fermentation, and then separates and purifies to obtain N-acetylglucosamine.

[0018] In one or more embodiments, the fermentation medium comprises: 80-120 g / L glucose, 90-110 mg / L magnesium sulfate, 30-40 g / L Angel Yeast Powder, 8-12 g / L ammonium sulfate, 10-12 mg / L FeSO4, 90-110 mg / L sodium citrate, 240-260 ul / L H3PO4 (85%), 450-550 mg / L defoamer, 55-65 mg / L calcium pantothenate, 15-20 mg / L nicotinamide, 45-55 mg / L thinamin·HCl, and 45-55 mg / L biotin.

[0019] In one or more embodiments, the method further comprises the step of adding feed during the fermentation of the recombinant Corynebacterium glutamicum.

[0020] In one or more embodiments, the feed medium comprises: 650-700 g / L glucose, 9-11 g / L ammonium sulfate, and 1-3 ml / L defoamer. The present invention is not limited to the defoamer, and any defoamer publicly used in biological fermentation processes can be used in this application, such as polyether defoamer PPE.

[0021] The present invention uses Corynebacterium glutamicum ATCC13032 as the starting strain, knocks out genes that have a negative impact on the synthesis of N-acetylglucosamine, adds a sugar transporter system, and expresses genes related to the synthesis of N-acetylglucosamine to obtain a recombinant Corynebacterium glutamicum that produces a high yield of N-acetylglucosamine. The recombinant strain of the present invention can significantly increase the yield of N-acetylglucosamine, making it suitable for industrial large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the structure of the plasmid pK18mobsacB-S10-galP-M9-glf in Example 2.

[0023] Figure 2 Schematic diagram of the structure of plasmid pK18mobsacB-S6-galP-S5-glf in Example 2.

[0024] Figure 3 Schematic diagram of the structure of plasmid pEKEx2-S10glmS-S5GNA1 in Example 3. DETAILED DESCRIPTION

[0025] The present invention is based on the existing technology (CN114250188B) and further performs gene editing on Corynebacterium glutamicum to obtain a high-yield N-acetylglucosamine recombinant strain.

[0026] CN114250188B knocks out related genes in Corynebacterium glutamicum, including the GlcNAc-specific phosphotransferase gene cgl2642 that transports extracellular GlcNAc to the cell, the 6p-GlcNAc deacetylase gene nagA of the acetylglucosamine catabolic pathway, the 6p-GlcNAc deaminase gene nagB, the gene ldh of the lactic acid synthesis pathway, the 6p-GlcNAc mannose epimerase gene nanE that converts 6p-GlcNAc into 6p-mannose, the related gene of the pentose phosphate pathway is gene zwf, and the related gene fba of the glycolytic pathway is also weakened. In contrast, the present invention does not weaken the related gene fba of the glycolytic pathway, but instead performs gene editing at other sites.

[0027] Specifically, the present invention also includes adding a sugar transporter system into the strain, and integrating the genes glmS and GNA1 into the strain to obtain the recombinant Corynebacterium glutamicum.

[0028] More specifically, the present invention integrates the glucose transporter encoding genes galP and glf into the ptsG knockout site of the bacterium, wherein the glucose transporter encoding gene galP is derived from Escherichia coli, and the glucose transporter encoding gene glf is derived from Zymomonas mobilis; the present invention also expresses the glucosamine-6-phosphate acetyltransferase encoding gene GNA1 derived from Saccharomyces cerevisiae BY4742 and the glutamine-fructose-6-phosphate transaminase encoding gene glmS derived from Escherichia coli in the bacterium.

[0029] Furthermore, the present invention screened and studied the promoters of the aforementioned related genes and found that inserting specific promoters before the related genes significantly increased N-acetylglucosamine production. Specifically, a promoter such as SEQ ID NO. 2 or SEQ ID NO. 3 was inserted before the galP gene; a promoter such as SEQ ID NO. 1 or SEQ ID NO. 4 was inserted before the glf gene; a promoter such as SEQ ID NO. 1 was inserted before the GNA1 gene; and a promoter such as SEQ ID NO. 3 or SEQ ID NO. 5 was inserted before the glmS gene.

