A mutant of n-acetylglutamate synthetase for n-acetylornithine production and application thereof

Abstract

The present application relates to the field of enzyme engineering and microbial fermentation production, and particularly relates to a N-acetylglutamate synthetase mutant for N-acetylglaucine production and application thereof. The present application, by studying the structure of N-acetylglutamate synthetase ArgA (EcargA) derived from Escherichia coli, and combining with the directed evolution technology, mutates the 323th and 435th sites of the EcargA protein, and constructs the N-acetylglutamate synthetase mutant EcargA V323A / S435A . The fermentation of the engineering strain of the N-acetylglutamate synthetase mutant for producing N-acetylglaucine can significantly improve the yield of N-acetylglaucine, reduce the generation of by-product glaucine, and simplify the fermentation process and reduce the production cost. The N-acetylglutamate synthetase mutant and the engineering strain provided by the present application have good industrial application value.

Description

Technical Field

[0001] This invention relates to the fields of enzyme engineering and microbial fermentation production, and in particular to a mutant N-acetylglutamate synthase for the production of N-acetylglucosamine and its application. Background Technology

[0002] Indigoidine is a natural blue pigment with broad biological activity and application potential. Its chemical name is 5,5'-diamino-4,4'-dihydroxy-3,3'-diazodibenzoquinone-(2,2'), and its molecular formula is C2. 10 H8N4O4. Blue violet is initially produced by certain bacteria and fungi, such as *Streptomyces* and *Pseudomonas*. These microorganisms produce blue violet through specific biosynthetic pathways, typically involving L-glutamine as a precursor, followed by a series of enzymatic reactions to generate the final product.

[0003] N-acetylated speckle is an acetylated derivative of speckle. Studies have shown that compared to speckle itself, N-acetylation modification can improve the water solubility of speckle, and N-acetylated speckle is more stable, less prone to fading, and has better color brightness, possessing great application potential. Current technologies for producing N-acetylated speckle each have their shortcomings. For example, patent CN2024107899337 discloses a recombinant strain that biosynthesizes N-acetylated speckle using glutamic acid as a substrate, but this recombinant strain also produces a large amount of speckle as a byproduct during the production of N-acetylated speckle. Patent CN202410826820.X discloses a fermentation process for producing N-acetylated speckle through a two-stage fermentation process. This process is relatively complex, requiring changes to the fermentation temperature and pH after 24 hours of fermentation, which would reduce fermentation yield, complicate the fermentation process, and increase production costs in industrial production. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the existing technology and provide an N-acetylglutamate synthase mutant that improves the yield of N-acetylglucosamine and its application.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides an N-acetylglutamate synthase mutant, wherein the N-acetylglutamate synthase mutant has at least 90% amino acid sequence identity with SEQ ID NO:1 and contains mutations at at least two sites; the mutation sites include positions 323 and 435 of SEQ ID NO:1; the mutations are substitution mutations.

[0007] N-acetylglucosamine is an acetylated derivative of glucosamine, and its structural formula is shown in formula (I).

[0008]

[0009] N-acetylglutamate synthase (NAGS) catalyzes the acetylation of glutamate. There are different types of NAGS, including ArgA, ArgJ, ArgO, and S-NAGS. Currently, there are no reports of increasing the yield of N-acetylglucosamine by modifying N-acetylglutamate synthase. In this invention, during the investigation of the protein structure of N-acetylglutamate synthase ArgA (hereinafter referred to as EcargA, encoded by the argA gene) derived from *Escherichia coli* BL21(DE3), it was discovered that positions 323 and 435 of the EcargA protein are located in the enzyme's catalytic pocket. Targeted mutation of these two amino acids can increase the yield of N-acetylglucosamine.

[0010] Preferably, the mutation includes the substitution of valine at position 323 of SEQ ID NO:1 with alanine, and the substitution of serine at position 435 with alanine.

