N-acetylglucosamine-producing strains and methods for their construction and use

Abstract

The present invention relates to a genetically modified strain capable of producing N-acetylglucosamine, as well as methods for constructing and using the same. The genetically modified strain is capable of fermenting N-acetylglucosamine at temperatures between 40°C and 50°C. The N-acetylglucosamine catabolic pathway is blocked by knocking out the parent bacterium's glucosamine-6-phosphate deaminase gene, N-acetylglucosamine-6-phosphate deacetylase gene, and N-acetylglucosamine transporter protein gene. Furthermore, the introduction of overexpression genes for glucosamine-6-phosphate synthase and glucosamine-6-phosphate acetylase enables extracellular accumulation of N-acetylglucosamine and high-temperature fermentation of N-acetylglucosamine at temperatures above 40°C.

Description

[Technical Field]

[0001] The present invention relates to the field of biotechnology and to genetically modified strains that produce N-acetylglucosamine, as well as methods for constructing and using the same. [Background technology]

[0002] N-acetylglucosamine (GlcNAc), a building block of various polysaccharides in living organisms, is formed by replacing the hydroxyl group of glucose with an amino group. It is particularly abundant in the exoskeletons of crustaceans and is widely used in the food, pharmaceutical, and cosmetic industries. In the food industry, it can be used as a food antioxidant, an additive for infant foods, and a sweetener for diabetics. In the pharmaceutical industry, it has been clinically used as a novel biochemical to treat rheumatism and rheumatoid arthritis. Other important applications include enhancing the function of the human immune system, inhibiting the excessive proliferation of cancer cells or fibroblasts, and suppressing or treating cancer and malignant tumors. N-acetylglucosamine also has therapeutic effects on osteoarthritis and joint pain. In the cosmetic industry, it can be synthesized from D-glucuronic acid polymeric mucopolysaccharides to produce hyaluronic acid. This application is expected to have broad market potential.

[0003] N-acetylglucosamine is mainly produced by chemical, enzymatic, and microbial fermentation approaches. The chemical approach involves extracting chitin from crab and shrimp shells and then acid-hydrolyzing it to produce glucosamine. Subsequently, N-acetylglucosamine is directly hydrolyzed and deacetylated with concentrated hydrochloric acid to produce glucosamine. Acetic anhydride can also be used to acetylate glucosamine to N-acetylglucosamine. However, this approach is not environmentally friendly, is highly polluting, and excludes patients with seafood allergies. Compared to the chemical approach, both enzymatic and microbial fermentation approaches are environmentally friendly. However, the enzymatic approach involves the use of enzymes to catalyze the degradation of chitin and the synthesis of N-acetylglucosamine. However, the enzymatic approach involves the complex pretreatment of shrimp and crab shells as substrates, the low activity of the key enzymes, and the difficulty of product separation and purification, making it very difficult to mass-produce. Microbial fermentation is currently the most promising method for producing N-acetylglucosamine and is the focus of research in the field to which the present invention pertains.

[0004] Compared with low-temperature fermentation below 40°C, high-temperature fermentation offers advantages such as minimizing the risk of contamination, accelerating raw material conversion, and reducing heat exchange costs. All previous reports on N-acetylglucosamine production by metabolically engineered microorganisms have utilized fermentation temperatures below 38°C, and there are still no patents or reports on high-temperature N-acetylglucosamine production. Therefore, there is a need to develop a low-cost, robust microbial platform capable of producing N-acetylglucosamine at high temperatures and in high yields. Thermophilic bacteria such as Bacillus coagulans, Bacillus licheniformis, and Bacillus stearothermophilus can grow under high-temperature conditions (above 40°C) and utilize organic carbon sources (e.g., glucose, xylose, and arabinose) for fermentation. For example, Bacillus licheniformis (B. licheniformis) ATCC 14580 is a facultatively anaerobic, Gram-positive, endospore-forming bacterium that can utilize a variety of pentose and hexose sugars. Its rapid cell growth rate necessitates a short fermentation cycle. This bacterium allows stable genetic manipulation and has been recognized by the U.S. Food and Drug Administration (FDA) as a "Generally Regarded as Safe" (GRAS) strain. Furthermore, this strain can be fermented at temperatures as high as 50°C. These advantages suggest that ATCC 14580 could be used as an ideal platform strain. Summary of the Invention [Problem to be solved by the invention]

[0005] To solve the above problems, the present invention provides a genetically modified strain capable of producing N-acetylglucosamine under high-temperature conditions, as well as methods for constructing and using the same. Taking a thermophilic B. licheniformis strain as an example, the present invention removes the N-acetylglucosamine catabolic pathway from the B. licheniformis strain and introduces an N-acetylglucosamine production metabolic pathway, enabling extracellular accumulation of N-acetylglucosamine and efficient production of N-acetylglucosamine under high-temperature conditions. The strain construction concept proposed in the present invention can be used to develop strains for high-temperature production of various products. The proposed strain and fermentation process have good potential for application in industrial high-temperature N-acetylglucosamine fermentation.

[0006] In one embodiment of the present invention, a genetically modified strain capable of producing N-acetylglucosamine and capable of fermenting N-acetylglucosamine at temperatures between 40°C and 50°C is provided.

[0007] Furthermore, the original strain of the genetically modified strain is Streptococcus thermophilus.

[0008] Furthermore, the original strain of the genetically modified strain is a bacterium of the genus Bacillus.

[0009] Preferably, the original strain of the genetically modified strain is Bacillus licheniformis, Bacillus coagulans, Bacillus methylotrophicus, thermophilic Bacillus inulinus, or Geobacillus stearothermophilus, etc.

[0010] Preferably, the original strain for the genetically modified strain is Bacillus licheniformis ATCC 14580, which is available directly from the ATCC website.

[0011] Furthermore, the genetically modified strain is Bacillus licheniformis BNGS1, which was deposited at the China Center for Type Culture Collection on March 14, 2020, under CCTCC number M2020054.

[0012] Furthermore, the catabolic pathways and intracellular transport pathways of N-acetylglucosamine and N-acetylglucosamine intermediates in the original strain of the genetically modified strain are blocked.

[0013] Preferably, the catabolic pathway of N-acetylglucosamine and N-acetylglucosamine intermediates is blocked by inactivation or deletion of one or more of the glucosamine-6-phosphate deaminase nagB gene and the gamma gene and the N-acetylglucosamine-6-phosphate deacetylase nagA gene, and the intracellular transport pathway of N-acetylglucosamine is blocked by inactivation or deletion of one or both of the N-acetylglucosamine transporter protein gamP gene and the nagP gene.

[0014] Furthermore, the overexpression genes for glucosamine-6-phosphate synthase and glucosamine-6-phosphate acetylase have been introduced into the genetically modified strain.

