WO2017174040A1 - 微生物发酵生产n-乙酰-d-氨基葡萄糖和/或d-氨基葡萄糖盐的方法 - Google Patents

微生物发酵生产n-乙酰-d-氨基葡萄糖和/或d-氨基葡萄糖盐的方法 Download PDF

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WO2017174040A1
WO2017174040A1 PCT/CN2017/080653 CN2017080653W WO2017174040A1 WO 2017174040 A1 WO2017174040 A1 WO 2017174040A1 CN 2017080653 W CN2017080653 W CN 2017080653W WO 2017174040 A1 WO2017174040 A1 WO 2017174040A1
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promoter
microorganism
glucosamine
acetyl
phosphate
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PCT/CN2017/080653
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English (en)
French (fr)
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孙镧
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孙镧
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Priority claimed from CN201710217604.5A external-priority patent/CN107267579B/zh
Application filed by 孙镧 filed Critical 孙镧
Priority to US16/091,865 priority Critical patent/US11466300B2/en
Publication of WO2017174040A1 publication Critical patent/WO2017174040A1/zh

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    • 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
    • C12P19/02—Monosaccharides
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • 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
    • 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
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the invention belongs to the field of microbial fermentation.
  • the invention relates to a process for the production of N-acetyl-D-glucosamine by microbial fermentation and the further preparation of D-glucosamine salts.
  • N-Acetyl-D-glucosamine also known as N-acetyl-glucosamine, N-acetylglucosamine, is the basic building block of many important polysaccharides in biological cells. It has important physiological functions in the living body. N-acetyl-D-glucosamine can be used clinically to: enhance the function of the human immune system; inhibit the growth of malignant tumors or fibroblasts; effectively treat various inflammations; as a low-calorie sweetener for diabetic patients and food additives for infants and young children, etc. .
  • N-acetyl-D-glucosamine can be used to produce D-glucosamine hydrochloride, which can be used as a food supplement for anti-cancer, anti-cancer, hypolipidemic and blood pressure lowering. It is the third in the chitosan health food series. Generation of functional food additives.
  • N-acetyl-D-glucosamine is the main raw material for synthesizing the anticancer drug chloramphenicol in the pharmaceutical industry; as a biochemical reagent, it can also be used as an immunoadjuvant for antibacterial infection and an activator for human anti-influenza virus.
  • N-acetyl-D-glucosamine is thought to have a similar effect as D-glucosamine, and it is known that uptake of N-acetyl-D-glucosamine can induce the production of new cartilage and prevent the onset of osteoarthritis, or in some cases Used to treat osteoarthritis. Since D-glucosamine has a bitter taste, and N-acetyl-D-glucosamine has a sweetness of 50% sucrose and is easily ingested, N-acetyl-D-glucosamine has been used as a substitute for D-glucosamine. attract attention.
  • the source of glucosamine at home and abroad is mainly based on biological extraction.
  • the biological extraction is mainly obtained by extracting chitin or chitosan from the shrimp and crab shell, and then preparing by hydrolysis with concentrated hydrochloric acid, or by extracting with acid and alkali using citric acid residue.
  • the annual production is about 20,000 tons.
  • extracting with shrimp and crab shells a large amount of waste residue and more than 100 tons of waste water will be produced for each ton of product obtained; when extracting with citric acid slag, 30-50 tons of waste acid slag will be produced for each ton of product obtained, which is a highly polluting process. It has been banned in many places.
  • glucosamine extracted from shrimp and crab shells is inevitably contaminated by heavy metals.
  • the bio-extraction method for producing glucosamine is difficult to meet people's needs in terms of quantity and quality, and a new alternative method must be opened up. If chemical synthesis is used to prepare, there are three disadvantages: high production cost; serious environmental pollution; and safety hazards. This method is currently not used at home and abroad. In comparison, the production of glucosamine by microbial fermentation is a good way.
  • the microbial fermentation method uses glucose and inorganic salts as raw materials, selects excellent strains for liquid fermentation, and directly produces glucosamine by separation, concentration and purification. No harmful gases are produced during the production process.
  • the glucosamine produced by the fermentation method has no fishy smell and the production is not limited by resources.
  • N-acetyl-D-glucosamine Conventional methods for the production of N-acetyl-D-glucosamine by microbial fermentation include a method involving the production of N-acetyl-D-glucosamine from chitin produced from shrimp shell material by an enzyme produced by a microorganism (for example, US 5998173, "Process for producing N-acetyl-D-glucosamine”); enzymatic hydrolysis by microorganisms (Trichoderma) or partial hydrolysis of acid to purify chitin from fungal residue (such as the slag of Aspergillus niger used in citric acid fermentation) to produce N- A method of acetyl-D-glucosamine (for example, US20030073666A1, "N-acetyl-D-glucosamine and process for producing N-acetyl-D-glucosamine”); direct use of glucose as a carbon source by Trichoderma does not require fungal residue Or
  • coli introduced with a gene derived from Chlorella virus Glucosyl-based method (for example, JP2004283144A, "Method for producing glucosamine and N-acetylglucosamine”); fermentation-produced D-glucosamine or N-acetyl-D-glucosamine using genetically modified microorganisms, particularly genetically modified Escherichia coli The method (for example, US 6,372,457, "Process and materials for production of glucosamine”; WO2004/003175, “Process and materials for production of glucosamine and N-acetylglucosamine”).
  • JP2004283144A Method for producing glucosamine and N-acetylglucosamine
  • fermentation-produced D-glucosamine or N-acetyl-D-glucosamine using genetically modified microorganisms, particularly genetically modified Escherichia coli
  • the method for example, US 6,372,457,
  • N-acetyl-D-glucosamine The use of enzymes produced by microorganisms or microorganisms to degrade chitin from crustaceans such as crabs and shrimps to produce N-acetyl-D-glucosamine is relatively traditional, and has low yield, high cost, and insufficient source of animals. problem.
  • a method for producing N-acetyl-D-glucosamine by culturing Chlorella cells infected with Chlorella virus involves a step of crushing cells to obtain N-acetyl-D-glucosamine, which has problems such as complicated operation.
  • a method for fermentative production of N-acetyl-D-glucosamine by directly using glucose as a carbon source and having the advantage of not using a carbon source such as chitin or chitin oligosaccharide produced from crustacean shell or fungal residue, but Fungi such as Trichoderma has low fermentation temperature (27 ° C), long time (10 days), and low yield (15 mg/ml), which has the disadvantages of long production cycle, high cost, easy contamination of bacteria, and severely limits the method.
  • Industrial application is provided.
  • N-acetyl-D-glucosamine by genetically modified microorganisms is an important application method for large-scale industrial production in view of the growing market demand for glucosamine.
  • New genetically modified microorganisms can be obtained in many ways, such as genetic recombination, gene transfer, gene mutation, gene deletion, gene overexpression, and metabolic pathway changes.
  • the invention includes genetically modified microorganisms for use in the method of producing glucosamine of the invention, as well as recombinant nucleic acid molecules and proteins produced by the recombinant nucleic acid molecules.
  • the genetically modified microorganism of the invention is primarily directed to genetic modifications that increase glucosamine-6-phosphate synthase activity, including multiple gene mutations or amino acid deletions and substitutions.
  • this patent does not involve an increase or decrease in glucosamine-6-phosphate synthase activity by changes such as endogenous glucosamine-6-phosphate synthase gene promoter replacement or deletion.
  • the patent mainly produces D-glucosamine by genetic modification of glucosamine-6-phosphate synthase, and does not involve N-acetyl-D-glucosamine production.
  • D-glucosamine is very unstable in the fermentation broth, the degradation products may be toxic to microorganisms.
  • the production of D-glucosamine by genetic modification has a low yield and has limitations in practical application.
  • Biosynthesis methods for the production of D-glucosamine and N-acetyl-D-glucosamine are disclosed in WO2004/003175.
  • the method modifies the microorganism by fermentation to produce glucosamine and/or N-acetyl-D-glucosamine.
  • the invention also discloses genetically modified microorganisms for the production of glucosamine and N-acetyl-D-glucosamine. Further, the invention also describes a method of recovering N-acetyl-D-glucosamine produced by a fermentation process, including a method of producing high-purity N-acetyl-D-glucosamine.
  • the invention also discloses a process for producing D-glucosamine from N-acetyl-D-glucosamine.
  • the genetically modified microorganism of the invention is primarily directed to a genetic modification that increases the activity of glucosamine-6-phosphate acetyltransferase.
  • coli can acetylate glucosamine-6-phosphate to acetylglucosamine-6-phosphate, which has also been reported and confirmed in previous literature (Mio T1, Yamada-Okabe T, Arisawa M, Yamada-Okabe H: Saccharomyces cerevisiae GNA1, an essential gene encoding a novel acetyltransferase involved in UDP-N-acetylglucosamine synthesis, J Biol Chem., 1999 Jan 1;274(1):424- 9.).
  • the present invention is directed to the metabolic pathway of N-acetyl-D-glucosamine, using a novel genetic modification method to transform microorganisms, and using the microorganism to produce N-acetyl-D-glucosamine (GlcNAc) and/or with higher efficiency and higher yield. Or D-glucosamine salt.
  • the present invention enhances N-acetyl-D-aminomannose (N-acetyl-D- in microorganisms) by increasing the action of N-acetylmannosamine kinase (NanK) in microorganisms.
  • NanK N-acetylmannosamine kinase
  • Mannosamine, ManNAc phosphorylates to N-acetyl-D-mannosamine-6-phosphate (ManNAc-6-P), making the microorganism more efficient and more High yields produce N-acetyl-D-glucosamine (GlcNAc) and/or D-glucosamine salts.
  • the present invention further relates to one or more of the following contents based on the above content:
  • N-acetyl-D-aminomannose in microorganisms by increasing the action of N-acetyl-mannosamine-6-phosphate epimerase (NanE) in microorganisms Conversion of 6-phosphate (N-acetyl-D-mannosamine-6-phosphate, ManNAc-6-P) to N-acetyl-D-glucosamine-6-phosphate, GlcNAc -6-P), excreted extracellularly to N-acetyl-D-glucosamine (GlcNAc), thereby allowing the microorganism to produce N-acetyl-D-glucosamine (GlcNAc) and/or D with higher efficiency and higher yield.
  • - Glucosamine salt N-acetyl-D-aminomannose in microorganisms by increasing the action of N-acetyl-mannosamine-6-phosphate epimerase (NanE) in microorganisms Conversion of 6-phosphate (N-
  • D-Glucosamine-6-phosphate deaminase NagB
  • Glucosamine-6-phosphate synthase Glucosamine-6-phosphate synthase
  • GlmS also known as L-glutamine-6-phosphate fructose aminotransferase
  • -6-P is aminated to D-glucosamine-6-phosphate (GlcN-6-P).
  • glucosamine-6-phosphate synthase also known as L-glutamine-6-phosphate fructose aminotransferase
  • L-glutamine D-fructose-6-phosphate
  • the reaction catalyzed by D-glucosamine-6-phosphate deaminase (NagB) is reversible.
  • N-acetyl-glucoseamine-6-phosphate deacetylase N-acetyl-glucoseamine-6-phosphate deacetylase
  • Phosphoric acid N-acetyl-D-glucosamine-6-phosphate, GlcNAc-6-P
  • D-glucosamine-6-phosphate GlcN-6-P
  • the invention relates to a method for the production of N-acetyl-D-glucosamine (GlcNAc) and/or D-glucosamine salt by microbial fermentation, the method comprising:
  • GlcNAc N-acetyl-D-glucosamine
  • the genetic modification for increasing the action of N-acetyl-D-aminomannose kinase (NanK) in the microorganism is selected from a) an increase in the enzymatic activity of N-acetyl-D-aminomannose kinase (NanK) in the microorganism; / or b) N-acetyl-D-aminomannose kinase (NanK) is overexpressed in the microorganism.
  • NanK N-acetyl-D-aminomannose kinase
  • N-acetyl-D-aminomannose kinase N-acetyl-D-aminomannose kinase (NanK) in microorganisms
  • D-aminomannose kinase NanK
  • the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification that enhances the action of N-acetyl-D-aminomannosyl kinase (NanK) in the microorganism.
  • the microorganism is transformed with at least one recombinant nucleic acid molecule comprising a nucleic acid sequence encoding N-acetyl-D-aminomannose kinase (NanK).
  • the nucleic acid sequence encoding N-acetyl-D-aminomannose kinase (NanK) contains at least one genetic modification that increases the enzymatic activity of N-acetyl-D-aminomannosyl kinase (NanK).
  • the genetic modification comprises one or more of substitutions at positions corresponding to the amino acid sequence of SEQ ID NO: 17: lysine at position 36 is replaced by arginine, and isomer at position 103 The acid is replaced by methionine and the 223th arginine is substituted with serine.
  • nucleic acid sequence encoding the N-acetyl-D-aminomannose kinase (NanK) is SEQ ID NO: 26; the amino acid sequence of the N-acetyl-D-aminomannose kinase (NanK) is SEQ ID NO: 27.
  • the N-acetyl-D-aminomannose kinase has at least about 30% identical, preferably at least about 50% identical, and even more preferably at least about 70% identical to the amino acid sequence of SEQ ID NO: 17. Similarly, further preferably at least about 80% identical, still more preferably at least about 90% identical, most preferably at least about 95% identical amino acid sequence, wherein said N-acetyl-D-aminomannose kinase (NanK) has enzymatic activity .
  • N-acetyl-D-aminomannose kinase has the amino acid sequence of SEQ ID NO:17.
  • the gene copy number encoding N-acetyl-D-aminomannose kinase is increased in the recombinant nucleic acid molecule.
  • the recombinant nucleic acid molecule comprises an endogenous natural promoter, having an endogenous day Promoters have higher expression levels of promoters, enhancers, fusion sequences, and the like.
  • the recombinant nucleic acid molecule comprises a promoter having a higher expression level than the endogenous natural promoter, such as an HCE promoter, a gap promoter, a trc promoter, a T7 promoter, etc.; more preferably, the recombinant nucleic acid molecule comprises a trc promoter. child.
  • the trc promoter is a split promoter of the trp promoter and the lac promoter, which has higher transcription efficiency than trp and strong promoter properties regulated by lacI repressor.
  • the recombinant nucleic acid molecule is transformed into a microorganism selected from the group consisting of a free form (i.e., a recombinant nucleic acid molecule is loaded into a plasmid) and an integrated type (i.e., a recombinant nucleic acid molecule is integrated into the genome of the microorganism).
  • a free form i.e., a recombinant nucleic acid molecule is loaded into a plasmid
  • an integrated type i.e., a recombinant nucleic acid molecule is integrated into the genome of the microorganism.
  • the recombinant nucleic acid molecule is integrated into the genome of the microorganism.
  • the microorganism comprises at least one genetic modification of an endogenous native promoter of a gene encoding N-acetyl-D-aminomannose kinase (NanK).
  • the endogenous native promoter of the gene encoding N-acetyl-D-aminomannosyl kinase (NanK) is replaced by a promoter with a higher expression level, such as the HCE promoter, gap promoter, trc promoter, T7 Promoter, etc.; more preferably, the endogenous native promoter of the gene encoding N-acetyl-D-aminomannosyl kinase (NanK) is replaced by a trc promoter.
  • the microorganism further comprises one or more of the following genetic modifications:
  • the genetic modification for enhancing the action of N-acetyl-D-aminomannose-6-phosphate isomerase (NanE) in the microorganism is selected from the group consisting of a) N-acetyl-D-aminomannose in the microorganism Increased enzymatic activity of sugar-6-phosphate isomerase (NanE); and/or b) N-acetyl-D-aminomannose-6-phosphate in microorganisms
  • the enzyme (NanE) is overexpressed.
  • NanE N-acetyl-D-aminomannose-6-phosphate isomerase
  • Screening of NanE gene mutants can be accomplished by error-producing PCR techniques to obtain high frequency mutant genes.
  • N-acetyl-D-aminomannose-6-phosphate isomerase (NanE) in microorganisms
  • the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification that enhances the action of N-acetyl-D-aminomannose-6-phosphate isomerase (NanE) in the microorganism.
  • the microorganism is transformed with at least one recombinant nucleic acid molecule comprising a nucleic acid sequence encoding N-acetyl-D-aminomannose-6-phosphate isomerase (NanE).
  • NeE N-acetyl-D-aminomannose-6-phosphate isomerase
  • the nucleic acid sequence encoding N-acetyl-D-aminomannose-6-phosphate isomerase contains at least one increase in N-acetyl-D-aminomannose-6-phosphate isomerase (NanE) Genetic modification of the enzyme activity).
  • the genetic modification comprises one or two of substitutions at positions corresponding to the amino acid sequence of SEQ ID NO: 29: 133th cysteine is replaced by arginine and 187th tyrosine The acid is replaced by histidine.
  • nucleic acid sequence encoding the N-acetyl-D-aminomannose-6-phosphate isomerase is SEQ ID NO: 56; the N-acetyl-D-aminomannose-6-phosphate The amino acid sequence of the isomerase (NanE) is SEQ ID NO:57.
  • the N-acetyl-D-aminomannose-6-phosphate isomerase has at least about 30% identical, preferably at least about 50% identical to the amino acid sequence of SEQ ID NO:29, Further preferably at least about 70% identical, further preferably at least about 80% identical, still more preferably at least about 90% identical, most preferably at least about 95% identical amino acid sequence, wherein said N-acetyl-D-aminomannose- 6-phosphate isomerase (NanE) has enzymatic activity.
  • N-acetyl-D-aminomannose-6-phosphate isomerase has the amino acid sequence of SEQ ID NO:29.
  • the gene copy number encoding the N-acetyl-D-aminomannose-6-phosphate isomerase is increased in the recombinant nucleic acid molecule.
  • the recombinant nucleic acid molecule comprises an endogenous native promoter, a promoter having a higher expression level than the endogenous native promoter, an enhancer, a fusion sequence, and the like.
  • the recombinant nucleic acid molecule A promoter having a higher expression level than an endogenous natural promoter, such as an HCE promoter, a gap promoter, a trc promoter, a T7 promoter, and the like, is included therein; more preferably, the recombinant nucleic acid molecule comprises a trc promoter.
  • the recombinant nucleic acid molecule is transformed into a microorganism selected from the group consisting of a free form (i.e., a recombinant nucleic acid molecule is loaded into a plasmid) and an integrated type (i.e., a recombinant nucleic acid molecule is integrated into the genome of the microorganism).
  • a free form i.e., a recombinant nucleic acid molecule is loaded into a plasmid
  • an integrated type i.e., a recombinant nucleic acid molecule is integrated into the genome of the microorganism.
  • the recombinant nucleic acid molecule is integrated into the genome of the microorganism.
  • the microorganism comprises at least one genetic modification of an endogenous native promoter of a gene encoding N-acetyl-D-aminomannose-6-phosphate isomerase (NanE).
  • an endogenous native promoter of the gene encoding N-acetyl-D-aminomannose-6-phosphate isomerase (NanE) is replaced by a promoter with a higher expression level, such as the HCE promoter, the gap promoter.
  • NeE N-acetyl-D-aminomannose-6-phosphate isomerase
  • the genetic modification for enhancing the action of D-glucosamine-6-phosphate deaminase (NagB) in the microorganism is selected from the group consisting of a) D-glucosamine-6-phosphate deaminase (NagB) in the microorganism.
  • the enzyme activity is increased; and/or b) D-glucosamine-6-phosphate deaminase (NagB) is overexpressed in the microorganism.
  • D-glucosamine-6-phosphate deaminase (NagB) in microorganisms
  • the enzyme activity encoding D-glucosamine-6-phosphate deaminase (NagB) can be screened. Increased gene mutants are achieved. Screening for NagB gene mutants can be accomplished by error-producing PCR techniques to obtain high frequency mutant genes.
  • D-glucosamine-6-phosphate deaminase (NagB) in microorganisms it is also possible to overexpress D-amino by increasing the number of copies of the gene and replacing a promoter with a higher expression level than the native promoter.
  • Glucose-6-phosphate deaminase (NagB) is achieved.
  • the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification that enhances the action of D-glucosamine-6-phosphate deaminase (NagB) in the microorganism.
  • the microorganism is transformed with at least one recombinant nucleic acid molecule comprising a nucleic acid sequence encoding D-glucosamine-6-phosphate deaminase (NagB).
  • the nucleic acid sequence encoding D-glucosamine-6-phosphate deaminase contains at least one genetic modification that increases the enzymatic activity of D-glucosamine-6-phosphate deaminase (NagB).
  • the gene copy number encoding D-glucosamine-6-phosphate deaminase is increased in the recombinant nucleic acid molecule.
  • the recombinant nucleic acid molecule comprises an endogenous native promoter, a promoter having a higher expression level than the endogenous native promoter, an enhancer, a fusion sequence, and the like.
  • the recombinant nucleic acid molecule A promoter having a higher expression level than an endogenous natural promoter, such as an HCE promoter, a gap promoter, a trc promoter, a T7 promoter, and the like, is included therein; more preferably, the recombinant nucleic acid molecule comprises a trc promoter.
  • the recombinant nucleic acid molecule is transformed into a microorganism selected from the group consisting of a free form (i.e., a recombinant nucleic acid molecule is loaded into a plasmid) and an integrated type (i.e., a recombinant nucleic acid molecule is integrated into the genome of the microorganism).
  • a free form i.e., a recombinant nucleic acid molecule is loaded into a plasmid
  • an integrated type i.e., a recombinant nucleic acid molecule is integrated into the genome of the microorganism.
  • the recombinant nucleic acid molecule is integrated into the genome of the microorganism.
  • the microorganism comprises at least one genetic modification of an endogenous native promoter of a gene encoding D-glucosamine-6-phosphate deaminase (NagB).
  • the endogenous native promoter of the gene encoding D-glucosamine-6-phosphate deaminase (NagB) is replaced by a promoter with a higher expression level, such as the HCE promoter, the gap promoter, the trc promoter, The T7 promoter or the like; more preferably, the endogenous natural promoter of the gene encoding D-glucosamine-6-phosphate deaminase (NagB) is replaced by the trc promoter.
  • the genetic modification for reducing the action of glucosamine-6-phosphate synthase (GlmS) in the microorganism is selected from a) a decrease in the enzymatic activity of glucosamine-6-phosphate synthase (GlmS) in the microorganism; and/or b) Reduced expression of glucosamine-6-phosphate synthase (GlmS) in microorganisms, including but not limited to: partial or complete deletion of the endogenous gene encoding glucosamine-6-phosphate synthase (GlmS) in the microorganism, or partial or Completely inactivated, and/or partially or completely deleted, or partially or completely inactivated, of an endogenous natural promoter encoding a glucosamine-6-phosphate synthase (GlmS) gene in a microorganism.
  • the genetic modification to reduce the action of glucosamine-6-phosphate synthase (GlmS) in the microorganism is that the endogenous natural promoter encoding the glucosamine-6-phosphate synthase (GlmS) gene in the microorganism is completely deleted, ie, deleted.
  • the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification that reduces the action of glucosamine-6-phosphate synthase (GlmS) in the microorganism.
  • GlmS glucosamine-6-phosphate synthase
  • the genetic modification for increasing the action of glucosamine-6-phosphate synthase (GlmS) in the microorganism is selected from a) an increase in the enzymatic activity of glucosamine-6-phosphate synthase (GlmS) in the microorganism; And/or b) the glucosamine-6-phosphate synthase (GlmS) is overexpressed in the microorganism.
  • glucosamine-6-phosphate synthase in order to enhance the action of glucosamine-6-phosphate synthase (GlmS) in microorganisms, it is possible to screen for a gene mutant encoding an increase in enzymatic activity of glucosamine-6-phosphate synthase (GlmS). achieve. Screening of GlmS gene mutants can be accomplished by error-producing PCR techniques to obtain high frequency mutant genes. In order to enhance the action of glucosamine-6-phosphate synthase (GlmS) in microorganisms, it is also possible to overexpress glucosamine-6-phosphate by increasing its gene copy number and replacing a promoter with a higher expression level than the native promoter.
  • the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification that enhances the action of glucosamine-6-phosphate synthase (GlmS) in the microorganism.
  • GlmS glucosamine-6-phosphate synthase
  • the microorganism is transformed with at least one recombinant nucleic acid molecule comprising a nucleic acid sequence encoding glucosamine-6-phosphate synthase (GlmS).
  • GlmS glucosamine-6-phosphate synthase
  • the nucleic acid sequence encoding glucosamine-6-phosphate synthase contains at least one genetic modification that increases the enzymatic activity of glucosamine-6-phosphate synthase (GlmS).
  • the gene copy number encoding the glucosamine-6-phosphate synthase is increased in the recombinant nucleic acid molecule.
  • the recombinant nucleic acid molecule comprises an endogenous native promoter, a promoter having a higher expression level than the endogenous native promoter, an enhancer, a fusion sequence, and the like.
  • the recombinant nucleic acid molecule comprises a promoter having a higher expression level than the endogenous natural promoter, such as an HCE promoter, a gap promoter, a trc promoter, a T7 promoter, etc.; more preferably, the recombinant nucleic acid molecule comprises a trc promoter. child.
  • the recombinant nucleic acid molecule is transformed into a microorganism selected from the group consisting of a free form (i.e., a recombinant nucleic acid molecule is loaded into a plasmid) and an integrated type (i.e., a recombinant nucleic acid molecule is integrated into the genome of the microorganism).
  • a free form i.e., a recombinant nucleic acid molecule is loaded into a plasmid
  • an integrated type i.e., a recombinant nucleic acid molecule is integrated into the genome of the microorganism.
  • the recombinant nucleic acid molecule is integrated into the genome of the microorganism.
  • the microorganism comprises at least one genetic modification of an endogenous native promoter of a gene encoding glucosamine-6-phosphate synthase (GlmS).
  • the endogenous native promoter of the gene encoding glucosamine-6-phosphate synthase (GlmS) is replaced by a promoter with a higher expression level, such as the HCE promoter, the gap promoter, the trc promoter, the T7 promoter.
  • the endogenous native promoter of the gene encoding glucosamine-6-phosphate synthase (GlmS) is replaced by a trc promoter.
  • the genetic modification for reducing the action of D-glucosamine-6-phosphate deaminase (NagB) in microorganisms is selected from a) the reduction of the enzyme activity of D-glucosamine-6-phosphate deaminase (NagB) in the microorganism.
  • D-glucosamine-6-phosphate deaminase (NagB) in the microorganism, including but not limited to: encoding D-glucosamine-6-phosphate deaminase (NagB) in the microorganism Partial or complete deletion, or partial or complete inactivation of the gene, and/or partial or complete deletion of the endogenous natural promoter of the D-glucosamine-6-phosphate deaminase (NagB) gene encoding the microorganism, Partially or completely inactivated.
  • the genetic modification to reduce the action of D-glucosamine-6-phosphate deaminase (NagB) in the microorganism is to completely encode the endogenous natural promoter of the D-glucosamine-6-phosphate deaminase (NagB) gene in the microorganism. Missing, that is, deleted.
  • the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification that reduces the action of D-glucosamine-6-phosphate deaminase (NagB) in the microorganism.
  • agB D-glucosamine-6-phosphate deaminase
  • the genetic modification for enhancing the action of UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) in the microorganism is selected from a) UDP-N-acetyl-D- in the microorganism.
  • the enzymatic activity of glucosamine-2-isomerase (WecB) is increased; and/or b) UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) is overexpressed in the microorganism.
  • the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification that enhances the action of UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) in the microorganism.
  • the microorganism is transformed with at least one recombinant nucleic acid molecule comprising a nucleic acid sequence encoding UDP-N-acetyl-D-glucosamine-2-isomerase (WecB).
  • WecB UDP-N-acetyl-D-glucosamine-2-isomerase
  • the nucleic acid sequence encoding UDP-N-acetyl-D-glucosamine-2-isomerase contains at least one increase in UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) Genetic modification of the enzyme activity).
  • the genetic modification comprises one or more of substitutions at positions corresponding to the amino acid sequence of SEQ ID NO: 50: the 34th cysteine is substituted with serine and the 145th histidine is Aspartic acid substitution, 226th cysteine substituted by phenylalanine and 245 valine substituted by glycine; more preferably, encoding UDP-N-acetyl-D-glucosamine-2-isomerase ( The nucleic acid sequence of WecB) is SEQ ID NO: 58; the amino acid sequence of UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) is SEQ ID NO: 59.
  • the UDP-N-acetyl-D-glucosamine-2-isomerase has at least about 30% identical, preferably at least about 50% identical to the amino acid sequence of SEQ ID NO:50, Further preferably at least about 70% identical, further preferably at least about 80% identical, still more preferably at least about 90% identical, most preferably at least about 95% identical amino acid sequence, wherein said UDP-N-acetyl-D-glucosamine -2-isomerase (WecB) has enzymatic activity.
  • the UDP-N-acetyl-D-glucosamine-2-isomerase has the amino acid sequence of SEQ ID NO:50.
  • the gene copy number encoding the UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) in the recombinant nucleic acid molecule is increased.
  • the recombinant nucleic acid molecule comprises an endogenous native promoter, a promoter having a higher expression level than the endogenous native promoter, an enhancer, a fusion sequence, and the like.
  • the recombinant nucleic acid molecule comprises a promoter having a higher expression level than the endogenous natural promoter, such as an HCE promoter, a gap promoter, a trc promoter, a T7 promoter, etc.; more preferably, the recombinant nucleic acid molecule comprises a trc promoter. child.
  • the recombinant nucleic acid molecule is transformed into a microorganism selected from the group consisting of a free form (i.e., a recombinant nucleic acid molecule is loaded into a plasmid) and an integrated type (i.e., a recombinant nucleic acid molecule is integrated into the genome of the microorganism).
  • a free form i.e., a recombinant nucleic acid molecule is loaded into a plasmid
  • an integrated type i.e., a recombinant nucleic acid molecule is integrated into the genome of the microorganism.
  • the recombinant nucleic acid molecule is integrated into the genome of the microorganism.
  • the microorganism comprises at least one genetic modification of an endogenous native promoter of a gene encoding UDP-N-acetyl-D-glucosamine-2-isomerase (WecB).
  • the endogenous native promoter of the gene encoding UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) is replaced by a promoter with a higher expression level, such as the HCE promoter, the gap promoter.
