WO2022016641A1 - 一种生产n-乙酰氨基葡萄糖的菌株及其构建方法和应用 - Google Patents

一种生产n-乙酰氨基葡萄糖的菌株及其构建方法和应用 Download PDF

Info

Publication number
WO2022016641A1
WO2022016641A1 PCT/CN2020/109480 CN2020109480W WO2022016641A1 WO 2022016641 A1 WO2022016641 A1 WO 2022016641A1 CN 2020109480 W CN2020109480 W CN 2020109480W WO 2022016641 A1 WO2022016641 A1 WO 2022016641A1
Authority
WO
WIPO (PCT)
Prior art keywords
acetylglucosamine
gene
genetically engineered
phosphate
fermentation
Prior art date
Application number
PCT/CN2020/109480
Other languages
English (en)
French (fr)
Inventor
许平
吴雨桐
陶飞
尚海涛
穆晓玲
张毅
段中岳
Original Assignee
上海交通大学
安徽银创生物科技股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 上海交通大学, 安徽银创生物科技股份有限公司 filed Critical 上海交通大学
Priority to EP20946455.1A priority Critical patent/EP4194544A1/en
Priority to JP2023505387A priority patent/JP2023534879A/ja
Publication of WO2022016641A1 publication Critical patent/WO2022016641A1/zh
Priority to US18/158,343 priority patent/US20230272443A1/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1096—Transferases (2.) transferring nitrogenous groups (2.6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01003—Glucosamine N-acetyltransferase (2.3.1.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01004—Glucosamine-phosphate N-acetyltransferase (2.3.1.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01157—Glucosamine-1-phosphate N-acetyltransferase (2.3.1.157)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y206/00—Transferases transferring nitrogenous groups (2.6)
    • C12Y206/01—Transaminases (2.6.1)
    • C12Y206/01016—Glutamine-fructose-6-phosphate transaminase (isomerizing) (2.6.1.16), i.e. glucosamine-6-phosphate-synthase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01097—Glycopeptide alpha-N-acetylgalactosaminidase (3.2.1.97)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
    • C12Y305/01025—N-Acetylglucosamine-6-phosphate deacetylase (3.5.1.25)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/99—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in other compounds (3.5.99)
    • C12Y305/99006—Glucosamine-6-phosphate deaminase (3.5.99.6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/10—Bacillus licheniformis