[0015] Furthermore, the gene sequence of glucosamine-6-phosphate acetylase is as shown in SEQ ID NO:1, and the gene sequence of glucosamine-6-phosphate synthase is as shown in SEQ ID NO:2.

[0016] Preferably, the glucosamine-6-phosphate synthase gene can be obtained by PCR amplification using the DNA sequence of the entire genome of B. licheniformis (GenBank number NC_006270.3) as a template.

[0017] Preferably, the gene for glucosamine-6-phosphate acetylase can be obtained by whole genome synthesis using codon optimization from the whole genome of Saccharomyces cerevisiae (GenBank number NM_001179949).

[0018] In another aspect of the present invention, there is provided a method for constructing the genetically modified strain defined above. In a specific embodiment, the method comprises inactivating or deleting one or more of the genes encoding glucosamine-6-phosphate deaminase, N-acetylglucosamine-6-phosphate deacetylase, and N-acetylglucosamine transporter protein in the N-acetylglucosamine catabolic pathway of the original strain, and introducing an overexpression gene for glucosamine-6-phosphate synthase and an overexpression gene for glucosamine-6-phosphate acetylase.

[0019] Furthermore, the method comprises: A. obtaining a knockout strain by knocking out the nagB gene and gamma gene encoding glucosamine-6-phosphate deaminase, the nagA gene encoding N-acetylglucosamine-6-phosphate deacetylase, and the gamP gene and nagP gene encoding N-acetylglucosamine transporter protein of the original strain; B. constructing a dual expression vector containing both the glmS gene for glucosamine-6-phosphate synthase and the GNA1 gene for glucosamine-6-phosphate acetylase; C. A step of introducing the dual expression vector into the knockout strain obtained in step A to obtain a genetically modified strain that produces N-acetylglucosamine. Includes:

[0020] Furthermore, a promoter is introduced to express an expression vector in which the promoter, the glmS gene, and the GNA1 gene are arranged in series, thereby constructing a dual expression vector.

[0021] Preferably, the expression vector is pHY300PLK.

[0022] Preferably, the promoter is P als , P 43 , P st , and P apre is.

[0023] Preferably, P als The promoter sequence is shown in SEQ ID NO: 3. 43 The promoter sequence is shown in SEQ ID NO: 4. st The sequence of the promoter is as shown in SEQ ID NO: 5, and P apre The sequence of the promoter is as shown in SEQ ID NO:6.

[0024] Preferably, the glmS gene for glucosamine-6-phosphate synthase is derived from Bacillus licheniformis, Bacillus coagulans, Bacillus methylotrophicus, thermophilic Bacillus inulinus, or Geobacillus stearothermophilus, etc.

[0025] Preferably, the GNA1 gene for glucosamine-6-phosphate acetylase is derived from Saccharomyces cerevisiae or another microorganism that produces a thermophilic enzyme with the same function, such as Kluyveromyces marxianus or Nadsonia fulvescens.

[0026] Preferably, the original strain is Bacillus licheniformis ATCC 14580.

[0027] In a further aspect of the present invention, there is provided the use of the genetically modified strain as defined above, in particular for the production of N-acetylglucosamine.

[0028] Furthermore, the fermentation temperature for production is 25°C to 50°C.

[0029] Preferably, the fermentation temperature for production is 40°C to 50°C.

[0030] Furthermore, the carbon source utilized in the production may be glucose, glycerol, xylose, or arabinose.

[0031] A seed culture is then obtained from the genetically modified strain, followed by secondary activation in a fermentation medium using glucose as the carbon source. Finally, the culture is transferred to a fermenter for fermentation of N-acetylglucosamine. Specifically, the steps include the following: 1) Seed culture: Inoculating the genetically modified strain into a seed medium to confer tetracycline resistance, and culturing at 50°C and 200 rpm for 12 to 16 hours to activate the seed; 2) Shake flask culture: The activated seeds from step 1) are transferred to a shake flask containing a fermentation medium at an inoculation rate of 5%, conferring tetracycline resistance, and then cultured at 50°C and 200 rpm for 12-16 hours for secondary activation; 3) Fermentation culture: This includes the steps of transferring the above to a fermenter at an inoculation rate of 4%, conferring tetracycline resistance, adding a carbon source, performing fed-batch fermentation at 40°C to 50°C, and culturing until fermentation is complete.

[0032] Preferably, the seed medium in step 1) comprises the following components: peptone, yeast powder, and sodium chloride. Preferably, the seed medium comprises 10 g / L of peptone, 5 g / L of yeast powder, and 10 g / L of sodium chloride.

[0033] Preferably, the fermentation medium in step 2) contains the following components: yeast powder, peptone, ammonium sulfate, dipotassium hydrogen phosphate trihydrate, potassium dihydrogen phosphate, and glucose, preferably 12 g / L yeast powder, 6 g / L peptone, 6 g / L ammonium sulfate, 18.75 g / L dipotassium hydrogen phosphate trihydrate, 2.5 g / L potassium dihydrogen phosphate, and 30 g / L glucose.

[0034] Preferably, in step 2), the shake flask is a 500 mL Erlenmeyer flask and contains 75 mL of fermentation medium.

[0035] Preferably, the fermentation medium in step 3) contains the following ingredients: yeast powder, corn steep liquor powder, ammonium sulfate, dipotassium hydrogen phosphate trihydrate, potassium dihydrogen phosphate, and glucose. Preferably, the fermentation medium contains 12 g / L yeast powder, 6 g / L corn steep liquor powder, 6 g / L ammonium sulfate, 18.75 g / L dipotassium hydrogen phosphate trihydrate, 2.5 g / L potassium dihydrogen phosphate, and 30 g / L glucose.

[0036] Preferably, the fermentation conditions in step 3) are a pH value maintained at 7.0 using a 3 mol / L hydrochloric acid solution and a 25% (v / v) ammonia solution, an aeration rate of 1.5 vvm, an initial stirring speed of 600 rpm, and a glucose concentration continuously adjusted to 30 g / L by replenishing glucose when 3-4 g / L of glucose is consumed.