  • WecB UDP-N-acetyl-D-glucosamine-2-isomerase
  • the microorganism further comprises one or more of the following genetic modifications:
  • (1) comprising at least one genetic modification capable of reducing the action of the mannose transporter EIIM, P/III man (ManXYZ) in the microorganism;
  • the genetic modification of reducing the action of the mannose transporter EIIM, P/III man (ManXYZ) in the microorganism includes, but is not limited to, encoding the mannose transporter EIIM, P/III man (ManXYZ) Partial or complete deletion, or partial or complete inactivation of the endogenous gene, and/or partial or complete encoding of the endogenous natural promoter of the mannose transporter EIIM, P/III man (ManXYZ) gene in the microorganism Missing, or partially or completely inactivated.
  • mannose transporter EIIM reducing microorganisms genetic P / III man (ManXYZ) acting microorganism modified to encode the mannose transporter EIIM, the complete absence of P / III man (ManXYZ) an endogenous gene, is deleted .
  • the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification that reduces the action of the mannose transporter EIIM, P/III man (ManXYZ) in the microorganism.
  • the genetic modification for reducing the action of N-acetylneuraminic acid lyase (NanA) in the microorganism includes, but is not limited to, encoding the endogenous source of N-acetylneuraminic acid lyase (NanA) in the microorganism. Partial or complete deletion, or partial or complete inactivation of a sex gene, and/or partial or complete deletion, or partial or complete, of an endogenous natural promoter of the N-acetylneuraminic acid lyase (NanA) gene in a microorganism Inactivated.
  • the genetic modification to reduce the action of N-acetylneuraminic acid lyase (NanA) in the microorganism is that the endogenous gene encoding the N-acetylneuraminic acid lyase (NanA) in the microorganism is completely deleted, ie, deleted.
  • the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification that reduces the action of N-acetylneuraminic acid lyase (NanA) in the microorganism.
  • the genetic modification for reducing the action of N-acetyl-D-glucosamine-6-phosphate deacetylase (NagA) in the microorganism includes, but is not limited to, encoding the N-acetyl-D-amino group in the microorganism. Partial or complete deletion, or partial or complete inactivation of the endogenous gene of glucose-6-phosphate deacetylase (NagA), and/or N-acetyl-D-glucosamine-6-phosphate deacetylase in the coding microorganism
  • the endogenous natural promoter of the (NagA) gene is partially or completely deleted, or partially or completely inactivated.
  • the genetic modification to reduce the action of N-acetyl-D-glucosamine-6-phosphate deacetylase (NagA) in the microorganism is to encode N-acetyl-D-glucosamine-6-phosphate deacetylase (NagA) in the microorganism.
  • the endogenous gene is completely deleted and is deleted.
  • the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification that reduces the action of N-acetyl-D-glucosamine-6-phosphate deacetylase (NagA) in the microorganism.
  • the genetic modification for reducing the action of the N-acetyl-D-glucosamine specific enzyme II Nag (NagE) in the microorganism includes, but is not limited to, encoding the N-acetyl-D-glucosamine specific enzyme in the microorganism Partial or complete deletion, or partial or complete inactivation of the endogenous gene of II Nag (NagE), and/or endogenous natural coding of the N-acetyl-D-glucosamine specific enzyme II Nag (NagE) gene in the microorganism Partial or complete deletion of the promoter, or partial or complete inactivation.
  • the reducing microbial genetic -D- N- acetyl-glucosamine-specific enzyme II Nag (NagE) acting microorganism modified to encode the complete absence of N- acetyl -D- glucosamine-specific enzyme II Nag (NagE) an endogenous gene That is deleted.
  • the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification that reduces the action of the N-acetyl-D-glucosamine specific enzyme II Nag (NagE) in the microorganism.
  • the genetic modification for increasing the action of the phosphoglucosamine mutase (GlmM) in the microorganism is selected from a) an increase in the enzymatic activity of the phosphoglucosamine mutase (GlmM) in the microorganism; and / Or b) the phosphoglucosamine mutase (GlmM) is overexpressed in the microorganism.
  • GlmM phosphoglucosamine mutase
  • GlmM phosphoglucosamine mutase
  • Screening for the GlmM gene mutant can be accomplished by error-producing PCR techniques to obtain high frequency mutant genes.
  • GlmM phosphoglucosamine mutase
  • the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification that enhances the action of phosphoglucosamine mutase (GlmM) in the microorganism.
  • GlmM phosphoglucosamine mutase
  • the microorganism is transformed with at least one recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a phosphoglucosamine mutase (GlmM).
  • GlmM phosphoglucosamine mutase
  • the nucleic acid sequence encoding a phosphoglucosamine mutase contains at least one genetic modification that increases the enzymatic activity of a phosphoglucosamine mutase (GlmM).
  • the gene copy number encoding the phosphoglucosamine mutase is increased in the recombinant nucleic acid molecule.
  • the recombinant nucleic acid molecule comprises an endogenous native promoter, a promoter having a higher expression level than the endogenous native promoter, an enhancer, a fusion sequence, and the like.
  • the recombinant nucleic acid molecule comprises a promoter having a higher expression level than the endogenous natural promoter, such as an HCE promoter, a gap promoter, a trc promoter, a T7 promoter, etc.; more preferably, the recombinant nucleic acid molecule comprises a trc promoter. child.
  • the recombinant nucleic acid molecule is transformed into a microorganism selected from the group consisting of free forms (ie, recombinant nucleic acid molecules are Loaded into the plasmid) and integrated (ie, the recombinant nucleic acid molecule is integrated into the genome of the microorganism).
  • the recombinant nucleic acid molecule is integrated into the genome of the microorganism.
  • the microorganism comprises at least one genetic modification of an endogenous native promoter of a gene encoding a phosphoglucosamine mutase (GlmM).
  • the endogenous native promoter encoding the gene for phosphoglucosamine mutase (GlmM) is replaced by a promoter with a higher expression level, such as the HCE promoter, gap promoter, trc promoter, T7 promoter, etc.
  • the endogenous native promoter of the gene encoding the phosphoglucosamine mutase (GlmM) is replaced by the trc promoter.
  • the genetic modification for enhancing the action of the bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU) in the microorganism is selected from a) the bifunctional enzyme N in the microorganism - an increase in the enzymatic activity of acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU); and/or b) bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine transfer in microorganisms
  • the enzyme (GlmU) is overexpressed.
  • N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU) in microorganisms
  • N-acetyl-D glucosamine-1-phosphate uridine acyltransferase
  • Screening for the GlmU gene mutant can be accomplished by error-producing PCR techniques to obtain high frequency mutant genes.
  • N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase GlmU
  • the promoter is expressed by overexpressing the bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU).
  • the microorganism comprises at least one genetically modified recombinant nucleic acid comprising at least one effect of enhancing the bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU) in the microorganism Molecular transformation.
  • GlmU N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase
  • the microorganism is transformed with at least one recombinant nucleic acid molecule comprising a nucleic acid sequence encoding the bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU).
  • GlmU N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase
  • the nucleic acid sequence encoding the bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU) contains at least one additional bifunctional enzyme N-acetyl-D-glucosamine-1- Genetic modification of the enzymatic activity of phosphouridine syltransferase (GlmU).
  • the gene copy number encoding the bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase is increased in the recombinant nucleic acid molecule.
  • the recombinant nucleic acid molecule comprises an endogenous native promoter, a promoter having a higher expression level than the endogenous native promoter, an enhancer, a fusion sequence, and the like.
  • the recombinant nucleic acid molecule A promoter having a higher expression level than an endogenous natural promoter, such as an HCE promoter, a gap promoter, a trc promoter, a T7 promoter, and the like, is included therein; more preferably, the recombinant nucleic acid molecule comprises a trc promoter.
  • the recombinant nucleic acid molecule is transformed into a microorganism selected from the group consisting of a free form (i.e., a recombinant nucleic acid molecule is loaded into a plasmid) and an integrated type (i.e., a recombinant nucleic acid molecule is integrated into the genome of the microorganism).
  • a free form i.e., a recombinant nucleic acid molecule is loaded into a plasmid
  • an integrated type i.e., a recombinant nucleic acid molecule is integrated into the genome of the microorganism.
  • the recombinant nucleic acid molecule is integrated into the genome of the microorganism.
  • the microorganism comprises at least one inheritance of an endogenous natural promoter of a gene encoding the bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU) Modification.
  • the endogenous native promoter of the gene encoding the bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU) is replaced by a promoter with a higher expression level, such as the HCE promoter.
  • a gap promoter a trc promoter, a T7 promoter, etc.; more preferably, an endogenous natural promoter encoding a gene of the bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU) Replaced by the trc promoter.
  • GlmU N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase
  • the invention relates to a process for the production of N-acetyl-D-glucosamine (GlcNAc) and/or D-glucosamine salt by microbial fermentation, the process comprising:
  • A) cultivating a microorganism in a fermentation medium comprising: at least one genetic modification capable of increasing the action of N-acetyl-D-aminomannose kinase (NanK) in the microorganism; and at least one capable of increasing N- in the microorganism Genetic modification of the action of acetyl-D-aminomannose-6-phosphate isomerase (NanE);
  • the present invention relates to a method for producing N-acetyl-D-glucosamine (GlcNAc) and/or D-glucosamine salt by microbial fermentation, the method comprising:
  • A) cultivating a microorganism in a fermentation medium comprising: at least one genetic modification capable of enhancing the action of N-acetyl-D-aminomannose kinase (NanK) in the microorganism; and at least one capable of increasing D- in the microorganism Genetic modification of the action of glucosamine-6-phosphate deaminase (NagB); and
  • the microorganism further comprises at least one genetic modification that reduces the action of glucosamine-6-phosphate synthase (GlmS).
  • GlmS glucosamine-6-phosphate synthase
  • the present invention relates to a method for producing N-acetyl-D-glucosamine (GlcNAc) and/or D-glucosamine salt by microbial fermentation, the method comprising:
  • A) cultivating a microorganism in a fermentation medium comprising: at least one genetic modification capable of increasing the action of N-acetyl-D-aminomannose kinase (NanK) in the microorganism; at least one capable of increasing glucosamine in the microorganism - Genetic modification of the action of 6-phosphate synthase (GlmS); and at least one genetic modification that reduces the action of D-glucosamine-6-phosphate deaminase (NagB);
  • the present invention relates to a method for producing N-acetyl-D-glucosamine (GlcNAc) and/or D-glucosamine salt by microbial fermentation, the method comprising:
  • A) cultivating a microorganism in a fermentation medium comprising: at least one genetic modification capable of enhancing the action of N-acetyl-D-aminomannose kinase (NanK) in the microorganism; at least one capable of increasing N-acetyl in the microorganism Genetic modification of the action of -D-aminomannose-6-phosphate isomerase (NanE); and at least one genetic modification that enhances the action of D-glucosamine-6-phosphate deaminase (NagB) in the microorganism;
  • the microorganism further comprises at least one genetic modification that reduces the action of glucosamine-6-phosphate synthase (GlmS).
  • GlmS glucosamine-6-phosphate synthase
  • the present invention relates to a method for producing N-acetyl-D-glucosamine (GlcNAc) and/or D-glucosamine salt by microbial fermentation, the method comprising:
  • A) cultivating a microorganism in a fermentation medium comprising: at least one genetic modification capable of enhancing the action of N-acetyl-D-aminomannose kinase (NanK) in the microorganism; at least one capable of increasing N-acetyl in the microorganism Genetic modification of the action of -D-aminomannose-6-phosphate isomerase (NanE); at least one genetic modification that enhances the action of glucosamine-6-phosphate synthase (GlmS) in the microorganism; and at least one Genetic modification of the action of D-glucosamine-6-phosphate deaminase (NagB); and
  • the present invention relates to a method for producing N-acetyl-D-glucosamine (GlcNAc) and/or D-glucosamine salt by microbial fermentation, the method comprising:
  • A) cultivating a microorganism in a fermentation medium comprising: at least one genetic modification capable of increasing the action of N-acetyl-D-aminomannos kinase (NanK) in the microorganism; and at least one capable of increasing UDP in the microorganism Genetic modification of the action of N-acetyl-D-glucosamine-2-isomerase (WecB);
  • the present invention relates to a method for producing N-acetyl-D-glucosamine (GlcNAc) and/or D-glucosamine salt by microbial fermentation, the method comprising:
  • A) cultivating a microorganism in a fermentation medium comprising: at least one genetic modification capable of enhancing the action of N-acetyl-D-aminomannose kinase (NanK) in the microorganism; at least one capable of increasing N-acetyl in the microorganism Genetic modification of the action of -D-aminomannose-6-phosphate isomerase (NanE); and at least one of which enhances the action of UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) in microorganisms Genetic modification; and
  • the present invention relates to a method for producing N-acetyl-D-glucosamine (GlcNAc) and/or D-glucosamine salt by microbial fermentation, the method comprising:
  • A) cultivating a microorganism in a fermentation medium comprising: at least one genetic modification capable of enhancing the action of N-acetyl-D-aminomannose kinase (NanK) in the microorganism; at least one capable of increasing D-amino group in the microorganism Genetic modification of the action of glucose-6-phosphate deaminase (NagB); and at least one genetic modification that enhances the action of UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) in the microorganism;
  • the microorganism further comprises at least one genetic modification that reduces the action of glucosamine-6-phosphate synthase (GlmS).
  • GlmS glucosamine-6-phosphate synthase
  • the present invention relates to a method for producing N-acetyl-D-glucosamine (GlcNAc) and/or D-glucosamine salt by microbial fermentation, the method comprising:
  • A) cultivating a microorganism in a fermentation medium comprising: at least one genetic modification capable of increasing the action of N-acetyl-D-aminomannose kinase (NanK) in the microorganism; at least one capable of increasing glucosamine in the microorganism - Genetic modification of the action of 6-phosphate synthase (GlmS); at least one genetic modification that reduces the action of D-glucosamine-6-phosphate deaminase (NagB); and at least one that enhances UDP-N-acetylation in microorganisms Genetic modification of the action of -D-glucosamine-2-isomerase (WecB);
  • the invention relates to a process for the production of N-acetyl-D-glucosamine (GlcNAc) and/or D-glucosamine salt by microbial fermentation, the process comprising:
  • A) cultivating a microorganism in a fermentation medium comprising: at least one genetic modification capable of enhancing the action of N-acetyl-D-aminomannose kinase (NanK) in the microorganism; at least one capable of increasing N-acetyl in the microorganism Genetic modification of the action of -D-aminomannose-6-phosphate isomerase (NanE); at least one genetic modification that enhances the action of D-glucosamine-6-phosphate deaminase (NagB) in microorganisms; and at least one Genetics that enhance the action of UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) in microorganisms Modification; and
  • the microorganism further comprises at least one genetic modification that reduces the action of glucosamine-6-phosphate synthase (GlmS).
  • GlmS glucosamine-6-phosphate synthase
  • the invention relates to a process for the production of N-acetyl-D-glucosamine (GlcNAc) and/or D-glucosamine salt by microbial fermentation, the process comprising:
  • A) cultivating a microorganism in a fermentation medium comprising: at least one genetic modification capable of enhancing the action of N-acetyl-D-aminomannose kinase (NanK) in the microorganism; at least one capable of increasing N-acetyl in the microorganism Genetic modification of the action of -D-aminomannose-6-phosphate isomerase (NanE); at least one genetic modification that enhances the action of glucosamine-6-phosphate synthase (GlmS) in microorganisms; at least one can reduce D - Genetic modification of the action of glucosamine-6-phosphate deaminase (NagB); and at least one genetic modification that enhances the action of UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) in microorganisms; with
  • the microorganism further comprises: at least one genetic modification capable of reducing the action of the mannose transporter EIIM, P/III man (ManXYZ) in the microorganism; at least one capable of reducing the N-acetyl nerve in the microorganism Genetic modification of the action of lyase (NanA); at least one genetic modification that reduces the action of N-acetyl-D-glucosamine-6-phosphate deacetylase (NagA) in microorganisms; and at least one that reduces microbial activity Genetic modification of the action of N-acetyl-D-glucosamine specific enzyme II Nag (NagE).
  • the expression of any of the above recombinant nucleic acid molecules is inducible, including, but not limited to, induced by lactose, for example, lactose-induced expression can be achieved by the addition of lactose or the like to the culture broth.
  • the fermentation medium contains a source of carbon.
  • the fermentation medium comprises a source of nitrogen.
  • the fermentation medium comprises a source of carbon and a source of nitrogen.
  • the fermentation medium contains a carbon source, a nitrogen source, and an inorganic salt.
  • the carbon source is selected from one or more of the group consisting of glucose, fructose, sucrose, galactose, dextrin, glycerin, starch, syrup, and molasses.
  • the concentration of the carbon source is maintained from about 0.1% to about 5%.
  • nitrogen sources known in the art can be used in the present invention, including organic nitrogen sources and/or inorganic nitrogen sources.
  • the nitrogen source is selected from the group consisting of ammonia, ammonium chloride, ammonium sulfate, ammonium nitrate, ammonium acetate, sodium nitrate, urea, yeast extract, meat extract, peptone, fish meal, soy flour, malt, corn syrup and cottonseed meal.
  • ammonia ammonium chloride, ammonium sulfate, ammonium nitrate, ammonium acetate, sodium nitrate, urea, yeast extract, meat extract, peptone, fish meal, soy flour, malt, corn syrup and cottonseed meal.
  • the present invention employs a fed fermentation process.
  • the sugar-retaining liquid comprises glucose and ribose, preferably, the glucose concentration is 10%-85% (w/v), the ribose concentration is 0.5%-15% (w/v), further preferably, the glucose concentration 55%-75% (w/v), ribose concentration is 5%-7% (w/v);
  • the sugar-containing solution comprises glucose and gluconate, preferably, the glucose concentration is 10 %-85% (w/v), gluconate concentration 0.5%-15% (w/v), further preferably, glucose concentration 55%-75% (w/v), gluconate concentration 2% - 3% (w/v);
  • the sugar-comprising solution comprises glucose, ribose and gluconate, preferably having a glucose concentration of 10%-85% (w/v) and a ribose concentration of 0.5.
  • gluconate concentration 0.5%-15% (w/v), further preferably, glucose concentration 55%-75% (w/v), ribose concentration 5%-7 %(w/v), gluconate concentration is 2%-3% (w/v).
  • the gluconate is sodium gluconate.
  • the culturing step is carried out at a temperature of from about 20 ° C to about 45 ° C, and more preferably, the culturing step is carried out at a temperature of from about 33 ° C to about 37 ° C.
  • the culturing step is carried out at a pH of from about 4.5 to about pH 8.5. Further preferably, the culturing step is carried out at a pH of from about 6.7 to about pH 7.2.
  • N-acetyl-D-glucosamine can be collected in the present invention using various conventional methods known in the art.
  • N-acetyl-D-glucosamine can be collected from the extracellular product in the fermentation medium.
  • the collecting step comprises the step of: (a) precipitating N-acetyl-D-glucosamine from the fermentation broth for removing microorganisms; (b) crystallizing N-acetyl-D- from the fermentation broth for removing microorganisms Glucosamine.
  • the collecting step further comprises the step of decolorizing the fermentation broth.
  • the decolorization step may include, but is not limited to, performing prior to precipitation or crystallization of the fermentation broth, after one or more precipitation or crystallization re-dissolution of the fermentation broth, including decolorization including activated carbon treatment and/or chromatographic decolorization.
  • the chromatographic decolorization package Including the step of contacting the fermentation broth with an ion exchange resin, the ion exchange resin includes, but is not limited to, an anion exchange resin and/or a cation exchange resin, for example, contacting the fermentation broth with a mixed bed of anion and cation exchange resin.
  • D-glucosamine salts can be obtained by deacetylating N-acetyl-D-glucosamine, including but not limited to hydrochlorides, sulfates, sodium salts, phosphates, hydrogen sulfates and the like.
  • N-acetyl-D-glucosamine can be deacetylated under acidic and heated conditions to obtain a D-glucosamine salt, preferably in a 30% to 37% hydrochloric acid solution at 60 ° C to 90 ° C for deacetylation hydrolysis.
  • N-acetyl-D-glucosamine gives D-glucosamine hydrochloride; it can also hydrolyze N-acetyl-D-glucosamine under the action of UDP-3-ON-acetylglucosamine deacetylase to obtain D-glucosamine. And further into salt.
  • the present invention relates to a microorganism comprising at least one genetic modification capable of enhancing the action of N-acetyl-D-aminomannose kinase (NanK) in a microorganism.
  • This genetic modification has been described in detail above.
  • the microorganism further comprises one or more of the following genetic modifications:
  • the microorganism further comprises one or more of the following genetic modifications:
  • (1) comprising at least one genetic modification capable of reducing the action of the mannose transporter EIIM, P/III man (ManXYZ) in the microorganism;
  • the present invention relates to a microorganism comprising: at least one genetic modification capable of enhancing the action of N-acetyl-D-aminomannos kinase (NanK) in a microorganism; and at least A genetic modification that enhances the action of N-acetyl-D-aminomannose-6-phosphate isomerase (NanE) in microorganisms.
  • NaK N-acetyl-D-aminomannos kinase
  • anE N-acetyl-D-aminomannose-6-phosphate isomerase
  • the present invention relates to a microorganism comprising: at least one genetic modification capable of enhancing the action of N-acetyl-D-aminomannose kinase (NanK) in a microorganism; At least one genetic modification that enhances the action of D-glucosamine-6-phosphate deaminase (NagB) in microorganisms.
  • NaK N-acetyl-D-aminomannose kinase
  • NagB D-glucosamine-6-phosphate deaminase
  • the microorganism further comprises at least one genetic modification that reduces the action of glucosamine-6-phosphate synthase (GlmS).
  • GlmS glucosamine-6-phosphate synthase
  • the present invention relates to a microorganism comprising: at least one genetic modification capable of enhancing the action of N-acetyl-D-aminomannos kinase (NanK) in a microorganism; A genetic modification that enhances the action of glucosamine-6-phosphate synthase (GlmS) in microorganisms; and at least one genetic modification that reduces the action of D-glucosamine-6-phosphate deaminase (NagB).
  • the invention relates to a microorganism, said microorganism Including: at least one genetic modification capable of increasing the action of N-acetyl-D-aminomannos kinase (NanK) in microorganisms; at least one capable of increasing N-acetyl-D-aminomannose-6-phosphate isomerase in microorganisms (NanE) genetic modification of the action; and at least one genetic modification that enhances the action of D-glucosamine-6-phosphate deaminase (NagB) in the microorganism.
  • NaK N-acetyl-D-aminomannos kinase
  • NaE N-acetyl-D-aminomannose-6-phosphate isomerase
  • NagB D-glucosamine-6-phosphate deaminase
  • the microorganism further comprises at least one genetic modification that reduces the action of glucosamine-6-phosphate synthase (GlmS).
  • GlmS glucosamine-6-phosphate synthase
  • the present invention relates to a microorganism comprising: at least one genetic modification capable of enhancing the action of N-acetyl-D-aminomannos kinase (NanK) in a microorganism; at least one Genetic modification of the action of N-acetyl-D-aminomannose-6-phosphate isomerase (NanE) in microorganisms; at least one inheritance that enhances the action of glucosamine-6-phosphate synthase (GlmS) in microorganisms Modification; and at least one genetic modification that reduces the action of D-glucosamine-6-phosphate deaminase (NagB).
  • NaK N-acetyl-D-aminomannos kinase
  • anE N-acetyl-D-aminomannose-6-phosphate isomerase
  • GlmS glucosamine-6-phosphate synthase
  • NagB D-glucosamine-6-phosphate de
  • the present invention relates to a microorganism comprising: at least one genetic modification capable of enhancing the action of N-acetyl-D-aminomannos kinase (NanK) in a microorganism; and at least A genetic modification that enhances the action of UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) in microorganisms.
  • NeK N-acetyl-D-aminomannos kinase
  • WecB UDP-N-acetyl-D-glucosamine-2-isomerase
  • the invention relates to a microorganism comprising: at least one genetic modification capable of enhancing the action of N-acetyl-D-aminomannos kinase (NanK) in a microorganism; at least one Genetic modification that enhances the action of N-acetyl-D-aminomannose-6-phosphate isomerase (NanE) in microorganisms; and at least one enhances UDP-N-acetyl-D-glucosamine-2- in microorganisms Genetic modification of the action of isomerase (WecB).
  • NaK N-acetyl-D-aminomannos kinase
  • NaE N-acetyl-D-aminomannose-6-phosphate isomerase
  • WecB Genetic modification of the action of isomerase
  • the present invention relates to a microorganism comprising: at least one genetic modification capable of enhancing the action of N-acetyl-D-aminomannose kinase (NanK) in a microorganism; a genetic modification capable of increasing the action of D-glucosamine-6-phosphate deaminase (NagB) in microorganisms; and at least one capable of increasing UDP-N-acetyl-D-glucosamine-2-isomerase in microorganisms ( Genetic modification of the action of WecB).
  • NaK N-acetyl-D-aminomannose kinase
  • NagB D-glucosamine-6-phosphate deaminase
  • the microorganism further comprises at least one genetic modification that reduces the action of glucosamine-6-phosphate synthase (GlmS).
  • GlmS glucosamine-6-phosphate synthase
  • the present invention relates to a microorganism comprising: at least one which enhances the action of N-acetyl-D-aminomannose kinase (NanK) in a microorganism Genetic modification; at least one genetic modification that enhances the action of glucosamine-6-phosphate synthase (GlmS) in the microorganism; at least one genetic modification that reduces the action of D-glucosamine-6-phosphate deaminase (NagB); And at least one genetic modification that enhances the action of UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) in the microorganism.
  • NaK N-acetyl-D-aminomannose kinase
  • the present invention relates to a microorganism comprising: at least one genetic modification capable of enhancing the action of N-acetyl-D-aminomannos kinase (NanK) in a microorganism; at least one Genetic modification of the action of N-acetyl-D-aminomannose-6-phosphate isomerase (NanE) in microorganisms; at least one can increase D-glucosamine-6-phosphate deaminase (NagB) in microorganisms Genetic modification of action; and at least one genetic modification that enhances the action of UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) in microorganisms.
  • NaK N-acetyl-D-aminomannos kinase
  • anE N-acetyl-D-aminomannose-6-phosphate isomerase
  • NagB D-glucosamine-6-phosphate deaminase
  • the microorganism further comprises at least one genetic modification that reduces the action of glucosamine-6-phosphate synthase (GlmS).
  • GlmS glucosamine-6-phosphate synthase
  • the invention relates to a microorganism comprising: at least one genetic modification capable of enhancing the action of N-acetyl-D-aminomannos kinase (NanK) in a microorganism; at least one Genetic modification of the action of N-acetyl-D-aminomannose-6-phosphate isomerase (NanE) in microorganisms; at least one inheritance that enhances the action of glucosamine-6-phosphate synthase (GlmS) in microorganisms Modification; at least one genetic modification that reduces the action of D-glucosamine-6-phosphate deaminase (NagB); and at least one that increases UDP-N-acetyl-D-glucosamine-2-isomerase in the microorganism Genetic modification of the role of (WecB).
  • the microorganism further comprises: at least one genetic modification capable of reducing the action of the mannose transporter EIIM, P/III man (ManXYZ) in the microorganism; at least one capable of reducing the N-acetyl nerve in the microorganism Genetic modification of the action of lyase (NanA); at least one genetic modification that reduces the action of N-acetyl-D-glucosamine-6-phosphate deacetylase (NagA) in microorganisms; and at least one that reduces microbial activity Genetic modification of the action of N-acetyl-D-glucosamine specific enzyme II Nag (NagE).
  • the present invention relates to an N-acetyl-D-aminomannose kinase (NanK) having a higher enzymatic activity, which has the amino acid sequence of SEQ ID NO:27.
  • the invention further relates to a nucleic acid molecule encoding the above N-acetyl-D-aminomannose kinase (NanK),
  • the nucleic acid molecule has the nucleic acid sequence set forth in SEQ ID NO:26.
  • the invention further relates to a vector comprising the above nucleic acid molecule.
  • the invention further relates to a microorganism comprising the above vector.
  • the invention further relates to a microorganism comprising the above nucleic acid molecule in the genome.
  • the microorganism may be any microorganism (for example, a bacterium, a protist, an alga, a fungus, or other microorganisms).
  • the microorganism includes, but is not limited to, bacteria, yeast or fungi.
  • the microorganism is selected from the group consisting of bacteria or yeast.
  • the bacterium includes, but is not limited to, a genus selected from the group consisting of Escherichia, Bacillus, Lactobacillus, Pseudomonas, or Streptomyces.
  • the bacteria include, but are not limited to, selected from the group consisting of Escherichia coli, Bacillus subtilis, Bacillus licheniformis, Lactobacillus brevis, Pseudomonas aeruginosa ( Pseudomonas aeruginosa) or a species of Streptomyces lividans.
  • the yeast includes, but is not limited to, selected from the group consisting of Saccharomyces, Schizosaccharomyces, Candida, Hansenula, Pichia, and gram.
  • yeast includes, but is not limited to, Saccharomyce scerevisiae, Schizosaccharo mycespombe, Candida. Albicans), Hansenula polymorpha, Pichia pastoris, Pichia canadensis, Kluyveromyces marxianus or Phaffia rohodozyma.
  • the microorganism is a fungus; further preferably, the fungus includes, but is not limited to, selected from the group consisting of Aspergillus, Absidia, Rhizopus, Chrysosporium, and Neurospora a fungus belonging to the genus Neurospora or Trichoderma; more preferably, the fungus includes, but is not limited to, selected from the group consisting of Aspergillus niger, Aspergillus nidulans, and Absidia coerulea. ), Rhizopus oryzae, Chrysosporium lucknowense, Neurospora crassa, Neurospora intermedia or Trichoderma reesei . Particularly preferred E.
  • coli strains include K-12, B and W, most preferably K-12.
  • E. coli is a preferred microorganism and is used as an example of various embodiments of the present invention, it is understood that N-acetyl-D-glucosamine can be used in the method of the invention and can be genetically modified to increase N-acetyl-D. - any other microorganism that produces glucosamine.
  • the microorganism used in the present invention may also be referred to as a production organism.