Definitions

  • the invention belongs to the field of bioengineering, and relates to a genetically engineered strain for producing N-acetylglucosamine and a construction method and application thereof.
  • GlcNAc N-Acetylglucosamine
  • N-acetylglucosamine also has a therapeutic effect on osteoarthritis and joint pain.
  • it can form macromolecular mucopolysaccharide with D-glucuronic acid to produce hyaluronic acid, which has broad prospects and huge market.
  • the production methods of N-acetylglucosamine mainly include chemical method, enzymatic catalysis method and microbial fermentation method.
  • the chemical method is to obtain N-acetylglucosamine by acid hydrolysis of chitin. It is necessary to extract chitin from crab shells and shrimp shells, and then undergo acid hydrolysis to obtain glucosamine, and then use concentrated hydrochloric acid to hydrolyze N-acetylglucosamine to deacetylate and directly generate amino groups.
  • Glucose and acetic anhydride can acetoxylate glucosamine to produce N-acetylglucosamine.
  • Thermophilic bacteria such as Bacillus coagulans, Bacillus licheniformis, Bacillus stearothermophilus, etc., can grow at high temperature (40°C and above) and use organic carbon sources (glucose, xylose, arabinose, etc.) for fermentation production .
  • Bacillus licheniformis (B.licheniformis) ATCC 14580 is a facultative anaerobic, gram-positive, endospore-type bacterium, which can utilize various five-carbon and six-carbon sugars, and has a fast cell growth rate, thus shortening the fermentation period. Its genetic manipulation is stable and it is a GRAS (generally regarded as safe) strain recognized by the U.S. Food and Drug Administration, and this strain can be fermented at high temperatures up to 50°C.
  • GRAS generally regarded as safe
  • the present invention provides a genetically engineered strain capable of producing N-acetylglucosamine under high temperature conditions, and a construction method and application thereof.
  • the present invention takes Bacillus licheniformis as an example, by eliminating the catabolic pathway of N-acetylglucosamine of Bacillus licheniformis, and introducing the metabolic pathway of N-acetylglucosamine production, the extracellular accumulation of N-acetylglucosamine is realized, So that N-acetylglucosamine can be efficiently produced under high temperature conditions.
  • the strain construction idea proposed by the invention can be applied to the development of high-temperature production strains of various products, and the proposed strain and fermentation process have good application prospects in the industrial high-temperature fermentation production of N-acetylglucosamine.
  • One aspect of the present invention provides a genetically engineered strain for producing N-acetylglucosamine, which can be fermented to produce N-acetylglucosamine at 40-50°C.
  • the starting bacteria of the genetically engineered strain is thermophilic bacteria.
  • the starting bacteria of the genetically engineered strain is Bacillus.
  • the starting bacteria of the genetically engineered strain are Bacillus licheniformis, Bacillus coagulans, Bacillus methylotroph, Bacillus thermophilus, Geobacillus stearothermophilus, and the like.
  • the starting bacteria of the genetically engineered strain is Bacillus licheniformis ATCC 14580, and the Bacillus licheniformis ATCC 14580 can be directly purchased from the ATCC website.
  • the genetically engineered strain is Bacillus licheniformis BNGS1, the preservation number is CCTCC NO: M2020054, and it was deposited in the China Center for Type Culture Collection on March 14, 2020.
  • the N-acetylglucosamine and N-acetylglucosamine intermediate product catabolism pathway through 6-phosphate glucosamine deaminase nagB and gamA gene, 6-phosphate-N-acetylglucosamine deacetylase nagA gene is blocked by inactivation or deletion of one or more of the N-acetylglucosamine transporters into the cell through inactivation of one or both of the N-acetylglucosamine transporters gamP and nagP genes or blocked by deletion. .
  • the genetically engineered strain was introduced to overexpress the 6-phosphate glucosamine synthase gene and the 6-phosphate glucosamine acetylase gene.
  • 6-phosphate glucosamine acetylase gene sequence is shown in SEQ ID NO.1
  • 6-phosphate glucosamine synthase gene sequence is shown in SEQ ID NO.2.
  • the 6-phosphate glucosamine synthase gene can be obtained by PCR amplification according to the whole genome DNA sequence of Bacillus licheniformis GenBank No.NC_006270.3 as a template.
  • the 6-phosphate glucosamine acetylation gene can be obtained according to the whole genome of Saccharomyces cerevisiae GenBank No. NM_001179949, and codon optimization is carried out for whole gene synthesis.
  • the method comprises: making the 6-phosphate glucosamine deaminase gene and the 6-phosphate-N-acetylglucosamine deacetylase gene in the N-acetylglucosamine catabolic pathway in the starting bacteria , and inactivation or deletion of one or more of the N-acetylglucosamine transporter genes; the introduction of overexpressed 6-phosphate glucosamine synthase gene and 6-phosphate glucosamine acetylase gene.
  • the method includes the following steps:
  • step C Transfer the double expression vector into the knockout strain obtained in step A to obtain the genetically engineered strain that produces N-acetylglucosamine.
  • the construction method of the dual expression vector is to introduce a promoter, and express the expression vector in series with the promoter, glmS gene and GNA1 gene.
  • the expression vector is pHY300PLK.
  • the promoters are P als, P 43, P st and P apre.
  • P als promoter sequence as shown in SEQ ID P 43 promoter sequence as shown in SEQ ID, P st promoter sequence as shown in SEQ ID NO.5 NO.3 NO.4, P apre promoter sequence As shown in SEQ ID NO.6.
  • the glucosamine-6-phosphate synthase glmS gene is derived from Bacillus licheniformis or other microorganisms with the same function as thermophilic enzymes, such as Bacillus coagulans, Bacillus methylotrophs, Bacillus thermophilus or Bacillus thermophilus Bacillus, etc.
  • the 6-phosphate glucosamine acetylase GNA1 gene is derived from Saccharomyces cerevisiae or other microorganisms with the same function as thermophilic enzymes, such as Kluyveromyces marxianus, Naxonospora rhodochrous and the like.
  • the starting bacteria is Bacillus licheniformis ATCC 14580.
  • Another aspect of the present invention also provides the application of the above-mentioned genetic engineering strain, especially the application in the production of N-acetylglucosamine.
  • the fermentation temperature of this production is 25°C-50°C.
  • the fermentation temperature for this production is between 40°C and 50°C.
  • the carbon source used in the production is glucose, glycerol, xylose or arabinose and the like.
  • genetic engineering strain is carried out seed culture to obtain seed culture liquid, then takes glucose as carbon source to carry out secondary activation in fermentation medium, and finally transfers into fermentor and ferments to obtain N-acetylglucosamine. Specifically include the following steps:
  • Seed culture connect the genetically engineered strain to the seed medium, add tetracycline resistance, and cultivate for 12-16 hours at 50°C and 200rpm to activate the seeds;
  • Fermentation culture 4% of the inoculum was transferred into a fermenter, added with tetracycline resistance, and a carbon source was added to carry out fed-batch fermentation at 40°C-50°C, and cultured until the fermentation was over.
  • the seed medium in step 1) includes the following components: peptone, yeast powder and sodium chloride, preferably: 10 g/L peptone, 5 g/L yeast powder, and 10 g/L sodium chloride.
  • the fermentation medium in step 2) includes the following components: yeast powder, peptone, ammonium sulfate, dipotassium hydrogen phosphate trihydrate, potassium dihydrogen phosphate and glucose, preferably containing: 12g/L yeast powder, 6g/L peptone , 6g/L ammonium sulfate, 18.75g/L dipotassium hydrogen phosphate trihydrate, 2.5g/L potassium dihydrogen phosphate, 30g/L glucose.
  • the shake flask in step 2) is a 500-milliliter conical flask, and the filling volume of the fermentation medium is 75 milliliters.
  • the fermentation medium in step 3) includes the following components: yeast powder, dry corn steep liquor powder, ammonium sulfate, dipotassium hydrogen phosphate trihydrate, potassium dihydrogen phosphate and glucose, preferably containing: 12g/L yeast powder, 6g/L L corn steep liquor dry powder, 6g/L ammonium sulfate, 18.75g/L dipotassium hydrogen phosphate trihydrate, 2.5g/L potassium dihydrogen phosphate, 30g/L glucose.
  • the fermentation conditions in step 3) are: 3mol/L hydrochloric acid solution and 25% (v/v) ammonia water are adjusted to maintain pH at 7.0, the ventilation rate is 1.5vvm, the initial stirring speed is 600rpm, and the glucose concentration is continuously controlled At 30g/L, supplement sugar when the sugar concentration is consumed to 3-4g/L.
  • the genetically engineered strain provided by the present application realizes the high temperature production of N-acetylglucosamine.
  • the fermentation process of genetically engineered strains since it is constitutive expression, it is not necessary to add inducers.
  • the demand for cooling during the fermentation process is very low, and the high temperature production greatly improves the fermentation rate.
  • the high temperature can prevent the growth of other microorganisms, open fermentation can be realized, which effectively reduces the production cost.
  • the invention realizes the fermentative production of N-acetylglucosamine at a higher temperature of 40 DEG C to 50 DEG C, and has higher industrial utilization value.
  • the strain construction idea proposed by the invention can be applied to the development of high-temperature production strains of various products, and the proposed strain and fermentation process have good application prospects in the industrial high-temperature fermentation production of N-acetylglucosamine.
  • Figure 1 shows the synthetic and metabolic pathways of N-acetylglucosamine in Bacillus licheniformis engineered bacteria.
  • Glutamine (Gln) is used as an amino acid donor, and glucosamine synthase converts fructose-6-phosphate (F 6-phosphate) through the glmS gene encoding.
  • -6-P glucosamine 6-phosphate
  • GlcN-6-P glucosamine 6-phosphate
  • NAA1 glucosamine 6-phosphate acetylase
  • -6-Glucosamine phosphate (GlcNAc-6-P), which is then dephosphorylated by phosphorylase to generate N-acetylglucosamine, which is then secreted to the outside of the cell. Due to the 6-phosphate glucosamine deaminase nagB and gamA genes, - The knockout of the phospho-N-acetylglucosamine deacetylase nagA gene inactivates or reduces the consumption of N-acetylglucosamine precursor in cells, and the N-acetylglucosamine transporter gamP gene and nagP gene are knocked out Therefore, the engineered strains are able to accumulate high concentrations of N-acetylglucosamine extracellularly in the absence of transport.
  • the expression of the rate-limiting enzyme gene in the synthesis pathway of N-acetylglucosamine is enhanced.
  • the gene for acetylglucosamine consumption and reflux prevents the reflux consumption of N-acetylglucosamine, allowing the engineered strain to accumulate high concentrations of N-acetylglucosamine.
  • One aspect of the present invention provides a genetically engineered strain for producing N-acetylglucosamine, which can be fermented to produce N-acetylglucosamine at 40-50°C.
  • the N-acetylglucosamine catabolic pathway is blocked in the starting strain of the genetically engineered strain.
  • the N-acetylglucosamine catabolic pathway through the 6-phosphoglucosamine deaminase nagB and gamA genes, the 6-phospho-N-acetylglucosamine deacetylase nagA gene, and the N- is blocked by inactivation or deletion of one or more of the acetylglucosamine transporter gamP and nagP genes.
  • Blocking refers to blocking a certain pathway through various genetic engineering methods. This involves the inactivation or deletion of the gene for one or more of the catalytic enzymes in the pathway, thereby rendering the pathway incapable of proceeding.
  • the starting strain may also not contain the above-mentioned deleted or inactivated enzymes or blocked pathways, such as 6-phosphate glucosamine deaminase gene, 6-phosphate-N-acetylglucosamine deacetylation Enzyme genes, N-acetylglucosamine transporter genes, etc., these genes or pathways are naturally deleted when constructing genetically engineered strains, and no additional genetic engineering operations are required to inactivate or delete or block them.
  • deleted or inactivated enzymes or blocked pathways such as 6-phosphate glucosamine deaminase gene, 6-phosphate-N-acetylglucosamine deacetylation Enzyme genes, N-acetylglucosamine transporter genes, etc.
  • Example 1 Knockout of Bacillus licheniformis nagP gene
  • Example 2 Continue to knock out the gamP gene of Bacillus licheniformis
  • Example 3 Continue to knock out Bacillus licheniformis gamA gene
  • Example 4 Continue to knock out the Bacillus licheniformis gene cluster nagAB
  • the knockout vector pKVM- ⁇ nagAB was transformed into Escherichia coli S17-1, and then conjugated with Bacillus licheniformis knockout strain MW3 ⁇ nagP ⁇ gamP ⁇ gamA, and the nagAB gene cluster was knocked out by homologous recombination.
  • nagAB-DR GCGTCGGGCGATATC CCTCTTCATATCAATGACGAA (SEQ ID NO. 22)
  • the fragment that can amplify to 1600bp proves that the correct double-crossover positive has been screened Colony, the gene knockout was successful, and the knockout strain MW3 ⁇ nagP ⁇ gamP ⁇ gamA ⁇ nagAB was obtained.
  • the whole gene synthesis was carried out according to the Pst promoter, and the designed primers were: the upstream primer was Pst- F: TATCGATAAGCTTGATATCG CATGATGTGGGGCGTTTTT (SEQ ID NO. 27) and the downstream primer was Pst- R: GTCCGGCAGGCTCAT AGCCCTCACTCCTCCATT (SEQ ID NO. 28). And the Pst promoter sequence was obtained by PCR amplification.
  • the whole gene was synthesized according to the P aprE promoter, and the designed primers were: the upstream primer was P aprE- F: TATCGATAAGCTTGATATCG CAGCATAATGAACATTTACTCATG (SEQ ID NO. 29) and the downstream primer was P aprE- R: GTCCGGCAGGCTCAT AGCCCTCACTCCTCCATT (SEQ ID NO. 30). And the P aprE promoter sequence was obtained by PCR amplification.
  • the 6-phosphate glucosamine acetylase GNA1 gene was obtained, and codon optimization was performed for whole gene synthesis, and PCR amplification was performed to obtain the optimized GNA1 sequence.
  • the designed primers are: the upstream primer is gna1-F: GGAGGATGAGGGCT ATGAGCCTGCCGGACG (SEQ ID NO. 31), the downstream primer is: gna1-R: TACAATACCACACAT TTTGCGGATCTGCATTTC (SEQ ID NO. 32).
  • Primers were designed according to the sequence of Bacillus licheniformis MW3 genome (GenBank No. NC_006270.3): the upstream primer was glmS -F: ATGCAGATCCGCAAA ATGTGTGGTATTGTAGGTTATATTG (SEQ ID NO. 33) and the downstream primer was glmS -R: CGGCCGCTCTAGAACTAGTG CTACTCCACCGTCACACTCTT (SEQ ID NO. 34) ).
  • the glmS gene sequence was obtained by PCR amplification with Bacillus licheniformis MW3 genomic DNA as template.
  • the P als promoter sequence, GNA1 and glmS three gene fragments recovered by PCR amplification were taken 1 microliter each, and were seamlessly cloned with the pHY300PLK vector to obtain the vector pHY300PLK-P als- GNA1-glmS.
  • the PCR amplified promoter sequence of P 43 was recovered, and GNA1 glmS gene fragment from each of three 1 l, pHY300PLK seamless cloning and vector construction, resulting in vector pHY300PLK-P 43 -GNA1-glmS.
  • the P aprE promoter sequence, GNA1 and glmS three gene fragments recovered by PCR amplification were taken 1 microliter each and were cloned seamlessly with the pHY300PLK vector to obtain the vector pHY300PLK-P aprE- GNA1-glmS.
  • Example 6 Construction of BNGS1, BNGS2, BNGS3 and BNGS4 engineering bacteria
  • pHY300PLK-P als -GNA1-glmS vector transfected by electroporation into MW3 ⁇ nagP ⁇ gamP ⁇ gamA ⁇ nagAB knockout strains, that is able to synthesize N- acetylglucosamine Bacillus licheniformis, named BNGS1.
  • pHY300PLK-P 43 -GNA1-glmS vector was introduced into the knockout strain MW3 ⁇ nagP ⁇ gamP ⁇ gamA ⁇ nagAB by electroporation, which was Bacillus licheniformis capable of synthesizing N-acetylglucosamine, named BNGS2.
  • pHY300PLK-P aprE- GNA1-glmS vector was introduced into the knockout strain MW3 ⁇ nagP ⁇ gamP ⁇ gamA ⁇ nagAB by electroporation, which is Bacillus licheniformis capable of synthesizing N-acetylglucosamine, named BNGS4.
  • Example 7 Shake flask fermentation of recombinant Bacillus licheniformis BNGS1
  • the seed medium was LB medium (the components were sodium chloride 10g/L, yeast powder 5g/L, peptone 10g/L), and tetracycline resistance 25mg/L was added during liquid culture.
  • the fermentation medium includes 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.
  • the seed solution of the BNGS1 strain was placed in 5 mL of LB, and cultured at 50 °C and 200 rpm for 12-16 h, and 25 mg/L of tetracycline resistance was added during the culture. Transfer to a 500 ml conical flask according to 5% of the inoculum volume, and carry out shake flask fermentation in 75 ml of fermentation medium. Add 25 mg/L of tetracycline resistance during the culture, the fermentation time is 50 h, and the culture temperature is 40 °C. After sampling 1 mL Centrifuge at 12,000 rpm for 5 min, filter with 0.22-micron aqueous filter, and detect by high performance liquid chromatography.