[0030] The present invention will be further described in detail below with reference to specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0031] Example

[0032] This article involves the addition amount, content and concentration of various substances, and the percentages mentioned therein, unless otherwise specified, refer to the percentage by mass.

[0033] Materials and methods

[0034] The whole gene synthesis, primer synthesis and sequencing in the examples were commissioned to General Bio (Anhui) Co., Ltd.

[0035] The molecular biology experiments in the examples, including plasmid construction, enzyme digestion, ligation, competent cell preparation, transformation, culture medium preparation, etc., were performed primarily with reference to the third edition of Molecular Cloning: A Laboratory Manual (J. Sambrook, D.W. Russell, USA, translated by Huang Peitang et al., Science Press, Beijing, 2002). Specific experimental conditions can be determined by simple experiments if necessary.

[0036] The primer sequences involved in the following examples are as follows:

[0037] Table 1 Primer sequences involved in the examples

[0038]

[0039]

[0040] PCR amplification experiments should be performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. These conditions can be adjusted through simple experiments if necessary.

[0041] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.2. (Add 20 g / L agar powder to LB solid medium.)

[0042] In the following embodiments, when a culture medium containing kanamycin (kan) is used, the final concentration of kanamycin in the culture medium is 50 μg / ml; when a culture medium containing chloramphenicol (cm) is used, the final concentration of chloramphenicol in the culture medium is 30 μg / ml; when a culture medium containing apramycin resistance (Apr) is used, the final concentration of apramycin in the culture medium is 50 μg / ml; when IPTG is used for induction, the final concentration of IPTG in the culture medium is 0.2 mM.

[0043] The HPLC detection method of N-acetylglucosamine in the following examples is as follows:

[0044] Detection column: Bio-Rad AMINEX HPX 87H Organic Analysis Column (300×7.8mm);

[0045] Column temperature: 60°C; mobile phase: 6 mM sulfuric acid, flow rate: 0.6 ml / min; detection wavelength: 210 nm.

[0046] It should be noted that, for the sake of convenience of description, in the embodiments, the strain number, plasmid number, enzyme number, and enzyme-encoding gene number may share one number, which is easily understood by those skilled in the art, that is, the same number can refer to different biological forms in different environments.

[0047] Example 1: Construction of gene knockout strains

[0048] The invention uses the rod-shaped glutamic acid rod ATCC13032 as the starting strain, and knocks out genes that have a negative impact on the synthesis of N-acetylglucosamine, including knocking out the genes nagA, nagB, zwf, ldhA, nanE and cgl2642 in the starting strain.

[0049] The information of the knocked-out genes is shown in the following table:

[0050] Table 2 Information of knockout genes

[0051]

[0052]

[0053] The specific method refers to the example in patent CN114250188B to obtain the gene knockout strain △nagA / B△ldh△cgl2642△zwf△nanE.

[0054] Example 2: Adding a sugar transporter system

[0055] The glucose transporter encoding genes galP and glf are integrated into the ptsG knockout site, wherein the glucose transporter encoding gene galP is derived from Escherichia coli (DNA sequence is shown in SEQ ID NO.6), and the glucose transporter encoding gene glf is derived from Zymomonas mobilis (DNA sequence is shown in SEQ ID NO.7).

[0056] 2.1 Construction of recombinant plasmid pK18mobsacB-S10-galP-M9-glf

[0057] Plasmid pK18mobsacB was double-digested with HindIII / EcoRI to recover the digested fragment 1; 1.5 kb fragment 2 was amplified by PCR using the primer pair S10_F / GalP10_R and the MG1655 genome as a template; 1.5 kb fragment 3 was amplified by PCR using the primer pair M9_F / Glf_R and the synthetic Zymomonas mobilis glf as a template; 1 kb fragments 4 and 5 were amplified by PCR using the primer pairs PtsG_UPF / PtsG10_UPR and PtsG5_DNF / PtsG_DNR and the Escherichia coli MG1655 genome as templates, respectively;

[0058] OE-PCR was performed using the S10_F / Glf_R primer pair with fragments 2 and 3 as templates to amplify the 3kb fragment 6; then fragments 1, 4, 5, and 6 were spliced ​​using the GIBSON kit (Nanjing Novezan), transformed into DH5a competent cells, and screened on LB-kan (50) solid plates to obtain the target plasmid pK18mobsacB-S10-galP-M9-glf.