[0037] The genetically modified strain provided in the present application enables high-temperature production of N-acetylglucosamine. Furthermore, due to constitutive expression during fermentation of the genetically modified strain, the addition of inducers is unnecessary. Furthermore, the fermentation process requires little temperature reduction, and fermentation can proceed at a significantly increased rate at high temperatures. Furthermore, high temperatures inhibit the growth of other microorganisms, enabling open fermentation and effectively reducing production costs. The present invention enables N-acetylglucosamine fermentation at relatively high temperatures of 40°C to 50°C, making it highly useful industrially. The strain construction concept proposed in the present invention can be used to develop strains for high-temperature production of various products. The proposed strain and fermentation process have good potential for application in industrial high-temperature N-acetylglucosamine fermentation. [Brief explanation of the drawings]

[0038] [Figure 1] FIG. 1 shows N-acetylglucosamine synthesis and metabolic pathways in engineered B. licheniformis strains. DETAILED DESCRIPTION OF THE INVENTION

[0039] The N-acetylglucosamine synthesis and metabolic pathway of the engineered strain described herein is shown in Figure 1. Glutamine (Gln) serves as the amino acid donor, and the glucosamine synthase encoded by the glmS gene converts fructose-6-phosphate (F-6-P) to glucosamine-6-phosphate (GlcN-6-P), which is then converted to N-acetyl-glucosamine-6-phosphate (GlcNAc-6-P) by the action of glucosamine-6-phosphate acetylase (GNA1). GlcNAc-6-P is dephosphorylated to N-acetylglucosamine by the catalytic action of phosphorylase, and then secreted extracellularly. The nagB and gamma genes encoding glucosamine-6-phosphate deaminase and the nagA gene encoding N-acetylglucosamine-6-phosphate deacetylase are knocked out to be inactivated or to reduce intracellular consumption of N-acetylglucosamine precursors. Furthermore, the gamP and nagP genes encoding N-acetylglucosamine transporter proteins are knocked out, so that N-acetylglucosamine produced in the engineered strain is not transported and accumulates at high concentrations outside the cell.

[0040] According to the present specification, a metabolic pathway for high-temperature fermentation of N-acetylglucosamine has been established in a thermophilic B. licheniformis strain, in which expression of genes encoding rate-limiting enzymes involved in the synthesis of N-acetylglucosamine has been enhanced and genes that can cause N-acetylglucosamine consumption and back-diffusion have been inactivated or knocked out. Inhibiting N-acetylglucosamine consumption and back-diffusion allows the N-acetylglucosamine produced by the engineered strain to accumulate to high concentrations.

[0041] In one embodiment of the present invention, a genetically modified strain capable of producing N-acetylglucosamine is provided, which can ferment N-acetylglucosamine at 40°C to 50°C.

[0042] In one particular embodiment, the N-acetylglucosamine catabolic pathway is blocked in the original strain of the genetically modified strain.

[0043] In another embodiment, the N-acetylglucosamine catabolic pathway is blocked as a result of inactivation or deletion of one or more of the nagB and gamma genes encoding glucosamine-6-phosphate deaminase, the nagA gene encoding N-acetylglucosamine-6-phosphate deacetylase, and the gamP and nagP genes encoding the N-acetylglucosamine transporter protein.

[0044] As used herein, the term "blocking" refers to blocking a pathway through various genetic engineering approaches, including rendering the pathway non-viable by inactivating or deleting genes encoding one or more catalytic enzymes required for the pathway.

[0045] Those skilled in the art will understand that it is also possible for the original strain to contain no deleted or inactivated genes or even blocked pathways, such as the gene for glucosamine-6-phosphate deaminase, the gene for N-acetylglucosamine-6-phosphate deacetylase, and the gene for N-acetylglucosamine transporter protein, in which case the construction of a genetically modified strain does not include inactivation, deletion, or blocking achieved by genetic engineering, since the genes or pathways are absent. [Example]

[0046] The present invention will be further described below with reference to specific examples.

[0047] The following examples are illustrative and do not limit the scope of the present invention in any way. All experimental methods referred to in the following examples are conventional methods unless otherwise specified. Materials and reagents referred to in the following examples are available from commercial sources unless otherwise specified.

[0048] Example 1: Knockout of the B. licheniformis nagP gene 1. The following primers were designed according to the sequence of the B. licheniformis MW3 genome (GenBank number NC_006270.3): [ka] An 800-bp fragment upstream of the nagP gene was obtained by PCR amplification using B. licheniformis MW3 genomic DNA as a template.

[0049] 2. The following primers were designed according to the sequence of the B. licheniformis MW3 genome (GenBank number NC_006270.3): [ka] An 800-bp fragment downstream of the nagP gene was obtained by PCR amplification using B. licheniformis MW3 genomic DNA as a template.

[0050] 3. After purifying the two PCR products, 1 μL of each was used as a template and inserted into the pKVM vector using seamless cloning technology, resulting in the △nagP knockout pKVM vector.

[0051] 4. The ΔnagP knockout pKVM vector was transformed into E. coli S17-1, and the nagP gene was knocked out by conjugation with B. licheniformis and homologous recombination. The primers used were as follows: [ka] The successful amplification of the 1600 bp fragment was considered to be an indication of successful screening of double-crossover positive clones and successful knockout of the gene. Finally, the knockout strain MW3ΔnagP was obtained.

[0052] Example 2: Additional knockout of the B. licheniformis gamP gene 1. The following primers were designed according to the sequence of the B. licheniformis MW3 genome (GenBank number NC_006270.3): [ka] An 800-bp fragment upstream of the gamP gene was obtained by PCR amplification using B. licheniformis MW3 genomic DNA as a template.

[0053] 2. The following primers were designed according to the sequence of the B. licheniformis MW3 genome (GenBank number NC_006270.3): [ka] An 800-bp fragment downstream of the gamP gene was obtained by PCR amplification using B. licheniformis MW3 genomic DNA as a template.

[0054] 3. After purifying the two PCR products, 1 μL of each was used as a template and inserted into the pKVM vector using seamless cloning technology, resulting in the △gamP knockout pKVM vector.

[0055] 4. The △gamP knockout pKVM vector was transformed into E. coli S17-1, and the gamP gene was knocked out by conjugation with the knockout B. licheniformis strain MW3△nagP and homologous recombination. The primers used were as follows: [ka] The successful amplification of the 1600 bp fragment was considered to be an indication of successful screening of double-crossover positive clones and successful knockout of the gene. Finally, the knockout strain MW3ΔnagPΔgamP was obtained.

[0056] Example 3: Additional knockout of the B. licheniformis gmA gene 1. The following primers were designed according to the sequence of the B. licheniformis MW3 genome (GenBank number NC_006270.3): [ka] An 800-bp fragment upstream of the gamA gene was obtained by PCR amplification using B. licheniformis MW3 genomic DNA as a template.

[0057] 2. The following primers were designed according to the sequence of the B. licheniformis MW3 genome (GenBank number NC_006270.3): [ka] An 800-bp fragment downstream of the gamA gene was obtained by PCR amplification using B. licheniformis MW3 genomic DNA as a template.

[0058] 3. After purifying the two PCR products, 1 μL of each was used as a template and inserted into the pKVM vector using seamless cloning technology, resulting in the △gamA knockout pKVM vector.