  • Example 8 Shake flask fermentation of recombinant Bacillus licheniformis BNGS2
  • the seed solution of the BNGS2 strain was placed in 5 mL of LB, and cultured at 50 °C and 200 rpm for 12-16 h, and 25 mg/L of tetracycline resistance was added during the culture. Transfer to a 500 ml conical flask according to 5% of the inoculum volume, and carry out shake flask fermentation in 75 ml of fermentation medium. Add 25 mg/L of tetracycline resistance during the culture, the fermentation time is 50 h, and the culture temperature is 40 °C. After sampling 1 mL Centrifuge at 12,000 rpm for 5 min, filter with 0.22-micron aqueous filter, and detect by high performance liquid chromatography.
  • Example 9 Shake flask fermentation of recombinant Bacillus licheniformis BNGS3
  • the seed solution of the BNGS3 strain was placed in 5 mL of LB, and cultured at 50 °C and 200 rpm for 12-16 h, and 25 mg/L of tetracycline resistance was added during the culture. Transfer to a 500 ml conical flask according to 5% of the inoculum volume, and carry out shake flask fermentation in 75 ml of fermentation medium. Add 25 mg/L of tetracycline resistance during the culture, the fermentation time is 50 h, and the culture temperature is 40 °C. After sampling 1 mL Centrifuge at 12,000 rpm for 5 min, filter with 0.22-micron aqueous filter, and detect by high performance liquid chromatography.
  • Example 10 Shake flask fermentation of recombinant Bacillus licheniformis BNGS4
  • the BNGS4 strain seed solution was placed in 5 mL of LB, and cultured at 50 °C and 200 rpm for 12-16 h, and 25 mg/L of tetracycline resistance was added during culture. Transfer to a 500 ml conical flask according to 5% of the inoculum volume, and carry out shake flask fermentation in 75 ml of fermentation medium. Add 25 mg/L of tetracycline resistance during the culture, the fermentation time is 50 h, and the culture temperature is 40 °C. After sampling 1 mL Centrifuge at 12,000 rpm for 5 min, filter with 0.22-micron aqueous filter, and detect by high performance liquid chromatography.
  • Example 11 Recombinant Bacillus licheniformis BNGS1 was fermented in batches at 37°C, 1L fermentor 1. Seed culture: The BNGS1 strain seed solution was placed in 5mL LB, and cultured at 50°C and 200rpm for 12-16h. 25mg/L of tetracycline resistance was added during culture.
  • Shake flask culture transfer to 75 ml of fermentation medium at 5% inoculation amount (shaking flask volume 500 ml), and add tetracycline resistance 25 mg/L during shaking flask culture. Incubate overnight at 50°C at 200 rpm.
  • Fermentation culture with 4% (v/v) inoculum, transfer the activated seed liquid to a 1L automatic fermenter, the culture temperature is 37°C, the working concentration of tetracycline resistance is 25mg/L, and 3mol ⁇ L -1 hydrochloric acid solution and 25% (v/v) ammonia water were adjusted to maintain pH at 7.0, ventilation volume was 1.5 vvm, and initial stirring speed was 600 r ⁇ min -1 .
  • the glucose concentration should be continuously controlled at 30g/L. When the sugar concentration is consumed to 3-4g/L, continuous constant-flow sugar supplementation is carried out, and the culture is carried out to the end of the fermentation, and the sampling is detected by high performance liquid chromatography.
  • the content of acetylglucosamine in the fermentation broth can reach 12g/L.
  • Example 12 Recombinant Bacillus licheniformis BNGS1 was fermented in fed-batch at 50°C, 1L fermentor 1. Seed culture: the same as in Example 11.
  • Fermentation culture with 4% (v/v) inoculum, transfer the activated seed liquid to a 1L automatic fermenter, the culture temperature is 50°C, the working concentration of tetracycline resistance is 25mg/L, and 3mol ⁇ L -1 hydrochloric acid solution and 25% (v/v) ammonia water were adjusted to maintain pH at 7.0, ventilation volume was 1.5 vvm, and initial stirring speed was 600 r ⁇ min -1 .
  • the glucose concentration should be continuously controlled at 30g/L. When the sugar concentration is consumed to 3-4g/L, continuous constant-flow sugar supplementation is carried out, and the culture is carried out to the end of the fermentation, and the sampling is detected by high performance liquid chromatography.
  • the content of acetylglucosamine in the fermentation broth can reach 2.8g/L.
  • Example 13 Fed-batch fermentation of recombinant Bacillus licheniformis BNGS1 at 37°C, 50L fermenter
  • Seed culture the same as in Example 11.
  • Fermentation culture transfer the activated seed liquid to a 50L automatic fermenter with an inoculum of 4% (v/v), the culture temperature is 37°C, the working concentration of tetracycline resistance is 25mg/L, and 3mol ⁇ L -1 hydrochloric acid solution and 25% (v/v) ammonia water were adjusted to maintain pH at 7.0, ventilation volume was 1.5 vvm, and initial stirring speed was 600 r ⁇ min -1 .
  • the glucose concentration should be continuously controlled at 30g/L. When the sugar concentration is consumed to 3-4g/L, continuous constant-flow sugar supplementation is carried out, and the culture is carried out to the end of the fermentation, and the sampling is detected by high performance liquid chromatography.
  • the content of acetylglucosamine in the fermentation broth can reach 50.9g/L.
  • Example 14 Fed-batch fermentation of recombinant Bacillus licheniformis BNGS1 at 37°C, 50L fermenter
  • Seed culture the same as in Example 11.
  • Fermentation culture transfer the activated seed liquid to a 50L automatic fermenter with an inoculum of 4% (v/v), the culture temperature is 37°C, the working concentration of tetracycline resistance is 25mg/L, and 3mol ⁇ L -1 hydrochloric acid solution and 25% (v/v) ammonia water were adjusted to maintain pH at 7.0, ventilation volume was 1.5 vvm, and initial stirring speed was 600 r ⁇ min -1 .
  • the glucose concentration should be continuously controlled at 30g/L. When the sugar concentration is consumed to 3-4g/L, continuous constant-flow sugar supplementation is carried out, and the culture is carried out to the end of the fermentation, and the sampling is detected by high performance liquid chromatography.
  • the content of acetylglucosamine in the fermentation broth can reach 52.5g/L.
  • Example 15 Fed-batch fermentation of recombinant Bacillus licheniformis BNGS1 at 40°C, 50L fermenter
  • Seed culture the same as in Example 11.
  • Fermentation culture transfer the activated seed liquid to a 50L automatic fermenter with an inoculum of 4% (v/v), the culture temperature is 40°C, the working concentration of tetracycline resistance is 25mg/L, and 3mol ⁇ L -1 hydrochloric acid solution and 25% (v/v) ammonia water were used to adjust the pH to maintain 7.0, the ventilation volume was 1.5vvm, and the initial stirring speed was 600r ⁇ min -1 .
  • the glucose concentration should be continuously controlled at 30g/L. When the sugar concentration is consumed to 3-4g/L, continuous constant-flow sugar supplementation is carried out, and the culture is carried out to the end of the fermentation, and the sampling is detected by high performance liquid chromatography.
  • the content of acetylglucosamine in the fermentation broth can reach 28.1g/L.
  • Example 16 Fed-batch fermentation of recombinant Bacillus licheniformis BNGS1 at 40°C, 50L fermenter
  • Seed culture the same as in Example 11.
  • Fermentation culture transfer the activated seed liquid to a 50L automatic fermenter with an inoculum of 4% (v/v), the culture temperature is 40°C, the working concentration of tetracycline resistance is 25mg/L, and 3mol ⁇ L -1 hydrochloric acid solution and 25% (v/v) ammonia water were adjusted to maintain pH at 7.0, ventilation volume was 1.5 vvm, and initial stirring speed was 600 r ⁇ min -1 .
  • the glucose concentration should be continuously controlled at 30g/L. When the sugar concentration is consumed to 3-4g/L, continuous constant-flow sugar supplementation is carried out, and the culture is carried out to the end of the fermentation, and the sampling is detected by high performance liquid chromatography.
  • the content of acetylglucosamine in the fermentation broth can reach 34.6g/L.
  • Example 17 Fed-batch fermentation of recombinant Bacillus licheniformis BNGS1 at 45°C, 50L fermenter
  • Seed culture the same as in Example 11.
  • Fermentation culture transfer the activated seed liquid to a 50L automatic fermenter with an inoculum of 4% (v/v), the culture temperature is 45°C, the working concentration of tetracycline resistance is 25mg/L, and 3mol ⁇ L -1 hydrochloric acid solution and 25% (v/v) ammonia water were adjusted to maintain pH at 7.0, ventilation volume was 1.5 vvm, and initial stirring speed was 600 r ⁇ min -1 .
  • the glucose concentration should be continuously controlled at 30g/L. When the sugar concentration is consumed to 3-4g/L, continuous constant-flow sugar supplementation is carried out, and the culture is carried out to the end of the fermentation, and the sampling is detected by high performance liquid chromatography.
  • the content of acetylglucosamine in the fermentation broth can reach 24g/L.
  • Example 18 Fed-batch fermentation of recombinant Bacillus licheniformis BNGS1 at 50°C, 50L fermenter
  • Fermentation culture transfer the activated seed solution to a 50L automatic fermenter with an inoculum of 4% (v/v), the culture temperature is 50°C, the working concentration of tetracycline resistance is 25mg/L, and 3mol ⁇ L -1 hydrochloric acid solution and 25% (v/v) ammonia water were adjusted to maintain pH at 7.0, ventilation volume was 1.5 vvm, and initial stirring speed was 600 r ⁇ min -1 .
  • the glucose concentration should be continuously controlled at 30g/L. When the sugar concentration is consumed to 3-4g/L, continuous constant-flow sugar supplementation is carried out, and the culture is carried out to the end of the fermentation, and the sampling is detected by high performance liquid chromatography.
  • the content of acetylglucosamine in the fermentation broth can reach 4.1g/L.
  • Example 19 Fed-batch fermentation of recombinant Bacillus licheniformis BNGS2 at 37°C, 50L fermenter
  • Seed culture connect the BNGS2 strain seed solution to 5mL of LB, and cultivate at 50°C and 200rpm for 12-16h. 25mg/L of tetracycline resistance was added during culture.
  • Shake flask culture transfer to a fermentation medium containing 75 ml of the inoculum at 5% (shaking flask volume 500 ml), and add tetracycline resistance 25 mg/L during shaking flask culture. Incubate overnight at 50°C at 200 rpm.
  • Fermentation culture transfer the activated seed liquid to a 50L automatic fermenter with an inoculum of 4% (v/v), the culture temperature is 37°C, the working concentration of tetracycline resistance is 25mg/L, and 3mol ⁇ L -1 hydrochloric acid solution and 25% (v/v) ammonia water were adjusted to maintain pH at 7.0, ventilation volume was 1.5vvm, and initial stirring speed was 600r ⁇ min -1 .
  • the glucose concentration should be continuously controlled at 30g/L. When the sugar concentration is consumed to 3-4g/L, continuous constant-flow sugar supplementation is carried out, and the culture is carried out to the end of the fermentation, and the sampling is detected by high performance liquid chromatography.
  • the content of acetylglucosamine in the fermentation broth can reach 23.6g/L.
  • Example 20 Fed-batch fermentation of recombinant Bacillus licheniformis BNGS2 at 45°C, 50L fermenter
  • Fermentation culture transfer the activated seed liquid to a 50L automatic fermenter with an inoculum of 4% (v/v), the culture temperature is 45°C, the working concentration of tetracycline resistance is 25mg/L, and 3mol ⁇
  • the pH of L-1 hydrochloric acid solution and 25% (v/v) ammonia water was adjusted to maintain at 7.0, the ventilation volume was 1.5vvm, and the initial stirring speed was 600r ⁇ min-1.
  • the glucose concentration should be continuously controlled at 30g/L. When the sugar concentration is consumed to 3-4g/L, continuous constant-flow sugar supplementation is carried out, and the culture is carried out to the end of the fermentation, and the sampling is detected by high performance liquid chromatography.
  • the content of acetylglucosamine in the fermentation broth can reach 11.6g/L.
  • Example 21 Fed-batch fermentation of recombinant Bacillus licheniformis BNGS3 at 37°C, 50L fermenter
  • Seed culture connect the BNGS3 strain seed solution to 5mL of LB, and cultivate at 50°C and 200rpm for 12-16h. 25mg/L of tetracycline resistance was added during culture.
  • Shake flask culture transfer to 75 ml of fermentation medium at 5% inoculation amount (shaking flask volume 500 ml), and add tetracycline resistance 25 mg/L during shaking flask culture. Incubate overnight at 50°C at 200 rpm.
  • Fermentation culture transfer the activated seed liquid to a 50L automatic fermenter with an inoculum of 4% (v/v), the culture temperature is 37°C, the working concentration of tetracycline resistance is 25mg/L, and 3mol ⁇ L -1 hydrochloric acid solution and 25% (v/v) ammonia water were adjusted to maintain pH at 7.0, ventilation volume was 1.5 vvm, and initial stirring speed was 600 r ⁇ min -1 .
  • the glucose concentration should be continuously controlled at 30g/L. When the sugar concentration is consumed to 3-4g/L, continuous constant-flow sugar supplementation is carried out, and the culture is carried out to the end of the fermentation, and the sampling is detected by high performance liquid chromatography.
  • the content of acetylglucosamine in the fermentation broth can reach 10.8g/L.
  • Example 22 Fed-batch fermentation of recombinant Bacillus licheniformis BNGS3 at 45°C, 50L fermenter
  • Fermentation culture transfer the activated seed liquid to a 50L automatic fermenter with an inoculum of 4% (v/v), the culture temperature is 45°C, the working concentration of tetracycline resistance is 25mg/L, and 3mol ⁇ L -1 hydrochloric acid solution and 25% (v/v) ammonia water were adjusted to maintain pH at 7.0, ventilation volume was 1.5 vvm, and initial stirring speed was 600 r ⁇ min -1 .
  • the glucose concentration should be continuously controlled at 30g/L. When the sugar concentration is consumed to 3-4g/L, continuous constant-flow sugar supplementation is carried out, and the culture is carried out to the end of the fermentation, and the sampling is detected by high performance liquid chromatography.
  • the content of acetylglucosamine in the fermentation broth can reach 7.2g/L.
  • Example 23 Fed-batch fermentation of recombinant Bacillus licheniformis BNGS4 at 37°C, 50L fermenter
  • Seed culture connect the BNGS4 strain seed solution to 5mL of LB, and cultivate at 50°C and 200rpm for 12-16h. 25mg/L of tetracycline resistance was added during culture.
  • Shake flask culture transfer the inoculum to 75ml of fermentation medium (shaking flask volume 500ml) according to 5% inoculum, add tetracycline resistance 25mg/L during shake flask culture. Incubate overnight at 50°C at 200 rpm.
  • Fermentation culture transfer the activated seed liquid to a 50L automatic fermenter with an inoculum of 4% (v/v), the culture temperature is 37°C, the working concentration of tetracycline resistance is 25mg/L, and 3mol ⁇ L -1 hydrochloric acid solution and 25% (v/v) ammonia water were adjusted to maintain pH at 7.0, ventilation volume was 1.5 vvm, and initial stirring speed was 600 r ⁇ min -1 .
  • the glucose concentration should be continuously controlled at 30g/L. When the sugar concentration is consumed to 3-4g/L, continuous constant-flow sugar supplementation is carried out, and the culture is carried out to the end of the fermentation, and the sampling is detected by high performance liquid chromatography.
  • the content of acetylglucosamine in the fermentation broth can reach 6.1g/L.
  • Example 24 Fed-batch fermentation of recombinant Bacillus licheniformis BNGS4 at 45°C, 50L fermenter
  • Seed culture the same as in Example 23.
  • Fermentation culture transfer the activated seed liquid to a 50L automatic fermenter with an inoculum of 4% (v/v), the culture temperature is 45°C, the working concentration of tetracycline resistance is 25mg/L, and 3mol ⁇ L -1 hydrochloric acid solution and 25% (v/v) ammonia water were adjusted to maintain pH at 7.0, ventilation volume was 1.5 vvm, and initial stirring speed was 600 r ⁇ min -1 .
  • the glucose concentration should be continuously controlled at 30g/L. When the sugar concentration is consumed to 3-4g/L, continuous constant-flow sugar supplementation is carried out, and the culture is carried out to the end of the fermentation, and the sampling is detected by high performance liquid chromatography.
  • the content of acetylglucosamine in the fermentation broth can reach 2.9g/L.
  • Example 25 Stability test of BNGS1 strain
  • the BNGS1 bacterial liquid after the fermentation was streaked on a 25 mg/mL LB solid plate, and after culturing at 50 °C for 18 h, 10 single colonies were picked and connected to a 500 mL shake flask containing 75 mL of fermentation medium, respectively.
  • the shaker temperature was 40°C
  • the rotational speed was 200rpm
  • the glucose concentration was 30g/L.
  • the yield stability of N-acetylglucosamine was detected by HPLC at 70h after inoculation, and the yield concentration was between 2.24g/L and 2.41g/L.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