  • N-acetyl-D-glucosamine may be referred to as 2-acetamido-2-deoxy-D-glucose.
  • the terms N-acetyl-D-glucosamine, N-acetyl-D-glucosamine-6-phosphate and N-acetyl-D-glucosamine-1-phosphate can be abbreviated as GlcNAc, GlcNAc-6-P and GlcNAc-1, respectively.
  • N-acetyl-D-glucosamine is also abbreviated as NAG.
  • D-glucosamine, D-glucosamine-6-phosphate and D-glucosamine-1-phosphate can be abbreviated as GlcN, GlcN-6-P and GlcN, respectively.
  • -1-P the terms N-acetyl-D-aminomannose, N-acetyl-D-aminomannose-6-phosphate, glucose, glucose-6-phosphate, fructose-6-phosphate can be abbreviated as ManNAc, ManNAc-6, respectively.
  • increasing the action of an enzyme in a microorganism means that the activity of the enzyme in the microorganism is increased and/or the enzyme is overexpressed, thereby increasing the amount of substrate-producing product catalyzed by the enzyme in the microorganism.
  • reducing the action of an enzyme in a microorganism means that the activity of the enzyme in the microorganism is reduced and/or the expression of the enzyme is reduced, thereby reducing the amount of substrate-producing product catalyzed by the enzyme in the microorganism.
  • increased enzyme activity refers to an increased ability of an enzyme to catalyze a certain chemical reaction. It covers an increase in the ability of the enzyme to self-catalyze a chemical reaction in the event that the enzyme is inhibited by the product and the enzyme has a constant affinity for the substrate, and/or because the enzyme is inhibited by product inhibition and/or the enzyme affinity for the substrate
  • the ability to increase the enzyme-catalyzed chemical reactions is increased.
  • enzyme reduced by product inhibition means that the activity of the enzyme catalyzing the reaction is reduced by the specific inhibition of its end product.
  • enzyme increased affinity for a substrate refers to an increase in the affinity of the enzyme for the substrate being catalyzed.
  • Figure 1 illustrates, in the case of E. coli, the main aspects of the genetic modification of the amino sugar metabolic pathway disclosed in the present invention for the large-scale production of N-acetyl-D-glucosamine.
  • bold arrows indicate that the present invention relates to genetically engineered production and/or increased metabolic flux.
  • Figure 1 discloses several different methods for synthesizing N-acetyl-D-glucosamine, including modifications to NanK, which may further include modifications to NanE, NagB, GlmS, WecB, or combinations thereof, and may further include ManXYZ Modification of NanA, NagA, NagE, GlmM, GlmU or a combination thereof.
  • Enzymes having the same biological activity are known in the art to have different names depending on which microorganism the enzyme is derived from. The following are some of the alternative names for the enzymes covered in this article and from some organisms. The specific gene name encoding the enzyme. The names of these enzymes may be used interchangeably or, if appropriate, for a given sequence or organism, but the invention is intended to include enzymes from a given function of any organism.
  • N-acetyl-D-aminomannose kinase catalyzes the phosphorylation of N-acetyl-D-aminomannose to N-acetyl-D-aminomannose-6-P.
  • N-acetyl-D-aminomannose kinase from E. coli is generally referred to as NanK.
  • N-acetyl-D-aminomannose kinases from various organisms are well known in the art and can be used in the genetic engineering strategies of the present invention.
  • N-acetyl-D-aminomannose kinase derived from Escherichia coli having the amino acid sequence encoded by the nucleic acid sequence represented by SEQ ID NO: 16, represented by SEQ ID NO: 17.
  • N-acetyl-D-aminomannose-6-P isomerase catalyzes the conversion of N-acetyl-D-aminomannose-6-P to N-acetyl-D-glucosamine- 6-P.
  • the N-acetyl-D-aminomannose-6-P isomerase from E. coli is generally referred to as NanE.
  • N-acetyl-D-aminomannose-6-P isomerases from various organisms are well known in the art and can be used in the genetic engineering strategies of the present invention.
  • N-acetyl-D-aminomannose-6-P isomerase from Escherichia coli has the amino acid sequence encoded by the nucleic acid sequence represented by SEQ ID NO: 28 and represented by SEQ ID NO: 29.
  • UDP-N-acetyl-D-glucosamine-2-isomerase catalyzes the conversion of UDP-N-acetyl-D-glucosamine to N-acetyl-D-aminomannose.
  • UDP-N-acetyl-D-glucosamine-2-isomerase from E. coli is generally referred to as WecB.
  • UDP-N-acetyl-D-glucosamine-2-isomerases from various organisms are well known in the art and can be used in the genetic engineering strategies of the present invention.
  • UDP-N-acetyl-D-glucosamine-2-isomerase from Escherichia coli has the amino acid sequence encoded by the nucleic acid sequence represented by SEQ ID NO: 49 and represented by SEQ ID NO:50.
  • D-glucosamine-6-phosphate deaminase catalyzes the reversible reaction of D-glucosamine-6-phosphate with water to form glucose-6-phosphate and ammonium.
  • the enzyme is also known as D-glucosamine-6-phosphate isomerase, GlcN6P deaminase, D-glucosamine isomerase, D-glucosamine isomerase, D-glucosamine phosphate deaminase and 2-amino-2-deoxy-D-glucose-6-phosphate ethyl ketone alcohol isomerase (deamination).
  • D-glucosamine-6-phosphate deaminase from various organisms is well known in the art and can be used in the genetic engineering strategies of the present invention. In E. coli and other bacteria, the enzyme is generally referred to as NagB.
  • D-glucosamine-6-phosphate synthase catalyzes the formation of D-glucosamine-6-phosphate and glutamic acid from glucose-6-phosphate and glutamine.
  • the enzyme is also called D-glucosamine-fructose-6-phosphate aminotransferase (isomerization), hexose phosphate aminotransferase, D-fructose-6-phosphate transamidase, D-glucosamine-6-phosphate Isomerase (formation of glutamine), L-glutamine-fructose-6-phosphate transamidase And GlcN6P synthase.
  • D-glucosamine-6-phosphate synthase from various organisms is well known in the art and can be used in the genetic engineering strategies of the present invention.
  • D-glucosamine-6-phosphate synthase from E. coli and other bacteria is generally referred to as GlmS.
  • N-acetyl-D-glucosamine-6-phosphate deacetylase hydrolyzes N-acetyl-D-glucosamine-6-phosphate to D-glucosamine-6-phosphate and acetate .
  • N-acetyl-D-glucosamine-6-phosphate deacetylases from various organisms are well known in the art and can be used in the genetic engineering strategies of the present invention. For example, a method from E. coli called NagA is described herein.
  • N-acetylneuraminic lyase catalyzes the degradation of N-acetyl-D-aminomannose to N-acetylneuraminic acid.
  • N-acetylneuraminic lyases from various organisms are well known in the art and can be used in the genetic engineering strategies of the present invention.
  • an N-acetylneuraminic lyase from E. coli is described herein as NanoA.
  • phosphoglucosamine mutase catalyzes the conversion of D-glucosamine-6-phosphate to D-glucosamine-1-phosphate.
  • Phospho D-glucosamine mutases from various organisms are well known in the art and can be used in the genetic engineering strategies of the present invention.
  • the phosphoglucosamine mutase of this enzyme in Escherichia coli and other bacteria is generally referred to as GlmM.
  • D-glucosamine-1-phosphate N-acetyltransferase converts D-glucosamine-1-phosphate and acetyl-CoA to N-acetyl-D-glucosamine-1-phosphate, and Release the CoA.
  • N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase also known as UDP-N-acetyl-D-glucosamine pyrophosphorylase
  • UDP- N-acetyl-D-glucosamine diphosphatase further converts N-acetyl-D-glucosamine-1-phosphate to UDP-N-acetyl-D-glucosamine.
  • D-glucosamine-1-phosphate N-acetyltransferase and N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase from various organisms are well known in the art and can be used in the inheritance of the present invention In the transformation strategy.
  • This enzyme is called GlmU in E. coli and other bacteria.
  • Trc Promoter has been subtly designed for prokaryotic expression, such as the E. coli expression system. Trc promoters are well known in the art and can be used in the genetic engineering strategies of the present invention. For example, the Trc promoter described herein has the nucleotide sequence represented by SEQ ID NO:32.
  • D-glucosamine is extremely unstable in the general pH range for E. coli growth. D-glucosamine and/or its degradation products have toxic effects on the strain. Toxicity was also observed even when D-glucosamine having a concentration as low as 20 g/L was preincubated for 3.5 hours in the medium (pH 7.0) before cell seeding. Toxicity is at least partly due to a starting pH of 7.0 Caused by D-glucosamine degradation products in the medium. GlcN is more stable at lower pH conditions and D-glucosamine does not degrade below pH 4.7. However, E. coli grows slowly at pH conditions below 6-7. Therefore, the scheme of performing D-glucosamine production in a fermentor at a relatively low pH is difficult to perform.
  • UDP-N-acetyl-D-glucosamine is catalyzed by D-glucosamine-6-P (GlcN-6-P) in GlmM and GlmU in a cell, in UDP-N -N-acetyl-D-aminomannose (ManNAc) catalyzed by acetyl-glucosamine-2-isomerase (WecB), further converted to N-acetyl-D-amino by overexpressing NanK and NanE Glucose-6-phosphate (GlcNAc-6-P) is phosphorylated by phosphatase and excreted as extracellular N-acetyl-D-glucosamine (GlcNAc).
  • the method of the present invention avoids the formation of D-glucosamine, thereby avoiding the toxic effects of D-glucosamine and/or its degradation products on the strain.
  • the present invention has the beneficial effects that the present invention proves that the completely natural N-acetyl-D-glucosamine can be directly produced by the microbial fermentation method; the new production method has no risk of heavy metal pollution, no antibiotics, drug residue risk, and production is not affected. Influence of raw material supply, long-term stable production, high yield and low cost; N-acetyl-D-glucosamine and D-glucosamine products produced are non-animal, chitin without shrimp shell, glucose, etc. Carbon source fermentation, is a vegetarian product, and an allergen of anhydrous products.
  • This example describes the construction of an E. coli mutant that blocks the metabolic pathway associated with uptake of N-acetyl-D-glucosamine and degradation of beneficial intermediates.
  • the parent strain of the production strain was AT-001 (Escherichia coli ATCC 27325) belonging to the E. coli K-12 derivative from the American Type Culture Collection.
  • Blocking the N-acetyl-D-glucosamine uptake and degradation of intermediate metabolites can reduce the loss in the metabolic process and increase the accumulation of the target product (N-acetyl-D-glucosamine).
  • Construction of such a mutant host strain can cause N-acetyl-D-glucosamine accumulation by completely or partially deleting the humanXYZ, nanA, nagA and nagE gene sequences on its chromosomal genome to disable its function.
  • Red recombination is a DNA homologous recombination technique mediated by the lambda phage Red operon and the Rac phage RecE/RecT system. By this technique, it is possible to easily and rapidly perform various modifications such as insertion, knockout, and mutation of any large DNA molecule.
  • Red Recombination Technology simply states that the pKD46 plasmid carrying the recombinase gene is first transferred into the cells, and then the linear DNA fragment for targeting is prepared by electroporation, and the positive clones are screened. Finally, the resistance in the recombinant strain is determined. Gene elimination.
  • the mannose transporter EIIM, P/III man can be used as the second transporter of N-acetyl-D-glucosamine, which can be used for N-acetyl-D-glucosamine, etc.
  • the hexose is transported into the cell, thereby transporting the extracellular and accumulated target product back into intracellular degradation. Deletion of the manXYZ gene sequence prevents extracellular N-acetyl-D-glucosamine from being transported back into the cell for degradation.
  • the fKanrf fragment that is, the FRT-Kanr-FRT fragment, refers to a base sequence of the FRT site specifically recognized by the FLP recombinase at both ends of the kanamycin resistance gene (Kanr).
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • fKanrf size 1.28kb. Its nucleotide sequence is SEQ ID No. 3.
  • the PCR product was separated and purified by 1% agarose gel electrophoresis.
  • Designing a homology arm primer Designing a homologous arm forward primer (manXYZKO-F) SEQ ID No. 5, reverse primer (manXYZKO-R) SEQ ID No. 6 to delete the manXYZ sequence according to the humanXYZ sequence SEQ ID No. 4. .
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • Amplification product homology arm + fKanrf + homology arm.
  • the PCR product was separated by agarose gel electrophoresis, purified and recovered, and 100 ng/ ⁇ l of linear DNA full-length PCR fragment was obtained for Red recombinant targeting.
  • the pKD46 vector was transferred into the E. coli AT-001 strain. Then, the electrical conversion is well prepared. Screening for positive clones using linear DNA fragments. Finally, the resistance gene is eliminated.
  • the pKD46 vector is a plasmid carrying the gene for expression of the Red recombinase, and expresses the three gene segments of Exo, Bet and Gam.
  • the three genes are placed under the arabinose promoter and can be expressed in a large amount by L-arabinose induction.
  • Competency preparation First, Escherichia coli ATCC 27325 stock solution stored at -20 ° C was inoculated in 10 ml of LB liquid medium at 1:50-100, and cultured at 37 ° C, 225 rpm, shaking for 2-3 hours. The culture solution was further added to a 10 ml centrifuge tube, 4000 g ⁇ 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl 2 5 ml for 5 min. Finally, it was centrifuged at 4000 g ⁇ 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl 2 5 ml.
  • Plasmid transformation 250 ⁇ l of naturally-precipitated cells were taken, and 5 ⁇ l of pKD46 plasmid was added at -4 ° C for 30 min. Then, in a 42 ° C water bath for 1.5 min, 0.7 ml of SOC medium was added, and the mixture was shaken at 30 ° C for 2 hours. Take 0.2 ml of bacterial solution and apply penicillin plate. Incubate overnight (12-16 hours) at 30 °C. Monoclones were picked, cultured in 5 ml of LB liquid medium, and plasmids were identified. Preserve positive strains for use.
  • pCP20 is a plasmid with ampicillin and chloramphenicol resistance gene, which can express FLP recombinase after heat induction.
  • the enzyme can specifically recognize the FRT site, and the sequence between FRT sites can be deleted by recombination, leaving only one FRT site.
  • pCP20 was transferred into the above-mentioned caramycin resistant clone, cultured at 30 ° C for 8 h, and then increased to 42 ° C overnight, heat-induced FLP recombinase expression, and the plasmid was gradually lost.
  • the antimony inoculum was plated on the antibiotic-free medium, and the grown monoclonal spot was picked onto the caramycin resistant plate, and the undeveloped clone in which the caramycin resistance gene had been deleted by the FLP recombinase. Clones with the disappearance of resistance to caramycin were identified by PCR using the identified primers.
  • N-acetylneuraminate lyase is capable of degrading N-acetyl-D-aminomannose (ManNAc) in microorganisms to N-acetyl-D-neuraminic acid (Neu5Ac). Deletion of the nanA gene sequence in the nanKETA operon prevents the degradation of N-acetyl-D-aminomannose (ManNAc) to N-acetyl-D-neuraminic acid (Neu5Ac).
  • Designing the homology arm primer Designing the homologous arm primer for deletion of the nanA sequence according to the nanE sequence of nanE-nanK SEQ ID No. 7, forward primer (nanAKO-F) SEQ ID No. 8, reverse primer (nanAKO-R) ) SEQ ID No. 9.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • Amplification product homology arm + fKanrf + homology arm.
  • the PCR product was separated by agarose gel electrophoresis, purified and recovered, and 100 ng/ ⁇ l of linear DNA full-length PCR fragment was obtained for Red recombinant targeting.
  • the pKD46 vector was transferred into the E. coli AT-002-02 strain. Then, a linear DNA fragment for targeting was prepared by electroporation, and positive clones were selected. Finally, the resistance gene is eliminated.
  • Competent preparation First, Escherichia coli AT-002-02 (AT-001, ⁇ manXYZ) stock solution stored at -20 ° C, inoculated in 10 ml LB liquid medium at 1:50-100, 37 ° C, Incubate at 225 rpm for 2-3 hours with shaking. The culture solution was further added to a 10 ml centrifuge tube, 4000 g ⁇ 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl 2 5 ml for 5 min. Finally, it was centrifuged at 4000 g ⁇ 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl 2 5 ml. It was allowed to stand at -4 ° C for 12 hours and settled naturally.
  • Plasmid transformation 250 ⁇ l of naturally-precipitated cells were taken, and 5 ⁇ l of pKD46 plasmid was added at -4 ° C for 30 min. Then, in a 42 ° C water bath for 1.5 min, 0.7 ml of SOC medium was added, and the mixture was shaken at 30 ° C for 2 hours. Take 0.2 ml of bacterial solution and apply penicillin plate. Incubate overnight (12-16 hours) at 30 °C. Monoclones were picked, cultured in 5 ml of LB liquid medium, and plasmids were identified. Preserve positive strains for use.
  • pCP20 was transferred into the above-mentioned caramycin resistant clone, cultured at 30 ° C for 8 h, and then increased to 42 ° C overnight, heat-induced FLP recombinase expression, and the plasmid was gradually lost.
  • the antimony inoculum was plated on the antibiotic-free medium, and the grown monoclonal spot was picked onto the caramycin resistant plate, and the undeveloped clone in which the caramycin resistance gene had been deleted by the FLP recombinase. Clones with the disappearance of resistance to caramycin were identified by PCR using the identified primers.
  • N-acetyl-D-glucosamine-6-phosphate deacetylase can treat N-acetyl-D-glucosamine-6-phosphate (GlcNAc-6-P) in microorganisms Conversion to D-glucosamine-6-phosphate (GlcN-6-P).
  • Deletion of the nagA gene sequence in the nag operon prevents the conversion of N-acetyl-D-glucosamine-6-phosphate (GlcNAc-6-P) to D-glucosamine-6-phosphate (GlcN- 6-P).
  • Designing the homology arm primer designing the homologous arm primer for deletion of the nagA sequence according to NCBI search NC_000913, Escherichia coli str. K-12N-acetyl-D-glucosamine-6-phosphate deacetylase gene nagA sequence SEQ ID No.10 : forward primer (nagAKO-F) SEQ ID No. 11, reverse primer (nagAKO-R) SEQ ID No. 12.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • Amplification product homology arm + fKanf + homology arm.
  • the PCR product was separated by agarose gel electrophoresis, purified and recovered, and 100 ng/ ⁇ l of linear DNA full-length PCR fragment was obtained for Red recombinant targeting.
  • the pKD46 vector was transferred into the E. coli AT-003-02 strain. Then, a linear DNA fragment for targeting was prepared by electroporation, and positive clones were selected. Finally, the resistance gene is eliminated.
  • Competent preparation First, the Escherichia coli AT-003-02 (AT-002-02, ⁇ nanA) stock solution stored at -20 °C was inoculated in 10 ml LB liquid medium at 1:50-100, 37 Incubate for 2-3 hours at 225 rpm with °C. The culture solution was further added to a 10 ml centrifuge tube, 4000 g ⁇ 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl 2 5 ml for 5 min. Finally, it was centrifuged at 4000 g ⁇ 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl 2 5 ml. It was allowed to stand at -4 ° C for 12 hours and settled naturally.
  • Plasmid transformation 250 ⁇ l of naturally-precipitated cells were taken, and 5 ⁇ l of pKD46 plasmid was added at -4 ° C for 30 min. Then, in a 42 ° C water bath for 1.5 min, 0.7 ml of SOC medium was added, and the mixture was shaken at 30 ° C for 2 hours. Take 0.2 ml of bacterial solution and apply penicillin plate. Incubate overnight (12-16 hours) at 30 °C. Monoclones were picked, cultured in 5 ml of LB liquid medium, and plasmids were identified. Preserve positive strains for use.
  • pCP20 was transferred into the above-mentioned caramycin resistant clone, cultured at 30 ° C for 8 h, and then increased to 42 ° C overnight, heat-induced FLP recombinase expression, and the plasmid was gradually lost.
  • the antimony inoculum was plated on the antibiotic-free medium, and the grown monoclonal spot was picked onto the caramycin resistant plate, and the undeveloped clone in which the caramycin resistance gene had been deleted by the FLP recombinase. Clones with the disappearance of resistance to caramycin were identified by PCR using the identified primers.
  • N- acetyl -D- glucosamine-specific enzyme II Nag N-acetyl-glucosamine -specific enzyme II Nag, NagE
  • nagE gene sequence deleted, can prevent the degradation of extracellular GlcNAc is transported back to the cell.
  • Designing the homology arm primer Designing the homologous arm forward primer (nagEKO-F1) SEQ ID No to delete the nagE gene sequence according to NCBI search NC_000913, Escherichia coli str. K-12nagB promoter and nagE gene sequence SEQ ID No. 13. .14, reverse primer (nagEKO-R1) SEQ ID No. 15.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • Amplification product homology arm + fKanrf + homology arm.
  • the PCR product was separated by agarose gel electrophoresis, purified and recovered, and 100 ng/ ⁇ l of linear DNA full-length PCR fragment was obtained for Red recombinant targeting.
  • the pKD46 vector was transferred into the E. coli AT-004-02 strain. Then, a linear DNA fragment for targeting was prepared by electroporation, and positive clones were selected. Finally, the resistance gene is eliminated.
  • Competent preparation First, the Escherichia coli AT-004-02 (AT-003-02, ⁇ nagA) stock solution stored at -20 ° C was inoculated in 10 ml LB liquid medium at 1:50-100, 37 Incubate for 2-3 hours at 225 rpm with °C. The culture solution was further added to a 10 ml centrifuge tube, 4000 g ⁇ 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl 2 5 ml for 5 min. Finally, it was centrifuged at 4000 g ⁇ 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl 2 5 ml. It was allowed to stand at -4 ° C for 12 hours and settled naturally.
  • Plasmid transformation 250 ⁇ l of naturally-precipitated cells were taken, and 5 ⁇ l of pKD46 plasmid was added at -4 ° C for 30 min. Then, in a 42 ° C water bath for 1.5 min, 0.7 ml of SOC medium was added, and the mixture was shaken at 30 ° C for 2 hours. Take 0.2 ml of bacterial solution and apply penicillin plate. Incubate overnight (12-16 hours) at 30 °C. Monoclones were picked, cultured in 5 ml of LB liquid medium, and plasmids were identified. Preserve positive strains for use.
  • pCP20 was transferred into the above-mentioned caramycin resistant clone, cultured at 30 ° C for 8 h, and then increased to 42 ° C overnight, heat-induced FLP recombinase expression, and the plasmid was gradually lost.
  • the antimony inoculum was plated on the antibiotic-free medium, and the grown monoclonal spot was picked onto the caramycin resistant plate, and the undeveloped clone in which the caramycin resistance gene had been deleted by the FLP recombinase. Clones with the disappearance of resistance to caramycin were identified by PCR using the identified primers.
  • This example describes the cloning of the gene nanK of N-acetyl-D-aminomannose kinase (NanK) and the transformation of the nanK/pTrc99A plasmid in E. coli, and the integration of the ptrc-nanK gene cassette into the E. coli chromosome.
  • NaK N-acetyl-D-aminomannose kinase
  • Escherichia coli NanK N-acetylmannosamine kinase, N-acetyl-D-aminoglycan
  • the nanK gene which is under the control of the Trc promoter, is overexpressed and can enhance ManNAc (N-Acetyl-D-mannosamine, N-acetyl-D-aminomannose or N-acetyl- D-mannosamine is phosphorylated to ManNAc-6-P (N-Acetyl-D-mannosamine-6-P, N-acetyl-D-aminomannose-6-phosphate).
  • the Uscherichia coli nanK gene nucleotide sequence SEQ ID No. 16 having the amino acid sequence of SEQ ID No. 17 was obtained by looking up U00096 from NCBI.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • Amplification product size 0.9 kb.
  • the PCR product was separated and purified by 1% agarose gel electrophoresis.
  • the obtained PCR amplified fragment and the pUC57-T vector were ligated and sequenced to obtain nanK/pUC57.
  • Plasmid construction The plasmid nanK/pUC57 was amplified, and the plasmid nanK/pUC57 and the vector pTrc99A were digested with Nco I and HindIII respectively. The nanK fragment and the pTrc99A fragment were separated and purified by agarose gel electrophoresis, and the T4 DNA ligase was used at 16 ° C. The mixture was ligated overnight and identified to give the nanK/pTrc99A plasmid.
  • Competency preparation First, the AT-005-02 bacterial solution stored at -20 ° C was inoculated into 10 ml of LB liquid medium at 1:50-100, and cultured at 37 ° C, 225 rpm, and shaken for 2-3 hours. The culture solution was further added to a 10 ml centrifuge tube, 4000 g ⁇ 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl 2 5 ml for 5 min. Finally, it was centrifuged at 4000 g ⁇ 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl 2 5 ml. It was allowed to stand at -4 ° C for 12 hours and settled naturally.
  • Plasmid transformation 250 ⁇ l of naturally-precipitated cells were taken, and 5 ⁇ l of nanK/pTrc99A plasmid was added at -4 ° C for 30 min. Then, in a 42 ° C water bath for 1.5 min, 0.7 ml of SOC medium was added, and the mixture was shaken at 30 ° C for 2 hours. Take 0.2 ml of bacterial solution and apply penicillin plate. Incubate overnight (12-16 hours) at 30 °C. Monoclones were picked, cultured in 5 ml of LB liquid medium, and plasmids were identified. Preserve positive strains for use. Recombinant bacteria nanK/pTrc99A(AT-005-02)
  • the recombinant strain nanK/pTrc99A (AT-005-02) and the control strain were subjected to a shake flask fermentation test.
  • the monoclonal strain on the freshly cultured LB plate medium was inoculated into a 3 ml LB liquid medium test tube (13 x 150 mm), and cultured at 30 ° C, 225 rpm for about 8 hours.
  • LB liquid medium composition 5 g / l yeast powder, 10 g / l peptone, 10 g / l NaCl. Then, the seed culture solution was taken, and 3% was inoculated into a 250 ml shake flask containing 50 ml of the fermentation broth (M9 medium).
  • the initial OD 600 was about 0.5, cultured at 225 rpm at 37 ° C, and the fermentation cycle was 72 hours.
  • the pH of the fermentation broth was adjusted to 7.0 with 10 M NaOH.
  • 65% glucose solution was added in portions to maintain the glucose concentration at 20 g/L.
  • 1 ml of the fermentation broth was taken and centrifuged.
  • the N-acetyl-D-glucosamine content was determined by HPLC.
  • Buffer Add 3.5g of dipotassium hydrogen phosphate to a 1L volumetric flask, add enough water to dissolve, add 0.25mL of ammonia water, add water to dilute and mix, adjust the pH to 7.5 with phosphoric acid, and dilute to volume with water.
  • Standard solution 1.0 mg/mL USP N-acetyl-D-glucosamine standard (RS) was dissolved in the diluent.
  • Sample solution 1.0 mg/mL N-acetyl-D-glucosamine sample was dissolved in the diluent.
  • Second prepare 5 ⁇ M9 medium add about 64g of Na 2 HPO 4 ⁇ 7H 2 O, 15g of KH 2 PO 4 , 2.5g of NaCl, 5.0g of NH 4 Cl in about 800ml of double distilled water (ddH 2 O), after dissolving Add water to 1000ml. Sterilize at 121 ° C for 30 minutes. 1 M MgSO 4 , 1 M CaCl 2 , 20% glucose were separately prepared and sterilized separately. Then, M9 culture solution was prepared according to Table 1, wherein 1000 ⁇ trace element solution was prepared according to Table 2.
  • the yield of shake flask fermentation is shown in Table 3. The results showed that the yield of the control strain AT-005-02 was very low and not detected, and the yield of the recombinant nanK/pTrc99A (AT-005-02) overexpressed by the nanK gene under the control of the Trc promoter was significantly increased.
  • the nagE gene locus is the integration site of the pTrc-nanK gene cassette on the chromosome.
  • the nanK fragment pTrc-nanK with the Trc promoter and the claranomycin with the FLP recombinase recognition site (FRT site) on both sides were amplified. Resistance gene fragment: FRT-Kanr-FRT (fKanrf), and spliced.
  • the primer for deleting the homologous arm of the nagE gene sequence was designed again, and the full-length linear DNA fragment of Red recombinant targeting was amplified by using the fragment fused by pTrc-nanK and fKanrf as a template.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • the PCR product was separated and purified by 1% agarose gel electrophoresis.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • fKanrf size 1.28kb. Its nucleotide sequence is SEQ ID No. 3.
  • the PCR product was separated and purified by 1% agarose gel electrophoresis.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • the size of the second amplified fKanrf was 1.3 kb.
  • the PCR product was separated and purified by 1% agarose gel electrophoresis.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • Amplification product homology arm + pTrc-nanK-fKanrf + homology arm.
  • the PCR product was separated by agarose gel electrophoresis, purified and recovered, and 100 ng/ ⁇ l of linear DNA full-length PCR fragment was obtained for Red recombinant targeting.
  • the pKD46 vector was transferred into the E. coli AT-004-02 strain. Then, a linear DNA fragment for targeting was prepared by electroporation, and positive clones were selected. Finally, the resistance gene is eliminated.
  • Competent preparation First, the Escherichia coli AT-004-02 stock solution stored at -20 ° C was inoculated in 10 ml LB liquid medium at 1:50-100, and cultured at 37 ° C, 225 rpm, shaking for 2-3 hours. . The culture solution was further added to a 10 ml centrifuge tube, 4000 g ⁇ 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl 2 5 ml for 5 min. Finally, it was centrifuged at 4000 g ⁇ 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl 2 5 ml. It was allowed to stand at -4 ° C for 12 hours and settled naturally.
  • Plasmid transformation 250 ⁇ l of naturally-precipitated cells were taken, and 5 ⁇ l of pKD46 plasmid was added at -4 ° C for 30 min. Then, in a 42 ° C water bath for 1.5 min, 0.7 ml of SOC medium was added, and the mixture was shaken at 30 ° C for 2 hours. Take 0.2 ml of bacterial solution and apply penicillin plate. Incubate overnight (12-16 hours) at 30 °C. Monoclones were picked, cultured in 5 ml of LB liquid medium, and plasmids were identified. Preserve positive strains for use.