[0011] This invention discovers that specific mutations can be performed at two specific sites through enzyme engineering: replacing valine and serine with alanine, respectively. Valine and serine have larger steric cavities, while alanine has less steric hindrance. Substitution facilitates the entry of glutamate into the active site, thereby improving the enzyme activity of EcargA. Based on these two sites, the resulting N-acetylglutamate synthase mutant is named EcargA. V323A / S435A This N-acetylglutamate synthase mutant can reduce the proportion of the byproduct sucralose while increasing the fermentation yield of N-acetylglucosamine, simplifying the fermentation process and reducing production costs.

[0012] Secondly, the present invention provides a gene that encodes the N-acetylglutamate synthase mutant.

[0013] Preferably, the nucleotide sequence of the gene is as shown in SEQ ID NO:2.

[0014] Thirdly, the present invention provides a recombinant vector comprising the aforementioned gene.

[0015] Preferably, the pRSFDuet plasmid is used to construct the recombinant vector.

[0016] Fourthly, the present invention provides an engineered strain that expresses the N-acetylglutamate synthase mutant; and / or contains the gene; and / or contains the recombinant vector.

[0017] Preferably, *Escherichia coli* is selected as the host bacterium to construct the engineered strain.

[0018] Preferably, the present invention introduces point mutations into strain HG-N-Idg06 (the construction method of which is described in Example 1 of patent CN 118360308 A). This strain has a clear genetic background, containing the gene encoding bpsA for safflower synthase, the gene encoding EntD for 4'-phosphopanylthioethylamine transferase, the gene encoding glnA for glutamine synthase, and the gene encoding argA for N-acetylglutamate synthase, facilitating fermentation process control. The introduction of EcargA... V323A / S435A The yield and content of N-acetylglucosamine were significantly increased after the mutation.

[0019] More preferably, during the construction process, a corresponding point mutation, EcargA, is introduced into the pRSFDuet-EcglnA-EcargA plasmid (which contains the glutamine synthase encoding gene glnA and the N-acetylglutamate synthase encoding gene argA). V323A / S435A The mutant strain was then co-transformed with pCDFDuet-bpsA-entD to finally obtain the engineered strain.

[0020] Fifthly, the present invention provides a method for constructing the engineered strain, comprising the following steps:

[0021] (a) Using the pRSFDuet-EcglnA-EcargA plasmid as the chassis strain, a point mutation was introduced at position 323 of the EcargA gene to mutate valine to alanine; then, a point mutation was introduced at position 435 of the EcargA gene to mutate serine to alanine, resulting in the mutant plasmid pRSFDuet-EcglnA-EcargA. V323A / S435A ;

[0022] (b) The mutant plasmid obtained in step (a) is co-transformed with pCDFDuet-bpsA-entD into competent cells, cultured on plates, and positive mutant plasmids are screened to obtain the engineered strain.

[0023] Preferably, the primer nucleotide sequences used to introduce the point mutation at position 323 are as shown in SEQ ID NO:3 and SEQ ID NO:4; and / or, the primer nucleotide sequences used to introduce the point mutation at position 435 are as shown in SEQ ID NO:5 and SEQ ID NO:6.

[0024] In a sixth aspect, the present invention provides the application of the N-acetylglutamate synthase mutant, or the engineered strain, in the production of N-acetylglucosamine.

[0025] In a seventh aspect, the present invention provides a method for producing N-acetylglucosamine by fermentation, wherein the engineered strain is used to produce N-acetylglucosamine by fermentation.

[0026] Preferably, the process includes the following steps: activating the engineered bacteria to obtain a seed culture, inoculating it into a fermentation medium, inducing fermentation, and cultivating to produce N-acetylglucosamine.

[0027] The engineered strain containing the N-acetylglutamate synthase mutant increased the yield of N-acetylglucosamine in the fermenter, increased its proportion in the fermentation product, and reduced the production of the byproduct glucosamine.