一种生产N-乙酰氨基葡萄糖的基因工程菌株及其构建方法和应用。该基因工程菌株可以在40-50℃条件下发酵生产N-乙酰氨基葡萄糖，通过敲除起始菌中的6-磷酸氨基葡萄糖脱氨酶基因，6-磷酸-N-乙酰氨基葡萄糖脱乙酰酶基因，N-乙酰氨基葡萄糖转运蛋白基因，阻断了N-乙酰氨基葡萄糖的分解代谢途径，并引入过表达6-磷酸氨基葡萄糖合成酶基因和6-磷酸氨基葡萄糖乙酰化酶基因，使得N-乙酰氨基葡萄糖可以在胞外积累，实现N-乙酰氨基葡萄糖在高于40℃条件下的高温发酵生产。

Description

一种生产N-乙酰氨基葡萄糖的菌株及其构建方法和应用 技术领域
本发明属于生物工程领域，涉及一种生产N-乙酰氨基葡萄糖的基因工程菌株及其构建方法和应用。
背景技术
N-乙酰氨基葡萄糖(N-Acetylglucosamine，GlcNAc)是生物体内多种多糖的组成单位，由葡萄糖的一个羟基被氨基取代形成，尤其是甲壳类动物的外骨骼含量最高，并广泛应用于食品、制药和化妆品行业。在食品行业，它可以作为食品抗氧化剂，婴幼儿食品添加剂，以及糖尿病患者甜味剂。在制药行业，它作为新型生化药品，在临床上是治疗风湿性及类风湿性关节炎的药物。同时它也主要用于临床增强人体免疫系统的功能，抑制癌细胞或纤维细胞的过度生长，对癌症和恶性肿瘤起到抑制和治疗作用。除此之外，N-乙酰氨基葡萄糖对于骨关节炎及关节疼痛也有治疗作用。在化妆品行业，它可以与D-葡萄糖醛酸形成高分子粘多糖生产透明质酸，有广阔的前景，市场巨大。
N-乙酰氨基葡萄糖的生产方法主要有化学法、酶催化法和微生物发酵法。化学法为通过酸水解甲壳素得到N-乙酰氨基葡萄糖，需要从蟹壳和虾壳中提取甲壳素，再经过酸水解得到氨基葡萄糖，之后利用浓盐酸水解N-乙酰氨基葡萄糖脱乙酰直接生成氨基葡萄糖，乙酸酐可使氨基葡萄糖乙酸化生产N-乙酰氨基葡萄糖。但该方法对环境不友好，同时污染大，且产品不适用于海鲜过敏症患者。与化学法相比，酶催化法和微生物发酵法属于环境友好型生产方法，但是酶催化法需要使用酶催化几丁质降解进而合成N-乙酰氨基葡萄糖，受制于底物虾蟹壳预处理困难、关键酶活性低、产物分离纯化困难，导致规模化生产难度较大。微生物发酵法生产N-乙酰氨基葡萄糖是目前最具前景的方法，也是本领域重点研究方向。
相对于40℃以下的低温发酵，高温发酵具有最小化污染的风险，提高原料转化速率，并且能够降低热交换成本等优势。到目前为止，关于微生物通过代谢工程改造生产N-乙酰氨基葡萄糖的报道中，发酵温度均为38℃及以下，没有高温生产N-乙酰氨基葡萄糖的专利及报道。因此，很有必要开发一种低成本的、强健的微生物平台来高温高产N-乙酰氨基葡萄糖。
嗜热细菌比如凝结芽孢杆菌、地衣芽孢杆菌、嗜热脂肪芽孢杆菌等，可以在高温条件下(40℃及以上)生长、并利用有机碳源(葡萄糖、木糖、阿拉伯糖等)进行发酵生产。例如其中的地衣芽孢杆菌(B.licheniformis)ATCC 14580是一株兼性厌氧、革兰氏阳性、内生孢子型细菌，它能够利用各种五碳糖和六碳糖，具有较快的细胞生 长速率，从而可以缩短发酵周期。它的遗传操作稳定并且是美国食品和药物管理局公认的GRAS(generally regarded as safe)菌株，并且该菌株可以在高达50℃的高温条件下进行高温发酵。这些优点表明菌株ATCC 14580可作为一株理想的平台菌株。
发明内容
为了解决上述问题，本发明提供一种可以在高温条件下生产N-乙酰氨基葡萄糖的基因工程菌株及其构建方法和应用。本发明以嗜热地衣芽孢杆菌为例，通过消除地衣芽孢杆菌的N-乙酰氨基葡萄糖的分解代谢途径，引入N-乙酰氨基葡糖生产的代谢途径，实现N-乙酰氨基葡萄糖的胞外积累，使得N-乙酰氨基葡萄糖可以在高温条件下高效生产。本发明所提出的菌株构建思路可应用于多种产品的高温生产菌株的开发，所提出的菌株和发酵工艺在N-乙酰氨基葡萄糖的工业高温发酵生产方面具有良好的应用前景。
本发明的一个方面提供了一种生产N-乙酰氨基葡萄糖的基因工程菌株，该基因工程菌株可以在40-50℃条件下发酵生产N-乙酰氨基葡萄糖。
进一步地，该基因工程菌株的起始菌为嗜热菌。
进一步地，该基因工程菌株的起始菌为芽孢杆菌。
优选地，该基因工程菌株的起始菌为地衣芽孢杆菌、凝结芽孢杆菌、甲基营养芽孢杆菌、嗜热菊糖芽孢杆菌或嗜热脂肪地芽孢杆菌等。
优选地，该基因工程菌株的起始菌为地衣芽孢杆菌(Bacillus licheniformis)ATCC 14580，所述地衣芽孢杆菌ATCC 14580可以从ATCC网站直接购买获得。
进一步地，该基因工程菌株为地衣芽孢杆菌(Bacillus licheniformis)BNGS1，保藏号为CCTCC NO:M2020054，于2020年3月14日保藏于中国典型培养物保藏中心。
进一步地，该基因工程菌株的起始菌中的N-乙酰氨基葡萄糖及N-乙酰氨基葡萄糖中间产物分解代谢和向胞内转运的途径被阻断。
优选地，该N-乙酰氨基葡萄糖及N-乙酰氨基葡萄糖中间产物分解代谢的途径，通过6-磷酸氨基葡萄糖脱氨酶nagB和gamA基因，6-磷酸-N-乙酰氨基葡萄糖脱乙酰酶nagA基因中一种或多种的失活或缺失而被阻断；向胞内转运N-乙酰氨基葡萄糖的途径，通过N-乙酰氨基葡萄糖转运蛋白gamP和nagP基因中的一种或两种的失活或缺失而被阻断。。
进一步地，该基因工程菌株中被引入过表达6-磷酸氨基葡萄糖合成酶基因和6-磷酸氨基葡萄糖乙酰化酶基因。
进一步地，该6-磷酸氨基葡萄糖乙酰化酶基因序列如SEQ ID NO.1所示，该6-磷酸氨基葡萄糖合成酶基因序列如SEQ ID NO.2所示。
优选地，该6-磷酸氨基葡萄糖合成酶基因可根据地衣芽孢杆菌全基因组DNA 序列GenBank No.NC_006270.3为模板通过PCR扩增得到。
优选地，该6-磷酸氨基葡萄糖乙酰化基因可根据酿酒酵母全基因组GenBank No.NM_001179949获得，并进行密码子优化进行全基因合成。
本发明的另一方面还提供了如上所述的基因工程菌株的构建方法。在一个具体实施例中，该方法包括，使起始菌中的N-乙酰氨基葡萄糖分解代谢途径中的6-磷酸氨基葡萄糖脱氨酶基因，6-磷酸-N-乙酰氨基葡萄糖脱乙酰酶基因，以及N-乙酰氨基葡萄糖转运蛋白基因中的一种或多种失活或缺失；引入过表达6-磷酸氨基葡萄糖合成酶基因和6-磷酸氨基葡萄糖乙酰化酶基因。
进一步地，该方法包括以下步骤：
A.敲除起始菌中的6-磷酸氨基葡萄糖脱氨酶nagB和gamA基因，6-磷酸-N-乙酰氨基葡萄糖脱乙酰酶nagA基因，以及N-乙酰氨基葡萄糖转运蛋白gamP基因和nagP基因，得到敲除菌株；
B.构建6-磷酸氨基葡萄糖合成酶glmS基因以及6-磷酸氨基葡萄糖乙酰化酶GNA1基因双表达载体；
C.将该双表达载体转入到步骤A所得到的敲除菌株中，得到生产N-乙酰氨基葡萄糖的该基因工程菌株。
进一步地，该双表达载体的构建方法为引入启动子，将表达载体与所述启动子，glmS基因，GNA1基因进行串联表达。
优选地，该表达载体为pHY300PLK。
优选地，该启动子分别为P als，P 43，P st以及P apre。
进一步地，P als启动子序列如SEQ ID NO.3所示，P 43启动子序列如SEQ ID NO.4所示，P st启动子序列如SEQ ID NO.5所示，P apre启动子序列如SEQ ID NO.6所示。
优选地，6-磷酸氨基葡萄糖合成酶glmS基因来源于地衣芽孢杆菌或其它具有相同功能嗜热酶的微生物，例如凝结芽孢杆菌、甲基营养芽孢杆菌、嗜热菊糖芽孢杆菌或嗜热脂肪地芽孢杆菌等。
优选地，6-磷酸氨基葡萄糖乙酰化酶GNA1基因来源于酿酒酵母或其它具有相同功能嗜热酶的微生物，例如马克思克鲁维酵母、红棕拿逊酵母等。
优选地，该起始菌为地衣芽孢杆菌(Bacillus licheniformis)ATCC 14580。
本发明的还有一方面还提供了如上所述的基因工程菌株的应用，尤其是在生产N-乙酰氨基葡萄糖中的应用。
进一步地，该生产的发酵温度为25℃-50℃。
优选地，该生产的发酵温度为40℃-50℃。
进一步地，该生产采用的碳源为葡萄糖、甘油、木糖或阿拉伯糖等。
进一步地，将基因工程菌株进行种子培养得到种子培养液，然后以葡萄糖为 碳源在发酵培养基中进行二次活化，最后转入发酵罐中进行发酵，得到N-乙酰氨基葡萄糖。具体包括如下步骤：
1)种子培养：将基因工程菌株液接到种子培养基中，加入四环素抗性，在50℃，200rpm条件下，培养12-16h，进行种子活化；
2)摇瓶培养：按5％接种量，将活化的种子转入装有发酵培养基的摇瓶中，加入四环素抗性，在50℃，200rpm条件下，过夜培养，进行二次活化；
3)发酵培养：按4％接种量，转入发酵罐中，加入四环素抗性，加入碳源在40℃-50℃进行分批补料发酵，培养至发酵结束。
优选地，步骤1)中的种子培养基包括以下成分：蛋白胨、酵母粉和氯化钠，优选含有：10g/L蛋白胨，5g/L酵母粉，10g/L氯化钠。
优选地，步骤2)中的发酵培养基包括以下成分：酵母粉、蛋白胨、硫酸铵、三水合磷酸氢二钾、磷酸二氢钾和葡萄糖，优选含有：12g/L酵母粉，6g/L蛋白胨，6g/L硫酸铵，18.75g/L三水合磷酸氢二钾，2.5g/L磷酸二氢钾，30g/L葡萄糖。
优选地，步骤2)中的摇瓶为500毫升锥形瓶，发酵培养基装液量为75毫升。
优选地，步骤3)中的发酵培养基包括以下成分：酵母粉、玉米浆干粉、硫酸铵、三水合磷酸氢二钾、磷酸二氢钾和葡萄糖，优选含有：12g/L酵母粉，6g/L玉米浆干粉，6g/L硫酸铵，18.75g/L三水合磷酸氢二钾，2.5g/L磷酸二氢钾，30g/L葡萄糖。
优选地，步骤3)中的发酵条件为：3mol/L盐酸溶液及25％(v/v)的氨水调节pH使其维持7.0，通气量为1.5vvm，初始搅拌转速为600rpm，葡萄糖浓度持续控制在30g/L，待糖浓度消耗至3-4g/L时进行补糖。
本申请提供的基因工程菌株，实现了N-乙酰氨基葡萄糖的高温生产。与此同时，在基因工程菌株进行发酵过程中，由于是组成型表达，所以不需要添加诱导剂。另外在发酵过程中对降温需求很低，且高温生产大大提升了发酵速率，同时因为高温可以阻止其它微生物生长，可以实现开放发酵，有效降低了生产成本。本发明实现了在40℃-50℃较高温度下发酵生产N-乙酰氨基葡萄糖，具有较高的工业化利用价值。本发明所提出的菌株构建思路可应用于多种产品的高温生产菌株的开发，所提出的菌株和发酵工艺在N-乙酰氨基葡萄糖的工业高温发酵生产方面具有良好的应用前景。
附图说明
图1为在地衣芽孢杆菌工程菌中N-乙酰氨基葡萄糖的合成和代谢途径。
具体实施方式
在本申请工程菌中的N-乙酰氨基葡萄糖的合成和代谢途径如附图1所示：谷胺酰胺(Gln)作为氨基酸供体，氨基葡萄糖合酶通过glmS基因编码转化6-磷酸果糖(F-6-P)生成6-磷酸氨基葡萄糖(GlcN-6-P)，6-磷酸氨基葡萄糖(GlcN-6-P)在6-磷酸氨基葡萄糖乙酰化酶(GNA1)作用下转化为N-乙酰-6-磷酸氨基葡萄糖(GlcNAc-6-P)，再经磷酸化酶催化脱磷酸生成N-乙酰氨基葡萄糖，然后分泌到细胞外，由于6-磷酸氨基葡萄糖脱氨酶nagB和gamA基因，6-磷酸-N-乙酰氨基葡萄糖脱乙酰酶nagA基因被敲除失活或敲除减少了N-乙酰氨基葡萄糖前体在细胞内的消耗，N-乙酰氨基葡萄糖转运蛋白gamP基因和nagP基因被敲除，所以在无法转运的情况下，工程菌株能够在细胞外积累高浓度的N-乙酰氨基葡萄糖。
本申请通过在嗜热地衣芽孢杆菌中构建一条高温发酵生产N-乙酰氨基葡萄糖的代谢通路，增强N-乙酰氨基葡萄糖合成途径中的限速酶基因表达，同时，失活或敲除导致N-乙酰氨基葡萄糖消耗和回流的基因，阻止N-乙酰氨基葡萄糖的回流消耗，使得工程菌株能积累高浓度的N-乙酰氨基葡萄糖。
本发明的一个方面提供了一种生产N-乙酰氨基葡萄糖的基因工程菌株，该基因工程菌株可以在40-50℃条件下发酵生产N-乙酰氨基葡萄糖。
在一个具体实施例中，该基因工程菌株的起始菌株中的N-乙酰氨基葡萄糖分解代谢途径被阻断。
在另一个具体实施例中，该N-乙酰氨基葡萄糖分解代谢途径，通过6-磷酸氨基葡萄糖脱氨酶nagB和gamA基因，6-磷酸-N-乙酰氨基葡萄糖脱乙酰酶nagA基因，以及N-乙酰氨基葡萄糖转运蛋白gamP和nagP基因中的一种或多种的失活或缺失而被阻断。
本文中“阻断”是指，通过各种基因工程的方法，将某一途径阻断。包括将该途径中的一种或多种催化酶的基因失活或缺失，从而使该途径不能进行。
本领域技术人员可知，起始菌株也可以不含上述被缺失或失活的酶或被阻断的途径，如6-磷酸氨基葡萄糖脱氨酶基因，6-磷酸-N-乙酰氨基葡萄糖脱乙酰酶基因，N-乙酰氨基葡萄糖转运蛋白基因等，那么在构建基因工程菌株时，这些基因或途径自然缺失，不需要额外进行基因工程操作使其失活或缺失或被阻断。
下面结合实施例对本发明的技术内容做进一步的说明。
下述实施例是说明性的，不是限定性的，不能以下述实施例来限定本发明的保护范围。下述实施例中所使用的实验方法如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1：敲除地衣芽孢杆菌nagP基因
1、根据地衣芽孢杆菌MW3基因组(GenBank No.NC_006270.3)序列设计引 物：上游引物为nagP-U-F: GGTACCCGGGAGCTCATGAATGAGGAGGATCACACAGTC(SEQ ID NO.7)和下游引物为nagP-U-R: GAAGGGGCTTATCTTAGTTAAAACCCCTTTCGATGATATT(SEQ ID NO.8)。以地衣芽孢杆菌MW3基因组DNA为模板经PCR扩增得到nagP基因上游800bp片段。