  • pCP20 was transferred into the above-mentioned caramycin resistant clone, cultured at 30 ° C for 8 h, and then increased to 42 ° C overnight, heat-induced FLP recombinase expression, and the plasmid was gradually lost.
  • the antimony inoculum was plated on the antibiotic-free medium, and the grown monoclonal spot was picked onto the caramycin resistant plate, and the undeveloped clone in which the caramycin resistance gene had been deleted by the FLP recombinase. Clones with the disappearance of resistance to caramycin were identified by PCR using the identified primers.
  • the recombinant strain AT-006-02 and the control strain in which the pTrc-nanK gene cassette was integrated at the chromosome nagE gene locus were subjected to a shake flask fermentation test.
  • the monoclonal strain on the freshly cultured LB plate medium was inoculated into a 3 ml LB liquid medium test tube (13 x 150 mm), and cultured at 30 ° C, 225 rpm for about 8 hours. Then, the seed culture solution was taken, and 3% was inoculated into a 250 ml shake flask containing 50 ml of the fermentation broth (M9 medium).
  • the initial OD600 was about 0.5, cultured at 225 rpm at 37 ° C, and the fermentation cycle was 72 hours.
  • the pH of the fermentation broth was adjusted to 7.0 with 10 M NaOH for 24 hours and 48 hours.
  • 65% glucose solution was added in portions to maintain the glucose concentration at 20 g/L.
  • 1 ml of the fermentation broth was taken and centrifuged.
  • the N-acetyl-D-glucosamine content was determined by HPLC.
  • This example describes the screening of a gene for the mutated N-acetyl-D-aminomannose kinase (NanK), which encodes an N-acetyl-D-aminomannose kinase (NanK) with increased enzymatic activity.
  • a gene mutant encoding N-acetyl-D-aminomannose kinase (NanK) having an increased enzyme activity was screened.
  • the cloned gene is amplified by error-prone PCR technology, and the gene is amplified by a DNA polymerase for amplification under conditions that cause high frequency mismatches to obtain high frequency mutations in the PCR product. .
  • Taq DNA polymerase does not have the 3'-5' proofreading property at high magnesium ion concentration (8mmol/L) and different concentrations of dNTP (where dATP and dGTP concentrations are 1.5mmol/L; dTTP and dCTP) The concentration was 3.0mmol/L) to control the frequency of random mutations, and random mutations were introduced into the target gene to construct a mutant library.
  • the template concentration of A260 was 1000 ng/mL, the enzyme concentration was 5 U/ ⁇ L, and the primer concentration was 100 ⁇ M.
  • Error-prone PCR reaction system 10 ⁇ l of PCR reaction buffer, 5 ⁇ l of dNTP (2.5 mM), 5 ⁇ l of MgCl 2 (2.5 mM), 1 ⁇ l of forward primer (nanK-F, SEQ ID No. 18), reversed Primer (nanK-R, SEQ ID No. 19) 1 ⁇ l, DNA template (nanK/pUC57) 0.1 ⁇ l, Taq DNA polymerase 0.5 ⁇ l, ddH 2 O 32.4 ⁇ l.
  • PCR procedure pre-denaturation at 96 °C for 4 min; denaturation at 94 °C for 1 min, annealing at 56 °C for 1 min, extension at 75 °C for 2 min, 45 cycles; final extension at 75 °C for 15 min, recovery of PCR product by gel recovery method (product size: 0.9 kb); 5 ⁇ l of the product was examined by 1% agarose gel electrophoresis and stored at -20 ° C until use.
  • the above PCR product was digested with restriction endonucleases Nco I and Hind III, and ligated with the pTrc99A plasmid digested with Nco I and Hind III endonuclease, and then transformed into E. coli AT-005 with the ligation product mixture. -02, a large number of cloned transformants were obtained, and a transformed mutant library was constructed.
  • N-acetyl-D-aminomannose kinase based on how much phosphorylation of N-acetyl-D-aminomannose (ManNAc) is based, ie N-acetyl-D-aminomannose is reduced to The mark is determined.
  • Enzyme unit definition The amount of enzyme required to reduce the reducing sugar equivalent to 1 ⁇ mol of N-acetyl-D-aminomannose per minute under enzymatic reaction conditions, defined as an enzyme activity unit (IU).
  • a 5 ml reaction system is used as an enzyme activity assay system, which comprises 500 mmol/L N-acetyl-D-aminomannose, 5 mmol/L glucose, 100 mmol/L Tris-HCl (pH 8.0) and 100 ⁇ l of crude enzyme solution. .
  • the enzyme reaction was carried out in a 37 ° C water bath, incubated for 4 h, and then the reaction was stopped at 70 ° C for 10 min. Centrifuge at 3000 rpm for 10 min and take the supernatant.
  • the N-acetyl-D-aminomannose content was determined by HPLC.
  • NanK was modified by error-prone PCR to obtain a mutant strain with an approximately 5-fold increase in enzyme activity. Pick The mutant strain with the highest activity of the enzyme was selected, and plasmid sequencing was performed. The results showed that the N-acetyl-D-aminomannos kinase mutant gene sequence is shown in SEQ ID No. 26, and the corresponding amino acid sequence is shown in SEQ ID No. 27.
  • mutant gene Compared with the wild-type N-acetyl-D-aminomannose kinase gene sequence, four base point mutations occurred: 107A/G, 309T/G, 669G/C, 783A/G; Mutant mutations, the mutation points are: Q36R (the 36th lysine becomes arginine), I103M (the 103th isoleucine becomes methionine), R223S (the 223th arginine becomes serine) .
  • the mutant gene was named nanKM.
  • the pTrc-nanKM gene cassette integrates into the nagE gene locus of E. coli
  • the nagE gene locus is the integration site of the pTrc-nanKM gene cassette on the chromosome.
  • the nanKM fragment pTrc-nanKM with the Trc promoter and the claratinmycin flanked by the FLP recombinase recognition site (FRT site) were amplified. Resistance gene fragment: FRT-Kanr-FRT (fKanrf), and spliced.
  • the primers for deleting the homologous arm of the nagE gene sequence were designed again, and the full-length linear DNA fragment of Red recombinant targeting was amplified by using the fragment of pTrc-nanKM and fKanrf splicing as a template.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • the PCR product was separated and purified by 1% agarose gel electrophoresis.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • fKanrf size 1.28kb. Its nucleotide sequence is SEQ ID No. 3.
  • the PCR product was separated and purified by 1% agarose gel electrophoresis.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • the size of the second amplified fKanrf was 1.3 kb.
  • the PCR product was separated and purified by 1% agarose gel electrophoresis.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • Amplification product homology arm + pTrc-nanKM-fKanrf + homology arm.
  • the PCR product was separated by agarose gel electrophoresis, purified and recovered, and 100 ng/ ⁇ l of linear DNA full-length PCR fragment was obtained for Red recombinant targeting.
  • the pKD46 vector was transferred into the E. coli AT-004-02 strain. Then, a linear DNA fragment for targeting was prepared by electroporation, and positive clones were selected. Finally, the resistance gene is eliminated.
  • Competent preparation First, the Escherichia coli AT-004-02 stock solution stored at -20 ° C was inoculated in 10 ml LB liquid medium at 1:50-100, and cultured at 37 ° C, 225 rpm, shaking for 2-3 hours. . The culture solution was further added to a 10 ml centrifuge tube, 4000 g ⁇ 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl 2 5 ml for 5 min. Finally, it was centrifuged at 4000 g ⁇ 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl 2 5 ml. It was allowed to stand at -4 ° C for 12 hours and settled naturally.
  • Plasmid transformation 250 ⁇ l of naturally-precipitated cells were taken, and 5 ⁇ l of pKD46 plasmid was added at -4 ° C for 30 min. Then, in a 42 ° C water bath for 1.5 min, 0.7 ml of SOC medium was added, and the mixture was shaken at 30 ° C for 2 hours. Take 0.2ml of bacteria Liquid, penicillin plate. Incubate overnight (12-16 hours) at 30 °C. Monoclones were picked, cultured in 5 ml of LB liquid medium, and plasmids were identified. Preserve positive strains for use.
  • pCP20 was transferred into the above-mentioned caramycin resistant clone, cultured at 30 ° C for 8 h, and then increased to 42 ° C overnight, heat-induced FLP recombinase expression, and the plasmid was gradually lost.
  • the antimony inoculum was plated on the antibiotic-free medium, and the grown monoclonal spot was picked onto the caramycin resistant plate, and the undeveloped clone in which the caramycin resistance gene had been deleted by the FLP recombinase. Clones with the disappearance of resistance to caramycin were identified by PCR using the identified primers.
  • the recombinant strain AT-007-02 and the control strain in which the pTrc-nanKM gene cassette was integrated at the chromosome nagE gene locus was subjected to a shake flask fermentation test.
  • the monoclonal strain on the freshly cultured LB plate medium was inoculated into 3 ml of LB liquid medium test tube (13 ⁇ 150 mm) at 30 ° C, 225 rpm. Cultivate for about 8 hours. Then, the seed culture solution was taken, and 3% was inoculated into a 250 ml shake flask containing 50 ml of the fermentation broth (M9 medium).
  • the initial OD600 was about 0.5, cultured at 225 rpm at 37 ° C, and the fermentation cycle was 72 hours.
  • the pH of the fermentation broth was adjusted to 7.0 with 10 M NaOH.
  • 65% glucose solution was added in portions to maintain the glucose concentration at 20 g/L.
  • 1 ml of the fermentation broth was taken and centrifuged.
  • the N-acetyl-D-glucosamine content was determined by HPLC.
  • This example describes an E. coli strain integrated with a pTrc-nanKM cassette in which the gene nanE overexpressing N-acetyl-D-aminomannose-6-P isomerase (NanE) and its N-acetyl-D-amino group The effect of glucose production.
  • NaE N-acetyl-D-aminomannose-6-P isomerase
  • AT-001 Erscherichia coli ATCC 27325 genome.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • Amplification product size 690 bp.
  • the PCR product was separated and purified by 1% agarose gel electrophoresis.
  • PCR amplified fragment and the vector pTrc99A were digested with Nco I and Hind III respectively, and the nanE fragment and the pTrc99A fragment were separated and purified by agarose gel electrophoresis, and ligated with T4 DNA ligase at 16 ° C overnight, and identified. nanE/pTrc99A plasmid.
  • the culture solution was added to a 10 ml centrifuge tube, 4000 g ⁇ 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl 2 5 ml for 5 min.
  • the mixture was heated at 242 ° C for 1.5 min, added to 0.7 ml of SOC medium, and shaken at 30 ° C for 2 hours.
  • Trc promoter sequence fragment and the fKanrf fragment were amplified and spliced. Then, a homology arm primer was designed to amplify a full-length linear DNA fragment for Red recombinant targeting.
  • Trc promoter sequence was found: SEQ ID No. 32.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • the PCR product was separated and purified by 1% agarose gel electrophoresis.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • fKanrf size 1.28kb. Its nucleotide sequence is SEQ ID No. 3.
  • the PCR product was separated and purified by 1% agarose gel electrophoresis.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • the size of the second amplified fKanrf was 1.3 kb.
  • the PCR product was separated and purified by 1% agarose gel electrophoresis.
  • Trc promoter PCR fragment and secondary amplified fKanrf PCR fragment, 1:1 mixing.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • Amplification product homology arm + fKanrf + Trc promoter + homology arm.
  • the PCR product was separated by agarose gel electrophoresis, purified and recovered, and 100 ng/ ⁇ l of linear DNA full-length PCR fragment was obtained for Red recombinant targeting.
  • the pKD46 vector was transferred into the E. coli AT-007-02 strain. Then, a linear DNA fragment for targeting was prepared by electroporation, and positive clones were selected. Finally, the resistance gene is eliminated.
  • AT-009 AT-007-02, ⁇ nanE promotor::Trc promoter.
  • a shake flask fermentation test was performed on a strain in which the pTrc-nanKM cassette was integrated, a strain in which NanoE was overexpressed (including a strain in which nanE/pTrc99A was transformed, and a strain in which the NanE promoter was replaced with a Trc promoter).
  • the monoclonal strain on the freshly cultured LB plate medium was inoculated into a 3 ml LB liquid medium test tube (13 x 150 mm), and cultured at 30 ° C, 225 rpm for about 8 hours. Then, the seed culture solution was taken, and 3% was inoculated into a 250 ml shake flask containing 50 ml of M9 medium.
  • the initial OD 600 was about 0.5, cultured at 225 rpm at 37 ° C, and the fermentation cycle was 72 hours.
  • the pH of the fermentation broth was adjusted to 7.0 with 10 M NaOH.
  • 65% glucose solution was added in portions to maintain the glucose concentration at 20 g/L.
  • 1 ml of the fermentation broth was taken and centrifuged.
  • the N-acetyl-D-glucosamine content was determined by HPLC.
  • the yield of recombinant shake flask fermentation is shown in Table 6.
  • Table 6 The results showed that overexpression of NanE, whether the replacement of the NanE promoter for the Trc promoter or the transformation of the nanE/pTrc99A plasmid, significantly increased yield.
  • the replacement of the NanE promoter for the Trc promoter recombinant strain was more significant than the transformed nanE/pTrc99A plasmid.
  • This example describes an E. coli strain integrated with a pTrc-nanKM cassette in which the gene of glucose-6-phosphate synthase (GlmS) gene glmS and/or D-glucosamine-6-phosphate deaminase (NagB) Effect of endogenous natural promoter replacement and/or deletion of nagB on N-acetyl-D-glucosamine production.
  • GlmS glucose-6-phosphate synthase
  • NagB D-glucosamine-6-phosphate deaminase
  • D-glucosamine-6-phosphate deaminase (NagB) gene in the nagE-nagBACD was deleted and replaced with the Trc promoter.
  • the reaction catalyzed by D-Glucosamine-6-phosphate deaminase (NagB) is reversible, over-expressing nagB, accelerating the forward catalytic reaction of NagB, and increasing D-glucosamine- 6-phosphoric acid (GlcN-6-P) purpose.
  • Trc promoter fragment and the fKanrf fragment were amplified and spliced. Then, a homology arm primer was designed to amplify a full-length linear DNA fragment for Red recombinant targeting.
  • Designing homology arm primers Designing the deletion of the nagB promoter homology arm according to NCBI search NC_000913, Escherichia coli str.K-12nagB promoter sequence and nagE gene sequence SEQ ID No.13 Forward primer (nagBKO-F1) SEQ ID No. 40, reverse primer (nagBKO-R1) SEQ ID No. 41.
  • Trc promoter PCR fragment and secondary amplified fKanrf PCR fragment, 1:1 mixing.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • Amplification product homology arm + fKanrf + Trc promoter + homology arm.
  • the PCR product was separated by agarose gel electrophoresis, purified and recovered, and 100 ng/ ⁇ l of linear DNA full-length PCR fragment was obtained for Red recombinant targeting.
  • the pKD46 vector was transferred into the E. coli AT-007-02 strain. Then, a linear DNA fragment for targeting was prepared by electroporation, and positive clones were selected. Finally, the resistance gene is eliminated.
  • Glucosamine-6-phosphate synthase (glmS) gene promoter sequence was deleted.
  • Glucosamine-6-phosphate synthase (GlmS) also known as L-glutamine-6-phosphate aminotransferase, catalyzes glucose-6-phosphate (Glc-6) -P) Amination to D-glucosamine-6-phosphate (GlcN-6-P), but with serious product inhibition problems, deletion of its promoter sequence, loss of expression of the enzyme, release of GlcN-6-P Product inhibition.
  • the fKanrf fragment was amplified, and then the homology arm primer was designed to amplify the full-length linear DNA fragment of Red recombinant targeting.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • fKanrf size 1.28kb. Its nucleotide sequence is SEQ ID No. 3.
  • the PCR product was separated and purified by 1% agarose gel electrophoresis.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • Amplification product homology arm + fKanf + homology arm.
  • the PCR product was separated by agarose gel electrophoresis, purified and recovered, and 100 ng/ ⁇ l of linear DNA full-length PCR fragment was obtained for Red recombinant targeting.
  • the pKD46 vector was transferred into the E. coli AT-010 strain. Then, a linear DNA fragment for targeting was prepared by electroporation, and positive clones were selected. Finally, the resistance gene is eliminated.
  • AT-011 AT-010, ⁇ glmS promotor.
  • the nagB promoter was replaced with a promoter of a higher expression level, and the recombinant strain in which the glmS promoter was further deleted was subjected to a shake flask fermentation test.
  • the monoclonal strain on the freshly cultured LB plate medium was inoculated into a 3 ml LB liquid medium test tube (13 x 150 mm), and cultured at 30 ° C, 225 rpm for about 8 hours. Then, the seed culture solution was taken, and 3% was inoculated into a 250 ml shake flask containing 50 ml of the fermentation broth (M9 medium).
  • the initial OD600 was about 0.5, cultured at 225 rpm at 37 ° C, and the fermentation cycle was 72 hours.
  • the pH of the fermentation broth was adjusted to 7.0 with 10 M NaOH.
  • 65% glucose solution was added in portions to maintain the glucose concentration at 20 g/L.
  • 1 ml of the fermentation broth was taken and centrifuged.
  • the N-acetyl-D-glucosamine content was determined by HPLC.
  • L-glutamine-6-phosphate aminotransferase gene promoter sequence was replaced with a Trc promoter sequence.
  • L-Glutamine-6-phosphate fructose aminotransferase also known as Glucosamine-6-phosphate synthase (GlmS) replaces its promoter sequence with the Trc promoter sequence, overexpressing glmS, accelerating GlmS catalyzes the function of increasing D-glucosamine-6-phosphate (GlcN-6-P).
  • Trc promoter sequence fragment and the fKanrf fragment were amplified and spliced. Then, a homology arm primer was designed to amplify a full-length linear DNA fragment for Red recombinant targeting.
  • Designing a homology arm primer designing a homologous arm forward primer (ProglmspTrc-F) SEQ ID No. 45, reverse primer (ProglmspTrc-R), which was replaced with the Trc promoter according to the glmS gene promoter sequence SEQ ID No. SEQ ID No. 46.
  • Trc promoter PCR fragment and secondary amplified fKanrf PCR fragment, 1:1 mixing.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • Amplification product homology arm + fKanrf + Trc promoter + homology arm.
  • the PCR product was separated by agarose gel electrophoresis, purified and recovered, and 100 ng/ ⁇ l of linear DNA full-length PCR fragment was obtained for Red recombinant targeting.
  • the pKD46 vector was transferred into the E. coli AT-007-02 strain. Then, a linear DNA fragment for targeting was prepared by electroporation, and positive clones were selected. Finally, the resistance gene is eliminated.
  • AT-012 AT-007-02, ⁇ glmS promotor::Trc promoter.
  • the fKanrf fragment was amplified, and then the homology arm primer was designed to prepare a full-length linear DNA fragment for Red recombinant targeting.
  • Designing a homology arm primer Designing a homologous arm forward primer (NagBKO-F2) SEQ ID No. 47, a reverse primer (NagBKO-), based on the nagB promoter and the nagE gene sequence SEQ ID No. 13, designed to delete the nagB promoter sequence. R2) SEQ ID No. 48.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • Amplification product homology arm + fKanrf + homology arm.
  • the PCR product was separated by agarose gel electrophoresis, purified and recovered, and 100 ng/ ⁇ l of linear DNA full-length PCR fragment was obtained for Red recombinant targeting.
  • the pKD46 vector was transferred into the E. coli AT-012 strain. Then, a linear DNA fragment for targeting was prepared by electroporation, and positive clones were selected. Finally, the resistance gene is eliminated.
  • AT-013 AT-012, ⁇ nagB promotor
  • the glmS promoter was replaced with a promoter of a higher expression level, and the recombinant strain which further deleted the nagB promoter was subjected to a shake flask fermentation test.
  • the monoclonal strain on the freshly cultured LB plate medium was inoculated into a 3 ml LB liquid medium test tube (13 x 150 mm), and cultured at 30 ° C, 225 rpm for about 8 hours. Then, the seed culture solution was taken, and 3% was inoculated into a 250 ml shake flask containing 50 ml of the fermentation broth (M9 medium).
  • This example describes the endogenous nature of the gglS gene integrated with the pTrc-nanKM cassette and the gene nagB of D-glucosamine-6-phosphate deaminase (NagB) and glucosamine-6-phosphate synthase (GlmS). Promoter replacement and/or deletion of E. coli strains, transformation of the nanE/pTrc99A plasmid or conversion of the nanE gene endogenous natural promoter to the Trc promoter, effect on N-acetyl-D-glucosamine production.
  • Competent state preparation First, the recombinant strain AT-011 was stored at -20 ° C, inoculated in 10 ml of LB liquid medium at 1:50-100, and cultured at 37 ° C, 225 rpm, shaking for 2-3 hours. The culture solution was further added to a 10 ml centrifuge tube, 4000 g ⁇ 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl 2 5 ml for 5 min. Finally, it was centrifuged at 4000 g ⁇ 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl 2 5 ml. It was allowed to stand at -4 ° C for 12 hours and settled naturally.
  • Plasmid transformation 250 ⁇ l of naturally-precipitated cells were added, and 5 ⁇ l of nanE/pTrc99A plasmid was added. -4 ° C, 30 min. Then, in a 42 ° C water bath for 1.5 min, 0.7 ml of SOC medium was added, and the mixture was shaken at 30 ° C for 2 hours. Take 0.2 ml of bacterial solution and apply penicillin plate. Incubate overnight (12-16 hours) at 30 °C. Monoclones were picked, cultured in 5 ml of LB liquid medium, and plasmids were identified. Save positive clones for use.
  • AT-014 (AT-011, nanE/pTrc99A).
  • the endogenous natural promoter of the nanE gene is integrated with the pTrc-nanKM cassette, and the endogenous natural promoter of the nagB gene is replaced with the Trc promoter and the E. coli strain which simultaneously deletes the endogenous natural promoter of the glmS gene. Switch to the Trc promoter
  • Trc promoter sequence fragment and the fKanrf fragment were amplified and spliced. Then, a homology arm primer was designed to amplify a full-length linear DNA fragment for Red recombinant targeting.
  • Trc promoter PCR fragment and secondary amplified fKanrf PCR fragment, 1:1 mixing.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • Amplification product homology arm + fKanrf + Trc promoter + homology arm.
  • the PCR product was separated by agarose gel electrophoresis, purified and recovered, and 100 ng/ ⁇ l of linear DNA full-length PCR fragment was obtained for Red recombinant targeting.
  • the pKD46 vector was transferred into the E. coli AT-011 strain. Then, a linear DNA fragment for targeting was prepared by electroporation, and positive clones were selected. Finally, the resistance gene is eliminated.
  • AT-015 AT-011, ⁇ nanE promotor::Trc promoter.
  • the recombinant E. coli strain AT-013 was prepared. Then, the NanE/pTrc99A plasmid was transformed into AT-013 by CaCl 2 transformation, and the monoclonal clone was picked and the positive clone was identified by plasmid extraction.
  • AT-016 AT-013, nanE/pTrc99A.
  • the endogenous natural promoter of the nanE gene is integrated with the pTrc-nanKM cassette, and the endogenous natural promoter of the glmS gene is replaced with the Trc promoter and the E. coli strain which simultaneously deletes the endogenous natural promoter of the nagB gene. Switch to the Trc promoter
  • Trc promoter sequence fragment and the fKanrf fragment were amplified and spliced. Then, a homology arm primer was designed to amplify a full-length linear DNA fragment for Red recombinant targeting.
  • the pKD46 vector was transferred into E. coli AT-013 strain. Then, a linear DNA fragment for targeting was prepared by electroporation, and positive clones were selected. Finally, the resistance gene is eliminated.
  • AT-017 AT-013, ⁇ nanE promotor::Trc promoter.
  • strains incorporating the pTrc-nanKM cassette wherein the glmS and nagB endogenous natural promoters are replaced and/or deleted, and the nanE/pTrc99A plasmid is transformed or the nanE gene endogenous natural promoter is replaced with a Trc promoter.
  • Different genotypes of recombinant bacteria were formed and subjected to shake flask fermentation test.
  • the monoclonal strain on the freshly cultured LB plate medium was inoculated into a 3 ml LB liquid medium test tube (13 x 150 mm), and cultured at 30 ° C, 225 rpm for about 8 hours.
  • the seed culture solution was taken, and 3% was inoculated into a 250 ml shake flask containing 50 ml of the fermentation broth (M9 medium).
  • the initial OD600 was about 0.5, cultured at 225 rpm at 37 ° C, and the fermentation cycle was 72 hours.
  • the pH of the fermentation broth was adjusted to 7.0 with 10 M NaOH.
  • 65% glucose solution was added in portions to maintain the glucose concentration at 20 g/L.
  • 1 ml of the fermentation broth was taken and centrifuged.
  • the N-acetyl-D-glucosamine content was determined by HPLC.
  • the yield of shake flask fermentation is shown in Table 9.
  • Table 9 The results showed that overexpression of NanE, whether the replacement of the NanE promoter with the Trc promoter or the transformation of the NanE/pTrc99A plasmid, resulted in substantial improvement in N-acetyl-D-glucosamine production, but the replacement of the NanE promoter for the Trc promoter recombination
  • the yield of N-acetyl-D-glucosamine was significantly increased by the transformed nanE/pTrc99A plasmid.
  • This example describes an E. coli strain integrated with a pTrc-nanKM cassette in which the gene wecB overexpressing UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) and its N-acetyl-D-amino group The effect of glucose production.
  • WecB UDP-N-acetyl-D-glucosamine-2-isomerase
  • the endogenous natural promoter of the gene was replaced with the Trc promoter to over-express, which enhanced UDP-GlcNAc (UDP-N-acetyl glucosamine, UDP-N-acetyl-D-glucosamine) into ManNAc (N-Acetyl- D-mannosamine, N-acetyl-D-aminomannose or N-acetyl-D-mannosamine).
  • UDP-GlcNAc UDP-N-acetyl glucosamine
  • UDP-N-acetyl-D-glucosamine UDP-N-acetyl-D-glucosamine
  • ManNAc N-Acetyl- D-mannosamine, N-acetyl-D-aminomannose
  • AT-001 Erscherichia coli ATCC 27325 genome.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • Amplification product size 1.13 kb.
  • the PCR product was separated and purified by 1% agarose gel electrophoresis.
  • the obtained PCR amplified fragment and the vector pTrc99A were digested with Nco I and Hind III respectively, and the wecB fragment and the pTrc99A fragment were separated and purified by agarose gel electrophoresis, and ligated with T4 DNA ligase at 16 ° C overnight, and identified. wecB/pTrc99A plasmid.
  • Competent preparation First, the recombinant strain AT-007-02 stored at -20 °C was inoculated into 10 ml of LB liquid medium at 1:50-100, and cultured at 37 ° C, 225 rpm, shaking 2-3 hour. The culture solution was further added to a 10 ml centrifuge tube, 4000 g ⁇ 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl 2 5 ml for 5 min. Finally, it was centrifuged at 4000 g ⁇ 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl 2 5 ml. It was allowed to stand at -4 ° C for 12 hours and settled naturally.
  • Plasmid transformation 250 ⁇ l of the naturally-precipitated cells were taken, and 5 ⁇ l of the wecB/pTrc99A plasmid was added at -4 ° C for 30 min. Then, in a 42 ° C water bath for 1.5 min, 0.7 ml of SOC medium was added, and the mixture was shaken at 30 ° C for 2 hours. Take 0.2 ml of bacterial solution and apply penicillin plate. Incubate overnight (12-16 hours) at 30 °C. Monoclones were picked, cultured in 5 ml of LB liquid medium, and plasmids were identified. Save positive clones for use.
  • strain number AT-018 (AT-007-02, wecB/pTrc99A).
  • Trc promoter sequence fragment and the fKanrf fragment were amplified and spliced. Then, a homology arm primer was designed to amplify a full-length linear DNA fragment for Red recombinant targeting.
  • Trc promoter PCR fragment and secondary amplified fKanrf PCR fragment, 1:1 mixing.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • Amplification product homology arm + fKanrf + Trc promoter + homology arm.
  • the PCR product was separated by agarose gel electrophoresis, purified and recovered, and 100 ng/ ⁇ l of linear DNA full-length PCR fragment was obtained for Red recombinant targeting.
  • the pKD46 vector was transferred into the E. coli AT-007-02 strain. Then, a linear DNA fragment for targeting was prepared by electroporation, and positive clones were selected. Finally, the resistance gene is eliminated.
  • AT-019 AT-007-02, ⁇ wecB promotor::Trc promoter.
  • the recombinant strain produced by the strain overexpressing the wecB (including the strain transformed with wecB/pTrc99A and the change of the wecB promoter to the Trc promoter) was subjected to a shake flask fermentation test.
  • the monoclonal strain on the freshly cultured LB plate medium was inoculated into a 3 ml LB liquid medium test tube (13 x 150 mm), and cultured at 30 ° C, 225 rpm for about 8 hours. Then, the seed culture solution was taken, and 3% was inoculated into a 250 ml shake flask containing 50 ml of M9 medium.
  • the initial OD 600 was about 0.5, cultured at 225 rpm at 37 ° C, and the fermentation cycle was 72 hours.
  • the pH of the fermentation broth was adjusted to 7.0 with 10 M NaOH.
  • 65% glucose solution was added in portions to maintain the glucose concentration at 20 g/L.
  • 1 ml of the fermentation broth was taken and centrifuged.
  • the N-acetyl-D-glucosamine content was determined by HPLC.
  • the shake flask fermentation yield is shown in Table 10.
  • Table 10 The results showed that compared with the control strain AT-007-02, the transformation of the wecB/pTrc99A plasmid significantly increased the yield of N-acetyl-D-glucosamine, and the recombinant strain that replaced the wecB promoter with the Trc promoter was N-acetyl- There is a greater increase in D-glucosamine production.
  • This example describes the integration of the pTrc-nanKM cassette and in which the endogenous native promoter of the nanE gene is replaced with the Trc promoter and the wecB/pTrc99A plasmid is transformed or the endogenous native promoter of the wecB gene is replaced with the Trc promoter. - The effect of acetyl-D-glucosamine production.
  • the wecB/pTrc99A plasmid was transformed into the E. coli strain AT-009 with the pTrc-NanKM cassette and the endogenous natural promoter of the nanE gene was replaced with the Trc promoter by CaCl 2 transformation.