[0059] 4. The ΔgamA knockout pKVM vector was transformed into E. coli S17-1, and the gamma gene was knocked out by conjugation and homologous recombination with the knockout B. licheniformis strain MW3 ΔnagP ΔgamP. The primers used were as follows: [ka] The successful amplification of the 1600 bp fragment was considered to be an indication of successful screening of double-crossover positive clones and successful knockout of the gene. Finally, the knockout strain MW3ΔnagPΔgamPΔgamA was obtained.

[0060] Example 4: Additional knockouts of the B. licheniformis nagAB gene cluster 1. The following primers were designed according to the sequence of the B. licheniformis MW3 genome (GenBank number NC_006270.3): [ka] An 800-bp fragment upstream of the nagAB gene cluster was obtained by PCR amplification using B. licheniformis MW3 genomic DNA as a template.

[0061] 2. The following primers were designed according to the sequence of the B. licheniformis MW3 genome (GenBank number NC_006270.3): [ka] An 800-bp fragment downstream of the nagAB gene cluster was obtained by PCR amplification using B. licheniformis MW3 genomic DNA as a template.

[0062] 3. After purifying the two PCR products, 1 μL of each was used as a template and inserted into the pKVM vector using seamless cloning technology, resulting in the △nagAB knockout pKVM vector.

[0063] 4. The △nagAB knockout pKVM vector was transformed into E. coli S17-1, and the nagAB gene cluster was knocked out by conjugation and homologous recombination with the knockout B. licheniformis strain MW3△nagP△gamP△gamA. The primers used were as follows: [ka] The successful amplification of the 1600 bp fragment was considered to be an indication of successful screening of double-crossover positive clones and successful knockout of the gene. Finally, the knockout strain MW3ΔnagPΔgamPΔgamAΔnagAB was obtained.

[0064] Example 5: Construction of a GNA1-glmS dual expression vector The following primers were designed according to the sequence of the B. licheniformis MW3 genome (GenBank number NC_006270.3). [ka] PCR amplification using B. licheniformis MW3 genomic DNA as a template identified P als The promoter sequence was obtained.

[0065] P 43 The entire gene was synthesized using the promoter. The primers used were designed as follows: [ka] Furthermore, PCR amplification revealed that P 43 The promoter sequence was obtained.

[0066] P st The entire gene was synthesized using the promoter. The primers used were designed as follows: [ka] Furthermore, PCR amplification revealed that P st The promoter sequence was obtained.

[0067] P aprE The entire gene was synthesized using the promoter. The primers used were designed as follows: [ka] Furthermore, PCR amplification revealed that P aprE The promoter sequence was obtained.

[0068] The GNA1 gene encoding glucosamine-6-phosphate acetylase was obtained from the whole genome of Saccharomyces cerevisiae (GenBank no. NM_001179949). Total gene synthesis with codon optimization was performed, and the optimized GNA1 sequence was obtained by PCR amplification. The primers used were designed as follows: [ka]

[0069] Primers were designed according to the sequence of the B. licheniformis MW3 genome (GenBank no. NC_006270.3). [ka] The glmS gene sequence was obtained by PCR amplification using B. licheniformis MW3 genomic DNA as a template.

[0070] P collected from PCR amplification als 1 μL of each of the three gene fragments, i.e., promoter sequence, GNA1, and glmS, was collected and inserted into the pHY300PLK vector by seamless cloning to create pHY300PLK-P. als -GNA1-glmS vector was constructed.

[0071] P collected from PCR amplification 43 1 μL of each of the three gene fragments, i.e., promoter sequence, GNA1, and glmS, was collected and inserted into the pHY300PLK vector by seamless cloning to create pHY300PLK-P. 43 -GNA1-glmS vector was constructed.

[0072] P collected from PCR amplification st1 μL of each of the three gene fragments, i.e., promoter sequence, GNA1, and glmS, was collected and inserted into the pHY300PLK vector by seamless cloning to create pHY300PLK-P. st -GNA1-glmS vector was constructed.

[0073] P collected from PCR amplification aprE 1 μL of each of the three gene fragments, i.e., promoter sequence, GNA1, and glmS, was collected and inserted into the pHY300PLK vector by seamless cloning to create pHY300PLK-P. aprE -GNA1-glmS vector was constructed.

[0074] Example 6: Construction of modified strains BNGS1, BNGS2, BNGS3, and BNGS4 pHY300PLK-P als The -GNA1-glmS vector was introduced into the knockout strain MW3△nagP△gamP△gamA△nagAB by electroporation, resulting in a new B. licheniformis strain capable of synthesizing N-acetylglucosamine, which we named BNGS1.

[0075] pHY300PLK-P 43 The -GNA1-glmS vector was introduced into the knockout strain MW3△nagP△gamP△gamA△nagAB by electroporation, resulting in a new B. licheniformis strain capable of synthesizing N-acetylglucosamine, which we named BNGS2.

[0076] pHY300PLK-P st The -GNA1-glmS vector was introduced into the knockout strain MW3△nagP△gamP△gamA△nagAB by electroporation, resulting in a new B. licheniformis strain capable of synthesizing N-acetylglucosamine, which we named BNGS3.

[0077] pHY300PLK-P aprEThe -GNA1-glmS vector was introduced into the knockout strain MW3△nagP△gamP△gamA△nagAB by electroporation, resulting in a new B. licheniformis strain capable of synthesizing N-acetylglucosamine, which we named BNGS4.

[0078] Example 7: Shake flask fermentation of recombinant B. licheniformis BNGS1 1. Seed medium and fermentation medium LB medium (containing 10 g / L sodium chloride, 5 g / L yeast powder, and 10 g / L peptone) was used as the seed medium. During liquid culture, 25 mg / L tetracycline resistance was conferred.

[0079] The fermentation medium contained the following components: 12 g / L yeast powder, 6 g / L peptone, 6 g / L ammonium sulfate, 18.75 g / L dipotassium hydrogen phosphate trihydrate, 2.5 g / L potassium dihydrogen phosphate, and 30 g / L glucose.

[0080] 2. Shake Flask Fermentation Process A seed solution containing strain BNGS1 was inoculated into 5 mL of LB medium and incubated at 50°C and 200 rpm for 12 to 16 hours. During cultivation, 25 mg / L tetracycline resistance was imparted. The strain was transferred to a 500 mL Erlenmeyer flask containing 75 mL of fermentation medium for shake flask fermentation at a 5% inoculation rate. During cultivation, 25 mg / L tetracycline resistance was imparted. Fermentation was carried out at 40°C for 50 hours, and a 1 mL sample was taken and centrifuged at 12,000 rpm for 5 minutes. The sample was filtered through a 0.22 μm aqueous membrane and detected by high-performance liquid chromatography.

[0081] 3. Detection Method Column: Aminex HPX-87H Mobile phase: 0.005 mol / L sulfuric acid solution Flow rate: 0.5mL / min Column temperature: 60.0℃ Injection volume: 20.00μL Detector: Differential refractive index detector Detector temperature: 35℃

[0082] The detection results showed that after 50 hours of shake flask fermentation, N-acetylglucosamine was present in the fermentation broth at 2.3 g / L.