2、根据地衣芽孢杆菌MW3基因组(GenBank No.NC_006270.3)序列设计引物：上游引物为nagP-D-F:AAAGGGGTTTTAACTAAGATAAGCCCCTTCTGAGGAAG(SEQ ID NO.9)和下游引物为nagP-D-R:GCGTCGGGCGATATCGAGGCGGACGAATACTTTGAC(SEQ ID NO.10)。以地衣芽孢杆菌MW3基因组DNA为模板经PCR扩增得到nagP基因下游800bp片段。
3、将上述两种PCR产物纯化后各取1微升为模板，以无缝克隆技术将其插入到载体pKVM，得到敲除载体pKVM-△nagP。
4、将敲除载体pKVM-△nagP转化到大肠杆菌S17-1中，与地衣芽孢杆菌进行接合转移，并通过同源重组敲除原理进行nagP基因的敲除，引物选择nagP-U-F: GGTACCCGGGAGCTCATGAATGAGGAGGATCACACAGTC(SEQ ID NO.7)，以及nagP-D-R: GCGTCGGGCGATATCGAGGCGGACGAATACTTTGAC(SEQ ID NO.10)，能扩增到1600bp的片段证明已经筛选到了正确的双交换阳性菌落，基因敲除成功，得到敲除菌株MW3△nagP。
实施例2：继续敲除地衣芽孢杆菌gamP基因
1、根据地衣芽孢杆菌MW3基因组(GenBank No.NC_006270.3)序列设计引物：上游引物为gamP-U-F: GGTACCCGGGAGCTCTAGGGTAAAACCGTATGCCGC(SEQ ID NO.11)和下游引物为gamP-U-R: AAGCAACTTCAGTTTTCCGGCATTCTCCTTATGTCAA(SEQ ID NO.12)。以地衣芽孢杆菌MW3基因组DNA为模板经PCR扩增得到gamP基因上游800bp片段。
2、根据地衣芽孢杆菌MW3基因组(GenBank No.NC_006270.3)序列设计引物：上游引物为gamP-D-F: AAGGAGAATGCCGGAAAACTGAAGTTGCTTTTGAGGAATC(SEQ ID NO.13)和下游引物为gamP-D-R: GCGTCGGGCGATATCGGAAATTTCTCTGCCAGCTGC(SEQ ID NO.14)。以地衣芽孢杆菌MW3基因组DNA为模板经PCR扩增得到gamP基因下游800bp片段。
3、将上述两种PCR产物纯化后各取1微升为模板，以无缝克隆技术将其插入到载体pKVM，得到敲除载体pKVM-△gamP。
4、将敲除载体pKVM-△gamP转化到大肠杆菌S17-1中，与地衣芽孢杆菌敲除菌株MW3△nagP进行接合转移，并通过同源重组敲除原理进行gamP基因的敲除，引物选择gamP-U-F: GGTACCCGGGAGCTCTAGGGTAAAACCGTATGCCGC(SEQ ID NO.11)，以及gamP-D-R: GCGTCGGGCGATATCGGAAATTTCTCTGCCAGCTGC(SEQ ID NO.14)，能扩增到1600bp的片段证明已经筛选到了正确的双交换阳性菌落，基因敲除成功，得到敲除菌株MW3△nagP△gamP。
实施例3：继续敲除地衣芽孢杆菌gamA基因
1、根据地衣芽孢杆菌MW3基因组(GenBank No.NC_006270.3)序列设计引物：上游引物为gamA-U-F: GGTACCCGGGAGCTCGGTCAAGAGGGAGGGTTCACTT(SEQ ID NO.15)和下游引物为gamA-U-R: TGTCAGTCATTCAATGTTTTTCTCCTTTCCACAAAATAAA(SEQ ID NO.16)。以地衣芽孢杆菌MW3基因组DNA为模板经PCR扩增得到gamA基因上游800bp片段。
2、根据地衣芽孢杆菌MW3基因组(GenBank No.NC_006270.3)序列设计引物：上游引物为gamA-D-F: GGAAAGGAGAAAAACATTGAATGACTGACAAAATCGGTTA(SEQ ID NO.17)和下游引物为gamA-D-R: GCGTCGGGCGATATCTCATATCGGGGATCGGCTT(SEQ ID NO.18)。以地衣芽孢杆菌MW3基因组DNA为模板经PCR扩增得到gamA基因下游800bp片段。
3、将上述两种PCR产物纯化后各取1微升为模板，以无缝克隆技术将其插入到载体pKVM，得到敲除载体pKVM-△gamA。
4、将敲除载体pKVM-△gamA转化到大肠杆菌S17-1中，与地衣芽孢杆菌敲除菌株MW3△nagP△gamP进行接合转移，并通过同源重组敲除原理进行gamA基因的敲除，引物选择gamA-U-F: GGTACCCGGGAGCTCGGTCAAGAGGGAGGGTTCACT(SEQ ID NO.15)T，以及gamA-D-R: GCGTCGGGCGATATCTCATATCGGGGATCGGCTT(SEQ ID NO.18)，能扩增到1600bp的片段证明已经筛选到正确的双交换阳性菌落，基因敲除成功，得到敲除菌株MW3△nagP△gamP△gamA。
实施例4：继续敲除地衣芽孢杆菌基因簇nagAB
1、根据地衣芽孢杆菌MW3基因组(GenBank No.NC_006270.3)序列设计引物：上游引物为nagAB-U-F: GGTACCCGGGAGCTCCCGCACGGTCAGCTTA(SEQ ID NO.19)和下游引物为nagAB-U-R: GGGAATCTTTTTTGATACAACTCTAGTTGTCTAGACCAAT(SEQ ID NO.20)。 以地衣芽孢杆菌MW3基因组DNA为模板经PCR扩增得到nagAB基因簇上游800bp片段。
2、根据地衣芽孢杆菌MW3基因组(GenBank No.NC_006270.3)序列设计引物：上游引物为nagAB-D-F: ACAACTAGAGTTGTATCAAAAAAGATTCCCACATT(SEQ ID NO.21)和下游引物为nagAB-D-R: GCGTCGGGCGATATCCCTCTTCATATCAATGACGAA(SEQ ID NO.22)。以地衣芽孢杆菌MW3基因组DNA为模板经PCR扩增得到nagAB基因簇下游800bp片段。
3、将上述两种PCR产物纯化后各取1微升为模板，以无缝克隆技术将其插入到载体pKVM，得到敲除载体pKVM-△nagAB。
4、将敲除载体pKVM-△nagAB转化到大肠杆菌S17-1中，与地衣芽孢杆菌敲除菌株MW3△nagP△gamP△gamA进行接合转移，并通过同源重组敲除原理进行nagAB基因簇的敲除，引物选择nagAB-U-F: GGTACCCGGGAGCTCCCGCACGGTCAGCTTA(SEQ ID NO.19)，以及nagAB-D-R: GCGTCGGGCGATATCCCTCTTCATATCAATGACGAA(SEQ ID NO.22)，能扩增到1600bp的片段证明已经筛选到了正确的双交换阳性菌落，基因敲除成功，得到敲除菌株MW3△nagP△gamP△gamA△nagAB。
实施例5：GNA1和glmS双表达载体的构建
根据地衣芽孢杆菌MW3基因组(GenBank No.NC_006270.3)序列设计引物：上游引物为P als-F: TATCGATAAGCTTGATATCGAAGGTGACGCCTATTTCACT(SEQ ID NO.23)和下游引物为P als-R: GTCCGGCAGGCTCATAGCCCTCACTCCTCCATT(SEQ ID NO.24)。以地衣芽孢杆菌MW3基因组DNA为模板经PCR扩增得到P als启动子序列。
根据P 43启动子进行全基因合成，设计引物为：上游引物为P 43-F: TATCGATAAGCTTGATATCGTGTCGACGTGCATGCAG(SEQ ID NO.25)和下游引物为P 43-R: GTCCGGCAGGCTCATAGCCCTCACTCCTCCTATAAT(SEQ ID NO.26)。并经PCR扩增得到P 43启动子序列。
根据P st启动子进行全基因合成，设计引物为：上游引物为P st-F: TATCGATAAGCTTGATATCGCATGATGTGGGCGTTTTT(SEQ ID NO.27)和下游引物为P st-R: GTCCGGCAGGCTCATAGCCCTCACTCCTCCATT(SEQ ID NO.28)。并经PCR扩增得到P st启动子序列。
根据P aprE启动子进行全基因合成，设计引物为：上游引物为P aprE-F: TATCGATAAGCTTGATATCGCAGCATAATGAACATTTACTCATG(SEQ ID NO.29)和下游引物为P aprE-R: GTCCGGCAGGCTCATAGCCCTCACTCCTCCATT(SEQ ID NO.30)。并经PCR扩增得到P aprE启动子序列。
根据酿酒酵母全基因组GenBank No.NM_001179949获得6-磷酸氨基葡萄糖乙酰化酶GNA1基因，并进行密码子优化进行全基因合成，并进行PCR扩增得到优化后的GNA1序列，设计引物为：上游引物为gna1-F: GGAGGAGTGAGGGCTATGAGCCTGCCGGACG(SEQ ID NO.31)，下游引物为：gna1-R: TACAATACCACACATTTTGCGGATCTGCATTTC(SEQ ID NO.32)。
根据地衣芽孢杆菌MW3基因组(GenBank No.NC_006270.3)序列设计引物：上游引物为glmS-F: ATGCAGATCCGCAAAATGTGTGGTATTGTAGGTTATATTG(SEQ ID NO.33)和下游引物为glmS-R: CGGCCGCTCTAGAACTAGTGCTACTCCACCGTCACACTCTT(SEQ ID NO.34)。以地衣芽孢杆菌MW3基因组DNA为模板经PCR扩增得到glmS基因序列。
将PCR扩增回收得到的P als启动子序列，GNA1以及glmS三个基因片段各取1微升，与pHY300PLK载体进行无缝克隆构建，得到载体pHY300PLK-P als-GNA1-glmS。
将PCR扩增回收得到的P 43启动子序列，GNA1以及glmS三个基因片段各取1微升，与pHY300PLK载体进行无缝克隆构建，得到载体pHY300PLK-P 43-GNA1-glmS。
将PCR扩增回收得到的P st启动子序列，GNA1以及glmS三个基因片段各取1微升，与pHY300PLK载体进行无缝克隆构建，得到载体pHY300PLK-P st-GNA1-glmS。
将PCR扩增回收得到的P aprE启动子序列，GNA1以及glmS三个基因片段各取1微升与pHY300PLK载体进行无缝克隆构建，得到载体pHY300PLK-P aprE-GNA1-glmS。
实施例6：BNGS1，BNGS2，BNGS3及BNGS4工程菌的构建
将pHY300PLK-P als-GNA1-glmS载体通过电转导入到敲除菌株MW3△nagP△gamP△gamA△nagAB中，即为能合成N-乙酰氨基葡萄糖的地衣芽孢杆菌，命名为BNGS1。
将pHY300PLK-P 43-GNA1-glmS载体通过电转导入到敲除菌株MW3△nagP△gamP△gamA△nagAB中，即为能合成N-乙酰氨基葡萄糖的地衣芽孢杆菌，命名为BNGS2。
将pHY300PLK-P st-GNA1-glmS载体通过电转导入到敲除菌株MW3△nagP△gamP△gamA△nagAB中，即为能合成N-乙酰氨基葡萄糖的地衣芽孢杆菌，命名为BNGS3。
将pHY300PLK-P aprE-GNA1-glmS载体通过电转导入到敲除菌株MW3△nagP△gamP△gamA△nagAB中，即为能合成N-乙酰氨基葡萄糖的地衣芽孢杆菌，命名 为BNGS4。
实施例7：重组地衣芽孢杆菌BNGS1摇瓶发酵
1、种子和发酵培养基
种子培养基为LB培养基(成分为氯化钠10g/L，酵母粉5g/L，蛋白胨10g/L)，液体培养时添加四环素抗性25mg/L。
发酵培养基包括以下成分：12g/L酵母粉，6g/L蛋白胨，6g/L硫酸铵，18.75g/L三水合磷酸氢二钾，2.5g/L磷酸二氢钾，30g/L葡萄糖。
2、摇瓶发酵过程
将BNGS1菌株种子液接到5mL LB中，在50℃，200rpm转速下培养12-16h，培养时加四环素抗性25mg/L。按5％接种量转接至500毫升锥形瓶，装液量75毫升发酵培养基中进行摇瓶发酵，培养时添加四环素抗性25mg/L，发酵时长50h，培养温度40℃，取样1mL后在12000rpm转速下，离心5min，并用0.22微米水相滤膜过滤，利用高效液相色谱进行检测。
3、检测方法
柱子型号：Aminex HPX-87H
流动相：0.005mol/L硫酸溶液
流速：0.5mL/min
柱温：60.0℃
进样量：20.00uL
检测器：示差检测器
检测器温度：35℃
经检测，50小时摇瓶发酵后，发酵液中的N-乙酰氨基葡萄糖达2.3g/L。
实施例8：重组地衣芽孢杆菌BNGS2摇瓶发酵