[0059] 2.2 Construction of recombinant plasmid pK18mobsacB-S6-galP-S5-glf

[0060] Using the same method, the S5_F / GalP6_R primer pair was used to amplify the 1.5kb fragment 7 using the MG1655 genome as a template; the S5_F / Glf_R primer pair was used to amplify the 1.5kb fragment 8 using the synthetic Zymomonas mobilis glf as a template; the PtsG_UPF / PtsG6_UPR primer pair was used to amplify the 1kb fragment 9 using the Escherichia coli MG1655 genome as a template; the S5_F / Glf_R primer pair was used to perform OE-PCR using fragments 7 and 8 as templates to amplify a 3kb fragment 10; fragments 1, 9, 10, and 5 were spliced ​​by GIBSON (Nanjing Novezan), transformed into DH5a competent cells, and screened on LB-kan (50) solid plates to obtain the target plasmid pK18mobsacB-S6-galP-S5-glf.

[0061] 2.3 Integration of recombinant plasmid into gene knockout strain

[0062] The above two plasmids were respectively transferred into the gene knockout strain △nagA / B△ldh△cgl2642△zwf△nanE constructed in Example 1 to obtain strain △nagA / B△ldh△cgl2642△zwf△nanE△ptsG::S10-galP-M9-glf and strain △nagA / B△ldh△cgl2642△zwf△nanE△ptsG::S6-galP-S5-glf.

[0063] Example 3: Expression of genes related to the synthesis of N-acetylglucosamine

[0064] The glucosamine-6-phosphate acetyltransferase encoding gene GNA1 (DNA sequence shown in SEQ ID NO.8) from Saccharomyces cerevisiae BY4742 and the glutamine-fructose-6-phosphate aminotransferase encoding gene glmS (DNA sequence shown in SEQ ID NO.9) from Escherichia coli were expressed in Corynebacterium glutamicum.

[0065] 3.1 Construction of recombinant plasmid pEKEx2-S10glmS-S5GNA1

[0066] The pEKEx2 plasmid was digested with PstI and EcoRI to recover fragment 1; the 2 kb fragment 2 was amplified by PCR using the S10-glmS-F2 and S10-glmS-R primer pairs with the Escherichia coli genome as a template; the 2 kb fragment 3 was obtained by PCR amplification using the S10-glmS-F1 / S10-glmS-R primer pair with fragment 2 as a template, and the promoter fragment was added to obtain a fragment 3 of about 2 kb; the 0.56 kb fragment 4 was amplified by PCR using the Saccharomyces cerevisiae BY4742 genome as a template using the S5-GNA1-F / S5-GNA1-R primer pair; fragments 1, 3, and 4 were spliced ​​by GIBSON (Nanjing Novizan), transformed into DH5a competent cells, and screened on LB-kan (50) solid plates to obtain the target plasmid pEKEx2-S10glmS-S5GNA1.

[0067] 3.2 Saturation mutagenesis of the S10 promoter-10 region of the recombinant plasmid pEKEx2-S10glmS-S5GNA1

[0068] PCR amplification was performed using primer pair S10mut_F / S10mut_R with pEKEx2-S10glmS-S5GNA1 as template. The PCR product was digested with DpnI and directly transformed into △nagA / B△ldh△cgl2642△zwf△nanE competent cells. Then, 96-well plate fermentation was carried out (LB solution + 20 g / L glucose). HPLC was used to detect product changes after 24 hours of fermentation.