  • the plasmid was identified as a positive clone.
  • strain number AT-020 (AT-009, wecB/pTrc99A).
  • the pKD46 vector was transferred into the E. coli AT-009 strain. Then, a linear DNA fragment for targeting was prepared by electroporation, and positive clones were selected. Finally, the resistance gene is eliminated.
  • AT-021 AT-009, ⁇ wecB promotor::Trc promoter.
  • the seed culture solution was taken, and 3% was inoculated into a 250 ml shake flask containing 50 ml of M9 medium.
  • the initial OD 600 was about 0.5, cultured at 225 rpm at 37 ° C, and the fermentation cycle was 72 hours.
  • the pH of the fermentation broth was adjusted to 7.0 with 10 M NaOH.
  • 65% glucose solution was added in portions to maintain the glucose concentration at 20 g/L.
  • 1 ml of the fermentation broth was taken and centrifuged.
  • the N-acetyl-D-glucosamine content was determined by HPLC.
  • This example describes an E. coli strain that incorporates the pTrc-nanKM cassette and replaces and/or deletes the endogenous natural promoter of the glmS gene and the nagB gene, transforms the wecB/pTrc99A plasmid or the wecB gene endogenous natural promoter. Effect of switching to Trc promoter on N-acetyl-D-glucosamine production
  • the wecB/pTrc99A plasmid was transformed into the integrated pTrc-NanKM cassette by CaCl 2 transformation, and the endogenous natural promoter of nagB gene was replaced with the Trc promoter and the endogenous natural promoter of glmS gene was deleted at the same time.
  • the monoclonal culture was picked and the positive clones were identified by plasmid extraction.
  • AT-022 AT-011, wecB/pTrc99A.
  • the endogenous natural promoter of the wecB gene is integrated with the pTrc-nanKM cassette, and the endogenous natural promoter of the nagB gene is replaced with the Trc promoter and the E. coli strain which simultaneously deletes the endogenous natural promoter of the glmS gene. Switch to the Trc promoter
  • the pKD46 vector was transferred into the E. coli AT-011 strain. Then, a linear DNA fragment for targeting was prepared by electroporation, and positive clones were selected. Finally, the resistance gene is eliminated.
  • AT-023 AT-011, ⁇ wecB promotor::Trc promoter.
  • the wecB/pTrc99A plasmid was transformed into a pTrc-nanKM cassette by CaCl 2 transformation, and the endogenous natural promoter of the glmS gene was replaced with the Trc promoter and the endogenous natural promoter of the nagB gene was simultaneously deleted.
  • the monoclonal culture was picked and the positive clones were identified by plasmid extraction.
  • AT-024 (AT-013, wecB/pTrc99A).
  • Trc promoter of the wecB gene was exchanged for the E. coli strain in which the pTrc-nanKM cassette was integrated and the endogenous natural promoter of the glmS gene was replaced with the Trc promoter and the endogenous natural promoter of the nagB gene was simultaneously deleted. Trc promoter
  • the pKD46 vector was transferred into E. coli AT-013 strain. Then, a linear DNA fragment for targeting was prepared by electroporation, and positive clones were selected. Finally, the resistance gene is eliminated.
  • AT-025 AT-013, ⁇ wecB promotor::Trc promoter.
  • the endogenous natural promoter of the glmS gene and the nagB gene is replaced and/or deleted
  • the wecB/pTrc99A plasmid is transformed or the endogenous natural promoter of the wecB gene is replaced with the Trc promoter.
  • the different genotypes of recombinant bacteria formed were used for shake flask fermentation experiments.
  • the monoclonal strain on the freshly cultured LB plate medium was inoculated into a 3 ml LB liquid medium test tube (13 x 150 mm), and cultured at 30 ° C, 225 rpm for about 8 hours.
  • the seed culture solution was taken, and 3% was inoculated into a 250 ml shake flask containing 50 ml of the fermentation broth (M9 medium).
  • the initial OD600 was about 0.5, cultured at 225 rpm at 37 ° C, and the fermentation cycle was 72 hours.
  • the pH of the fermentation broth was adjusted to 7.0 with 10 M NaOH.
  • 65% glucose solution was added in portions to maintain the glucose concentration at 20 g/L.
  • 1 ml of the fermentation broth was taken and centrifuged.
  • the N-acetyl-D-glucosamine content was determined by HPLC.
  • This example describes an E. coli strain in which a pTrc-NanKM cassette is integrated, an endogenous natural promoter of the glmS gene and the NagB gene is replaced and/or deleted, and an endogenous natural promoter of the nanE gene is replaced with a Trc promoter. Effect of wecB/pTrc99A plasmid or the endogenous natural promoter of wecB gene on Trc promoter on N-acetyl-D-glucosamine production
  • the wecB/pTrc99A plasmid was transformed into the integrated pTrc-nanKM cassette by the CaCl 2 transformation, the endogenous natural promoter of the nagB gene was replaced with the Trc promoter, the endogenous natural promoter of the glmS gene was deleted, and nanE was deleted.
  • the endogenous natural promoter of the gene was replaced with the Escherichia coli strain AT-015 of the Trc promoter, and the monoclonal clone was picked and the positive clone was identified by plasmid extraction.
  • AT-026 AT-015, wecB/pTrc99A.
  • the pKD46 vector was transferred into E. coli AT-015 strain. Then, a linear DNA fragment for targeting was prepared by electroporation, and positive clones were selected. Finally, the resistance gene is eliminated.
  • AT-027 AT-015, ⁇ wecB promotor::Trc promoter.
  • the wecB/pTrc99A plasmid was transformed into the integrated pTrc-nanKM cassette by the CaCl 2 method, the endogenous natural promoter of the glmS gene was replaced with the Trc promoter, the endogenous natural promoter of the nagB gene was deleted, and the nanE gene was deleted.
  • the endogenous natural promoter was replaced with the Escherichia coli strain AT-017 of the Trc promoter, and the monoclonal clone was picked and the positive clone was identified by plasmid extraction.
  • AT-028 AT-017, wecB/pTrc99A.
  • the pKD46 vector was transferred into the E. coli AT-017 strain. Then, a linear DNA fragment for targeting was prepared by electroporation, and positive clones were selected. Finally, the resistance gene is eliminated.
  • AT-029 AT-017, ⁇ wecB promotor::Trc promoter.
  • E. coli strains that have been integrated with the pTrc-nanKM cassette and replaced and/or deleted the endogenous natural promoter of the glmS gene and the nagB gene and replaced the endogenous natural promoter of the nanE gene with the Trc promoter, transforming wecB/
  • the pTrc99A plasmid and the replacement wecB promoter were recombinant strains produced by the Trc promoter and subjected to a shake flask fermentation test.
  • the monoclonal strain on the freshly cultured LB plate medium was inoculated into a 3 ml LB liquid medium test tube (13 x 150 mm), and cultured at 30 ° C, 225 rpm for about 8 hours.
  • the seed culture solution was taken, and 3% was inoculated into a 250 ml shake flask containing 50 ml of the fermentation broth (M9 medium).
  • the initial OD600 was about 0.5, cultured at 225 rpm at 37 ° C, and the fermentation cycle was 72 hours.
  • the pH of the fermentation broth was adjusted to 7.0 with 10 M NaOH.
  • 65% glucose solution was added in portions to maintain the glucose concentration at 20 g/L.
  • 1 ml of the fermentation broth was taken and centrifuged.
  • the N-acetyl-D-glucosamine content was determined by HPLC.
  • the yield of shake flask fermentation is shown in Table 13.
  • Table 13 The results showed that compared with the control strains AT-015 or AT-017, the transformation of wecB/pTrc99A plasmid resulted in a significant increase in N-acetyl-D-glucosamine production, and the replacement of the wecB promoter was a recombinant strain of the Trc promoter against N- There is a greater increase in acetyl-D-glucosamine production.
  • This example describes the production of N-acetyl-D-glucosamine fermentation in a 10 L fermentor.
  • the recombinant engineering strain AT-029 was used as the production strain, and the N-acetyl-D-glucosamine fermentation test was carried out in a 10 L fermentor.
  • the fermentation medium was prepared according to Table 14, wherein the trace element solution was prepared according to Table 15, and the multivitamin solution was prepared according to Table 16.
  • K 2 HPO 4 1.30g KH 2 PO 4 1.00g MgSO 4 .7H 2 O 0.10g NH 4 Cl 0.02g (NH 4 ) 2 SO 4 0.20g NaH 2 PO 4 0.60g Polyether defoamer 10ml Trace element solution 4ml Multivitamin solution 4ml glucose 6.00g
  • the trace element solution is separately sterilized and added, and the vitamin solution is filtered and added;
  • Fermentation medium is the basic medium before the addition of glucose, the initial liquid volume of the basic medium
  • the secondary seed solution was inoculated to the fermentor at 40 ml/L, inoculating amount: 2.5-5% by volume, and the initial OD 600 was 0.3-0.5.
  • the high-density fermentation was carried out by using a 10L self-controlled fermenter, and the data was collected by the machine belt software to realize online computer control.
  • the control parameters are: air flow rate of 0.5-1vvm.; dissolved oxygen ⁇ 20%, to increase the speed and ventilation adjustment; temperature 37 ° C; pH value of 7.0, automatic flow of saturated ammonia to maintain constant.
  • the glucose is consumed in the basal medium, that is, the sugar is added when the dissolved oxygen rises.
  • the sugar-filling rate is based on the control of the residual sugar concentration of 0.45 g/L or less.
  • the sugar solution glucose was at a concentration of 65% (w/v) and was added with 2.5% sodium gluconate or 6% ribose. The fermentation was stopped at 60-72h. Total liquid volume: 75-80%.
  • the inoculum size was 200 mL.
  • the residual sugar concentration is controlled to be 0.45 g/L or less.
  • Tracking index OD 600 and residual sugar content (residual glucose in fermentation broth).
  • This example describes the separation and purification treatment process of N-acetyl-D-glucosamine and D-glucosamine hydrochloride.
  • Solid-liquid separation Centrifugation at 4000-8000 rpm, discarding the slag and protein, and taking the fermentation broth. It can also be filtered with a ceramic membrane.
  • the initial salt concentration of the concentrated chamber tank into the fermentation liquid is 0.01-0.05 mol/L.
  • the flow rate of the fermentation broth of the light room is 40-80 L/h, the flow rate of the fermentation broth of the concentrated chamber is 40-80 L/h, and the voltage of the single membrane pair is 0.5-1.4 V.
  • Deionization can also be carried out using an anion-cation exchange resin.
  • Solid-liquid separation Centrifugation at 4000-8000 rpm, discarding the slag and protein, and taking the fermentation broth. It can also be filtered with a ceramic membrane.
  • the initial salt concentration of the concentrated chamber tank into the fermentation liquid is 0.01-0.05 mol/L.
  • the flow rate of the fermentation broth of the light room is 40-80 L/h, the flow rate of the fermentation broth of the concentrated chamber is 40-80 L/h, and the voltage of the single membrane pair is 0.5-1.4 V.
  • Deionization can also be carried out using an anion-cation exchange resin.
  • Crystallization firstly cool the water at 25 ° C to 25-35 ° C, and then use 0 ° C water to cool 1-3 h to 4 ° C.
  • This example describes the screening of a gene for the mutated N-acetyl-D-aminomannose-6-P isomerase (NanE), which encodes an N-acetyl-D-aminomannose-6-P with increased enzymatic activity. Isomerase.
  • a gene mutant encoding an increase in enzyme activity was screened.
  • the cloned gene is amplified by error-prone PCR technology, and the gene is amplified by a DNA polymerase for amplification under conditions that cause high frequency mismatches to obtain high frequency mutations in the PCR product. .
  • Taq DNA polymerase does not have the 3'-5' proofreading property at high magnesium ion concentration (8mmol/L) and different concentrations of dNTP (where dATP and dGTP concentrations are 1.5mmol/L; dTTP and dCTP) The concentration is 3.0mmol/L) to control the frequency of random mutations and introduce into the target gene. Random mutations were constructed to construct a mutant library; the template concentration of A260 was 1000 ng/mL, the enzyme concentration was 5 U/ ⁇ L, and the primer concentration was 100 ⁇ M.
  • Error-prone PCR reaction system 10 ⁇ l of PCR reaction buffer, 5 ⁇ l of dNTP (2.5 mM), 5 ⁇ l of MgCl 2 (2.5 mM), 1 ⁇ l of forward primer (nanE-F, SEQ ID No. 30), reversed Primer (nanE-R, SEQ ID No. 31) 1 ⁇ l, DNA template (nanE/pUC57) 0.1 ⁇ l, Taq DNA polymerase 0.5 ⁇ l, ddH 2 O 32.4 ⁇ l.
  • PCR procedure pre-denaturation at 96 °C for 4 min; denaturation at 94 °C for 1 min, annealing at 56 °C for 1 min, extension at 75 °C for 2 min, 45 cycles; final extension at 75 °C for 15 min, recovery of PCR product by gel recovery method (product size: 0.7 kb); 5 ⁇ l of the product was examined by 1% agarose gel electrophoresis and stored at -20 ° C until use.
  • the above PCR product was digested with restriction endonucleases Nco I and Hind III, and ligated with the pTrc99A plasmid digested with Nco I and Hind III endonuclease, and then transformed into E. coli AT-005 with the ligation product mixture. -02, a large number of cloned transformants were obtained, and a transformed mutant library was constructed.
  • Activity assay of N-acetyl-D-aminomannose-6-P isomerase conversion to N-acetyl-D-amino group with N-acetyl-D-aminomannose-6-phosphate (ManNAc-6-P) Based on the amount of glucose-6-phosphate (GlcNAc-6-P), that is, N-acetyl-D-aminomannose-6-phosphate is reduced to the assay label.
  • Enzyme unit definition The amount of enzyme required to reduce the equivalent of 1 ⁇ mol of N-acetyl-D-aminomannose-6-phosphate per minute under enzymatic reaction conditions, defined as an enzyme activity unit (IU).
  • an isotope-labeled ManNAc-6-P is prepared as a substrate.
  • the reaction was terminated by the addition of 350 ul of ethanol.
  • the product was eluted with water and lyophilized.
  • reaction solution a total volume of 26.5 ul of the reaction solution was prepared as an enzyme activity assay system containing 1 mM isotope-labeled ManNAc-6-P, 37 mM Tris-HCl, pH 8.0 and 19 mM MgCl 2 .
  • the reaction was boiled for 3 min, then 0.1 volume of alkaline phosphatase buffer was added to adjust the pH and 20 units of alkaline phosphatase. After incubation at 37 ° C for 1 hour, samples were taken onto dry chromatography paper and pre-soaked with 1% sodium tetraborate.
  • the solvent system used was ethyl acetate: isopropanol: pyridine: water (50:22:14:14).
  • the radioactive compound was separated by chromatography on paper. The radioactivity was measured by a liquid scintillation counter, and the activity unit of N-acetyl-D-aminomannose-6-P isomerase was calculated from the amount of ManNAc-6-P converted to GlcNAc-6-P.
  • the NanE was modified by error-prone PCR to obtain a mutant strain with greatly improved enzyme activity.
  • the mutant strain with the highest activity of the enzyme was selected and plasmid sequencing was performed.
  • the pTrc-nanEM gene cassette integrates into the nagE gene locus of E. coli
  • the nagE gene locus is the integration site of the pTrc-nanEM gene cassette on the chromosome.
  • the nanEM fragment pTrc-nanEM with the Trc promoter and the claratinmycin flanked by the FLP recombinase recognition site (FRT site) were amplified. Resistance gene fragment: FRT-Kanr-FRT (fKanrf), and spliced.
  • the primers for deleting the homologous arm of the nagE gene sequence were designed again, and the full-length linear DNA fragment of Red recombinant targeting was amplified by using the fragment of pTrc-nanEM and fKanrf splicing as a template.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, Cycle 30 times
  • third step extend at 72 ° C for 10 min.
  • the PCR product was separated and purified by 1% agarose gel electrophoresis.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • fKanrf size 1.28kb. Its nucleotide sequence is SEQ ID No. 3.
  • the PCR product was separated and purified by 1% agarose gel electrophoresis.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • the size of the second amplified fKanrf was 1.3 kb.
  • the PCR product was separated and purified by 1% agarose gel electrophoresis.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • Amplification product homology arm + pTrc-nanEM-fKanrf + homology arm.
  • the PCR product was separated by agarose gel electrophoresis, purified and recovered, and 100 ng/ ⁇ l of linear DNA full-length PCR fragment was obtained for Red recombinant targeting.
  • the pKD46 vector was transferred into the E. coli AT-004-02 strain. Then, electrotransformation preparation Good targeting linear DNA fragments were used to screen positive clones. Finally, the resistance gene is eliminated.
  • Competent preparation First, the Escherichia coli AT-004-02 stock solution stored at -20 ° C was inoculated in 10 ml LB liquid medium at 1:50-100, and cultured at 37 ° C, 225 rpm, shaking for 2-3 hours. . The culture solution was further added to a 10 ml centrifuge tube, 4000 g ⁇ 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl 2 5 ml for 5 min. Finally, it was centrifuged at 4000 g ⁇ 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl 2 5 ml. It was allowed to stand at -4 ° C for 12 hours and settled naturally.
  • Plasmid transformation 250 ⁇ l of naturally-precipitated cells were taken, and 5 ⁇ l of pKD46 plasmid was added at -4 ° C for 30 min. Then, in a 42 ° C water bath for 1.5 min, 0.7 ml of SOC medium was added, and the mixture was shaken at 30 ° C for 2 hours. Take 0.2 ml of bacterial solution and apply penicillin plate. Incubate overnight (12-16 hours) at 30 °C. Monoclones were picked, cultured in 5 ml of LB liquid medium, and plasmids were identified. Preserve positive strains for use.
  • strain AT-030-01 (AT-004-02, ⁇ nagE::pTrc-nanE-fKanrf) was prepared in the same manner as above.
  • pCP20 was transferred into the above-mentioned caramycin resistant clone, cultured at 30 ° C for 8 h, and then increased to 42 ° C overnight, heat-induced FLP recombinase expression, and the plasmid was gradually lost.
  • the antimony inoculum was plated on the antibiotic-free medium, and the grown monoclonal spot was picked onto the caramycin resistant plate, and the undeveloped clone in which the caramycin resistance gene had been deleted by the FLP recombinase. Clones with the disappearance of resistance to caramycin were identified by PCR using the identified primers.
  • strain AT-030-02 (AT-004-02, ⁇ nagE::pTrc-nanE) was prepared in the same manner as above.
  • the recombinant strains AT-030-02, AT-031-02 and the control strains in which the pTrc-nanE and pTrc-nanEM gene cassettes were integrated at the chromosome nagE gene locus were subjected to a shake flask fermentation test.
  • the monoclonal strain on the freshly cultured LB plate medium was inoculated into a 3 ml LB liquid medium test tube (13 x 150 mm), and cultured at 30 ° C, 225 rpm for about 8 hours. Then, the seed culture solution was taken, and 3% was inoculated into a 250 ml shake flask containing 50 ml of the fermentation broth (M9 medium).
  • the initial OD600 was about 0.5, cultured at 225 rpm at 37 ° C, and the fermentation cycle was 72 hours.
  • the pH of the fermentation broth was adjusted to 7.0 with 10 M NaOH.
  • 65% glucose solution was added in portions to maintain the glucose concentration at 20 g/L.
  • 1 ml of the fermentation broth was taken and centrifuged.
  • the N-acetyl-D-glucosamine content was determined by HPLC.
  • the yield of shake flask fermentation is shown in Table 17.
  • the results showed that the yield of the control strain AT-005-02 was very low, and the yield of the mutant pTrc-nanEM gene cassette integrated recombinant strain AT-031-02 was significantly increased, and compared with the unmutated control strain AT-030- 02 production has also increased significantly.
  • This example describes the screening of a gene for the mutated UDP-N-acetylglucosamine-2-isomerase (WecB), which encodes a UDP-N-acetylglucosamine-2-isomerase with increased enzymatic activity.
  • WecB mutated UDP-N-acetylglucosamine-2-isomerase
  • a gene mutant encoding UDP-N-acetylglucosamine-2-isomerase having an increased enzyme activity was screened.
  • the cloned gene is amplified by error-prone PCR technology, and the gene is amplified by a DNA polymerase for amplification under conditions that cause high frequency mismatches to obtain high frequency mutations in the PCR product. .
  • Taq DNA polymerase does not have the 3'-5' proofreading property at high magnesium ion concentration (8mmol/L) and different concentrations of dNTP (where dATP and dGTP concentrations are 1.5mmol/L; dTTP and dCTP) The concentration was 3.0mmol/L) to control the frequency of random mutations, and random mutations were introduced into the target gene to construct a mutant library.
  • the template concentration of A260 was 1000 ng/mL, the enzyme concentration was 5 U/ ⁇ L, and the primer concentration was 100 ⁇ M.
  • Error-prone PCR reaction system 50 ⁇ l: 5 ⁇ l of PCR reaction buffer, 5 ⁇ l of dNTP (2.5 mM), 5 ⁇ l of MgCl 2 (2.5 mM), 1 ⁇ l of forward primer (Trcwec B-F, SEQ ID No. 51), reversed Primer (TrcwecB-R, SEQ ID No. 52) 1 ⁇ l, DNA template (wecB/pUC57) 0.1 ⁇ l, Taq DNA polymerase 0.5 ⁇ l, ddH 2 O 32.4 ⁇ l.
  • PCR procedure pre-denaturation at 96 °C for 4 min; denaturation at 94 °C for 1 min, annealing at 56 °C for 1 min, extension at 75 °C for 2 min, 45 cycles; final extension at 75 °C for 15 min, recovery of PCR product by gel recovery method (product size: 1.13 kb); 5 ⁇ l of the product was examined by 1% agarose gel electrophoresis and stored at -20 ° C until use.
  • the above PCR product was digested with restriction endonucleases Nco I and Hind III, and ligated with the pTrc99A plasmid digested with Nco I and Hind III endonuclease, and then transformed into E. coli AT-005 with the ligation product mixture. -02, a large number of cloned transformants were obtained, and a transformed mutant library was constructed.
  • 640 mutant clones were randomly picked and wild-type WecB/pTrc99A (AT-005-02) was used as control.
  • the cells were inoculated into 5 ml LB medium containing 50 ⁇ g/mL penicillin (Amp). After incubation at 37 ° C, 150 rpm for 18 h, the cells were collected by centrifugation at 10,000 rpm at 5 mM. After discarding the supernatant, resuspend in 1 ml of PBS (pH 7.5, 10 mmol/L) at 4 ° C, select 300 V under ice bath conditions, ultrasonically disrupt it for 10 min after 6 s of ultrasonic, and centrifuge for supernatant. The enzyme activity was measured as a crude extract of the enzyme.
  • Activity detection of UDP-N-acetylglucosamine-2-isomerase based on the conversion of UDP-N-acetyl-D-glucosamine to N-acetyl-D-aminomannose. That is, UDP-N-acetyl-D-glucosamine is reduced to the assay marker.
  • Enzyme unit definition The amount of enzyme required to reduce the equivalent of 1 ⁇ mol of UDP-N-acetyl-D-glucosamine per minute under enzymatic reaction conditions, defined as an enzyme activity unit (IU).
  • the specific operation was as follows: a 20 ml reaction system was used as an enzyme activity assay system, which contained 45 mmol/L phosphate buffer (pH 7.5), 10 mM MgCl2 and 100 nCi of UDPGlcNAc, and 5 mg of crude enzyme solution. The enzyme was incubated for 30 min in a 37 ° C water bath. The reaction was terminated by the addition of ethanol. The radioactive compound was separated by chromatography on paper. The radioactivity was measured by a liquid scintillation counter. The solvent system used was n-propanol: 1 M sodium acetate, pH 5.0: water (7:1:2). The activity unit of UDP-N-acetylglucosamine-2-isomerase was calculated based on how much UDPGlcNAc was converted to ManNAc.
  • the pTrc-wecBM gene cassette integrates into the nagE gene locus of E. coli
  • the nagE gene locus is the integration site of the pTrc-wecBM gene cassette on the chromosome.
  • the nagE gene locus is the integration site of the pTrc-wecBM gene cassette on the chromosome.
  • the amplification of the Trc promoter The wecBM fragment pTrc-wecBM, and the carrageenin resistance gene fragment flanked by the FLP recombinase recognition site (FRT site): FRT-Kanr-FRT (fKanrf), and spliced.
  • the primer for deleting the homologous arm of the nagE gene sequence was designed again, and the full-length linear DNA fragment of Red recombinant targeting was amplified by using the fragment fused with pTrc-wecBM and fKanrf as a template.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • the PCR product was separated and purified by 1% agarose gel electrophoresis.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • fKanrf size 1.28kb. Its nucleotide sequence is SEQ ID No. 3.
  • the PCR product was separated and purified by 1% agarose gel electrophoresis.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • the size of the second amplified fKanrf was 1.3 kb.
  • the PCR product was separated and purified by 1% agarose gel electrophoresis.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • Amplification product homology arm + pTrc-wecBM-fKanrf + homology arm.
  • the PCR product was separated by agarose gel electrophoresis, purified and recovered, and 100 ng/ ⁇ l of linear DNA full-length PCR fragment was obtained for Red recombinant targeting.
  • the pKD46 vector was transferred into the E. coli AT-004-02 strain. Then, a linear DNA fragment for targeting was prepared by electroporation, and positive clones were selected. Finally, the resistance gene is eliminated.
  • Competent preparation First, the Escherichia coli AT-004-02 stock solution stored at -20 ° C was inoculated in 10 ml LB liquid medium at 1:50-100, and cultured at 37 ° C, 225 rpm, shaking for 2-3 hours. . The culture solution was further added to a 10 ml centrifuge tube, 4000 g ⁇ 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl 2 5 ml for 5 min. Finally, it was centrifuged at 4000 g ⁇ 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl 2 5 ml. It was allowed to stand at -4 ° C for 12 hours and settled naturally.
  • Plasmid transformation 250 ⁇ l of naturally-precipitated cells were taken, and 5 ⁇ l of pKD46 plasmid was added at -4 ° C for 30 min. Then, in a 42 ° C water bath for 1.5 min, 0.7 ml of SOC medium was added, and the mixture was shaken at 30 ° C for 2 hours. Take 0.2 ml of bacterial solution and apply penicillin plate. Incubate overnight (12-16 hours) at 30 °C. Monoclones were picked, cultured in 5 ml of LB liquid medium, and plasmids were identified. Preserve positive strains for use.
  • strain AT-042-01 (AT-004-02, ⁇ nagE::pTrc-wecB-fKanrf) was prepared in the same manner as above.
  • pCP20 was transferred into the above-mentioned caramycin resistant clone, cultured at 30 ° C for 8 h, and then increased to 42 ° C overnight, heat-induced FLP recombinase expression, and the plasmid was gradually lost.
  • the antimony inoculum was plated on the antibiotic-free medium, and the grown monoclonal spot was picked onto the caramycin resistant plate, and the undeveloped clone in which the caramycin resistance gene had been deleted by the FLP recombinase. Clones with the disappearance of resistance to caramycin were identified by PCR using the identified primers.
  • strain AT-042-02 (AT-004-02, ⁇ nagE::pTrc-wecB) was prepared in the same manner as above.
  • the recombinant strains AT-042-02, AT-043-02 and the control strains in which the pTrc-wecB and pTrc-wecBM gene cassettes were integrated at the chromosome nagE gene locus were subjected to a shake flask fermentation test.
  • the monoclonal strain on the freshly cultured LB plate medium was inoculated into a 3 ml LB liquid medium test tube (13 x 150 mm), and cultured at 30 ° C, 225 rpm for about 8 hours. Then, the seed culture solution was taken, and 3% was inoculated into a 250 ml shake flask containing 50 ml of the fermentation broth (M9 medium).
  • the initial OD600 was about 0.5, cultured at 225 rpm at 37 ° C, and the fermentation cycle was 72 hours.
  • the pH of the fermentation broth was adjusted to 7.0 with 10 M NaOH.
  • 65% glucose solution was added in portions to maintain the glucose concentration at 20 g/L.
  • 1 ml of the fermentation broth was taken and centrifuged.
  • the N-acetyl-D-glucosamine content was determined by HPLC.
  • the yield of shake flask fermentation is shown in Table 18. The results showed that the yield of the control strain AT-005-02 was very low and was not detected. The yield of the recombinant pTrc-wecBM gene cassette integrated recombinant strain AT-043-02 was significantly improved, and compared with the unmutated control strain AT-042- 02 production has also increased significantly.