将BNGS2菌株种子液接到5mL LB中，在50℃，200rpm转速下培养12-16h，培养时加四环素抗性25mg/L。按5％接种量转接至500毫升锥形瓶，装液量75毫升发酵培养基中进行摇瓶发酵，培养时添加四环素抗性25mg/L，发酵时长50h，培养温度40℃，取样1mL后在12000rpm转速下，离心5min，并用0.22微米水相滤膜过滤，利用高效液相色谱进行检测。
经检测，50小时摇瓶发酵后，发酵液中的N-乙酰氨基葡萄糖达1.5g/L。
实施例9：重组地衣芽孢杆菌BNGS3摇瓶发酵
将BNGS3菌株种子液接到5mL LB中，在50℃，200rpm转速下培养12-16h，培养时加四环素抗性25mg/L。按5％接种量转接至500毫升锥形瓶，装液量75毫升发酵培养基中进行摇瓶发酵，培养时添加四环素抗性25mg/L，发酵时长50h，培养温度40℃，取样1mL后在12000rpm转速下，离心5min，并用0.22微米水相滤膜过滤，利用高效液相色谱进行检测。
经检测，50小时摇瓶发酵后，发酵液中的N-乙酰氨基葡萄糖达0.57g/L。
实施例10：重组地衣芽孢杆菌BNGS4摇瓶发酵
将BNGS4菌株种子液接到5mL LB中，在50℃，200rpm转速下培养12-16h，培养时加四环素抗性25mg/L。按5％接种量转接至500毫升锥形瓶，装液量75毫升发酵培养基中进行摇瓶发酵，培养时添加四环素抗性25mg/L，发酵时长50h，培养温度40℃，取样1mL后在12000rpm转速下，离心5min，并用0.22微米水相滤膜过滤，利用高效液相色谱进行检测。
经检测，50小时摇瓶发酵后，发酵液中的N-乙酰氨基葡萄糖达0.32g/L。
实例11：重组地衣芽孢杆菌BNGS1在37℃条件下分批补料发酵，1L发酵罐1、种子培养：将BNGS1菌株种子液接到5mL LB中，在50℃，200rpm转速下培养12-16h。培养时加四环素抗性25mg/L。
2、摇瓶培养：按5％接种量转接到装有75毫升的发酵培养基中(摇瓶体积500mL)，摇瓶培养时添加四环素抗性25mg/L。在50℃，200rpm转速下过夜培养。
3、发酵培养：以4％(v/v)的接种量，将活化的种子液转接至1L全自动发酵罐中，培养温度37℃，添加四环素抗性工作浓度25mg/L，添加3mol·L -1的盐酸溶液及25％(v/v)的氨水调节pH使其维持在7.0，通气量为1.5vvm，初始搅拌转速600r·min -1。葡萄糖浓度要持续控制在30g/L，待糖浓度消耗至3-4g/L时进行连续恒流补糖，培养至发酵结束，取样利用高效液相色谱检测。
经检测，发酵70小时，发酵液中乙酰氨基葡萄糖含量可达到12g/L。
实例12：重组地衣芽孢杆菌BNGS1在50℃条件下分批补料发酵，1L发酵罐1、种子培养：同实施例11。
2、摇瓶培养：同实施例11。
3、发酵培养：以4％(v/v)的接种量，将活化的种子液转接至1L全自动发酵罐中，培养温度50℃，添加四环素抗性工作浓度25mg/L，添加3mol·L -1的盐酸溶液及25％(v/v)的氨水调节pH使其维持在7.0，通气量为1.5vvm，初始搅拌转速600r·min -1。葡萄糖浓度要持续控制在30g/L，待糖浓度消耗至3-4g/L时进行连续恒流补糖，培养至发酵结束，取样利用高效液相色谱检测。
经检测，发酵70小时，发酵液中乙酰氨基葡萄糖含量可达到2.8g/L。
实例13：重组地衣芽孢杆菌BNGS1在37℃条件下分批补料发酵，50L发酵罐
1、种子培养：同实施例11。
2、摇瓶培养：同实施例11。
3、发酵培养：以4％(v/v)的接种量，将活化的种子液转接至50L全自动发酵罐中，培养温度37℃，添加四环素抗性工作浓度25mg/L，添加3mol·L -1的盐酸 溶液及25％(v/v)的氨水调节pH使其维持在7.0，通气量为1.5vvm，初始搅拌转速600r·min -1。葡萄糖浓度要持续控制在30g/L，待糖浓度消耗至3-4g/L时进行连续恒流补糖，培养至发酵结束，取样利用高效液相色谱检测。
经检测，发酵69.5小时，发酵液中乙酰氨基葡萄糖含量可达到50.9g/L。
实例14：重组地衣芽孢杆菌BNGS1在37℃条件下分批补料发酵，50L发酵罐
1、种子培养：同实施例11。
2、摇瓶培养：同实施例11。
3、发酵培养：以4％(v/v)的接种量，将活化的种子液转接至50L全自动发酵罐中，培养温度37℃，添加四环素抗性工作浓度25mg/L，添加3mol·L -1的盐酸溶液及25％(v/v)的氨水调节pH使其维持在7.0，通气量为1.5vvm，初始搅拌转速600r·min -1。葡萄糖浓度要持续控制在30g/L，待糖浓度消耗至3-4g/L时进行连续恒流补糖，培养至发酵结束，取样利用高效液相色谱检测。
经检测，发酵73小时，发酵液中乙酰氨基葡萄糖含量可达到52.5g/L。
实例15：重组地衣芽孢杆菌BNGS1在40℃条件下分批补料发酵，50L发酵罐
1、种子培养：同实施例11。
2、摇瓶培养：同实施例11。
3、发酵培养：以4％(v/v)的接种量，将活化的种子液转接至50L全自动发酵罐中，培养温度40℃，添加四环素抗性工作浓度25mg/L，添加3mol·L -1的盐酸溶液及25％(v/v)的氨水调节pH使其维持7.0，通气量1.5vvm，初始搅拌转速600r·min -1。葡萄糖浓度要持续控制在30g/L，待糖浓度消耗至3-4g/L时进行连续恒流补糖，培养至发酵结束，取样利用高效液相色谱检测。
经检测，发酵54小时，发酵液中乙酰氨基葡萄糖含量可达到28.1g/L。
实例16：重组地衣芽孢杆菌BNGS1在40℃条件下分批补料发酵，50L发酵罐
1、种子培养：同实施例11。
2、摇瓶培养：同实施例11。
3、发酵培养：以4％(v/v)的接种量，将活化的种子液转接至50L全自动发酵罐中，培养温度40℃，添加四环素抗性工作浓度25mg/L，添加3mol·L -1的盐酸溶液及25％(v/v)的氨水调节pH使其维持在7.0，通气量为1.5vvm，初始搅拌转速600r·min -1。葡萄糖浓度要持续控制在30g/L，待糖浓度消耗至3-4g/L时进行连续恒流补糖，培养至发酵结束，取样利用高效液相色谱检测。
经检测，发酵56小时，发酵液中乙酰氨基葡萄糖含量可达到34.6g/L。
实例17：重组地衣芽孢杆菌BNGS1在45℃条件下分批补料发酵，50L发酵 罐
1、种子培养：同实施例11。
2、摇瓶培养：同实施例11。
3、发酵培养：以4％(v/v)的接种量，将活化的种子液转接至50L全自动发酵罐中，培养温度45℃，添加四环素抗性工作浓度25mg/L，添加3mol·L -1的盐酸溶液及25％(v/v)的氨水调节pH使其维持在7.0，通气量为1.5vvm，初始搅拌转速600r·min -1。葡萄糖浓度要持续控制在30g/L，待糖浓度消耗至3-4g/L时进行连续恒流补糖，培养至发酵结束，取样利用高效液相色谱检测。
经检测，发酵50小时，发酵液中乙酰氨基葡萄糖含量可达到24g/L。
实例18：重组地衣芽孢杆菌BNGS1在50℃条件下分批补料发酵，50L发酵罐
1、种子培养：同实施例11。
2、摇瓶培养：同实施例11。
3、发酵培养：以4％(v/v)的接种量，将活化的种子液转接至50L全自动发酵罐中，培养温度50℃，添加四环素抗性工作浓度25mg/L，添加3mol·L -1的盐酸溶液及25％(v/v)的氨水调节pH使其维持在7.0，通气量为1.5vvm，初始搅拌转速600r·min -1。葡萄糖浓度要持续控制在30g/L，待糖浓度消耗至3-4g/L时进行连续恒流补糖，培养至发酵结束，取样利用高效液相色谱检测。
经检测，发酵24小时，发酵液中乙酰氨基葡萄糖含量可达4.1g/L。
实例19：重组地衣芽孢杆菌BNGS2在37℃条件下分批补料发酵，50L发酵罐
1、种子培养：将BNGS2菌株种子液接到5mL LB中，在50℃，200rpm转速下培养12-16h。培养时加四环素抗性25mg/L。
2、摇瓶培养：按5％接种量转接到装有75毫升的发酵培养基中(摇瓶体积500mL)，摇瓶培养时添加四环素抗性25mg/L。在50℃，200rpm转速下过夜培养。
3、发酵培养：以4％(v/v)的接种量，将活化的种子液转接至50L全自动发酵罐中，培养温度37℃，添加四环素抗性工作浓度25mg/L，添加3mol·L -1的盐酸溶液及25％(v/v)的氨水调节pH使其维持在7.0，通气量为1.5vvm，初始搅拌转速600r·min -1。葡萄糖浓度要持续控制在30g/L，待糖浓度消耗至3-4g/L时进行连续恒流补糖，培养至发酵结束，取样利用高效液相色谱检测。
经检测，发酵64小时，发酵液中乙酰氨基葡萄糖含量可达23.6g/L。
实例20：重组地衣芽孢杆菌BNGS2在45℃条件下分批补料发酵，50L发酵罐
1、种子培养：同实施例19。
2、摇瓶培养：同实施例19。
3、发酵培养：以4％(v/v)的接种量，将活化的种子液转接至50L全自动发酵罐中，培养温度45℃，添加四环素抗性工作浓度25mg/L，添加3mol·L-1的盐酸溶液及25％(v/v)的氨水调节pH使其维持在7.0，通气量为1.5vvm，初始搅拌转速600r·min-1。葡萄糖浓度要持续控制在30g/L，待糖浓度消耗至3-4g/L时进行连续恒流补糖，培养至发酵结束，取样利用高效液相色谱检测。
经检测，发酵49小时，发酵液中乙酰氨基葡萄糖含量可达11.6g/L。
实例21：重组地衣芽孢杆菌BNGS3在37℃条件下分批补料发酵，50L发酵罐
1、种子培养：将BNGS3菌株种子液接到5mL LB中，在50℃，200rpm转速下培养12-16h。培养时加四环素抗性25mg/L。
2、摇瓶培养：按5％接种量转接到装有75毫升的发酵培养基中(摇瓶体积500mL)，摇瓶培养时添加四环素抗性25mg/L。在50℃，200rpm转速下过夜培养。
3、发酵培养：以4％(v/v)的接种量，将活化的种子液转接至50L全自动发酵罐中，培养温度37℃，添加四环素抗性工作浓度25mg/L，添加3mol·L -1的盐酸溶液及25％(v/v)的氨水调节pH使其维持在7.0，通气量为1.5vvm，初始搅拌转速600r·min -1。葡萄糖浓度要持续控制在30g/L，待糖浓度消耗至3-4g/L时进行连续恒流补糖，培养至发酵结束，取样利用高效液相色谱检测。
经检测，发酵64小时，发酵液中乙酰氨基葡萄糖含量可达10.8g/L。
实例22：重组地衣芽孢杆菌BNGS3在45℃条件下分批补料发酵，50L发酵罐
1、种子培养：同实施例21。
2、摇瓶培养：同实施例21。
3、发酵培养：以4％(v/v)的接种量，将活化的种子液转接至50L全自动发酵罐中，培养温度45℃，添加四环素抗性工作浓度25mg/L，添加3mol·L -1的盐酸溶液及25％(v/v)的氨水调节pH使其维持在7.0，通气量为1.5vvm，初始搅拌转速600r·min -1。葡萄糖浓度要持续控制在30g/L，待糖浓度消耗至3-4g/L时进行连续恒流补糖，培养至发酵结束，取样利用高效液相色谱检测。
经检测，发酵58小时，发酵液中乙酰氨基葡萄糖含量可达7.2g/L。
实例23：重组地衣芽孢杆菌BNGS4在37℃条件下分批补料发酵，50L发酵罐
1、种子培养：将BNGS4菌株种子液接到5mL LB中，在50℃，200rpm转速下培养12-16h。培养时加四环素抗性25mg/L。
2、摇瓶培养：按5％接种量转接到装有75毫升的发酵培养基中(摇瓶体积 500mL)，摇瓶培养时添加四环素抗性25mg/L。在50℃，200rpm转速下过夜培养。
3、发酵培养：以4％(v/v)的接种量，将活化的种子液转接至50L全自动发酵罐中，培养温度37℃，添加四环素抗性工作浓度25mg/L，添加3mol·L -1的盐酸溶液及25％(v/v)的氨水调节pH使其维持在7.0，通气量为1.5vvm，初始搅拌转速600r·min -1。葡萄糖浓度要持续控制在30g/L，待糖浓度消耗至3-4g/L时进行连续恒流补糖，培养至发酵结束，取样利用高效液相色谱检测。
经检测，发酵64小时，发酵液中乙酰氨基葡萄糖含量可达6.1g/L。
实例24：重组地衣芽孢杆菌BNGS4在45℃条件下分批补料发酵，50L发酵罐
1、种子培养：同实施例23。
2、摇瓶培养：同实施例23。
3、发酵培养：以4％(v/v)的接种量，将活化的种子液转接至50L全自动发酵罐中，培养温度45℃，添加四环素抗性工作浓度25mg/L，添加3mol·L -1的盐酸溶液及25％(v/v)的氨水调节pH使其维持在7.0，通气量为1.5vvm，初始搅拌转速600r·min -1。葡萄糖浓度要持续控制在30g/L，待糖浓度消耗至3-4g/L时进行连续恒流补糖，培养至发酵结束，取样利用高效液相色谱检测。
经检测，发酵58小时，发酵液中乙酰氨基葡萄糖含量可达2.9g/L。
实施例25：BNGS1菌种稳定性测试
将发酵结束后的BNGS1菌液在25mg/mL的LB固体平板上划线，在50℃条件下培养18h后，挑取10个单菌落分别接到装有75mL发酵培养基的500mL摇瓶中，摇床温度为40℃，转速为200rpm，葡萄糖浓度为30g/L，在接种后70h用HPLC检测N-乙酰氨基葡萄糖产量稳定性，其产量浓度在2.24g/L-2.41g/L之间。
以上详细描述了本发明的较佳具体实施例。应当理解，本领域的普通技术无需创造性劳动就可以根据本发明的构思作出诸多修改和变化。因此，凡本技术领域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的实验可以得到的技术方案，皆应在由权利要求书所确定的保护范围内。