[0028] Preferably, the fermentation medium used in the fermentation process has the following formula: glucose 5g / L, glycerol 7g / L, wheat peptone 3g / L, corn steep liquor powder 2g / L, disodium hydrogen phosphate dodecahydrate 2g / L, potassium dihydrogen phosphate 1.5g / L, ammonium chloride 2g / L, magnesium sulfate heptahydrate 0.8g / L, water as solvent, and ammonia water is used to adjust the pH to 7.0.

[0029] Preferably, the fermentation conditions are: a rotation speed of 100 rpm, an aeration rate of 1 vvm, a dissolved oxygen (DO) of 35%, and a fermentation temperature of 25°C.

[0030] Preferably, fed medium is added after 12 hours of fermentation to provide nitrogen and carbon sources for the fermentation strain; and a synthase inducer is added after 16 hours of fermentation to induce the expression of the product synthase and improve the fermentation yield of N-acetylglucosamine.

[0031] Preferably, the supplemental culture medium comprises the following components at the following mass concentrations: glucose 550 g / L, ammonium chloride 10 g / L, wheat peptone 3 g / L, zinc sulfate 2.5 g / L, and water as the solvent.

[0032] Preferably, the synthase inducer comprises IPTG at a concentration of 0.1 mmol / L.

[0033] The beneficial effects of this invention are as follows:

[0034] This invention, through structural studies of the N-acetylglutamate synthase EcargA and combined with directed evolution technology, mutated positions 323 and 435 of the EcargA protein to construct the N-acetylglutamate synthase mutant EcargA. V323A / S435A Fermentation of N-acetylglucosamine using the engineered strain of the N-acetylglutamate synthase mutant can significantly improve the yield of N-acetylglucosamine, reduce the generation of the byproduct glucosamine, simplify the fermentation process, and reduce production costs. The N-acetylglutamate synthase mutant and engineered strain provided by this invention have good industrial application value. Attached Figure Description

[0035] Figure 1 This is a high-performance liquid chromatography (HPLC) chromatogram of the fermentation broth from Example 1.

[0036] Figure 2 This is a high-performance liquid chromatography (HPLC) chromatogram of the control culture medium.

[0037] Figure 3 This is a high-performance liquid chromatography (HPLC) chromatogram of the fermentation broth from Comparative Example 1.

[0038] Figure 4 This is a high-performance liquid chromatography (HPLC) chromatogram of the fermentation broth from Comparative Example 2.

[0039] Figure 5 This is a high-performance liquid chromatography (HPLC) chromatogram of the fermentation broth from Comparative Example 3.

[0040] Figure 6 This is a high-performance liquid chromatography (HPLC) chromatogram of the fermentation broth from Comparative Example 4. Detailed Implementation

[0041] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0042] The strain HG-N-Idg06 (i.e., BL21(DE3) / pRSFDuet-EcglnA-EcargA+pCDFDuet-bpsA-entD), plasmids pRSFDuet-EcglnA-EcargA, and pCDFDuet-bpsA-entD used in this invention were all provided by Nanjing Hegu Life Biotechnology Co., Ltd., and their construction was the same as Example 1 in the specification of patent CN118360308A (Construction method and application of an engineered bacterium for the biosynthesis of N-acetylglucosamine using glutamic acid as a substrate).

[0043] Example 1: Construction of mutants and engineered strains

[0044] 1. In this embodiment, the pRSFDuet-EcglnA-EcargA plasmid (provided by Nanjing Hegu Life Biotechnology Co., Ltd.) containing the glutamine synthase encoding gene glnA and the N-acetylglutamate synthase encoding gene argA was used as the chassis strain. The construction method is based on step two of Example 1 in the specification of patent CN118360308A: construction of pRSFDuet-EcglnA-EcargA plasmid.

[0045] Using point mutation technology, point mutations were introduced at positions 323 and 435 of the EcargA gene in the chassis plasmid pRSFDuet-EcglnA-EcargA: valine at position 323 was replaced by alanine (V323A), and serine at position 435 was replaced by alanine (S435A).