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Abstract

一种通过微生物发酵生产N-乙酰-D-氨基葡萄糖和/或D-氨基葡萄糖盐的方法，主要通过提高微生物中N-乙酰-D-氨基甘露糖激酶的作用，以更高效率和更高产量生产N-乙酰-D-氨基葡萄糖和/或D-氨基葡萄糖盐。

Description

微生物发酵生产N-乙酰-D-氨基葡萄糖和/或D-氨基葡萄糖盐的方法 技术领域
本发明属于微生物发酵领域。具体地说，本发明涉及微生物发酵生产N-乙酰-D-氨基葡萄糖以及进一步制备D-氨基葡萄糖盐的方法。
背景技术
N-乙酰-D-氨基葡萄糖(N-Acetyl-D-glucosamine，NAG或GlcNAc)，又称N-乙酰-氨基葡萄糖、N-乙酰葡糖胺，是生物细胞内许多重要多糖的基本组成单位，在生物体内具有重要生理功能。N-乙酰-D-氨基葡萄糖在临床上可用于：增强人体免疫系统的功能；抑制恶性肿瘤或纤维细胞的生长；有效治疗各种炎症；作为糖尿病患者低热量甜味剂和婴幼儿食品添加剂等。水解N-乙酰-D-氨基葡萄糖可用于生产D-氨基葡萄糖盐酸盐，后者可作为抗癌、防癌、降血脂、降血压的食品补充剂，是目前壳多糖保健食品系列中第三代保健功能性食品添加剂。此外，N-乙酰-D-氨基葡萄糖在医药行业中是合成抗癌药物氯脲霉素的主要原料；作为生化试剂还可用作抗细菌感染的免疫佐剂和人体抗流感病毒的活化剂。
当今世界各地，有大量患者忍受不同程度关节炎痛苦，仅美国就有3300万人忍受骨关节炎及关节疼痛，我国有1.5亿人以上。由于D-氨基葡萄糖产品在关节炎及关节疼痛的治疗与保健方面具有特别功效，因而得到广泛应用，已成为目前国外市场非常重要的原料药品种。
N-乙酰-D-氨基葡萄糖据认为具有与D-氨基葡萄糖相似的效果，已知摄取N-乙酰-D-氨基葡萄糖能诱导产生新的软骨，并阻止骨关节炎的发作，或在一些病例中，用来治疗骨关节炎。由于D-氨基葡萄糖有苦味，而N-乙酰-D-氨基葡萄糖有蔗糖50％的甜味，并且很容易被摄取，因此，N-乙酰-D-氨基葡萄糖作为D-氨基葡萄糖的替代物质已引起关注。
目前，国内外氨基葡萄糖的来源主要以生物提取为主。生物提取主要是从虾蟹壳中提取甲壳素或壳聚糖，再经浓盐酸水解制备，或用柠檬酸渣经酸碱提取而得。年生产量在2万吨左右。但是，用虾蟹壳提取时，每获得1吨产品将产生大量废渣和100吨以上废水；用柠檬酸渣提取时，每获得1吨产品将产生30-50吨废酸渣，是高污染工艺，在许多地方已禁止使用。而且，来自水产品壳 提取的氨基葡萄糖对许多有水产品过敏的患者不适宜，有水产品过敏的人使用后可能会造成严重的过敏问题，甚至危及生命。此外，生物提取纯化工艺复杂，产品有鱼腥味，不稳定。另外，由于环境污染，从虾蟹壳中提取得到的氨基葡萄糖不可避免的受到重金属污染。
因此，生物提取方法生产氨基葡萄糖，从数量和质量上都难以满足人们的需求，须开辟新的替代方法。若采用化学合成方法来制备，更存在如下三个缺点：生产成本高；严重的环境污染；有安全性隐患。该方法目前国内外已不采用。相比较而言，微生物发酵法生产氨基葡萄糖是一条良好的途径，微生物发酵法是以葡萄糖和无机盐为原料，选用优良菌种进行液体发酵，并经分离、浓缩、纯化直接生产氨基葡萄糖。生产过程中无有害气体产生。发酵法生产的氨基葡萄糖无鱼腥味，生产不受资源限制。而且利用代谢工程进行菌种改良，产量高，工业化大生产潜力大。因此，微生物发酵法生产氨基葡萄糖在技术工艺上有重大变革，替代传统的生物提取，不仅在成本方面占有优势，在减少三废污染方面也具有一定的环保贡献。
微生物发酵生产N-乙酰-D-氨基葡萄糖常规方法包括：涉及用微生物产生的酶降解来自虾壳原料产生的甲壳素生产N-乙酰-D-氨基葡萄糖的方法(例如，US5998173，“Process for producing N-acetyl-D-glucosamine”)；用微生物(木霉菌)产生的酶酶解或用酸部分水解纯化来自真菌渣(如柠檬酸发酵使用的黑曲霉菌的菌渣)的甲壳素生产N-乙酰-D-氨基葡萄糖的方法(例如，US20030073666A1，“N-acetyl-D-glucosamine and process for producing N-acetyl-D-glucosamine”)；用木霉菌直接使用葡萄糖为碳源，不需要来自真菌渣或虾壳产生的甲壳素和壳多糖寡糖为碳源，发酵生产N-乙酰-D-氨基葡萄糖的方法(例如，US20110059489A1，“Method for fermentative production of N-acetyl-D-glucosamine by microorganism”)；通过培养绿藻病毒(Chlorovirus)感染的小球藻细胞或者导入了源自绿藻病毒的基因的重组大肠杆菌来生产N-乙酰-D-氨基葡萄糖的方法(例如，JP2004283144A，“Method for producing glucosamine and N-acetylglucosamine”)；使用遗传修饰的微生物，特别是遗传修饰的大肠杆菌，发酵生产D-氨基葡萄糖或N-乙酰-D-氨基葡萄糖的方法(例如，US6,372,457，“Process and materials for production of glucosamine”；WO2004/003175，“Process and materials for production of glucosamine and N-acetylglucosamine”)。
用微生物或微生物产生的酶来降解源自甲壳类动物如蟹、虾类的壳的甲壳素生产N-乙酰-D-氨基葡萄糖的方法，较为传统，存在产量低、成本高和动物源不足等问题。通过培养用绿藻病毒感染的小球藻细胞生产N-乙酰-D-氨基葡萄糖的方法，涉及压碎细胞获取N-乙酰-D-氨基葡萄糖的步骤，存在操作复杂等问题。用木霉菌直接使用葡萄糖为碳源发酵生产N-乙酰-D-氨基葡萄糖的方法，具有不需要使用来自甲壳类动物壳或真菌渣产生的甲壳素或壳多糖寡糖等碳源的优点，但木霉菌等真菌发酵温度低(27℃)、时间长(10天)、产量偏低(15mg/ml)，从而存在生产周期长、成本高、易污染杂菌等缺点，严重限制了该方法的工业化应用。
显然，针对氨基葡萄糖不断增长的市场需求，用遗传修饰的微生物生产N-乙酰-D-氨基葡萄糖，是实现大规模工业化生产的一条有应用前景的重要方法。而新的遗传修饰的微生物可通过基因重组、基因转移、基因突变、基因删除、基因超表达、代谢途径改变等许多方式获得。
美国专利US6,372,457中公开了通过微生物发酵生产D-氨基葡萄糖的方法和材料。该发明包括用于该发明生产氨基葡萄糖之方法的遗传修饰的微生物，以及重组核酸分子和由所述重组核酸分子产生的蛋白质。该发明所述遗传修饰的微生物，主要针对能提高氨基葡萄糖-6-磷酸合酶活性的遗传修饰，包括多种基因突变或氨基酸缺失和取代。但该专利未涉及通过内源性氨基葡萄糖-6-磷酸合酶基因启动子更换或缺失等改变，导致氨基葡萄糖-6-磷酸合酶活性的提高或降低。另外，该专利主要通过氨基葡萄糖-6-磷酸合酶的遗传修饰生产的目的产物仅为D-氨基葡萄糖，未涉及N-乙酰-D-氨基葡萄糖生产。而且，由于D-氨基葡萄糖在发酵液中很不稳定，降解产物可能对微生物有毒性，这种通过遗传修饰生产D-氨基葡萄糖的方式，其产量很低，实际应用存在局限性。
WO2004/003175中公开了用于生产D-氨基葡萄糖和N-乙酰-D-氨基葡萄糖的生物合成方法。该方法通过发酵基因修饰微生物以产生氨基葡萄糖和/或N-乙酰-D-氨基葡萄糖。该发明还公开了用于生产氨基葡萄糖和N-乙酰-D-氨基葡萄糖的基因修饰微生物。此外，该发明还描述了回收通过发酵法生产的N-乙酰-D-氨基葡萄糖的方法，包括产生高纯度N-乙酰-D-氨基葡萄糖的方法。该发明还公开了由N-乙酰-D-氨基葡萄糖生产D-氨基葡萄糖的方法。该发明所述遗传修饰的微生物,主要针对增加氨基葡萄糖-6-磷酸乙酰转移酶活性的遗传修饰。酵母氨 基葡萄糖-6-磷酸乙酰转移酶基因(GNA1)在大肠杆菌中表达可将氨基葡萄糖-6-磷酸乙酰化为乙酰氨基葡萄糖-6-磷酸，在先前的文献中也已有报道和证实(Mio T1,Yamada-Okabe T,Arisawa M,Yamada-Okabe H:Saccharomyces cerevisiae GNA1,an essential gene encoding a novel acetyltransferase involved in UDP-N-acetylglucosamine synthesis,J Biol Chem.,1999Jan 1；274(1):424-9.)。
发明内容
本发明针对N-乙酰-D-氨基葡萄糖的代谢途径，采用新的遗传修饰方法改造微生物，并利用该微生物以更高效率和更高产量生产N-乙酰-D-氨基葡萄糖(GlcNAc)和/或D-氨基葡萄糖盐。
具体而言，本发明通过提高微生物中N-乙酰-D-氨基甘露糖激酶(N-acetylmannosamine kinase，NanK)的作用，加强微生物中N-乙酰-D-氨基甘露糖(N-acetyl-D-mannosamine，ManNAc)磷酸化为N-乙酰-D-氨基甘露糖-6-磷酸(N-acetyl-D-mannosamine-6-phosphate，ManNAc-6-P)，从而使该微生物以更高效率和更高产量生产N-乙酰-D-氨基葡萄糖(GlcNAc)和/或D-氨基葡萄糖盐。
本发明在上述内容的基础上还进一步涉及如下内容中的一种或多种：
1.通过提高微生物中N-乙酰-D-氨基甘露糖-6-磷酸异构酶(N-acetylmannosamine-6-phosphate epimerase，NanE)的作用，加强微生物中N-乙酰-D-氨基甘露糖-6-磷酸(N-acetyl-D-mannosamine-6-phosphate，ManNAc-6-P)转变为N-乙酰-D-氨基葡萄糖-6-磷酸(N-acetyl-D-glucosamine-6-phosphate，GlcNAc-6-P)，排出细胞外成为N-乙酰-D-氨基葡萄糖(GlcNAc)，从而使该微生物以更高效率和更高产量生产N-乙酰-D-氨基葡萄糖(GlcNAc)和/或D-氨基葡萄糖盐。
2.通过提高微生物中D-氨基葡萄糖-6-磷酸脱氨酶(D-Glucosamine-6-phosphate deaminase，NagB)的作用，优选同时降低氨基葡萄糖-6-磷酸合成酶(Glucosamine-6-phosphate synthase，GlmS，又称作L-谷氨酰胺-6-磷酸果糖转氨酶，L-glutamine:D-fructose-6-phosphate aminotransferase)的作用，加强微生物中葡萄糖-6-磷酸(Glucose-6-phosphate，Glc-6-P)氨基化为D-氨基葡萄糖-6-磷酸(D-glucosamine-6-phosphate，GlcN-6-P)。D-氨基葡萄糖 -6-磷酸脱氨酶(NagB)所催化的反应是可逆的，氨基葡萄糖-6-磷酸合成酶(GlmS)所催化的反应虽是不可逆的，但有严重的产物抑制问题，当NagB催化的反应向由Glc-6-P生成GlcN-6-P的方向进行时，其和GlmS功能相同，可替代GlmS，而且无产物抑制问题。提高NagB的作用，加快NagB催化反应向由Glc-6-P生成GlcN-6-P的方向进行，优选同时降低GlmS的作用，减弱GlmS的产物抑制问题，达到增加GlcN-6-P的目的，从而使该微生物以更高效率和更高产量生产N-乙酰-D-氨基葡萄糖(GlcNAc)和/或D-氨基葡萄糖盐。
3.通过提高微生物中氨基葡萄糖-6-磷酸合成酶(Glucosamine-6-phosphate synthase，GlmS，又称作L-谷氨酰胺-6-磷酸果糖转氨酶，L-glutamine:D-fructose-6-phosphate aminotransferase)的作用，并同时降低D-氨基葡萄糖-6-磷酸脱氨酶(D-Glucosamine-6-phosphate deaminase，NagB)的作用，加强微生物中葡萄糖-6-磷酸(Glucose-6-phosphate，Glc-6-P)氨基化为D-氨基葡萄糖-6-磷酸(D-glucosamine-6-phosphate，GlcN-6-P)。D-氨基葡萄糖-6-磷酸脱氨酶(NagB)所催化的反应是可逆的，当NagB催化的反应向由GlcN-6-P生成Glc-6-P的方向进行时，其和GlmS功能相反，会抵消GlmS的作用。降低NagB的作用，阻止NagB催化反应向由GlcN-6-P生成Glc-6-P的方向进行，并同时超表达GlmS，加快GlmS催化Glc-6-P氨基化为GlcN-6-P，达到增加GlcN-6-P的目的，从而使该微生物以更高效率和更高产量生产N-乙酰-D-氨基葡萄糖(GlcNAc)和/或D-氨基葡萄糖盐。
4.通过提高微生物中UDP-N-乙酰-D-氨基葡萄糖-2-异构酶(UDP-N-acetyl-D-glucosamine-2-epimerase，WecB)的作用，加强微生物中UDP-N-乙酰-D-氨基葡萄糖(UDP-N-acetyl-D-glucosamine，UDP-GlcNAc)转变为N-乙酰-D-氨基甘露糖(N-acetyl-D-mannosamine，ManNAc)，从而使该微生物以更高效率和更高产量生产N-乙酰-D-氨基葡萄糖(GlcNAc)和/或D-氨基葡萄糖盐。
5.降低微生物中与目标产物再次被摄入细胞内或有益中间产物被降解相关的酶或者蛋白的作用，提高微生物中糖转化率和N-乙酰-D-氨基葡萄糖产量，从而使该微生物以更高效率和更高产量生产N-乙酰-D-氨基葡萄糖(GlcNAc)和/或D-氨基葡萄糖盐。包括但不限于如下内容中的一种或多种：
(1)降低微生物中甘露糖转运蛋白EIIM，P/IIIman(Mannose transporter EIIM， P/IIIMan，ManXYZ)的作用，阻止N-乙酰-D-氨基葡萄糖(GlcNAc)等己糖转运回细胞内降解。
(2)降低微生物中N-乙酰神经氨酸裂解酶(N-acetylneuraminate lyase，NanA)的作用，阻止微生物中N-乙酰-D-氨基甘露糖(N-acetyl-D-mannosamine，ManNAc)降解。
(3)降低微生物中N-乙酰-D-氨基葡萄糖-6-磷酸脱乙酰酶(N-acetylglucosamine-6-phosphate deacetylase，NagA)的作用，阻止微生物中N-乙酰-D-氨基葡萄糖-6-磷酸(N-acetyl-D-glucosamine-6-phosphate，GlcNAc-6-P)转变为D-氨基葡萄糖-6-磷酸(D-glucosamine-6-phosphate，GlcN-6-P)。
(4)降低微生物中N-乙酰-D-氨基葡萄糖特异酶IINag(N-acetylglucosamine specific enzyme IINag，NagE)的作用，阻止N-乙酰-D-氨基葡萄糖(GlcNAc)被转入微生物细胞内降解。
(5)通过提高微生物中磷酸葡糖胺变位酶(phosphoglucosamine mutase，GlmM)的作用，加强微生物中D-氨基葡萄糖-6-磷酸(D-glucosamine-6-phosphate，GlcN-6-P)转变为D-氨基葡萄糖-1-磷酸(D-glucosamine-1-phosphate，GlcN-1-P)。
(6)通过提高微生物中双功能酶N-乙酰-D-氨基葡萄糖-1-磷酸尿苷酰转移酶(bifunctional N-acetyl glucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyl transferase，GlmU)的作用，加强微生物中D-氨基葡萄糖-1-磷酸(D-glucosamine-1-phosphate，GlcN-1-P)转变为N-乙酰-D-氨基葡萄糖-1-磷酸(N-acetyl-D-glucosamine-1-phosphate，GlcNAc-1-P)，并进一步转变为UDP-N-乙酰-D-氨基葡萄糖(UDP-N-acetyl-D-glucosamine，UDP-GlcNAc)。
根据本发明的一个实施方案，本发明涉及一种通过微生物发酵生产N-乙酰-D-氨基葡萄糖(GlcNAc)和/或D-氨基葡萄糖盐的方法，该方法包括：
A)在发酵培养基中培养微生物，所述微生物包含至少一种能提高微生物中N-乙酰-D-氨基甘露糖激酶(NanK)作用的遗传修饰；和
B)收集从培养步骤A)中产生的N-乙酰-D-氨基葡萄糖(GlcNAc)。
优选，进一步包括C)由N-乙酰-D-氨基葡萄糖(GlcNAc)脱乙酰化得到D-氨基葡萄糖盐。
在本发明中，提高微生物中N-乙酰-D-氨基甘露糖激酶(NanK)作用的遗传修饰选自a)微生物中N-乙酰-D-氨基甘露糖激酶(NanK)的酶活性增加；和/或b)微生物中N-乙酰-D-氨基甘露糖激酶(NanK)被过量表达。
本领域技术人员可以理解，为提高微生物中N-乙酰-D-氨基甘露糖激酶(NanK)的作用，可以通过筛选编码具有N-乙酰-D-氨基甘露糖激酶(NanK)的酶活性增加的基因突变体来实现。筛选NanK基因突变体可以通过易错PCR技术得到高频突变基因来完成。为提高微生物中N-乙酰-D-氨基甘露糖激酶(NanK)的作用，也可以通过增加其基因拷贝数、更换比天然启动子有更高表达水平的启动子等方式过量表达N-乙酰-D-氨基甘露糖激酶(NanK)来实现。在具体的实施方案中，微生物用至少一种包含至少一种能提高微生物中N-乙酰-D-氨基甘露糖激酶(NanK)作用的遗传修饰的重组核酸分子转化。
在一个优选的实施方案中，微生物用至少一种包含编码N-乙酰-D-氨基甘露糖激酶(NanK)的核酸序列的重组核酸分子转化。
在一个方面，编码N-乙酰-D-氨基甘露糖激酶(NanK)的核酸序列含有至少一种增加N-乙酰-D-氨基甘露糖激酶(NanK)的酶活性的遗传修饰。优选，所述遗传修饰包括在对应于氨基酸序列SEQ ID NO：17的下述位置处的取代中的一种或多种：第36位赖氨酸被精氨酸取代、第103位异亮氨酸被蛋氨酸取代和第223位精氨酸被丝氨酸取代。进一步优选，编码所述N-乙酰-D-氨基甘露糖激酶(NanK)的核酸序列为SEQ ID NO：26；所述N-乙酰-D-氨基甘露糖激酶(NanK)的氨基酸序列为SEQ ID NO：27。
在另一个方面，所述的N-乙酰-D-氨基甘露糖激酶(NanK)具有与SEQ ID NO：17的氨基酸序列至少约30％相同，优选至少约50％相同，进一步优选至少约70％相同，进一步优选至少约80％相同，更进一步优选至少约90％相同，最优选至少约95％相同的氨基酸序列，其中所述的N-乙酰-D-氨基甘露糖激酶(NanK)具有酶活性。
在另一个方面，所述的N-乙酰-D-氨基甘露糖激酶(NanK)具有SEQ ID NO：17的氨基酸序列。
在另一个方面，重组核酸分子中编码N-乙酰-D-氨基甘露糖激酶(NanK)的基因拷贝数增加。
在另一个方面，重组核酸分子中包含内源性天然启动子、具有比内源性天 然启动子更高表达水平的启动子、增强子、融合序列等。优选，重组核酸分子中包含具有比内源性天然启动子更高表达水平的启动子，例如HCE启动子、gap启动子、trc启动子、T7启动子等；更优选，重组核酸分子中包含trc启动子。trc启动子是trp启动子和lac启动子的拼合启动子，具有比trp更高的转录效率和受lacI阻遏蛋白调控的强启动子特性。
在本发明中，重组核酸分子转化微生物，选自游离型(即重组核酸分子被装入质粒中)和整合型(即重组核酸分子被整合到微生物的基因组中)。优选，重组核酸分子被整合到微生物的基因组中。
在另一个优选的实施方案中，微生物包括至少一种对编码N-乙酰-D-氨基甘露糖激酶(NanK)的基因的内源性天然启动子的遗传修饰。优选，编码N-乙酰-D-氨基甘露糖激酶(NanK)的基因的内源性天然启动子被具有更高表达水平的启动子替换，例如HCE启动子、gap启动子、trc启动子、T7启动子等；更优选，编码N-乙酰-D-氨基甘露糖激酶(NanK)的基因的内源性天然启动子被trc启动子替换。
根据本发明的优选实施方案，所述微生物进一步包含下述遗传修饰中的一种或多种：
(1)包含至少一种能提高微生物中N-乙酰-D-氨基甘露糖-6-磷酸异构酶(NanE)作用的遗传修饰；
(2)包含至少一种能提高微生物中D-氨基葡萄糖-6-磷酸脱氨酶(NagB)作用的遗传修饰，优选同时包含至少一种能降低氨基葡萄糖-6-磷酸合成酶(GlmS)作用的遗传修饰；
(3)包含至少一种能提高微生物中氨基葡萄糖-6-磷酸合成酶(GlmS)作用的遗传修饰，并同时包含至少一种能降低D-氨基葡萄糖-6-磷酸脱氨酶(NagB)作用的遗传修饰；
(4)包含至少一种能提高微生物中UDP-N-乙酰-D-氨基葡萄糖-2-异构酶(WecB)作用的遗传修饰。
在上述第(1)个方面中，提高微生物中N-乙酰-D-氨基甘露糖-6-磷酸异构酶(NanE)作用的遗传修饰选自a)微生物中N-乙酰-D-氨基甘露糖-6-磷酸异构酶(NanE)的酶活性增加；和/或b)微生物中N-乙酰-D-氨基甘露糖-6-磷酸异 构酶(NanE)被过量表达。
本领域技术人员可以理解，为提高微生物中N-乙酰-D-氨基甘露糖-6-磷酸异构酶(NanE)的作用，可以通过筛选编码具有N-乙酰-D-氨基甘露糖-6-磷酸异构酶(NanE)的酶活性增加的基因突变体来实现。筛选NanE基因突变体可以通过易错PCR技术得到高频突变基因来完成。为提高微生物中N-乙酰-D-氨基甘露糖-6-磷酸异构酶(NanE)的作用，也可以通过增加其基因拷贝数、更换比天然启动子有更高表达水平的启动子等方式过量表达N-乙酰-D-氨基甘露糖-6-磷酸异构酶(NanE)来实现。在具体的实施方案中，微生物用至少一种包含至少一种能提高微生物中N-乙酰-D-氨基甘露糖-6-磷酸异构酶(NanE)作用的遗传修饰的重组核酸分子转化。
在一个优选的实施方案中，微生物用至少一种包含编码N-乙酰-D-氨基甘露糖-6-磷酸异构酶(NanE)的核酸序列的重组核酸分子转化。
在一个方面，编码N-乙酰-D-氨基甘露糖-6-磷酸异构酶(NanE)的核酸序列含有至少一种增加N-乙酰-D-氨基甘露糖-6-磷酸异构酶(NanE)的酶活性的遗传修饰。优选，所述遗传修饰包括在对应于氨基酸序列SEQ ID NO：29的下述位置处的取代中的一种或两种：第133位半胱氨酸被精氨酸取代和第187位酪氨酸被组氨酸取代。进一步优选，编码所述N-乙酰-D-氨基甘露糖-6-磷酸异构酶(NanE)的核酸序列为SEQ ID NO：56；所述N-乙酰-D-氨基甘露糖-6-磷酸异构酶(NanE)的氨基酸序列为SEQ ID NO：57。
在另一个方面，所述的N-乙酰-D-氨基甘露糖-6-磷酸异构酶(NanE)具有与SEQ ID NO：29的氨基酸序列至少约30％相同，优选至少约50％相同，进一步优选至少约70％相同，进一步优选至少约80％相同，更进一步优选至少约90％相同，最优选至少约95％相同的氨基酸序列，其中所述的N-乙酰-D-氨基甘露糖-6-磷酸异构酶(NanE)具有酶活性。
在另一个方面中，所述的N-乙酰-D-氨基甘露糖-6-磷酸异构酶(NanE)具有SEQ ID NO：29的氨基酸序列。
在另一个方面，重组核酸分子中编码N-乙酰-D-氨基甘露糖-6-磷酸异构酶(NanE)的基因拷贝数增加。
在另一个方面，重组核酸分子中包含内源性天然启动子、具有比内源性天然启动子更高表达水平的启动子、增强子、融合序列等。优选，重组核酸分子 中包含具有比内源性天然启动子更高表达水平的启动子，例如HCE启动子、gap启动子、trc启动子、T7启动子等；更优选，重组核酸分子中包含trc启动子。
在本发明中，重组核酸分子转化微生物，选自游离型(即重组核酸分子被装入质粒中)和整合型(即重组核酸分子被整合到微生物的基因组中)。优选，重组核酸分子被整合到微生物的基因组中。
在另一个优选的实施方案中，微生物包括至少一种对编码N-乙酰-D-氨基甘露糖-6-磷酸异构酶(NanE)的基因的内源性天然启动子的遗传修饰。优选，编码N-乙酰-D-氨基甘露糖-6-磷酸异构酶(NanE)的基因的内源性天然启动子被具有更高表达水平的启动子替换，例如HCE启动子、gap启动子、trc启动子、T7启动子等；更优选，编码N-乙酰-D-氨基甘露糖-6-磷酸异构酶(NanE)的基因的内源性天然启动子被trc启动子替换。
在上述第(2)个方面中，提高微生物中D-氨基葡萄糖-6-磷酸脱氨酶(NagB)作用的遗传修饰选自a)微生物中D-氨基葡萄糖-6-磷酸脱氨酶(NagB)的酶活性增加；和/或b)微生物中D-氨基葡萄糖-6-磷酸脱氨酶(NagB)被过量表达。
本领域技术人员可以理解，为提高微生物中D-氨基葡萄糖-6-磷酸脱氨酶(NagB)的作用，可以通过筛选编码具有D-氨基葡萄糖-6-磷酸脱氨酶(NagB)的酶活性增加的基因突变体来实现。筛选NagB基因突变体可以通过易错PCR技术得到高频突变基因来完成。为提高微生物中D-氨基葡萄糖-6-磷酸脱氨酶(NagB)的作用，也可以通过增加其基因拷贝数、更换比天然启动子有更高表达水平的启动子等方式过量表达D-氨基葡萄糖-6-磷酸脱氨酶(NagB)来实现。在具体的实施方案中，微生物用至少一种包含至少一种能提高微生物中D-氨基葡萄糖-6-磷酸脱氨酶(NagB)作用的遗传修饰的重组核酸分子转化。
在一个优选的实施方案中，微生物用至少一种包含编码D-氨基葡萄糖-6-磷酸脱氨酶(NagB)的核酸序列的重组核酸分子转化。
在一个方面，编码D-氨基葡萄糖-6-磷酸脱氨酶(NagB)的核酸序列含有至少一种增加D-氨基葡萄糖-6-磷酸脱氨酶(NagB)的酶活性的遗传修饰。
在另一个方面，重组核酸分子中编码D-氨基葡萄糖-6-磷酸脱氨酶(NagB)的基因拷贝数增加。
在另一个方面，重组核酸分子中包含内源性天然启动子、具有比内源性天然启动子更高表达水平的启动子、增强子、融合序列等。优选，重组核酸分子 中包含具有比内源性天然启动子更高表达水平的启动子，例如HCE启动子、gap启动子、trc启动子、T7启动子等；更优选，重组核酸分子中包含trc启动子。
在本发明中，重组核酸分子转化微生物，选自游离型(即重组核酸分子被装入质粒中)和整合型(即重组核酸分子被整合到微生物的基因组中)。优选，重组核酸分子被整合到微生物的基因组中。
在另一个优选的实施方案中，微生物包括至少一种对编码D-氨基葡萄糖-6-磷酸脱氨酶(NagB)的基因的内源性天然启动子的遗传修饰。优选，编码D-氨基葡萄糖-6-磷酸脱氨酶(NagB)的基因的内源性天然启动子被具有更高表达水平的启动子替换，例如HCE启动子、gap启动子、trc启动子、T7启动子等；更优选，编码D-氨基葡萄糖-6-磷酸脱氨酶(NagB)的基因的内源性天然启动子被trc启动子替换。