Claims (28)

  1. 一种生产N-乙酰氨基葡萄糖的基因工程菌株，其特征在于，所述基因工程菌株在40-50℃条件下发酵生产所述N-乙酰氨基葡萄糖。
  2. 根据权利要求1所述的基因工程菌株，其特征在于，所述基因工程菌株的起始菌为嗜热菌。
  3. 根据权利要求1所述的基因工程菌株，其特征在于，所述基因工程菌株的起始菌为芽孢杆菌。
  4. 根据权利要求1所述的基因工程菌株，其特征在于，所述基因工程菌株的起始菌为地衣芽孢杆菌、凝结芽孢杆菌、甲基营养芽孢杆菌、嗜热菊糖芽孢杆菌或嗜热脂肪地芽孢杆菌。
  5. 根据权利要求1所述的基因工程菌株，其特征在于，所述基因工程菌株的起始菌为地衣芽孢杆菌(Bacillus licheniformis)ATCC 14580。
  6. 根据权利要求1所述的基因工程菌株，其特征在于，所述基因工程菌株为地衣芽孢杆菌(Bacillus licheniformis)BNGS1，保藏号为CCTCC NO:M2020054，于2020年3月14日保藏于中国典型培养物保藏中心。
  7. 根据权利要求1所述的基因工程菌株，其特征在于，所述基因工程菌株的起始菌中的N-乙酰氨基葡萄糖及N-乙酰氨基葡萄糖中间产物分解代谢途径和向胞内转运的途径被阻断。
  8. 根据权利要求7所述的基因工程菌株，其特征在于，所述N-乙酰氨基葡萄糖及所述N-乙酰氨基葡萄糖中间产物分解代谢的途径，通过6-磷酸氨基葡萄糖脱氨酶nagB和gamA基因，6-磷酸-N-乙酰氨基葡萄糖脱乙酰酶nagA基因中一种或多种的失活或缺失而被阻断；向胞内转运N-乙酰氨基葡萄糖的途径，通过N-乙酰氨基葡萄糖转运蛋白gamP和nagP基因中的一种或两种的失活或缺失而被阻断。
  9. 根据权利要求7所述的基因工程菌株，其特征在于，所述基因工程菌株中被引入过表达6-磷酸氨基葡萄糖合成酶基因和6-磷酸氨基葡萄糖乙酰化酶基因。
  10. 根据权利要求8所述的基因工程菌株，其特征在于，所述6-磷酸氨基葡萄糖乙酰化酶基因序列如SEQ ID NO.1所示。
  11. 根据权利要求9所述的基因工程菌株，其特征在于，所述6-磷酸氨基葡萄糖合成酶基因序列如SEQ ID NO.2所示。
  12. 一种如权利要求1-11任一项所述的基因工程菌株的构建方法，其特征在于，所述方法包括，使起始菌中的N-乙酰氨基葡萄糖分解代谢途径中的6-磷酸氨基葡萄糖脱氨酶基因，6-磷酸-N-乙酰氨基葡萄糖脱乙酰酶基因，以及N-乙酰氨基葡萄糖转运蛋白基因中的一种或多种失活或缺失；引入过表达6-磷酸氨基葡萄糖合成酶基因和6-磷酸氨基葡萄糖乙酰化酶基因。
  13. 一种如权利要求12所述的构建方法，其特征在于，所述包括以下步骤：
    A.敲除起始菌中的6-磷酸氨基葡萄糖脱氨酶nagB和gamA基因，6-磷酸-N-乙酰氨基葡萄糖脱乙酰酶nagA基因，以及N-乙酰氨基葡萄糖转运蛋白gamP基因和nagP基因，得到敲除菌株；
    B.构建6-磷酸氨基葡萄糖合成酶glmS基因以及6-磷酸氨基葡萄糖乙酰化酶GNA1基因双表达载体；
    C.将所述双表达载体转入到步骤A所得到的所述敲除菌株中，得到生产N-乙酰氨基葡萄糖的所述基因工程菌株。
  14. 根据权利要求13所述的构建方法，其特征在于，所述双表达载体的构建方法为引入启动子，将表达载体与所述启动子，glmS基因，GNA1基因进行串联表达。
  15. 根据权利要求14所述的构建方法，其特征在于，所述表达载体为pHY300PLK。
  16. 根据权利要求14所述的构建方法，其特征在于，所述启动子分别为P als，P 43，P st以及P apre。
  17. 根据权利要求16所述的构建方法，其特征在于，所述P als启动子序列如SEQ ID NO.3所示，所述P 43启动子序列如SEQ ID NO.4所示，所述P st启动子序列如SEQ ID NO.5所示，所述P apre启动子序列如SEQ ID NO.6所示。
  18. 根据权利要求13所述的构建方法，其特征在于，所述6-磷酸氨基葡萄糖合成 酶glmS基因来源于地衣芽孢杆菌、凝结芽孢杆菌、甲基营养芽孢杆菌、嗜热菊糖芽孢杆菌或嗜热脂肪地芽孢杆菌。
  19. 根据权利要求13所述的构建方法，其特征在于，所述6-磷酸氨基葡萄糖乙酰化酶GNA1基因来源于酿酒酵母、马克思克鲁维酵母或红棕拿逊酵母。
  20. 根据权利要求12或13所述的构建方法，其特征在于，所述起始菌为地衣芽孢杆菌(Bacillus licheniformis)ATCC 14580。
  21. 根据权利要求1-11中任一项所述的基因工程菌株的应用，其特征在于，在生产N-乙酰氨基葡萄糖中的应用。
  22. 根据权利要求21所述的应用，其特征在于，所述生产的发酵温度为40℃-50℃。
  23. 根据权利要求21所述的应用，其特征在于，所述生产采用的碳源为葡萄糖、甘油、木糖或阿拉伯糖。
  24. 根据权利要求21所述的应用，其特征在于，所述生产包括以下步骤：
    1)种子培养：将所述基因工程菌株液接到种子培养基中，加入四环素抗性，在50℃，200rpm条件下，培养12-16h，进行种子活化；
    2)摇瓶培养：按5％接种量，将步骤1)中活化的种子转入装有发酵培养基的摇瓶中，加入四环素抗性，在50℃，200rpm条件下，培养12-16h，进行二次活化；
    3)发酵培养：按4％接种量，将步骤3)中二次活化的种子转入发酵罐中，加入四环素抗性，加入碳源在40℃-50℃进行分批补料发酵，培养至发酵结束。
  25. 根据权利要求24所述的应用，其特征在于，所述步骤1)中的种子培养基包括以下成分：蛋白胨、酵母粉和氯化钠。
  26. 根据权利要求24所述的应用，其特征在于，所述步骤2)中的发酵培养基包括以下成分：酵母粉、蛋白胨、硫酸铵、三水合磷酸氢二钾、磷酸二氢钾和葡萄糖。
  27. 根据权利要求24所述的应用，其特征在于，所述步骤3)中的发酵培养基包 括以下成分：酵母粉、玉米浆干粉、硫酸铵、三水合磷酸氢二钾、磷酸二氢钾和葡萄糖。
  28. 根据权利要求24所述的应用，其特征在于，所述步骤3)中的发酵条件为：3mol/L盐酸溶液及体积比为25％的氨水调节pH使其维持7.0，通气量为1.5vvm，初始搅拌转速为600rpm，葡萄糖浓度持续控制在30g/L，待糖浓度消耗至3-4g/L时进行补糖。
PCT/CN2020/109480 2020-07-24 2020-08-17 一种生产n-乙酰氨基葡萄糖的菌株及其构建方法和应用 WO2022016641A1 (zh)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP20946455.1A EP4194544A1 (en) 2020-07-24 2020-08-17 Strain for producing n-acetylglucosamine, and construction method therefor and use thereof
JP2023505387A JP2023534879A (ja) 2020-07-24 2020-08-17 Ｎ－アセチルグルコサミン産生菌株並びにその構築方法及び使用
US18/158,343 US20230272443A1 (en) 2020-07-24 2023-01-23 N-Acetylglucosamine-Producing Bacterial Strain As Well As Method Of Construction And Use Thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010725842.9A CN113969256A (zh) 2020-07-24 2020-07-24 一种生产n-乙酰氨基葡萄糖的菌株及其构建方法和应用
CN202010725842.9 2020-07-24