[0083] Example 8: Shake flask fermentation of recombinant B. licheniformis BNGS2 A seed solution containing the BNGS2 strain was inoculated into 5 mL of LB medium and incubated at 50°C and 200 rpm for 12 to 16 hours. During cultivation, 25 mg / L tetracycline resistance was imparted. The strain was transferred to a 500 mL Erlenmeyer flask containing 75 mL of fermentation medium for shake flask fermentation at a 5% inoculation rate. During cultivation, 25 mg / L tetracycline resistance was imparted. Fermentation was carried out at 40°C for 50 hours, and a 1 mL sample was taken and centrifuged at 12,000 rpm for 5 minutes. The sample was filtered through a 0.22 μm aqueous membrane and detected by high-performance liquid chromatography.

[0084] The detection results showed that after 50 hours of shake flask fermentation, N-acetylglucosamine was present in the fermentation broth at 1.5 g / L.

[0085] Example 9: Shake flask fermentation of recombinant B. licheniformis BNGS3 A seed solution containing strain BNGS3 was inoculated into 5 mL of LB medium and incubated at 50°C and 200 rpm for 12 to 16 hours. During cultivation, 25 mg / L tetracycline resistance was imparted. The strain was transferred to a 500 mL Erlenmeyer flask containing 75 mL of fermentation medium for shake flask fermentation at a 5% inoculation rate. During cultivation, 25 mg / L tetracycline resistance was imparted. Fermentation was carried out at 40°C for 50 hours, and a 1 mL sample was taken and centrifuged at 12,000 rpm for 5 minutes. The sample was filtered through a 0.22 μm aqueous membrane and detected by high-performance liquid chromatography.

[0086] The detection results showed that after 50 hours of shake flask fermentation, N-acetylglucosamine was present in the fermentation broth at 0.57 g / L.

[0087] Example 10: Shake flask fermentation of recombinant B. licheniformis BNGS4 A seed solution containing strain BNGS4 was inoculated into 5 mL of LB medium and incubated at 50°C and 200 rpm for 12 to 16 hours. During cultivation, 25 mg / L tetracycline resistance was imparted. The strain was transferred at a 5% inoculation rate to a 500 mL Erlenmeyer flask containing 75 mL of fermentation medium for shake flask fermentation. During cultivation, 25 mg / L tetracycline resistance was imparted. Fermentation was carried out at 40°C for 50 hours, and a 1 mL sample was taken and centrifuged at 12,000 rpm for 5 minutes. The sample was filtered through a 0.22 μm aqueous membrane and detected by high-performance liquid chromatography.

[0088] The detection results showed that after 50 hours of shake flask fermentation, N-acetylglucosamine was present in the fermentation broth at 0.32 g / L.

[0089] Example 11: Fed-batch fermentation of recombinant B. licheniformis BNGS1 in a 1 L fermentor at 37°C 1. Seed culture: 5 mL of the seed solution containing the BNGS1 strain was inoculated into LB medium and incubated at 50°C and 200 rpm for 12 to 16 hours. During the culture, 25 mg / L tetracycline resistance was imparted.

[0090] 2. Shake flask cultivation: The strain was transferred to 75 mL of fermentation medium (contained in a 500 mL shake flask) at a 5% inoculation rate. Tetracycline resistance of 25 mg / L was imparted during shake flask cultivation. The cultivation was carried out overnight at 50°C and 200 rpm.

[0091] 3. Fermentation: The activated seed solution was transferred to a 1 L automatic fermenter at an inoculation rate of 4% (v / v) and incubated at 37 °C. Tetracycline resistance was imparted until the effective concentration reached 25 mg / L, and 3 mol L -1 The pH was adjusted and maintained at 7.0 by adding hydrochloric acid solution and 25% (v / v) ammonia solution. The aeration rate was kept at 1.5 vvm and the initial speed was 600 r·min -1The mixture was stirred at a rate of 0.01 s. The glucose concentration had to be continuously adjusted to 30 g / L. For this purpose, glucose was continuously replenished at a constant flow rate when glucose was consumed to 3-4 g / L. The culture was continued until the end of the fermentation. Samples were taken and detected by high-performance liquid chromatography.

[0092] The detection results showed that after 70 hours of fermentation, acetylglucosamine was present in the fermentation broth at 12 g / L.

[0093] Example 12: Fed-batch fermentation of recombinant B. licheniformis BNGS1 in a 1 L fermentor at 50°C 1. Seed culture: Same as in Example 11. 2. Shake flask culture: same as in Example 11. 3. Fermentation: The activated seed solution was transferred to a 1 L automatic fermenter at an inoculation rate of 4% (v / v) and incubated at a temperature of 50 °C. Tetracycline resistance was imparted until the effective concentration reached 25 mg / L, and 3 mol L -1 The pH was adjusted and maintained at 7.0 by adding hydrochloric acid solution and 25% (v / v) ammonia solution. The aeration rate was kept at 1.5 vvm and the initial speed was 600 r·min -1 The mixture was stirred at a rate of 0.01 s. The glucose concentration had to be continuously adjusted to 30 g / L. For this purpose, glucose was continuously replenished at a constant flow rate when glucose was consumed to 3-4 g / L. The culture was continued until the end of the fermentation. Samples were taken and detected by high-performance liquid chromatography.

[0094] The detection results showed that after 70 hours of fermentation, acetylglucosamine was present in the fermentation broth at 2.8 g / L.

[0095] Example 13: Fed-batch fermentation of recombinant B. licheniformis BNGS1 in a 50 L fermentor at 37°C 1. Seed culture: Same as in Example 11. 2. Shake flask culture: same as in Example 11. 3. Fermentation: The activated seed solution was transferred to a 50 L automatic fermenter at an inoculation rate of 4% (v / v) and incubated at 37 °C. Tetracycline resistance was imparted until the effective concentration reached 25 mg / L, and 3 mol L -1 The pH was adjusted and maintained at 7.0 by adding hydrochloric acid solution and 25% (v / v) ammonia solution. The aeration rate was kept at 1.5 vvm and the initial speed was 600 r·min -1 The mixture was stirred at a rate of 0.01 s. The glucose concentration had to be continuously adjusted to 30 g / L. For this purpose, glucose was continuously replenished at a constant flow rate when glucose was consumed to 3-4 g / L. The culture was continued until the end of the fermentation. Samples were taken and detected by high-performance liquid chromatography.

[0096] The detection results showed that after 69.5 hours of fermentation, acetylglucosamine was present in the fermentation broth at 50.9 g / L.