[0046] The specific method is as follows:

[0047] Using pRSFDuet-EcglnA-EcargA plasmid as a template, the mutation sequence was V323A and S435A, and the primers were designed as shown in Table 1.

[0048] Table 1

[0049]

[0050] Recombinant plasmids containing the mutated gene were constructed using reverse PCR technology, while the original template DNA was removed using DpnI.

[0051] Next, the mutant plasmid was transformed into DH5α competent cells, and positive plasmids were screened using kanamycin agar plates. Then, plasmid extraction and sequencing were performed to verify the mutant plasmid pRSFDuet-EcglnA-EcargA. V323A / S435A .

[0052] The formula for kanamycin plate culture medium is: yeast extract 5g / L, tryptone 10g / L, sodium chloride 10g / L, agar powder 15g / L, and kanamycin 25mg / L.

[0053] 2. The mutant plasmid pRSFDuet-EcglnA-EcargA was... V323A / S435A The strain was co-transformed into BL21(DE3) competent cells with pCDFDuet-bpsA-entD (provided by Nanjing Hegu Life Biotechnology Co., Ltd.) and plated on a double antibiotic plate medium containing kanamycin and streptomycin to successfully construct the engineered strain, named HG-N-Idg2024.

[0054] The formulation of the double antibiotic plate medium for kanamycin and streptomycin is as follows: yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L, agar powder 15 g / L, kanamycin 25 mg / L, and streptomycin 50 mg / L.

[0055] Example 2: Production of N-acetylglucosamine by fermentation using engineered strain HG-N-Idg2024

[0056] 1. Take out the frozen glycerol bacterial suspension of recombinant Escherichia coli strain HG-N-Idg2024, and after thawing, use the streak plate method to streak it on a double antibiotic agar plate containing kanamycin and streptomycin. After streaking, place the plate in a constant temperature incubator at 37℃ for 12 hours.

[0057] 2. Select single colonies with a diameter of 0.12-0.15 mm, inoculate them into 10 mL of LB medium, and add kanamycin to a final concentration of 25 mg / L and streptomycin to 50 mg / L. Incubate in a shaker at 37℃ and 220 rpm for 8 hours until the OD of the bacterial culture reaches a certain level. 600 A value between 3.0 and 4.0 indicates the presence of activated primary seed culture.

[0058] 3. Take 10 mL of the activated primary seed culture and inoculate it into a 5 L fermenter containing 2.4 L LB medium. Add kanamycin and streptomycin to a final concentration of 25 mg / L and 50 mg / L respectively. Culture conditions: pH 7.0, temperature 37℃, initial aeration ratio 1 vvm, initial rotation speed 220 rpm, fermenter pressure 0.05 MPa, stirring and maintaining dissolved oxygen (DO) at 30%. Culture for 8-10 h. The OD of the bacterial culture is then recorded. 600 =8.0, to obtain the activated secondary seed solution;

[0059] 4. At 2m 3 Add 1.2m to the fermentation tank 3 A basic fermentation medium containing glucose; the secondary seed culture obtained in step (3) is introduced into a 2m medium via a transfer tube. 3 Fermentation was carried out in a fermenter with pH controlled at 7.0, temperature at 25℃, aeration ratio of 1 vvm, initial rotation speed of 100 rpm, dissolved oxygen (DO) controlled by stirring and maintained at 35%, and tank pressure of 0.05 MPa. Fed medium was added after 12 hours of fermentation to provide nitrogen and carbon sources for the fermentation strain. At 16 hours of fermentation, a synthase inducer was added to induce the expression of the product synthase and improve the fermentation yield of N-acetylglucosamine.

[0060] 5. After 16 hours of fermentation, control the glucose concentration within the range of 0-5 g / L; when the N-acetylglucosamine production stops increasing after 72 hours of fermentation, the fermentation is terminated.