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US18/158,343 Continuation-In-Part US20230272443A1 (en) 2020-07-24 2023-01-23 N-Acetylglucosamine-Producing Bacterial Strain As Well As Method Of Construction And Use Thereof

Publications (1)

Publication Number Publication Date
WO2022016641A1 true WO2022016641A1 (zh) 2022-01-27

Family

ID=79585850

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/109480 WO2022016641A1 (zh) 2020-07-24 2020-08-17 一种生产n-乙酰氨基葡萄糖的菌株及其构建方法和应用

Country Status (5)

Country Link
US (1) US20230272443A1 (zh)
EP (1) EP4194544A1 (zh)
JP (1) JP2023534879A (zh)
CN (1) CN113969256A (zh)
WO (1) WO2022016641A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115287314A (zh) * 2022-09-15 2022-11-04 深圳柏垠生物科技有限公司 可拉酸发酵放大工艺

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114836361B (zh) * 2022-04-11 2023-09-12 扬州日兴生物科技股份有限公司 一种高产氨基葡萄糖的解淀粉芽孢杆菌工程菌株及其应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104195094A (zh) * 2014-08-01 2014-12-10 张帆 一种生产n-乙酰氨基葡萄糖的枯草芽孢杆菌及其构建方法和应用
CN105176879A (zh) * 2015-10-14 2015-12-23 江南大学 一种敲除argCJBD提高重组枯草芽孢杆菌乙酰氨基葡萄糖产量的方法
CN106479945A (zh) * 2015-12-29 2017-03-08 山东润德生物科技有限公司 一种高效合成乙酰氨基葡萄糖的重组枯草芽孢杆菌
CN108546668A (zh) * 2018-05-15 2018-09-18 大自然生物集团有限公司 重组枯草芽孢杆菌及其过表达6-磷酸氨基葡萄糖合成酶促进乙酰氨基葡萄糖合成的方法
CN108570441A (zh) * 2018-05-15 2018-09-25 大自然生物集团有限公司 重组枯草芽孢杆菌及其过表达谷氨酰胺合成酶促进合成乙酰氨基葡萄糖的方法
CN108998402A (zh) * 2018-08-28 2018-12-14 江南大学 一种重组枯草芽孢杆菌及其构建方法与应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104195094A (zh) * 2014-08-01 2014-12-10 张帆 一种生产n-乙酰氨基葡萄糖的枯草芽孢杆菌及其构建方法和应用
CN105176879A (zh) * 2015-10-14 2015-12-23 江南大学 一种敲除argCJBD提高重组枯草芽孢杆菌乙酰氨基葡萄糖产量的方法
CN106479945A (zh) * 2015-12-29 2017-03-08 山东润德生物科技有限公司 一种高效合成乙酰氨基葡萄糖的重组枯草芽孢杆菌
CN108546668A (zh) * 2018-05-15 2018-09-18 大自然生物集团有限公司 重组枯草芽孢杆菌及其过表达6-磷酸氨基葡萄糖合成酶促进乙酰氨基葡萄糖合成的方法
CN108570441A (zh) * 2018-05-15 2018-09-25 大自然生物集团有限公司 重组枯草芽孢杆菌及其过表达谷氨酰胺合成酶促进合成乙酰氨基葡萄糖的方法
CN108998402A (zh) * 2018-08-28 2018-12-14 江南大学 一种重组枯草芽孢杆菌及其构建方法与应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
"GenBank", Database accession no. NM_001179949
AOUNALLAH MOHAMED AMINE; SLIMENE-DEBEZ IMEN BEN; DJEBALI KAIS; GHARBI DORRA; HAMMAMI MAJDI; AZAIEZ SANA; LIMAM FERID; TABBENE OLFA: "Enhancement of Exochitinase Production byBacillus licheniformisAT6 Strain and Improvement of N-Acetylglucosamine Production", APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY, HUMANA PRESS INC, NEW YORK, vol. 181, no. 2, 17 September 2016 (2016-09-17), New York , pages 650 - 666, XP036146074, ISSN: 0273-2289, DOI: 10.1007/s12010-016-2239-9 *
LIU YANFENG, ZHU YANQIU, LI JIANGHUA, SHIN HYUN-DONG, CHEN RACHEL R., DU GUOCHENG, LIU LONG, CHEN JIAN: "Modular pathway engineering of Bacillus subtilis for improved N-acetylglucosamine production", METABOLIC ENGINEERING, ACADEMIC PRESS, AMSTERDAM, NL, vol. 23, 1 May 2014 (2014-05-01), AMSTERDAM, NL, pages 42 - 52, XP055814951, ISSN: 1096-7176, DOI: 10.1016/j.ymben.2014.02.005 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115287314A (zh) * 2022-09-15 2022-11-04 深圳柏垠生物科技有限公司 可拉酸发酵放大工艺

Also Published As

Publication number Publication date
EP4194544A1 (en) 2023-06-14
JP2023534879A (ja) 2023-08-14
CN113969256A (zh) 2022-01-25
US20230272443A1 (en) 2023-08-31

Similar Documents

Publication Publication Date Title
CN111712570A (zh) 一种生产阿洛酮糖及其衍生物的工程菌株及其构建方法和应用
CN108102995B (zh) 一种d-阿洛酮糖3-差向异构酶生产菌株及其固定化方法
CN103468624B (zh) 一种用于高效生产海藻糖的基因工程菌
US20230272443A1 (en) N-Acetylglucosamine-Producing Bacterial Strain As Well As Method Of Construction And Use Thereof
CN108410783B (zh) 一种培养大肠杆菌发酵生产氨基葡萄糖的方法
US10633658B2 (en) Method for producing N-acetylglucosamine by co-utilizing glucose and xylose based on CRISPRi
EP4276171A1 (en) Bacillus subtilis genetically engineered bacterium for producing tagatose and method for preparing tagatose
CN104498517A (zh) 一种高产n-乙酰氨基葡萄糖大肠杆菌的构建及应用方法
CN112795569B (zh) 一种新型组成型启动子、重组地衣芽孢杆菌及其应用
CN105734092B (zh) 一种酶法制备d-塔格糖的方法
CN113073074B (zh) 一种高效合成核黄素的基因工程菌及其应用
CN102154190B (zh) 一种高效产透明质酸工程大肠杆菌及其制备方法
WO2022217827A1 (zh) 一种用于制备β-烟酰胺单核苷酸的酶组合物及其应用
CN111394410B (zh) 一种高催化活性神经氨酸合酶及应用
CN111411066B (zh) 一种双途径复合产神经氨酸枯草芽孢杆菌及构建方法
CN111662832A (zh) 一种高温好氧条件下产木糖醇的耐热酵母工程菌株的构建方法和应用
CN115838682A (zh) 一种利用甘露聚糖高效生产2′-岩藻糖基乳糖的地衣芽孢杆菌工程菌株
CN112458022B (zh) 高产几丁质脱乙酰酶的地衣芽孢杆菌Bl22及其相关产品和应用
US11618902B2 (en) Bacillus subtilis for producing N-acetylneuraminic acid and application thereof
CN112899248B (zh) 氨基葡萄糖-6磷酸合成酶突变体及其应用
CN113403332B (zh) 一种α-琼胶酶基因及其编码酶的应用
CN114317476B (zh) 葡萄糖基甘油的生物催化生产工艺及其蔗糖磷酸化酶
CN114395542B (zh) 一种蔗糖磷酸化酶及其应用
CN114164192B (zh) 一种蔗糖磷酸化酶及其在制备葡萄糖-1-磷酸中的应用
CN114231509B (zh) 一种蔗糖磷酸化酶及葡萄糖基甘油生产工艺

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20946455

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2023505387

Country of ref document: JP

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 2020946455

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2020946455

Country of ref document: EP

Effective date: 20230224