[0097] Example 14: Fed-batch fermentation of recombinant B. licheniformis BNGS1 in a 50 L fermentor at 37°C 1. Seed culture: Same as in Example 11. 2. Shake flask culture: same as in Example 11. 3. Fermentation: The activated seed solution was transferred to a 50 L automatic fermenter at an inoculation rate of 4% (v / v) and incubated at 37 °C. Tetracycline resistance was imparted until the effective concentration reached 25 mg / L, and 3 mol L -1 The pH was adjusted and maintained at 7.0 by adding hydrochloric acid solution and 25% (v / v) ammonia solution. The aeration rate was kept at 1.5 vvm and the initial speed was 600 r·min -1 The mixture was stirred at a rate of 0.01 s. The glucose concentration had to be continuously adjusted to 30 g / L. For this purpose, glucose was continuously replenished at a constant flow rate when glucose was consumed to 3-4 g / L. The culture was continued until the end of the fermentation. Samples were taken and detected by high-performance liquid chromatography.

[0098] The detection results showed that after 73 hours of fermentation, acetylglucosamine was present in the fermentation broth at 52.5 g / L.

[0099] Example 15: Fed-batch fermentation of recombinant B. licheniformis BNGS1 in a 50 L fermentor at 40°C 1. Seed culture: Same as in Example 11. 2. Shake flask culture: same as in Example 11. 3. Fermentation: The activated seed solution was transferred to a 50 L automatic fermenter at an inoculation rate of 4% (v / v) and incubated at 40 °C. Tetracycline resistance was added until the effective concentration reached 25 mg / L, and 3 mol L -1 The pH was adjusted and maintained at 7.0 by adding hydrochloric acid solution and 25% (v / v) ammonia solution. The aeration rate was kept at 1.5 vvm and the initial speed was 600 r·min -1 The mixture was stirred at a rate of 0.01 s. The glucose concentration had to be continuously adjusted to 30 g / L. For this purpose, glucose was continuously replenished at a constant flow rate when glucose was consumed to 3-4 g / L. The culture was continued until the end of the fermentation. Samples were taken and detected by high-performance liquid chromatography.

[0100] The detection results showed that after 54 hours of fermentation, acetylglucosamine was present in the fermentation broth at 28.1 g / L.

[0101] Example 16: Fed-batch fermentation of recombinant B. licheniformis BNGS1 in a 50 L fermentor at 40°C 1. Seed culture: Same as in Example 11. 2. Shake flask culture: same as in Example 11. 3. Fermentation: The activated seed solution was transferred to a 50 L automatic fermenter at an inoculation rate of 4% (v / v) and incubated at 40 °C. Tetracycline resistance was added until the effective concentration reached 25 mg / L, and 3 mol L -1 The pH was adjusted and maintained at 7.0 by adding hydrochloric acid solution and 25% (v / v) ammonia solution. The aeration rate was kept at 1.5 vvm and the initial speed was 600 r·min -1The mixture was stirred at a rate of 0.01 s. The glucose concentration had to be continuously adjusted to 30 g / L. For this purpose, glucose was continuously replenished at a constant flow rate when glucose was consumed to 3-4 g / L. The culture was continued until the end of the fermentation. Samples were taken and detected by high-performance liquid chromatography.

[0102] The detection results showed that after 56 hours of fermentation, acetylglucosamine was present in the fermentation broth at 34.6 g / L.

[0103] Example 17: Fed-batch fermentation of recombinant B. licheniformis BNGS1 in a 50 L fermentor at 45°C 1. Seed culture: Same as in Example 11. 2. Shake flask culture: same as in Example 11. 3. Fermentation: The activated seed solution was transferred to a 50 L automatic fermenter at an inoculation rate of 4% (v / v) and incubated at 45 °C. Tetracycline resistance was added until the effective concentration reached 25 mg / L, and 3 mol L -1 The pH was adjusted and maintained at 7.0 by adding hydrochloric acid solution and 25% (v / v) ammonia solution. The aeration rate was kept at 1.5 vvm and the initial speed was 600 r·min -1 The mixture was stirred at a rate of 0.01 s. The glucose concentration had to be continuously adjusted to 30 g / L. For this purpose, glucose was continuously replenished at a constant flow rate when glucose was consumed to 3-4 g / L. The culture was continued until the end of the fermentation. Samples were taken and detected by high-performance liquid chromatography.

[0104] The detection results showed that after 50 hours of fermentation, acetylglucosamine was present in the fermentation broth at 24 g / L.

[0105] Example 18: Fed-batch fermentation of recombinant B. licheniformis BNGS1 in a 50 L fermentor at 50°C 1. Seed culture: Same as in Example 11. 2. Shake flask culture: same as in Example 11. 3. Fermentation: The activated seed solution was transferred to a 50 L automatic fermenter at an inoculation rate of 4% (v / v) and incubated at 50 °C. Tetracycline resistance was added until the effective concentration reached 25 mg / L, and 3 mol L -1 The pH was adjusted and maintained at 7.0 by adding hydrochloric acid solution and 25% (v / v) ammonia solution. The aeration rate was kept at 1.5 vvm and the initial speed was 600 r·min -1 The mixture was stirred at a rate of 0.01 s. The glucose concentration had to be continuously adjusted to 30 g / L. For this purpose, glucose was continuously replenished at a constant flow rate when glucose was consumed to 3-4 g / L. The culture was continued until the end of the fermentation. Samples were taken and detected by high-performance liquid chromatography.

[0106] The detection results showed that after 24 hours of fermentation, acetylglucosamine was present in the fermentation broth at 4.1 g / L.

[0107] Example 19: Fed-batch fermentation of recombinant B. licheniformis BNGS2 in a 50 L fermentor at 37°C 1. Seed culture: 5 mL of the seed solution containing the BNGS2 strain was inoculated into LB medium and incubated at 50°C and 200 rpm for 12 to 16 hours. During the culture, 25 mg / L tetracycline resistance was imparted.

[0108] 2. Shake flask cultivation: The strain was transferred to 75 mL of fermentation medium (contained in a 500 mL shake flask) at a 5% inoculation rate. Tetracycline resistance of 25 mg / L was imparted during shake flask cultivation. The cultivation was carried out overnight at 50°C and 200 rpm.

[0109] 3. Fermentation: The activated seed solution was transferred to a 50 L automatic fermenter at an inoculation rate of 4% (v / v) and incubated at 37 °C. Tetracycline resistance was imparted until the effective concentration reached 25 mg / L, and 3 mol L -1 The pH was adjusted and maintained at 7.0 by adding hydrochloric acid solution and 25% (v / v) ammonia solution. The aeration rate was kept at 1.5 vvm and the initial speed was 600 r·min -1The mixture was stirred at a rate of 0.01 s. The glucose concentration had to be continuously adjusted to 30 g / L. For this purpose, glucose was continuously replenished at a constant flow rate when glucose was consumed to 3-4 g / L. The culture was continued until the end of the fermentation. Samples were taken and detected by high-performance liquid chromatography.