[0061] The LB medium comprises the following components at the following mass concentrations: yeast extract 5 g / L, tryptone 10 g / L, and sodium chloride 10 g / L.

[0062] The fermentation medium comprises the following components in the following mass concentrations: glucose 5 g / L, glycerol 7 g / L, wheat peptone 3 g / L, corn steep liquor powder 2 g / L, disodium hydrogen phosphate dodecahydrate 2 g / L, potassium dihydrogen phosphate 1.5 g / L, ammonium chloride 2 g / L, magnesium sulfate heptahydrate 0.8 g / L, with water as the solvent, and the pH adjusted to 7.0 using ammonia.

[0063] The fed culture medium comprises the following components at the following mass concentrations: glucose 550 g / L, ammonium chloride 10 g / L, wheat peptone 3 g / L, zinc sulfate 2.5 g / L, and water as the solvent.

[0064] The synthase inducer is IPTG, with an optimal concentration of 0.1 mmol / L.

[0065] Comparative Example 1

[0066] This comparative example provides an engineered strain, HG-N-Idg06, provided by Nanjing Hegu Biotechnology Co., Ltd., whose construction method is described in Example 1 of the specification of patent CN 118360308 A. The only difference between this comparative example engineered strain HG-N-Idg06 and the engineered strain constructed in Example 1 is that HG-N-Idg06 does not introduce two site mutations: the V323A and S435A mutations of EcargA.

[0067] The fermentation process of the engineered strain described in this comparative example is the same as that in Example 2.

[0068] Comparative Example 2

[0069] This comparative example provides an engineered strain. The only difference between this comparative example engineered strain and the engineered strain constructed in Example 1 is that this comparative example engineered strain does not introduce the S435A point mutation.

[0070] The only difference between the construction method of the engineered strain described in this comparative example and that in Example 1 is that the S435A point mutation was not introduced, and the mutant plasmid obtained during the construction process was pRSFDuet-EcglnA-EcargA. V323A .

[0071] The fermentation process of the engineered strain described in this comparative example is the same as that in Example 2.

[0072] Comparative Example 3

[0073] This comparative example provides an engineered strain. The only difference between this comparative example engineered strain and the engineered strain constructed in Example 1 is that this comparative example engineered strain does not introduce the V323A point mutation.

[0074] The only difference between the construction method of the engineered strain described in this comparative example and that in Example 1 is that the V323A point mutation was not introduced, and the mutant plasmid obtained during the construction process was pRSFDuet-EcglnA-EcargA. S435A .

[0075] The fermentation process of the engineered strain described in this comparative example is the same as that in Example 2.

[0076] Comparative Example 4

[0077] This comparative example provides an engineered strain, HG-N-Idg06, provided by Nanjing Hegu Biotechnology Co., Ltd., whose construction method is described in Example 1 of the specification of patent CN 118360308 A. The only difference between this comparative example engineered strain HG-N-Idg06 and the engineered strain constructed in Example 1 is that HG-N-Idg06 does not introduce two site mutations: the V323A and S435A mutations of EcargA.

[0078] Compared with Example 2, the fermentation process of the engineered strain described in this comparative example adds a first fermentation stage (refer to patent CN118562910A - A two-stage fermentation production process for N-acetylglucosamine), which specifically includes the following steps:

[0079] 1. Take out the frozen glycerol bacterial suspension of recombinant Escherichia coli strain HG-N-Idg06, and after thawing, streak it onto a double antibiotic plate medium containing kanamycin and streptomycin. After streaking, incubate the plate at 37°C for 12 hours.

[0080] 2. Select single colonies with a diameter of 0.12-0.15 mm, inoculate them into 10 mL of LB medium, and add kanamycin to a final concentration of 25 mg / L and streptomycin to 50 mg / L. Incubate in a shaker at 37℃ and 220 rpm for 8 hours. The OD of the bacterial culture should be... 600 The activated primary seed solution was obtained between 3.0 and 4.0.