[0069] After fermentation and sequencing testing, it was found that 5-10 mutants had increased bacterial yield. The specific mutations are shown in the following table:

[0070] Table 3 Mutations in the S10 promoter-10 region of the pEKEx2-S10glmS-S5GNA1 plasmid

[0071] serial number -10 zone sequence NAG g / L Improvement rate original TAGCAT 5.8 / Mut_1 CTACAT 8.3 43％ Mut_2 ATTGCA 7.2 24％ Mut_3 TCAAAT 7.0 21％ Mut_4 TACCAT 6.8 17％ Mut_5 ATATCA 6.3 9％

[0072] 3.3 Transformation of mutant plasmids

[0073] The screened mutant plasmid Mut1 was transformed into the strains △nagA / B△ldh△cgl2642△zwf△nanE△ptsG::S10-galP-M9-glf and △nagA / B△ldh△cgl2642△zwf△nanE△ptsG::S6-galP-S5-glf constructed in Example 2, respectively, to obtain NAG-P1 and NAG-P2 strains, which were preserved in glycerol tubes.

[0074] Example 4: Shake flask fermentation of recombinant strains

[0075] The recombinant strains NAG-P1 and NAG-P2 obtained in Example 3 were verified by shake flask fermentation.

[0076] The NAG-P1 and NAG-P2 strains were activated by non-resistant plate streaking, and single clones were picked and transferred into seed culture medium test tubes and cultured overnight at 30°C and 220rpm. The cells were transferred to fermentation shake flasks (25 / 250ml) at a transfer volume of 5%, and cultured with shaking at 30°C and 220rpm for 6 hours. The pH of the shake flasks was observed every 6 hours during the fermentation process and adjusted to about 7.0 with ammonia water.

[0077] The shake flask fermentation seed medium is composed of: 10 g / L peptone, 5 g / L beef extract, 5 g / L yeast powder, 2.5 g / L sodium chloride, 2 g / L urea, and 10 g / L glucose. Sterilize at 115°C for 15 minutes.

[0078] Among them, the shake flask fermentation medium is shown in the following table:

[0079] Table 4 Shake flask fermentation medium formula

[0080]

[0081] After 48 hours of shake flask fermentation, NAG in the product was detected by HPLC. The results showed that the NAG yield in the fermentation broth of strain NAG-P1 reached 27.8 g / L, and the NAG yield in the fermentation broth of strain NAG-P2 reached 28.7 g / L, which were significantly higher than the NAG yield in patent CN114250188B.

[0082] Example 5: Fermentation of recombinant strains

[0083] The recombinant strains NAG-P1 and NAG-P2 obtained in Example 3 were fermented in a 5 L fermenter (Shanghai Baoxing).

[0084] Streak the above strains on plates and incubate in a constant temperature incubator at 30°C for approximately 18 hours. Pick 2-3 single colonies and transfer them to a 5ml LB tube. Incubate in a shake flask at 30°C at 230 rpm for 8 hours. Inoculate a 5% inoculum into a 100ml / 500ml LB shake flask and incubate in a shake flask at 30°C at 230 rpm for 16-18 hours. Inoculate a 5% inoculum into a 2L / 5L fermentor.

[0085] The fermentation tank was regulated to pH 7.0 with ammonia water, initially stirred at 400 rpm, aerated at 1 vvm, and the dissolved oxygen content was controlled at approximately 30%. The fermentation temperature was 30°C. The fermentation tank culture medium is shown in the table below:

[0086] Table 5 Fermentation tank culture medium formula

[0087]

[0088] The feed medium during the fermentation process was: 670 g / L glucose, 10 g / L ammonium sulfate (sterilized separately), 2 ml / L antifoaming agent (PPE).

[0089] The results showed that through fermentation verification in fermentation tanks, after 72 hours of fermentation, the accumulation of N-acetylglucosamine in the fermentation product of strain NAG-P1 reached 135g / L, and the accumulation of N-acetylglucosamine in the fermentation product of strain NAG-P2 reached 143g / L, which were significantly higher than the concentrations reported in the prior art.