[0110] The detection results showed that after 64 hours of fermentation, acetylglucosamine was present in the fermentation broth at 23.6 g / L.

[0111] Example 20: Fed-batch fermentation of recombinant B. licheniformis BNGS2 in a 50 L fermentor at 45°C 1. Seed culture: Same as in Example 19. 2. Shake flask culture: same as in Example 19. 3. Fermentation: The activated seed solution was transferred to a 50 L automatic fermenter at an inoculation rate of 4% (v / v) and incubated at 45 °C. Tetracycline resistance was added until the effective concentration reached 25 mg / L, and 3 mol L -1 The pH was adjusted and maintained at 7.0 by adding hydrochloric acid solution and 25% (v / v) ammonia solution. The aeration rate was kept at 1.5 vvm and the initial speed was 600 r·min -1 The mixture was stirred at a rate of 0.01 s. The glucose concentration had to be continuously adjusted to 30 g / L. For this purpose, glucose was continuously replenished at a constant flow rate when glucose was consumed to 3-4 g / L. The culture was continued until the end of the fermentation. Samples were taken and detected by high-performance liquid chromatography.

[0112] The detection results showed that after 49 hours of fermentation, acetylglucosamine was present in the fermentation broth at 11.6 g / L.

[0113] Example 21: Fed-batch fermentation of recombinant B. licheniformis BNGS3 in a 50 L fermentor at 37°C 1. Seed culture: 5 mL of the seed solution containing the BNGS3 strain was inoculated into LB medium and incubated at 50°C and 200 rpm for 12 to 16 hours. During the culture, 25 mg / L tetracycline resistance was imparted.

[0114] 2. Shake flask cultivation: The strain was transferred to 75 mL of fermentation medium (contained in a 500 mL shake flask) at a 5% inoculation rate. Tetracycline resistance of 25 mg / L was imparted during shake flask cultivation. The cultivation was carried out overnight at 50°C and 200 rpm.

[0115] 3. Fermentation: The activated seed solution was transferred to a 50 L automatic fermenter at an inoculation rate of 4% (v / v) and incubated at 37 °C. Tetracycline resistance was added until the effective concentration reached 25 mg / L, and 3 mol L -1 The pH was adjusted and maintained at 7.0 by adding hydrochloric acid solution and 25% (v / v) ammonia solution. The aeration rate was kept at 1.5 vvm and the initial speed was 600 r·min -1 The mixture was stirred at a rate of 0.01 s. The glucose concentration had to be continuously adjusted to 30 g / L. For this purpose, glucose was continuously replenished at a constant flow rate when glucose was consumed to 3-4 g / L. The culture was continued until the end of the fermentation. Samples were taken and detected by high-performance liquid chromatography.

[0116] The detection results showed that after 64 hours of fermentation, acetylglucosamine was present in the fermentation broth at 10.8 g / L.

[0117] Example 22: Fed-batch fermentation of recombinant B. licheniformis BNGS3 in a 50 L fermentor at 45°C 1. Seed culture: Same as in Example 21. 2. Shake flask culture: same as in Example 21. 3. Fermentation: The activated seed solution was transferred to a 50 L automatic fermenter at an inoculation rate of 4% (v / v) and incubated at 45 °C. Tetracycline resistance was added until the effective concentration reached 25 mg / L, and 3 mol L -1 The pH was adjusted and maintained at 7.0 by adding hydrochloric acid solution and 25% (v / v) ammonia solution. The aeration rate was kept at 1.5 vvm and the initial speed was 600 r·min -1The mixture was stirred at a rate of 0.01 s. The glucose concentration had to be continuously adjusted to 30 g / L. For this purpose, glucose was continuously replenished at a constant flow rate when glucose was consumed to 3-4 g / L. The culture was continued until the end of the fermentation. Samples were taken and detected by high-performance liquid chromatography.

[0118] The detection results showed that after 58 hours of fermentation, acetylglucosamine was present in the fermentation broth at 7.2 g / L.

[0119] Example 23: Fed-batch fermentation of recombinant B. licheniformis BNGS4 in a 50 L fermentor at 37°C 1. Seed culture: 5 mL of the seed solution containing the BNGS4 strain was inoculated into LB medium and incubated at 50°C and 200 rpm for 12 to 16 hours. During the culture, 25 mg / L tetracycline resistance was imparted.

[0120] 2. Shake flask cultivation: The strain was transferred to 75 mL of fermentation medium (contained in a 500 mL shake flask) at a 5% inoculation rate. Tetracycline resistance of 25 mg / L was imparted during shake flask cultivation. The cultivation was carried out overnight at 50°C and 200 rpm.

[0121] 3. Fermentation: The activated seed solution was transferred to a 50 L automatic fermenter at an inoculation rate of 4% (v / v) and incubated at 37 °C. Tetracycline resistance was added until the effective concentration reached 25 mg / L, and 3 mol L -1 The pH was adjusted and maintained at 7.0 by adding hydrochloric acid solution and 25% (v / v) ammonia solution. The aeration rate was kept at 1.5 vvm and the initial speed was 600 r·min -1 The mixture was stirred at a rate of 0.01 s. The glucose concentration had to be continuously adjusted to 30 g / L. For this purpose, glucose was continuously replenished at a constant flow rate when glucose was consumed to 3-4 g / L. The culture was continued until the end of the fermentation. Samples were taken and detected by high-performance liquid chromatography.

[0122] The detection results showed that after 64 hours of fermentation, acetylglucosamine was present in the fermentation broth at 6.1 g / L.

[0123] Example 24: Fed-batch fermentation of recombinant B. licheniformis BNGS4 in a 50 L fermentor at 45°C 1. Seed culture: Same as in Example 23. 2. Shake flask culture: same as in Example 23. 3. Fermentation: The activated seed solution was transferred to a 50 L automatic fermenter at an inoculation rate of 4% (v / v) and incubated at 45 °C. Tetracycline resistance was added until the effective concentration reached 25 mg / L, and 3 mol L -1 The pH was adjusted and maintained at 7.0 by adding hydrochloric acid solution and 25% (v / v) ammonia solution. The aeration rate was kept at 1.5 vvm and the initial speed was 600 r·min -1 The mixture was stirred at a rate of 0.01 s. The glucose concentration had to be continuously adjusted to 30 g / L. For this purpose, glucose was continuously replenished at a constant flow rate when glucose was consumed to 3-4 g / L. The culture was continued until the end of the fermentation. Samples were taken and detected by high-performance liquid chromatography.

[0124] The detection results showed that after 58 hours of fermentation, acetylglucosamine was present in the fermentation broth at 2.9 g / L.