[0081] 3. Take 10 mL of the activated primary seed culture and inoculate it into a 5 L fermenter containing 2.4 L LB medium. Add kanamycin to a final concentration of 25 mg / L and streptomycin to a final concentration of 50 mg / L. Culture in the fermenter under the following conditions: pH 7.0, temperature 37℃, initial aeration ratio of 1 vvm, initial rotation speed of 220 rpm, fermenter pressure 0.05 MPa, stirring and maintaining dissolved oxygen (DO) at 30%. Culture for 8-10 hours. The OD of the bacterial culture is then recorded. 600 =8.0, to obtain the activated secondary seed solution;

[0082] 4. At 2m 3 Add 1.2m to the fermentation tank 3 A basic fermentation medium containing glucose; the secondary seed culture obtained in step (3) is introduced into a 2m medium via a transfer tube. 3Fermentation was carried out in a fermenter with pH controlled at 7.2, temperature at 37℃, aeration ratio of 1 vvm, initial rotation speed of 50 rpm, dissolved oxygen (DO) controlled at 25%, and tank pressure of 0.05 MPa. The fermentation time was 0-24 h, and a synthase inducer was added at 16 h of fermentation to induce the expression of the product synthase and improve the fermentation yield of N-acetylglucosamine. The first stage fermentation broth was obtained.

[0083] 5. After 24 hours, the second stage of culture was carried out, with the pH controlled at 7.0, the temperature at 25℃, the aeration ratio at 1 vvm, the initial rotation speed at 100 rpm, the dissolved oxygen (DO) controlled at 35%, and the tank pressure at 0.05 MPa. After 12 hours of fermentation, fed medium was added to provide nitrogen and carbon sources for the fermentation strain.

[0084] Except for the addition of a first-stage fermentation, the rest of the process is the same as in Example 2.

[0085] Test Example 1:

[0086] In all examples and comparative strains, the yield of N-acetylglucosamine stopped increasing after 72 hours of fermentation, at which point fermentation was terminated. The fermentation broth was collected, and the yields of N-acetylglucosamine and the byproduct glucosamine were determined using high-performance liquid chromatography (HPLC). The detection method was as follows: wavelength 600 nm, flow rate 1.0 mL / min, sample solution: DMSO, injection volume: 10 μL, column temperature 35℃, run time 20 min, and column used... C18 (5 μm) HPLC columns, 4.6 mm × 25 cm, were used for isocratic elution with a mobile phase of methanol:pure water (v / v) = 30:70.

[0087] 1 mg / mL solutions of styrax and N-acetyrax were prepared and serially diluted to obtain graded concentrations of standard solutions. High-performance liquid chromatography (HPLC) analysis was performed. Standard curves were plotted with the peak areas of styrax and N-acetyrax as the ordinates and the concentrations of styrax and N-acetyrax as the abscissas, respectively. The obtained styrax standard curve was Y = 163421X + 14728, R0 2 =0.9997; the resulting N-acetylglucosamine standard curve is Y = 184687X + 28179, R 2 =0.9999, and the yields of Guanlan and N-acetylanlan were calculated based on the standard curve.

[0088] The test results of the fermentation broth obtained in Example 2 and Comparative Examples 1-4 are shown in Table 1. Figure 1-6 As shown.

[0089] Table 1

[0090]

[0091] Testing showed that, compared to Comparative Examples 1-3, the N-acetylglucosamine yield in the fermentation broth of Example 2 was 31.39 g / L. Figure 1 In the fermentation products, the content of N-acetylglucosamine was 95.2%, while the content of the byproduct glucosamine was only 4.8%. N-acetylglucosamine was not detected in the control (blank fermentation medium group). Figure 2 The yield of N-acetylglucosamine in the fermentation broth of Comparative Example 1 was 18.40 g / L. Figure 3 The fermentation product contained 52.7% N-acetylglucosamine, and the byproduct glucosamine content was 47.3%. The N-acetylglucosamine yield in the fermentation broth of Comparative Example 2 was 23.30 g / L. Figure 4 The fermentation product contained 71.5% N-acetylglucosamine, while the byproduct glucosamine content was 28.5%. The N-acetylglucosamine yield in the fermentation broth of Comparative Example 3 was 22.74 g / L. Figure 5 The fermentation product contained 65.7% N-acetylglucosamine, while the byproduct glucosamine content was 34.3%. This indicates that the present invention can effectively increase the yield of N-acetylglucosamine through specific point mutation modification of Ecarga.