在本发明中，降低微生物中氨基葡萄糖-6-磷酸合成酶(GlmS)作用的遗传修饰选自a)微生物中氨基葡萄糖-6-磷酸合成酶(GlmS)的酶活性降低；和/或b)微生物中氨基葡萄糖-6-磷酸合成酶(GlmS)的表达减少，包括但不限于：编码微生物中氨基葡萄糖-6-磷酸合成酶(GlmS)的内源性基因的部分或完全缺失、或部分或完全失活，和/或编码微生物中氨基葡萄糖-6-磷酸合成酶(GlmS)基因的内源性天然启动子的部分或完全缺失、或部分或完全失活。优选，降低微生物中氨基葡萄糖-6-磷酸合成酶(GlmS)作用的遗传修饰为编码微生物中氨基葡萄糖-6-磷酸合成酶(GlmS)基因的内源性天然启动子完全缺失，即被删除。
在具体的实施方案中，微生物用至少一种包含至少一种能降低微生物中氨基葡萄糖-6-磷酸合成酶(GlmS)作用的遗传修饰的重组核酸分子转化。
在上述第(3)个方面中，提高微生物中氨基葡萄糖-6-磷酸合成酶(GlmS)作用的遗传修饰选自a)微生物中氨基葡萄糖-6-磷酸合成酶(GlmS)的酶活性增加；和/或b)微生物中氨基葡萄糖-6-磷酸合成酶(GlmS)被过量表达。
本领域技术人员可以理解，为提高微生物中氨基葡萄糖-6-磷酸合成酶(GlmS)的作用，可以通过筛选编码具有氨基葡萄糖-6-磷酸合成酶(GlmS)的酶活性增加的基因突变体来实现。筛选GlmS基因突变体可以通过易错PCR技术得到高频突变基因来完成。为提高微生物中氨基葡萄糖-6-磷酸合成酶(GlmS)的作用，也可以通过增加其基因拷贝数、更换比天然启动子有更高表达水平的启动子等方式过量表达氨基葡萄糖-6-磷酸合成酶(GlmS)来实现。在具体的实 施方案中，微生物用至少一种包含至少一种能提高微生物中氨基葡萄糖-6-磷酸合成酶(GlmS)作用的遗传修饰的重组核酸分子转化。
在一个优选的实施方案中，微生物用至少一种包含编码氨基葡萄糖-6-磷酸合成酶(GlmS)的核酸序列的重组核酸分子转化。
在一个方面，编码氨基葡萄糖-6-磷酸合成酶(GlmS)的核酸序列含有至少一种增加氨基葡萄糖-6-磷酸合成酶(GlmS)的酶活性的遗传修饰。
在另一个方面，重组核酸分子中编码氨基葡萄糖-6-磷酸合成酶(GlmS)的基因拷贝数增加。
在另一个方面，重组核酸分子中包含内源性天然启动子、具有比内源性天然启动子更高表达水平的启动子、增强子、融合序列等。优选，重组核酸分子中包含具有比内源性天然启动子更高表达水平的启动子，例如HCE启动子、gap启动子、trc启动子、T7启动子等；更优选，重组核酸分子中包含trc启动子。
在本发明中，重组核酸分子转化微生物，选自游离型(即重组核酸分子被装入质粒中)和整合型(即重组核酸分子被整合到微生物的基因组中)。优选，重组核酸分子被整合到微生物的基因组中。
在另一个优选的实施方案中，微生物包括至少一种对编码氨基葡萄糖-6-磷酸合成酶(GlmS)的基因的内源性天然启动子的遗传修饰。优选，编码氨基葡萄糖-6-磷酸合成酶(GlmS)的基因的内源性天然启动子被具有更高表达水平的启动子替换，例如HCE启动子、gap启动子、trc启动子、T7启动子等；更优选，编码氨基葡萄糖-6-磷酸合成酶(GlmS)的基因的内源性天然启动子被trc启动子替换。
在本发明中，降低微生物中D-氨基葡萄糖-6-磷酸脱氨酶(NagB)作用的遗传修饰选自a)微生物中D-氨基葡萄糖-6-磷酸脱氨酶(NagB)的酶活性降低；和/或b)微生物中D-氨基葡萄糖-6-磷酸脱氨酶(NagB)的表达减少，包括但不限于：编码微生物中D-氨基葡萄糖-6-磷酸脱氨酶(NagB)的内源性基因的部分或完全缺失、或部分或完全失活，和/或编码微生物中D-氨基葡萄糖-6-磷酸脱氨酶(NagB)基因的内源性天然启动子的部分或完全缺失、或部分或完全失活。优选，降低微生物中D-氨基葡萄糖-6-磷酸脱氨酶(NagB)作用的遗传修饰为编码微生物中D-氨基葡萄糖-6-磷酸脱氨酶(NagB)基因的内源性天然启动子完全缺失，即被删除。
在具体的实施方案中，微生物用至少一种包含至少一种能降低微生物中D-氨基葡萄糖-6-磷酸脱氨酶(NagB)作用的遗传修饰的重组核酸分子转化。
在上述第(4)个方面中，提高微生物中UDP-N-乙酰-D-氨基葡萄糖-2-异构酶(WecB)作用的遗传修饰选自a)微生物中UDP-N-乙酰-D-氨基葡萄糖-2-异构酶(WecB)的酶活性增加；和/或b)微生物中UDP-N-乙酰-D-氨基葡萄糖-2-异构酶(WecB)被过量表达。
本领域技术人员可以理解，为提高微生物中UDP-N-乙酰-D-氨基葡萄糖-2-异构酶(WecB)的作用，可以通过筛选编码具有UDP-N-乙酰-D-氨基葡萄糖-2-异构酶(WecB)的酶活性增加的基因突变体来实现。筛选WecB基因突变体可以通过易错PCR技术得到高频突变基因来完成。为提高微生物中UDP-N-乙酰-D-氨基葡萄糖-2-异构酶(WecB)的作用，也可以通过增加其基因拷贝数、更换比天然启动子有更高表达水平的启动子等方式过量表达UDP-N-乙酰-D-氨基葡萄糖-2-异构酶(WecB)来实现。在具体的实施方案中，微生物用至少一种包含至少一种能提高微生物中UDP-N-乙酰-D-氨基葡萄糖-2-异构酶(WecB)作用的遗传修饰的重组核酸分子转化。
在一个优选的实施方案中，微生物用至少一种包含编码UDP-N-乙酰-D-氨基葡萄糖-2-异构酶(WecB)的核酸序列的重组核酸分子转化。
在一个方面，编码UDP-N-乙酰-D-氨基葡萄糖-2-异构酶(WecB)的核酸序列含有至少一种增加UDP-N-乙酰-D-氨基葡萄糖-2-异构酶(WecB)的酶活性的遗传修饰。优选，所述遗传修饰包括在对应于氨基酸序列SEQ ID NO：50的下述位置处的取代中的一种或多种：第34位半胱氨酸被丝氨酸取代、第145位组氨酸被天冬氨酸取代、第226位半胱氨酸被苯丙氨酸取代和245位缬氨酸被甘氨酸取代；更优选，编码UDP-N-乙酰-D-氨基葡萄糖-2-异构酶(WecB)的核酸序列为SEQ ID NO：58；所述UDP-N-乙酰-D-氨基葡萄糖-2-异构酶(WecB)的氨基酸序列为SEQ ID NO：59。
在另一个方面，所述的UDP-N-乙酰-D-氨基葡萄糖-2-异构酶(WecB)具有与SEQ ID NO：50的氨基酸序列至少约30％相同，优选至少约50％相同，进一步优选至少约70％相同，进一步优选至少约80％相同，更进一步优选至少约90％相同，最优选至少约95％相同的氨基酸序列，其中所述的UDP-N-乙酰-D-氨基葡萄糖-2-异构酶(WecB)具有酶活性。
在另一个方面中，所述的UDP-N-乙酰-D-氨基葡萄糖-2-异构酶(WecB)具有SEQ ID NO：50的氨基酸序列。
在另一个方面，重组核酸分子中编码UDP-N-乙酰-D-氨基葡萄糖-2-异构酶(WecB)的基因拷贝数增加。
在另一个方面，重组核酸分子中包含内源性天然启动子、具有比内源性天然启动子更高表达水平的启动子、增强子、融合序列等。优选，重组核酸分子中包含具有比内源性天然启动子更高表达水平的启动子，例如HCE启动子、gap启动子、trc启动子、T7启动子等；更优选，重组核酸分子中包含trc启动子。
在本发明中，重组核酸分子转化微生物，选自游离型(即重组核酸分子被装入质粒中)和整合型(即重组核酸分子被整合到微生物的基因组中)。优选，重组核酸分子被整合到微生物的基因组中。
在另一个优选的实施方案中，微生物包括至少一种对编码UDP-N-乙酰-D-氨基葡萄糖-2-异构酶(WecB)的基因的内源性天然启动子的遗传修饰。优选，编码UDP-N-乙酰-D-氨基葡萄糖-2-异构酶(WecB)的基因的内源性天然启动子被具有更高表达水平的启动子替换，例如HCE启动子、gap启动子、trc启动子、T7启动子等；更优选，编码UDP-N-乙酰-D-氨基葡萄糖-2-异构酶(WecB)的基因的内源性天然启动子被trc启动子替换。
根据本发明的优选实施方案，所述微生物进一步包含下述遗传修饰中的一种或多种：
(1)包含至少一种能降低微生物中甘露糖转运蛋白EIIM，P/IIIman(ManXYZ)作用的遗传修饰；
(2)包含至少一种能降低微生物中N-乙酰神经氨酸裂解酶(NanA)作用的遗传修饰；
(3)包含至少一种能降低微生物中N-乙酰-D-氨基葡萄糖-6-磷酸脱乙酰酶(NagA)作用的遗传修饰；
(4)包含至少一种能降低微生物中N-乙酰-D-氨基葡萄糖特异酶IINag(NagE)作用的遗传修饰；
(5)包含至少一种能提高微生物中磷酸葡糖胺变位酶(GlmM)作用的遗传修饰；
(6)包含至少一种能提高微生物中双功能酶N-乙酰-D-氨基葡萄糖-1-磷酸尿苷酰转移酶(GlmU)作用的遗传修饰。
在上述第(1)个方面中，降低微生物中甘露糖转运蛋白EIIM，P/IIIman(ManXYZ)作用的遗传修饰包括但不限于：编码微生物中甘露糖转运蛋白EIIM，P/IIIman(ManXYZ)的内源性基因的部分或完全缺失、或部分或完全失活，和/或编码微生物中甘露糖转运蛋白EIIM，P/IIIman(ManXYZ)基因的内源性天然启动子的部分或完全缺失、或部分或完全失活。优选，降低微生物中甘露糖转运蛋白EIIM，P/IIIman(ManXYZ)作用的遗传修饰为编码微生物中甘露糖转运蛋白EIIM，P/IIIman(ManXYZ)的内源性基因完全缺失，即被删除。在具体的实施方案中，微生物用至少一种包含至少一种能降低微生物中甘露糖转运蛋白EIIM，P/IIIman(ManXYZ)作用的遗传修饰的重组核酸分子转化。
在上述第(2)个方面中，降低微生物中N-乙酰神经氨酸裂解酶(NanA)作用的遗传修饰包括但不限于：编码微生物中N-乙酰神经氨酸裂解酶(NanA)的内源性基因的部分或完全缺失、或部分或完全失活，和/或编码微生物中N-乙酰神经氨酸裂解酶(NanA)基因的内源性天然启动子的部分或完全缺失、或部分或完全失活。优选，降低微生物中N-乙酰神经氨酸裂解酶(NanA)作用的遗传修饰为编码微生物中N-乙酰神经氨酸裂解酶(NanA)的内源性基因完全缺失，即被删除。在具体的实施方案中，微生物用至少一种包含至少一种能降低微生物中N-乙酰神经氨酸裂解酶(NanA)作用的遗传修饰的重组核酸分子转化。
在上述第(3)个方面中，降低微生物中N-乙酰-D-氨基葡萄糖-6-磷酸脱乙酰酶(NagA)作用的遗传修饰包括但不限于：编码微生物中N-乙酰-D-氨基葡萄糖-6-磷酸脱乙酰酶(NagA)的内源性基因的部分或完全缺失、或部分或完全失活，和/或编码微生物中N-乙酰-D-氨基葡萄糖-6-磷酸脱乙酰酶(NagA)基因的内源性天然启动子的部分或完全缺失、或部分或完全失活。优选，降低微生物中N-乙酰-D-氨基葡萄糖-6-磷酸脱乙酰酶(NagA)作用的遗传修饰为编码微生物中N-乙酰-D-氨基葡萄糖-6-磷酸脱乙酰酶(NagA)的内源性基因完全缺失，即被删除。在具体的实施方案中，微生物用至少一种包含至少一种能降低微生物中N-乙酰-D-氨基葡萄糖-6-磷酸脱乙酰酶(NagA)作用的遗传修饰的重组核酸分子转化。
在上述第(4)个方面中，降低微生物中N-乙酰-D-氨基葡萄糖特异酶IINag (NagE)作用的遗传修饰包括但不限于：编码微生物中N-乙酰-D-氨基葡萄糖特异酶IINag(NagE)的内源性基因的部分或完全缺失、或部分或完全失活，和/或编码微生物中N-乙酰-D-氨基葡萄糖特异酶IINag(NagE)基因的内源性天然启动子的部分或完全缺失、或部分或完全失活。优选，降低微生物中N-乙酰-D-氨基葡萄糖特异酶IINag(NagE)作用的遗传修饰为编码微生物中N-乙酰-D-氨基葡萄糖特异酶IINag(NagE)的内源性基因完全缺失，即被删除。在具体的实施方案中，微生物用至少一种包含至少一种能降低微生物中N-乙酰-D-氨基葡萄糖特异酶IINag(NagE)作用的遗传修饰的重组核酸分子转化。
在上述第(5)个方面中，提高微生物中磷酸葡糖胺变位酶(GlmM)作用的遗传修饰选自a)微生物中磷酸葡糖胺变位酶(GlmM)的酶活性增加；和/或b)微生物中磷酸葡糖胺变位酶(GlmM)被过量表达。
本领域技术人员可以理解，为提高微生物中磷酸葡糖胺变位酶(GlmM)的作用，可以通过筛选编码具有磷酸葡糖胺变位酶(GlmM)的酶活性增加的基因突变体来实现。筛选GlmM基因突变体可以通过易错PCR技术得到高频突变基因来完成。为提高微生物中磷酸葡糖胺变位酶(GlmM)的作用，也可以通过增加其基因拷贝数、更换比天然启动子有更高表达水平的启动子等方式过量表达磷酸葡糖胺变位酶(GlmM)来实现。在具体的实施方案中，微生物用至少一种包含至少一种能提高微生物中磷酸葡糖胺变位酶(GlmM)作用的遗传修饰的重组核酸分子转化。
在一个优选的实施方案中，微生物用至少一种包含编码磷酸葡糖胺变位酶(GlmM)的核酸序列的重组核酸分子转化。
在一个方面，编码磷酸葡糖胺变位酶(GlmM)的核酸序列含有至少一种增加磷酸葡糖胺变位酶(GlmM)的酶活性的遗传修饰。
在另一个方面，重组核酸分子中编码磷酸葡糖胺变位酶(GlmM)的基因拷贝数增加。
在另一个方面，重组核酸分子中包含内源性天然启动子、具有比内源性天然启动子更高表达水平的启动子、增强子、融合序列等。优选，重组核酸分子中包含具有比内源性天然启动子更高表达水平的启动子，例如HCE启动子、gap启动子、trc启动子、T7启动子等；更优选，重组核酸分子中包含trc启动子。
在本发明中，重组核酸分子转化微生物，选自游离型(即重组核酸分子被 装入质粒中)和整合型(即重组核酸分子被整合到微生物的基因组中)。优选，重组核酸分子被整合到微生物的基因组中。
在另一个优选的实施方案中，微生物包括至少一种对编码磷酸葡糖胺变位酶(GlmM)的基因的内源性天然启动子的遗传修饰。优选，编码磷酸葡糖胺变位酶(GlmM)的基因的内源性天然启动子被具有更高表达水平的启动子替换，例如HCE启动子、gap启动子、trc启动子、T7启动子等；更优选，编码磷酸葡糖胺变位酶(GlmM)的基因的内源性天然启动子被trc启动子替换。
在上述第(6)个方面中，提高微生物中双功能酶N-乙酰-D-氨基葡萄糖-1-磷酸尿苷酰转移酶(GlmU)作用的遗传修饰选自a)微生物中双功能酶N-乙酰-D-氨基葡萄糖-1-磷酸尿苷酰转移酶(GlmU)的酶活性增加；和/或b)微生物中双功能酶N-乙酰-D-氨基葡萄糖-1-磷酸尿苷酰转移酶(GlmU)被过量表达。
本领域技术人员可以理解，为提高微生物中双功能酶N-乙酰-D-氨基葡萄糖-1-磷酸尿苷酰转移酶(GlmU)的作用，可以通过筛选编码具有双功能酶N-乙酰-D-氨基葡萄糖-1-磷酸尿苷酰转移酶(GlmU)的酶活性增加的基因突变体来实现。筛选GlmU基因突变体可以通过易错PCR技术得到高频突变基因来完成。为提高微生物中双功能酶N-乙酰-D-氨基葡萄糖-1-磷酸尿苷酰转移酶(GlmU)的作用，也可以通过增加其基因拷贝数、更换比天然启动子有更高表达水平的启动子等方式过量表达双功能酶N-乙酰-D-氨基葡萄糖-1-磷酸尿苷酰转移酶(GlmU)来实现。在具体的实施方案中，微生物用至少一种包含至少一种能提高微生物中双功能酶N-乙酰-D-氨基葡萄糖-1-磷酸尿苷酰转移酶(GlmU)作用的遗传修饰的重组核酸分子转化。
在一个优选的实施方案中，微生物用至少一种包含编码双功能酶N-乙酰-D-氨基葡萄糖-1-磷酸尿苷酰转移酶(GlmU)的核酸序列的重组核酸分子转化。
在一个方面，编码双功能酶N-乙酰-D-氨基葡萄糖-1-磷酸尿苷酰转移酶(GlmU)的核酸序列含有至少一种增加双功能酶N-乙酰-D-氨基葡萄糖-1-磷酸尿苷酰转移酶(GlmU)的酶活性的遗传修饰。
在另一个方面，重组核酸分子中编码双功能酶N-乙酰-D-氨基葡萄糖-1-磷酸尿苷酰转移酶(GlmU)的基因拷贝数增加。
在另一个方面，重组核酸分子中包含内源性天然启动子、具有比内源性天然启动子更高表达水平的启动子、增强子、融合序列等。优选，重组核酸分子 中包含具有比内源性天然启动子更高表达水平的启动子，例如HCE启动子、gap启动子、trc启动子、T7启动子等；更优选，重组核酸分子中包含trc启动子。
在本发明中，重组核酸分子转化微生物，选自游离型(即重组核酸分子被装入质粒中)和整合型(即重组核酸分子被整合到微生物的基因组中)。优选，重组核酸分子被整合到微生物的基因组中。
在另一个优选的实施方案中，微生物包括至少一种对编码双功能酶N-乙酰-D-氨基葡萄糖-1-磷酸尿苷酰转移酶(GlmU)的基因的内源性天然启动子的遗传修饰。优选，编码双功能酶N-乙酰-D-氨基葡萄糖-1-磷酸尿苷酰转移酶(GlmU)的基因的内源性天然启动子被具有更高表达水平的启动子替换，例如HCE启动子、gap启动子、trc启动子、T7启动子等；更优选，编码双功能酶N-乙酰-D-氨基葡萄糖-1-磷酸尿苷酰转移酶(GlmU)的基因的内源性天然启动子被trc启动子替换。
本发明进一步涉及如下优选的实施方案：
1.根据本发明的一个优选实施方案，本发明涉及一种通过微生物发酵生产N-乙酰-D-氨基葡萄糖(GlcNAc)和/或D-氨基葡萄糖盐的方法，该方法包括：
A)在发酵培养基中培养微生物，所述微生物包含：至少一种能提高微生物中N-乙酰-D-氨基甘露糖激酶(NanK)作用的遗传修饰；和至少一种能提高微生物中N-乙酰-D-氨基甘露糖-6-磷酸异构酶(NanE)作用的遗传修饰；和
B)收集从培养步骤A)中产生的N-乙酰-D-氨基葡萄糖(GlcNAc)。
2.根据本发明的另一个优选实施方案，本发明涉及一种通过微生物发酵生产N-乙酰-D-氨基葡萄糖(GlcNAc)和/或D-氨基葡萄糖盐的方法，该方法包括：
A)在发酵培养基中培养微生物，所述微生物包含：至少一种能提高微生物中N-乙酰-D-氨基甘露糖激酶(NanK)作用的遗传修饰；和至少一种能提高微生物中D-氨基葡萄糖-6-磷酸脱氨酶(NagB)作用的遗传修饰；和
B)收集从培养步骤A)中产生的N-乙酰-D-氨基葡萄糖(GlcNAc)。
优选，所述微生物还包含至少一种能降低氨基葡萄糖-6-磷酸合成酶(GlmS)作用的遗传修饰。
3.根据本发明的另一个优选实施方案，本发明涉及一种通过微生物发酵生产N-乙酰-D-氨基葡萄糖(GlcNAc)和/或D-氨基葡萄糖盐的方法，该方法包括：
A)在发酵培养基中培养微生物，所述微生物包含：至少一种能提高微生物中N-乙酰-D-氨基甘露糖激酶(NanK)作用的遗传修饰；至少一种能提高微生物中氨基葡萄糖-6-磷酸合成酶(GlmS)作用的遗传修饰；和至少一种能降低D-氨基葡萄糖-6-磷酸脱氨酶(NagB)作用的遗传修饰；和
B)收集从培养步骤A)中产生的N-乙酰-D-氨基葡萄糖(GlcNAc)。
4.根据本发明的另一个优选实施方案，本发明涉及一种通过微生物发酵生产N-乙酰-D-氨基葡萄糖(GlcNAc)和/或D-氨基葡萄糖盐的方法，该方法包括：
A)在发酵培养基中培养微生物，所述微生物包含：至少一种能提高微生物中N-乙酰-D-氨基甘露糖激酶(NanK)作用的遗传修饰；至少一种能提高微生物中N-乙酰-D-氨基甘露糖-6-磷酸异构酶(NanE)作用的遗传修饰；和至少一种能提高微生物中D-氨基葡萄糖-6-磷酸脱氨酶(NagB)作用的遗传修饰；和
B)收集从培养步骤A)中产生的N-乙酰-D-氨基葡萄糖(GlcNAc)。
优选，所述微生物还包含至少一种能降低氨基葡萄糖-6-磷酸合成酶(GlmS)作用的遗传修饰。
5.根据本发明的另一个优选实施方案，本发明涉及一种通过微生物发酵生产N-乙酰-D-氨基葡萄糖(GlcNAc)和/或D-氨基葡萄糖盐的方法，该方法包括：
A)在发酵培养基中培养微生物，所述微生物包含：至少一种能提高微生物中N-乙酰-D-氨基甘露糖激酶(NanK)作用的遗传修饰；至少一种能提高微生物中N-乙酰-D-氨基甘露糖-6-磷酸异构酶(NanE)作用的遗传修饰；至少一种能提高微生物中氨基葡萄糖-6-磷酸合成酶(GlmS)作用的遗传修饰；和至少一种能降低D-氨基葡萄糖-6-磷酸脱氨酶(NagB)作用的遗传修饰；和
B)收集从培养步骤A)中产生的N-乙酰-D-氨基葡萄糖(GlcNAc)。
6.根据本发明的另一个优选实施方案，本发明涉及一种通过微生物发酵生产N-乙酰-D-氨基葡萄糖(GlcNAc)和/或D-氨基葡萄糖盐的方法，该方法包括：
A)在发酵培养基中培养微生物，所述微生物包含：至少一种能提高微生物中N-乙酰-D-氨基甘露糖激酶(NanK)作用的遗传修饰；和至少一种能提高微生物中UDP-N-乙酰-D-氨基葡萄糖-2-异构酶(WecB)作用的遗传修饰；和
B)收集从培养步骤A)中产生的N-乙酰-D-氨基葡萄糖(GlcNAc)。
7.根据本发明的另一个优选实施方案，本发明涉及一种通过微生物发酵生产N-乙酰-D-氨基葡萄糖(GlcNAc)和/或D-氨基葡萄糖盐的方法，该方法包括：
A)在发酵培养基中培养微生物，所述微生物包含：至少一种能提高微生物中N-乙酰-D-氨基甘露糖激酶(NanK)作用的遗传修饰；至少一种能提高微生物中N-乙酰-D-氨基甘露糖-6-磷酸异构酶(NanE)作用的遗传修饰；和至少一种能提高微生物中UDP-N-乙酰-D-氨基葡萄糖-2-异构酶(WecB)作用的遗传修饰；和
B)收集从培养步骤A)中产生的N-乙酰-D-氨基葡萄糖(GlcNAc)。
8.根据本发明的另一个优选实施方案，本发明涉及一种通过微生物发酵生产N-乙酰-D-氨基葡萄糖(GlcNAc)和/或D-氨基葡萄糖盐的方法，该方法包括：
A)在发酵培养基中培养微生物，所述微生物包含：至少一种能提高微生物中N-乙酰-D-氨基甘露糖激酶(NanK)作用的遗传修饰；至少一种能提高微生物中D-氨基葡萄糖-6-磷酸脱氨酶(NagB)作用的遗传修饰；和至少一种能提高微生物中UDP-N-乙酰-D-氨基葡萄糖-2-异构酶(WecB)作用的遗传修饰；和
B)收集从培养步骤A)中产生的N-乙酰-D-氨基葡萄糖(GlcNAc)。
优选，所述微生物还包含至少一种能降低氨基葡萄糖-6-磷酸合成酶(GlmS)作用的遗传修饰。
9.根据本发明的另一个优选实施方案，本发明涉及一种通过微生物发酵生产N-乙酰-D-氨基葡萄糖(GlcNAc)和/或D-氨基葡萄糖盐的方法，该方法包括：
A)在发酵培养基中培养微生物，所述微生物包含：至少一种能提高微生物中N-乙酰-D-氨基甘露糖激酶(NanK)作用的遗传修饰；至少一种能提高微生物中氨基葡萄糖-6-磷酸合成酶(GlmS)作用的遗传修饰；至少一种能降低D-氨基葡萄糖-6-磷酸脱氨酶(NagB)作用的遗传修饰；和至少一种能提高微生物中UDP-N-乙酰-D-氨基葡萄糖-2-异构酶(WecB)作用的遗传修饰；和
B)收集从培养步骤A)中产生的N-乙酰-D-氨基葡萄糖(GlcNAc)。
10.根据本发明的另一个优选实施方案，本发明涉及一种通过微生物发酵生产N-乙酰-D-氨基葡萄糖(GlcNAc)和/或D-氨基葡萄糖盐的方法，该方法包括：
A)在发酵培养基中培养微生物，所述微生物包含：至少一种能提高微生物中N-乙酰-D-氨基甘露糖激酶(NanK)作用的遗传修饰；至少一种能提高微生物中N-乙酰-D-氨基甘露糖-6-磷酸异构酶(NanE)作用的遗传修饰；至少一种能提高微生物中D-氨基葡萄糖-6-磷酸脱氨酶(NagB)作用的遗传修饰；和至少一种能提高微生物中UDP-N-乙酰-D-氨基葡萄糖-2-异构酶(WecB)作用的遗传 修饰；和
B)收集从培养步骤A)中产生的N-乙酰-D-氨基葡萄糖(GlcNAc)。
优选，所述微生物还包含至少一种能降低氨基葡萄糖-6-磷酸合成酶(GlmS)作用的遗传修饰。
11.根据本发明的另一个优选实施方案，本发明涉及一种通过微生物发酵生产N-乙酰-D-氨基葡萄糖(GlcNAc)和/或D-氨基葡萄糖盐的方法，该方法包括：
A)在发酵培养基中培养微生物，所述微生物包含：至少一种能提高微生物中N-乙酰-D-氨基甘露糖激酶(NanK)作用的遗传修饰；至少一种能提高微生物中N-乙酰-D-氨基甘露糖-6-磷酸异构酶(NanE)作用的遗传修饰；至少一种能提高微生物中氨基葡萄糖-6-磷酸合成酶(GlmS)作用的遗传修饰；至少一种能降低D-氨基葡萄糖-6-磷酸脱氨酶(NagB)作用的遗传修饰；和至少一种能提高微生物中UDP-N-乙酰-D-氨基葡萄糖-2-异构酶(WecB)作用的遗传修饰；和
B)收集从培养步骤A)中产生的N-乙酰-D-氨基葡萄糖(GlcNAc)。
在上述优选的实施方案中，进一步包括C)由N-乙酰-D-氨基葡萄糖(GlcNAc)脱乙酰化得到D-氨基葡萄糖盐。
在上述优选的实施方案中，所述微生物进一步包含：至少一种能降低微生物中甘露糖转运蛋白EIIM，P/IIIman(ManXYZ)作用的遗传修饰；至少一种能降低微生物中N-乙酰神经氨酸裂解酶(NanA)作用的遗传修饰；至少一种能降低微生物中N-乙酰-D-氨基葡萄糖-6-磷酸脱乙酰酶(NagA)作用的遗传修饰；和至少一种能降低微生物中N-乙酰-D-氨基葡萄糖特异酶IINag(NagE)作用的遗传修饰。
在上述任意实施方案的一个方面中，上述任意重组核酸分子的表达是可诱导的，包括但不限于被乳糖诱导，例如，通过在培养液中添加乳糖等可实现被乳糖诱导表达。
本领域技术人员可以理解，本发明中可以使用本领域已知的各种常规发酵培养基。在一个方面中，发酵培养基中包含碳源。在另一个方面中，发酵培养基中包含氮源。在另一个方面中，发酵培养基中包含碳源和氮源。在另一个方 面中，发酵培养基中包含碳源、氮源和无机盐。
本领域技术人员可以理解，本领域已知的各种碳源均可用于本发明，包括有机碳源和/或无机碳源。优选，碳源选自葡萄糖、果糖、蔗糖、半乳糖、糊精、甘油、淀粉、糖浆和糖蜜中的一种或多种。优选，碳源的浓度维持在约0.1％-约5％。本领域技术人员可以理解，本领域已知的各种氮源均可用于本发明，包括有机氮源和/或无机氮源。优选，氮源选自氨水、氯化铵、硫酸铵、硝酸铵、醋酸铵、硝酸钠、尿素、酵母浸膏、肉类浸膏、蛋白胨、鱼粉、豆粉、麦芽、玉米浆和棉籽粉中的一种或多种。
优选，本发明采用补料发酵法。根据本发明的一个方面，补糖液包含葡萄糖和核糖，优选，葡萄糖浓度为10％-85％(w/v)，核糖浓度为0.5％-15％(w/v)，进一步优选，葡萄糖浓度为55％-75％(w/v)，核糖浓度为5％-7％(w/v)；根据本发明的另一个方面，补糖液包含葡萄糖和葡萄糖酸盐，优选，葡萄糖浓度为10％-85％(w/v)，葡萄糖酸盐浓度为0.5％-15％(w/v)，进一步优选，葡萄糖浓度为55％-75％(w/v)，葡萄糖酸盐浓度为2％-3％(w/v)；根据本发明的另一个方面，补糖液包含葡萄糖、核糖和葡糖酸盐，优选，葡萄糖浓度为10％-85％(w/v)，核糖浓度为0.5％-15％(w/v)，葡萄糖酸盐浓度为0.5％-15％(w/v)，进一步优选，葡萄糖浓度为55％-75％(w/v)，核糖浓度为5％-7％(w/v)，葡萄糖酸盐浓度为2％-3％(w/v)。优选，葡萄糖酸盐为葡萄糖酸钠。
在优选的实施方案中，在约20℃-约45℃进行所述培养步骤，进一步优选，在约33℃-约37℃进行所述培养步骤。
在优选的实施方案中，在约pH4.5-约pH8.5进行所述培养步骤。进一步优选，在约pH6.7-约pH7.2进行所述培养步骤。