[0125] Example 25: Stability assay of BNGS1 strain A 25 mg / mL solid LB medium plate was streaked with fermentation broth containing strain BNGS1. After 18 hours of incubation at 50°C, 10 single colonies were picked and inoculated into 500 mL shake flasks containing 75 mL of fermentation medium. The flasks were shaken at 40°C and 200 rpm, and glucose was present at a concentration of 30 g / L. N-acetylglucosamine production stability was assessed using HPLC 70 hours after inoculation, and the product was determined to be present at concentrations ranging from 2.24 g / L to 2.41 g / L.

[0126] The above has described in detail the preferred specific embodiments of the present invention. It should be understood that those skilled in the art can make various modifications and changes based on the concept of the present invention without any creative efforts. Therefore, all technical solutions that those skilled in the art can obtain through logical analysis, reasoning, or limited experiments according to the concept of the present invention based on the prior art are intended to be included in the protection scope defined in the claims.

Claims

1. A genetically modified strain that produces N-acetylglucosamine, The genetically modified strain produces N-acetylglucosamine at a fermentation temperature of 40°C to 50°C; The catabolic pathway and intracellular transport pathway of N-acetylglucosamine and N-acetylglucosamine intermediates are blocked in the genetically modified strain, wherein the catabolic pathway of N-acetylglucosamine and N-acetylglucosamine intermediates is blocked by inactivation or deletion of the nagB gene and gamma gene for glucosamine-6-phosphate deaminase and the nagA gene for N-acetylglucosamine-6-phosphate deacetylase, and the intracellular transport pathway of N-acetylglucosamine is blocked by inactivation or deletion of the gamP gene and nagP gene for N-acetylglucosamine transporter proteins. The genetically modified strain has an overexpressed glucosamine-6-phosphate synthase glmS gene and an overexpressed glucosamine-6-phosphate acetylase GNA1 gene, wherein the sequence of the glucosamine-6-phosphate acetylase GNA1 gene is as set forth in SEQ ID NO: 1, and the sequence of the glucosamine-6-phosphate synthase glmS gene is as set forth in SEQ ID NO:

2. The genetically modified strain, wherein the original strain of the genetically modified strain is Bacillus licheniformis.

2. The genetically modified strain according to claim 1, wherein the original strain is Bacillus licheniformis ATCC 14580.

3. The genetically modified strain was deposited at the China Center for Type Culture Collection (CCTCC) on March 14, 2020.

2. The genetically modified strain according to claim 1, characterized in that it is Bacillus licheniformis BNGS1, deposited under the number M2020054.

4. A method for constructing a genetically modified strain according to claim 1 or 2, Step A. Obtaining a knockout strain by knocking out the nagB and gamma genes for glucosamine-6-phosphate deaminase, the nagA gene for N-acetylglucosamine-6-phosphate deacetylase, and the gamP and nagP genes for N-acetylglucosamine transporter protein of the original strain; Step B. Constructing a dual expression vector containing both the glucosamine-6-phosphate synthase glmS gene and the glucosamine-6-phosphate acetylase GNA1 gene, wherein the sequence of the glucosamine-6-phosphate acetylase GNA1 gene is as set forth in SEQ ID NO:1 and the sequence of the glucosamine-6-phosphate synthase glmS gene is as set forth in SEQ ID NO:2; and Step C. A step of introducing the dual expression vector into the knockout strain obtained in Step A to obtain the genetically modified strain that produces N-acetylglucosamine. Including, The method as described above, wherein the original strain is Bacillus licheniformis.

5. 5. The method of claim 4, wherein the dual expression vector is constructed by introducing a promoter and expressing an expression vector in which the promoter, the glmS gene, and the GNA1 gene are in series.

6. 6. The method of claim 5, wherein the expression vector is pHY300PLK.

7. The promoter is P ａｌｓ , P ４３ , P ｓｔ , or P ａｐｒｅ The method according to claim 5, wherein

8. The P ａｌｓ The promoter sequence is as shown in SEQ ID NO:

3. ４３ The promoter sequence is as shown in SEQ ID NO:

4. ｓｔ The sequence of the promoter is as shown in SEQ ID NO: 5, and ａｐｒｅ The method of claim 7, wherein the sequence of the promoter is as shown in SEQ ID NO:

6.

9. 5. The method of claim 4, wherein the glmS gene for glucosamine-6-phosphate synthase is derived from Bacillus licheniformis.

10. 5. The method of claim 4, wherein the glucosamine-6-phosphate acetylase GNA1 gene is derived from Saccharomyces cerevisiae.

11. The method according to claim 4, characterized in that the original strain is Bacillus licheniformis ATCC 14580.

12. 4. Use of the genetically modified strain according to claim 1 in the production of N-acetylglucosamine, characterized in that the fermentation temperature for the production is 40°C to 50°C.

13. 13. The use according to claim 12, characterized in that the carbon source utilized in the production is glucose, glycerol, xylose or arabinose.

14. The production 1) Seed culture: inoculating the genetically modified strain into a seed medium to confer tetracycline resistance, and culturing at 50 ° C. and 200 rpm for 12 to 16 hours to activate the seed; 2) Shake flask cultivation: the activated seeds from step 1) are transferred to a shake flask containing a fermentation medium at an inoculation rate of 5%, conferring tetracycline resistance, and then cultivated at 50°C and 200 rpm for 12-16 hours for secondary activation; 3) Fermentation culture: The secondary activated seed from step 3) is transferred to a fermenter at an inoculation rate of 4%, tetracycline resistance is imparted, a carbon source is added, fed-batch fermentation is carried out at 40°C to 50°C, and the culture is cultured until the fermentation is completed.

13. The use according to claim 12, characterized in that it comprises:

15. 15. The use according to claim 14, characterized in that the seed medium in step 1) comprises the following components: peptone, yeast powder, and sodium chloride.

16. 15. The use according to claim 14, characterized in that the fermentation medium in step 2) comprises the following components: yeast powder, peptone, ammonium sulfate, dipotassium hydrogen phosphate trihydrate, potassium dihydrogen phosphate, and glucose.

17. 15. The use according to claim 14, characterized in that the fermentation medium in step 3) comprises the following ingredients: yeast powder, corn steep liquor powder, ammonium sulfate, dipotassium hydrogen phosphate trihydrate, potassium dihydrogen phosphate, and glucose.

18. 15. The use according to claim 14, wherein the fermentation conditions in step 3) are a pH value maintained at 7.0 using a 3 mol / L hydrochloric acid solution and a 25% by volume ammonia solution, an aeration rate of 1.5 vvm, an initial stirring speed of 600 rpm, and a glucose concentration continuously adjusted to 30 g / L by supplementing with glucose when 3 to 4 g / L of glucose is consumed.

Images