[0092] Compared with Comparative Example 4, the yield of N-acetylglucosamine in the fermentation broth of Comparative Example 4 was 28.45 g / L (Example 2). Figure 6 In the fermentation product, the N-acetylglucosamine content was 95%, and the byproduct glucosamine content was 5%. Although the fermentation product of Comparative Example 4 had a higher N-acetylglucosamine content, its fermentation process was complex. The engineered strains of this invention not only improve the N-acetylglucosamine fermentation yield and reduce the generation of byproduct glucosamine, but also further simplify the fermentation process and reduce production costs, facilitating the industrial production of N-acetylglucosamine in fermenters.

[0093] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. An N-acetylglutamate synthase mutant, characterized in that, The amino acid sequence of the N-acetylglutamate synthase mutant is shown in SEQ ID NO:

1.

2. A gene characterized in that, The gene encodes the N-acetylglutamate synthase mutant as described in claim 1.

3. The gene as described in claim 2, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO:

2.

4. A recombinant vector, characterized in that, The recombinant vector includes the gene as described in claim 2.

5. An engineered bacterial strain, characterized in that, The engineered strain expresses the N-acetylglutamate synthase mutant of claim 1; and / or contains the gene of claim 2; and / or contains the recombinant vector of claim 4; the engineered strain is expressed via pRSFDuet- Ec glnA- Ec The corresponding point mutation was introduced into the argA plasmid and co-transformed with pCDFDuet-bpsA-entD into E. coli to construct the plasmid. The pRSFDuet- Ec glnA- Ec argA and pCDFDuet-bpsA-entD are both derived from Example 1 of the specification of patent number CN118360308A.

6. A method for constructing the engineered strain as described in claim 5, characterized in that, Includes the following steps: (a) pRSFDuet- Ec glnA- Ec Using the argA plasmid as a base, a point mutation was introduced at position 323 of the EcargA gene to mutate valine to alanine; then, a point mutation was introduced at position 435 of the EcargA gene to mutate serine to alanine, resulting in the mutant plasmid pRSFDuet-EcglnA-EcargA. V323A / S435A ; (b) The mutant plasmid obtained in step (a) and pCDFDuet-bpsA-entD were co-transformed into Escherichia coli, cultured on plates, and positive mutant plasmids were screened to obtain the engineered strain; The pRSFDuet- Ec glnA- Ec argA and pCDFDuet-bpsA-entD are both derived from Example 1 of the specification of patent number CN118360308A.

7. The method for constructing the engineered strain as described in claim 6, characterized in that, The primer nucleotide sequences used to introduce the point mutation at position 323 are shown in SEQ ID NO:3 and SEQ ID NO:4; and / or, the primer nucleotide sequences used to introduce the point mutation at position 435 are shown in SEQ ID NO:5 and SEQ ID NO:

6.

8. The application of the N-acetylglutamate synthase mutant as described in claim 1, or the engineered strain as described in claim 5, in the production of N-acetylglucosamine.

9. A method for producing N-acetylglucosamine by fermentation, characterized in that, N-acetylglucosamine is produced by fermentation using the engineered strain described in claim 5.

10. The method for producing N-acetylglucosamine by fermentation as described in claim 9, characterized in that, Specifically, the steps include: activating the engineered bacteria to obtain seed liquid, inoculating it into a fermentation medium, inducing fermentation, and cultivating to produce N-acetylglucosamine.

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