[0090] In summary, the present invention obtains two recombinant Corynebacterium glutamicum strains with high N-acetylglucosamine production through gene editing, which can significantly increase the production of N-acetylglucosamine.

[0091] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by any technician familiar with this technical field within the technical scope disclosed by the present invention should be covered by the scope of protection of the present invention.

Claims

1. A recombinant Corynebacterium glutamicum with high N-acetylglucosamine production, characterized in that: The recombinant strain uses Corynebacterium glutamicum ATCC13032 as the starting strain, The genes encoding N-acetylglucosamine-6-phosphate deacetylase nagA, glucosamine-6-phosphate deaminase nagB, glucose-6-phosphate 1-dehydrogenase zwf, lactate dehydrogenase ldhA, N-acetylmanosamine-6-phosphate 2-epimerase nanE, N-acetylglucosamine-specific phosphotransferase cgl2642, and glucose transporter ptsG were knocked out. The glucosamine-6-phosphate acetyltransferase encoding gene GNA1 from Saccharomyces cerevisiae BY4742, the glutamine-fructose-6-phosphate aminotransferase encoding gene glmS from Escherichia coli, the glucose transporter encoding gene galP from Escherichia coli, and the glucose transporter encoding gene glf from Zymomonas mobilis were expressed.

2. The recombinant Corynebacterium glutamicum according to claim 1, wherein A promoter as shown in sequence SEQ ID NO.2 or SEQ ID NO.3 is inserted before the gene galP; a promoter as shown in sequence SEQ ID NO.1 or SEQ ID NO.4 is inserted before the gene glf; a promoter as shown in sequence SEQ ID NO.1 is inserted before the gene GNA1; and a promoter as shown in sequence SEQ ID NO.3 or SEQ ID NO.5 is inserted before the gene glmS.

3. The method for constructing the recombinant Corynebacterium glutamicum according to claim 1 or 2, characterized in that: The method comprises the following steps: (1) Knockout of genes that have a negative impact on the synthesis of N-acetylglucosamine: knockout of genes nagA, nagB, zwf, ldhA, nanE, and cgl2642 in the starting strain; (2) adding a sugar transporter system: integrating the glucose transporter encoding genes galP and glf at the ptsG knockout site; (3) Expression of genes related to the synthesis of N-acetylglucosamine: Integrating the genes glmS and GNA1 into the strain described in step (2) to obtain the recombinant Corynebacterium glutamicum.

4. The construction method according to claim 3, wherein: In the step (2), a recombinant plasmid containing the genes galP and glf is constructed based on the plasmid pK18mobsacB, and is transferred into the strain described in step (1).

5. The construction method according to claim 3, wherein: In the step (3), a recombinant plasmid containing genes glmS and GNA1 is constructed based on plasmid pEKEx2, and is transferred into the strain described in step (2).

6. Use of the recombinant Corynebacterium glutamicum according to claim 1 or 2 in the production of N-acetylglucosamine.

7. A method for efficiently producing N-acetylglucosamine, characterized in that: The recombinant Corynebacterium glutamicum according to claim 1 or 2 is used for fermentation, and then N-acetylglucosamine is obtained by separation and purification.

8. The method according to claim 7, wherein The fermentation culture medium comprises: 80-120 g / L glucose, 90-110 mg / L magnesium sulfate, 30-40 g / L Angel Yeast Powder, 8-12 g / L ammonium sulfate, 10-12 mg / L FeSO4, 90-110 mg / L sodium citrate, 240-260 ul / L H3PO4 (85%), 450-550 mg / L defoamer, 55-65 mg / L calcium pantothenate, 15-20 mg / L nicotinamide, 45-55 mg / L thinamin·HCl and 45-55 mg / L biotin.

9. The method according to claim 8, wherein The method further comprises the step of adding feed during the fermentation process of the recombinant Corynebacterium glutamicum.

10. The method according to claim 9, wherein The feed medium comprises: 650-700 g / L glucose, 9-11 g / L ammonium sulfate and 1-3 ml / L defoamer.

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