本领域技术人员可以理解，本发明中可以使用本领域已知的各种常规方法收集N-乙酰-D-氨基葡萄糖(GlcNAc)。优选，可以从发酵培养基中的胞外产物中收集N-乙酰-D-氨基葡萄糖。进一步优选，该收集步骤包括选自如下的步骤：(a)从去除微生物的发酵液中沉淀N-乙酰-D-氨基葡萄糖；(b)从去除微生物的发酵液中结晶N-乙酰-D-氨基葡萄糖。
根据本发明，收集步骤进一步包括将发酵液脱色的步骤。脱色步骤可以包括但不限于在对发酵液进行沉淀或结晶之前、在对发酵液进行一次或多次沉淀或结晶重溶解之后进行，脱色包括活性炭处理和/或色谱脱色。所述色谱脱色包 括使所述发酵液与离子交换树脂接触的步骤，离子交换树脂包括但不限于阴离子交换树脂和/或阳离子交换树脂，例如使发酵液与阴离子和阳离子交换树脂的混合床接触。
根据本发明，可以通过使N-乙酰-D-氨基葡萄糖脱乙酰化得到D-氨基葡萄糖盐，所述盐包括但不限于盐酸盐、硫酸盐、钠盐、磷酸盐和硫酸氢盐等。例如，可以在酸性和加热条件下脱乙酰化水解N-乙酰-D-氨基葡萄糖得到D-氨基葡萄糖盐，优选，在30％-37％盐酸溶液中、60℃-90℃下脱乙酰化水解N-乙酰-D-氨基葡萄糖得到D-氨基葡萄糖盐酸盐；也可以在UDP-3-O-N-乙酰葡萄糖胺脱乙酰基酶作用下水解N-乙酰-D-氨基葡萄糖得到D-氨基葡萄糖，并进一步成盐。
根据本发明的另一个实施方案，本发明涉及一种微生物，所述微生物包含至少一种能提高微生物中N-乙酰-D-氨基甘露糖激酶(NanK)作用的遗传修饰。上文已经对这一遗传修饰进行了详细描述。
根据本发明的优选实施方案，所述微生物进一步包含下述遗传修饰中的一种或多种：
(1)包含至少一种能提高微生物中N-乙酰-D-氨基甘露糖-6-磷酸异构酶(NanE)作用的遗传修饰；
(2)包含至少一种能提高微生物中D-氨基葡萄糖-6-磷酸脱氨酶(NagB)作用的遗传修饰，优选同时包含至少一种能降低氨基葡萄糖-6-磷酸合成酶(GlmS)作用的遗传修饰；
(3)包含至少一种能提高微生物中氨基葡萄糖-6-磷酸合成酶(GlmS)作用的遗传修饰，并同时包含至少一种能降低D-氨基葡萄糖-6-磷酸脱氨酶(NagB)作用的遗传修饰；
(4)包含至少一种能提高微生物中UDP-N-乙酰-D-氨基葡萄糖-2-异构酶(WecB)作用的遗传修饰。上文已经对这些遗传修饰进行了详细描述。
根据本发明的优选实施方案，所述微生物进一步包含下述遗传修饰中的一种或多种：
(1)包含至少一种能降低微生物中甘露糖转运蛋白EIIM，P/IIIman (ManXYZ)作用的遗传修饰；
(2)包含至少一种能降低微生物中N-乙酰神经氨酸裂解酶(NanA)作用的遗传修饰；
(3)包含至少一种能降低微生物中N-乙酰-D-氨基葡萄糖-6-磷酸脱乙酰酶(NagA)作用的遗传修饰；
(4)包含至少一种能降低微生物中N-乙酰-D-氨基葡萄糖特异酶IINag(NagE)作用的遗传修饰；
(5)包含至少一种能提高微生物中磷酸葡糖胺变位酶(GlmM)作用的遗传修饰；
(6)包含至少一种能提高微生物中双功能酶N-乙酰-D-氨基葡萄糖-1-磷酸尿苷酰转移酶(GlmU)作用的遗传修饰。上文已经对这些遗传修饰进行了详细描述。
本发明进一步涉及如下优选的实施方案：
1.根据本发明的一个优选实施方案，本发明涉及一种微生物，所述微生物包含：至少一种能提高微生物中N-乙酰-D-氨基甘露糖激酶(NanK)作用的遗传修饰；和至少一种能提高微生物中N-乙酰-D-氨基甘露糖-6-磷酸异构酶(NanE)作用的遗传修饰。
2.根据本发明的另一个优选实施方案，本发明涉及一种微生物，所述微生物包含：至少一种能提高微生物中N-乙酰-D-氨基甘露糖激酶(NanK)作用的遗传修饰；和至少一种能提高微生物中D-氨基葡萄糖-6-磷酸脱氨酶(NagB)作用的遗传修饰。
优选，所述微生物还包含至少一种能降低氨基葡萄糖-6-磷酸合成酶(GlmS)作用的遗传修饰。
3.根据本发明的另一个优选实施方案，本发明涉及一种微生物，所述微生物包含：至少一种能提高微生物中N-乙酰-D-氨基甘露糖激酶(NanK)作用的遗传修饰；至少一种能提高微生物中氨基葡萄糖-6-磷酸合成酶(GlmS)作用的遗传修饰；和至少一种能降低D-氨基葡萄糖-6-磷酸脱氨酶(NagB)作用的遗传修饰。
4.根据本发明的一个优选实施方案，本发明涉及一种微生物，所述微生物 包含：至少一种能提高微生物中N-乙酰-D-氨基甘露糖激酶(NanK)作用的遗传修饰；至少一种能提高微生物中N-乙酰-D-氨基甘露糖-6-磷酸异构酶(NanE)作用的遗传修饰；和至少一种能提高微生物中D-氨基葡萄糖-6-磷酸脱氨酶(NagB)作用的遗传修饰。
优选，所述微生物还包含至少一种能降低氨基葡萄糖-6-磷酸合成酶(GlmS)作用的遗传修饰。
5.根据本发明的一个优选实施方案，本发明涉及一种微生物，所述微生物包含：至少一种能提高微生物中N-乙酰-D-氨基甘露糖激酶(NanK)作用的遗传修饰；至少一种能提高微生物中N-乙酰-D-氨基甘露糖-6-磷酸异构酶(NanE)作用的遗传修饰；至少一种能提高微生物中氨基葡萄糖-6-磷酸合成酶(GlmS)作用的遗传修饰；和至少一种能降低D-氨基葡萄糖-6-磷酸脱氨酶(NagB)作用的遗传修饰。
6.根据本发明的一个优选实施方案，本发明涉及一种微生物，所述微生物包含：至少一种能提高微生物中N-乙酰-D-氨基甘露糖激酶(NanK)作用的遗传修饰；和至少一种能提高微生物中UDP-N-乙酰-D-氨基葡萄糖-2-异构酶(WecB)作用的遗传修饰。
7.根据本发明的一个优选实施方案，本发明涉及一种微生物，所述微生物包含：至少一种能提高微生物中N-乙酰-D-氨基甘露糖激酶(NanK)作用的遗传修饰；至少一种能提高微生物中N-乙酰-D-氨基甘露糖-6-磷酸异构酶(NanE)作用的遗传修饰；和至少一种能提高微生物中UDP-N-乙酰-D-氨基葡萄糖-2-异构酶(WecB)作用的遗传修饰。
8.根据本发明的另一个优选实施方案，本发明涉及一种微生物，所述微生物包含：至少一种能提高微生物中N-乙酰-D-氨基甘露糖激酶(NanK)作用的遗传修饰；至少一种能提高微生物中D-氨基葡萄糖-6-磷酸脱氨酶(NagB)作用的遗传修饰；和至少一种能提高微生物中UDP-N-乙酰-D-氨基葡萄糖-2-异构酶(WecB)作用的遗传修饰。
优选，所述微生物还包含至少一种能降低氨基葡萄糖-6-磷酸合成酶(GlmS)作用的遗传修饰。
9.根据本发明的另一个优选实施方案，本发明涉及一种微生物，所述微生物包含：至少一种能提高微生物中N-乙酰-D-氨基甘露糖激酶(NanK)作用的 遗传修饰；至少一种能提高微生物中氨基葡萄糖-6-磷酸合成酶(GlmS)作用的遗传修饰；至少一种能降低D-氨基葡萄糖-6-磷酸脱氨酶(NagB)作用的遗传修饰；和至少一种能提高微生物中UDP-N-乙酰-D-氨基葡萄糖-2-异构酶(WecB)作用的遗传修饰。
10.根据本发明的一个优选实施方案，本发明涉及一种微生物，所述微生物包含：至少一种能提高微生物中N-乙酰-D-氨基甘露糖激酶(NanK)作用的遗传修饰；至少一种能提高微生物中N-乙酰-D-氨基甘露糖-6-磷酸异构酶(NanE)作用的遗传修饰；至少一种能提高微生物中D-氨基葡萄糖-6-磷酸脱氨酶(NagB)作用的遗传修饰；和至少一种能提高微生物中UDP-N-乙酰-D-氨基葡萄糖-2-异构酶(WecB)作用的遗传修饰。
优选，所述微生物还包含至少一种能降低氨基葡萄糖-6-磷酸合成酶(GlmS)作用的遗传修饰。
11.根据本发明的一个优选实施方案，本发明涉及一种微生物，所述微生物包含：至少一种能提高微生物中N-乙酰-D-氨基甘露糖激酶(NanK)作用的遗传修饰；至少一种能提高微生物中N-乙酰-D-氨基甘露糖-6-磷酸异构酶(NanE)作用的遗传修饰；至少一种能提高微生物中氨基葡萄糖-6-磷酸合成酶(GlmS)作用的遗传修饰；至少一种能降低D-氨基葡萄糖-6-磷酸脱氨酶(NagB)作用的遗传修饰；和至少一种能提高微生物中UDP-N-乙酰-D-氨基葡萄糖-2-异构酶(WecB)作用的遗传修饰。
在上述优选的实施方案中，所述微生物进一步包含：至少一种能降低微生物中甘露糖转运蛋白EIIM，P/IIIman(ManXYZ)作用的遗传修饰；至少一种能降低微生物中N-乙酰神经氨酸裂解酶(NanA)作用的遗传修饰；至少一种能降低微生物中N-乙酰-D-氨基葡萄糖-6-磷酸脱乙酰酶(NagA)作用的遗传修饰；和至少一种能降低微生物中N-乙酰-D-氨基葡萄糖特异酶IINag(NagE)作用的遗传修饰。
根据本发明的另一个实施方案，本发明涉及一种具有更高酶活性的N-乙酰-D-氨基甘露糖激酶(NanK)，其具有SEQ ID NO：27所示的氨基酸序列。本发明进一步涉及编码上述N-乙酰-D-氨基甘露糖激酶(NanK)的核酸分子，所述 核酸分子具有SEQ ID NO：26所示的核酸序列。本发明进一步涉及包含上述核酸分子的载体。本发明进一步涉及包含上述载体的微生物。本发明进一步涉及基因组中包含上述核酸分子的微生物。
在本发明中，微生物可以是任意的微生物(例如细菌、原生生物、藻类、真菌或其它微生物)。在优选的实施方案中，微生物包括但不限于细菌、酵母或真菌。优选，所述的微生物选自细菌或酵母。进一步优选，细菌包括但不限于选自埃希氏菌属(Escherichia)、芽孢杆菌属(Bacillus)、乳杆菌属(Lactobacillus)、假单胞菌属(Pseudomonas)或链霉菌属(Streptomyces)的属的细菌；更优选，细菌包括但不限于选自大肠杆菌(Escherichia coli)、枯草芽孢杆菌(Bacillus subtilis)、地衣芽孢杆菌(Bacillus licheniformis)、短乳杆菌(Lactobacillus brevis)、铜绿假单胞菌(Pseudomonas aeruginosa)或浅青紫链霉菌(Streptomyces lividans)的种的细菌。进一步优选，酵母包括但不限于选自糖酵母属(Saccharomyces)、裂殖糖酵母属(Schizosaccharomyces)、念珠菌属(Candida)、汉逊酵母属(Hansenula)、毕赤酵母属(Pichia)、克鲁维酵母属(Kluveromyces)和红法夫属(Phaffia)的酵母；更优选，酵母包括但不限于选自酿酒酵母(Saccharomyce scerevisiae)、粟酒裂殖酵母(Schizosaccharo mycespombe)、白色念珠菌(Candida albicans)、多形汉逊酵母(Hansenulapolymorpha)、巴氏毕赤酵母(Pichia pastoris)、加拿大毕赤酵母(Pichia canadensis)、马克斯克鲁维酵母(Kluyveromyces marxianus)或红法夫酵母(Phaffia rohodozyma)。优选，所述的微生物为真菌；进一步优选，真菌包括但不限于选自曲霉属(Aspergillus)、犁头霉属(Absidia)、根霉属(Rhizopus)、金孢子菌属(Chrysosporium)、脉孢霉属(Neurospora)或木霉属(Trichoderma)的属的真菌；更优选，真菌包括但不限于选自黑曲霉(Aspergillus niger)、构巢曲霉(Aspergillus nidulans)、蓝色犁头霉(Absidia coerulea)、米根霉(Rhizopus oryzae)、劳肯诺温斯金孢子菌(Chrysosporium lucknowense)、粗糙脉孢霉(Neurospora crassa)、间型脉孢霉(Neurospora intermedia)或里氏木霉(Trichoderma reesei)。特别优选的大肠杆菌菌株包括K-12、B和W，最优选K-12。尽管大肠杆菌作为优选的微生物且用作本发明各种实施方案的实例，但是应理解在本发明方法中可以使用产生N-乙酰-D-氨基葡萄糖且可以通过遗传修饰以提高N-乙酰-D-氨基葡萄糖产量的任意其它微生物。用于本发明的微生物也可以称作生产生物体。
在本发明中，术语N-乙酰-D-氨基葡萄糖可以称作2-乙酰氨基-2-脱氧-D-葡萄糖。术语N-乙酰-D-氨基葡萄糖、N-乙酰-D-氨基葡萄糖-6-磷酸和N-乙酰-D-氨基葡萄糖-1-磷酸可以分别缩写为GlcNAc、GlcNAc-6-P和GlcNAc-1-P。N-乙酰-D-氨基葡萄糖也缩写为NAG。与N-乙酰-D-氨基葡萄糖和衍生物类似，术语D-氨基葡萄糖、D-氨基葡萄糖-6-磷酸和D-氨基葡萄糖-1-磷酸可以分别缩写为GlcN、GlcN-6-P和GlcN-1-P。类似地，术语N-乙酰-D-氨基甘露糖、N-乙酰-D-氨基甘露糖-6-磷酸、葡萄糖、葡萄糖-6-磷酸、果糖-6-磷酸可以分别缩写为ManNAc、ManNAc-6-P、Glc、Glc-6-P、Fru-6-P。
术语提高微生物中酶的作用是指微生物中该酶的活性增加和/或该酶被过量表达，从而提高了微生物中由该酶所催化的底物生成产物的量。
术语降低微生物中酶的作用是指微生物中该酶的活性降低和/或该酶的表达减少，从而降低了微生物中由该酶所催化的底物生成产物的量。
术语酶活性增加是指酶催化一定化学反应的能力增加。其涵盖了在酶受产物抑制作用和酶对底物亲合力不变的情况下酶自身催化化学反应的能力增加，和/或由于酶受产物抑制作用降低导致和/或酶对底物亲合力增加所导致的酶催化化学反应的能力增加。术语酶受产物抑制作用降低是指催化反应的酶的活性受其终产物特异抑制作用而降低。术语酶对底物亲合力增加是指酶对所催化的底物的亲合力增加。
图1以大肠杆菌为例解释了本发明公开的用于大规模生产N-乙酰-D-氨基葡萄糖的氨基糖代谢途经中遗传修饰的主要方面。就图1而言，粗体箭头表示本发明涉及的通过遗传改造产生和/或增加代谢流量。图1公开了数种用于合成N-乙酰-D-氨基葡萄糖的不同方法，包括对NanK的修饰，可进一步包括对NanE、NagB、GlmS、WecB或其组合的修饰，还可以进一步包括对ManXYZ、NanA、NagA、NagE、GlmM、GlmU或其组合的修饰。本领域技术人员可以理解，其它微生物具有类似的糖代谢途经，且在这类途经中基因和蛋白质具有类似的结构和功能。因此，本发明所讨论的除适用于大肠杆菌外同样适用于其它微生物且其它微生物显然包括在本发明中。
本领域中已知具有相同生物活性的酶可以具有不同的名称，这取决于该酶来源于什么样的微生物。下面是本文涉及的许多酶的可选名称和来自一些生物 体的编码这类酶的具体基因名称。这些酶的名称可以互换使用或如果合适用于给定的序列或生物体，但本发明意图包括来自任意生物体的指定功能的酶。
例如，本文一般称作“N-乙酰-D-氨基甘露糖激酶”的酶催化由N-乙酰-D-氨基甘露糖磷酸化为N-乙酰-D-氨基甘露糖-6-P。来自大肠杆菌的N-乙酰-D-氨基甘露糖激酶一般称作NanK。来自各种生物体的N-乙酰-D-氨基甘露糖激酶是本领域中公知的，且可用于本发明遗传改造策略中。例如，本文描述了来自大肠杆菌的N-乙酰-D-氨基甘露糖激酶具有由SEQ ID NO：16表示的核酸序列编码、由SEQ ID NO：17表示的氨基酸序列。
本文一般称作“N-乙酰-D-氨基甘露糖-6-P异构酶”的酶催化由N-乙酰-D-氨基甘露糖-6-P转变为N-乙酰-D-氨基葡萄糖-6-P。来自大肠杆菌的N-乙酰-D-氨基甘露糖-6-P异构酶一般称作NanE。来自各种生物体的N-乙酰-D-氨基甘露糖-6-P异构酶是本领域中公知的，且可用于本发明遗传改造策略中。例如，本文描述了来自大肠杆菌的N-乙酰-D-氨基甘露糖-6-P异构酶具有由SEQ ID NO：28表示的核酸序列编码、由SEQ ID NO：29表示的氨基酸序列。
本文一般称作“UDP-N-乙酰-D-氨基葡萄糖-2-异构酶”的酶催化由UDP-N-乙酰-D-氨基葡萄糖转变为N-乙酰-D-氨基甘露糖。来自大肠杆菌的UDP-N-乙酰-D-氨基葡萄糖-2-异构酶一般称作WecB。来自各种生物体的UDP-N-乙酰-D-氨基葡萄糖-2-异构酶是本领域中公知的，且可用于本发明遗传改造策略中。例如，本文描述了来自大肠杆菌的UDP-N-乙酰-D-氨基葡萄糖-2-异构酶具有由SEQ ID NO：49表示的核酸序列编码、由SEQ ID NO：50表示的氨基酸序列。
本文一般称作“D-氨基葡萄糖-6-磷酸脱氨酶”的酶催化D-氨基葡萄糖-6-磷酸和水形成葡萄糖-6-磷酸和铵的可逆反应。该酶也称作D-氨基葡萄糖-6-磷酸异构酶、GlcN6P脱氨酶、磷酸D-氨基葡萄糖异构酶、磷酸D-氨基葡萄糖异构酶、D-氨基葡萄糖磷酸酯脱氨酶和2-氨基-2-脱氧-D-葡萄糖-6-磷酸乙酮醇异构酶(脱氨)。来自各种生物体的D-氨基葡萄糖-6-磷酸脱氨酶是本领域中公知的，且可用于本发明遗传改造策略中。在大肠杆菌和其它细菌中，该酶一般称作NagB。
本文一般称作“D-氨基葡萄糖-6-磷酸合酶”的酶催化由葡萄糖-6-磷酸和谷氨酰胺形成D-氨基葡萄糖-6-磷酸和谷氨酸。该酶也称作D-氨基葡萄糖-果糖-6-磷酸氨基转移酶(异构化)、磷酸己糖氨基转移酶、D-果糖-6-磷酸转酰胺酶、D-氨基葡萄糖-6-磷酸异构酶(形成谷氨酰胺)、L-谷氨酰胺-果糖-6-磷酸转酰胺酶 和GlcN6P合酶。来自各种生物体的D-氨基葡萄糖-6-磷酸合酶是本领域中公知的，且可用于本发明遗传改造策略中。来自大肠杆菌和其它细菌的D-氨基葡萄糖-6-磷酸合酶一般称作GlmS。
本文一般称作“N-乙酰-D-氨基葡萄糖-6-磷酸脱乙酰酶”的酶将N-乙酰-D-氨基葡萄糖-6-磷酸水解成D-氨基葡萄糖-6-磷酸和乙酸酯。来自各种生物体的N-乙酰-D-氨基葡萄糖-6-磷酸脱乙酰酶是本领域中公知的且可用于本发明的遗传改造策略中。例如，本文描述了来自大肠杆菌的称作NagA。
本文一般称作“N-乙酰神经氨酸裂解酶”的酶催化N-乙酰-D-氨基甘露糖降解为N-乙酰神经氨酸。来自各种生物体的N-乙酰神经氨酸裂解酶是本领域中公知的且可用于本发明的遗传改造策略中。例如，本文描述了来自大肠杆菌的N-乙酰神经氨酸裂解酶称作NanA。
本文一般称作“磷酸葡糖胺变位酶”的酶催化D-氨基葡萄糖-6-磷酸转化为D-氨基葡萄糖-1-磷酸。来自各种生物体的磷酸D-氨基葡萄糖变位酶是本领域中公知的且可用于本发明的遗传改造策略中。该酶在大肠杆菌和其它细菌的磷酸葡糖胺变位酶一般称作GlmM。
本文一般称作“D-氨基葡萄糖-1-磷酸N-乙酰转移酶”的酶将D-氨基葡萄糖-1-磷酸和乙酰辅酶A转化成N-乙酰-D-氨基葡萄糖-1-磷酸，并释放CoA。作为一种双功能酶，它还具有N-乙酰-D-氨基葡萄糖-1-磷酸尿苷酰转移酶的功能，也称作UDP-N-乙酰-D-氨基葡萄糖焦磷酸化酶、UDP-N-乙酰-D-氨基葡萄糖二磷酸化酶，将N-乙酰-D-氨基葡萄糖-1-磷酸进一步转化为UDP-N-乙酰-D-氨基葡萄糖。来自各种生物体的D-氨基葡萄糖-1-磷酸N-乙酰转移酶和N-乙酰-D-氨基葡萄糖-1-磷酸尿苷酰转移酶是本领域中公知的且可用于本发明的遗传改造策略中。该酶在大肠杆菌和其它细菌中称作GlmU。
“Trc启动子”经过巧妙的设计可用于原核表达，例如大肠杆菌表达系统。Trc启动子是本领域中公知的且可用于本发明的遗传改造策略中。例如，本文描述了的Trc promoter具有SEQ ID NO：32表示的核苷酸序列。
正如WO2004/003175发明公开的，D-氨基葡萄糖在用于大肠杆菌生长的一般pH范围内极不稳定。D-氨基葡萄糖和/或其降解产物对菌株产生毒性作用。甚至当在进行细胞接种前将浓度低至20g/L的D-氨基葡萄糖在培养基(pH7.0)中预保温3.5小时的时候也观察到毒性。毒性至少部分是由于起始pH为7.0的 培养基中的D-氨基葡萄糖降解产物所致。GlcN在较低pH条件下更为稳定，D-氨基葡萄糖在pH4.7以下不会降解。但是，大肠杆菌在低于6-7的pH条件下生长缓慢。因此，在相对低pH下在发酵罐内进行D-氨基葡萄糖生产的方案难以施行。
根据本发明，在细胞内将由D-氨基葡萄糖-6-P(GlcN-6-P)在GlmM和GlmU催化生成UDP-N-乙酰基-D-氨基葡萄糖(UDP-GlcNAc)，在UDP-N-乙酰-氨基葡萄糖-2-异构酶(WecB)催化下变成N-乙酰基-D-氨基甘露糖(ManNAc)，通过超表达NanK和NanE，进一步转变为N-乙酰基-D-氨基葡萄糖-6-磷酸(GlcNAc-6-P)，在磷酸酶作用下去磷酸化，排出细胞外成为N-乙酰基-D-氨基葡萄糖(GlcNAc)。本发明的方法，避免了D-氨基葡萄糖的生成，从而避免了D-氨基葡萄糖和/或其降解产物对菌株产生毒性作用。
因此，本发明的有益效果在于：本发明证实可通过微生物发酵方法直接生产完全天然的N-乙酰-D-氨基葡萄糖；该生产新方法无重金属污染风险，无抗生素、药物残留风险，生产不受原料供应影响，可长期稳定生产，且产量高、成本低；所生产的N-乙酰-D-氨基葡萄糖和D-氨基葡萄糖产品具有非动物源性，不使用虾壳的甲壳素，使用葡萄糖等碳源发酵，属于素食产品，且无水产品过敏源。
将本文引述或描述的各公开文献和参考文献的全部内容引入本文作为参考。
附图说明
图1大肠杆菌中N-乙酰-D-氨基葡萄糖生物合成途径和代谢工程策略图
具体实施方式
下文将结合具体实施例对本发明做更进一步的详细说明。下列实施例仅为示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。凡基于本发明内容所实现的技术均涵盖在本发明旨在保护的范围内。
除非另有说明，实施例中使用的原料和试剂均为市售商品。
下面是本发明涉及和/或所述的各种基因修饰微生物的目录。
Figure PCTCN2017080653-appb-000001
Figure PCTCN2017080653-appb-000002
实施例1
本实施例描述了构建阻断与N-乙酰-D-氨基葡萄糖被摄入及有益中间产物被降解有关的代谢途径的大肠杆菌突变株
所述生产菌株的亲本菌株是AT-001(Escherichia coli ATCC 27325)，属于大肠杆菌K-12衍生株，来自美国模式培养物保藏中心(American Type Culture Collection)。
阻断菌种对N-乙酰-D-氨基葡萄糖摄入及中间代谢产物降解，可以减少代谢过程中的损耗，增加目标产物(N-乙酰-D-氨基葡萄糖)的积累。
构建这种突变型宿主菌株，可通过将其染色体基因组上manXYZ、nanA、nagA和nagE基因序列完全或部分删除，使其功能失效，从而使N-乙酰-D-氨基葡萄糖累积。
这种染色体上基因序列删除，可以采用Red重组技术完成。Red重组是一种基于λ噬菌体Red操纵子和Rac噬菌体RecE/RecT系统介导的DNA同源重组技术。通过该技术可以简单、快速地对任意大的DNA分子进行插入、敲除、突变等多种修饰。Red重组技术简单地说，首先向菌体中转入带有表达重组酶基因的pKD46质粒，然后电转化制备好的打靶用线性DNA片段，筛选阳性克隆，最后，将重组后菌种中抗性基因消除。
以下描述了具体的操作过程：
1、删除manXYZ基因序列
甘露糖转运蛋白EIIM，P/IIIman(mannose transporter EIIM，P/IIIMan，ManXYZ)可以用作N-乙酰-D-氨基葡萄糖的第二转运蛋白，能将N-乙酰-D-氨基葡萄糖等己糖转运入细胞，从而使排出胞外并积累的目标产物运回胞内降解。将manXYZ基因序列删除，可以阻止细胞外N-乙酰-D-氨基葡萄糖被转运回细胞内降解。
(1)制备Red重组打靶用线性DNA全长PCR片段
1)PCR扩增fKanrf片段
fKanrf片段，即FRT-Kanr-FRT片段，是指将卡拉霉素抗性基因(Kanr)的两端装有FLP重组酶特异性识别的FRT位点碱基序列。
设计引物：正向引物(mfKanf-F)SEQ ID No.1，反向引物(mfKanf-R)SEQ ID No.2。
模板：pPic9K。
PCR反应条件：第一步：94℃变性1min；第二步：94℃30s，55℃30s，72℃40s，循环30次；第三步：72℃延伸10min。
fKanrf大小：1.28kb。其核苷酸序列SEQ ID No.3。
PCR产物经1％琼脂糖凝胶电泳分离、纯化回收片段。
2)PCR扩增Red重组打靶用线性DNA全长片段
设计同源臂引物：根据manXYZ序列SEQ ID No.4，设计删除manXYZ序列的同源臂正向引物(manXYZKO-F)SEQ ID No.5，反向引物(manXYZKO-R)SEQ ID No.6。
模板：扩增的fKanrf PCR片段。
PCR反应条件：第一步：94℃变性1min；第二步：94℃30s，55℃30s，72℃40s，循环30次；第三步：72℃延伸10min。
扩增产物：同源臂+fKanrf+同源臂。
将PCR产物琼脂糖凝胶电泳分离、纯化回收，得到100ng/μl的线性DNA全长PCR片段用于Red重组打靶。
(2)Red重组操作
首先，将pKD46载体转入大肠杆菌AT-001菌株中。然后，电转化制备好 的打靶用线性DNA片段，筛选阳性克隆。最后，消除抗性基因。
1)转化pKD46质粒
pKD46载体是带有表达Red重组酶基因的质粒，表达Exo、Bet和Gam三基因片段，3个基因置于阿拉伯糖启动子下，经L-阿拉伯糖诱导就可以大量表达。为达到通过Red重组修饰染色体上目标基因的目的，有必要将pKD46质粒转化到大肠杆菌中。
①感受态制备：首先，将保存于-20℃的Escherichia coli ATCC 27325菌液，按1:50-100接种于10ml LB液体培养基中，37℃，225rpm，振荡培养2-3小时。再将培养液加入到10ml离心管中，4000g×5min，弃去上清，用冰浴的0.1M CaCl25ml悬浮5min。最后，4000g×5min离心，弃去上清，用冰浴的0.1M CaCl2 5ml悬浮。-4℃静置12小时，自然沉降。其中，0.1M CaCl2的制备：用无水CaCl2配1M的CaCl2，用蒸气压力为15lbf/in2的高压灭菌20min，分装1.5ml于-20℃保存，用时融化后按1:10比例稀释配成0.1M的CaCl2。
②质粒转化：取自然沉降的菌体250μl，加入5μl pKD46质粒，-4℃，30min。然后，42℃水浴1.5min，加入SOC培养基0.7ml，30℃摇2小时。取0.2ml菌液，涂青霉素平板。30℃过夜(12-16小时)培养。挑单克隆，加入5ml LB液体培养基中培养，抽质粒鉴定。保存阳性菌种备用。
2)电转化制备好的打靶用线性DNA片段，筛选阳性克隆
①电转感受态的制备：将含pKD46Escherichia coli ATCC 27325菌种AT-001接种于含有氨苄青霉素(Amp)LB培养基的试管，250rpm摇床过夜，第二天以1％的量接种至含有Amp的LB培养基中，30℃培养，待OD600达到0.2左右后，加入0.2％的L-阿拉伯糖，30℃诱导35分钟，直至OD600达到0.4左右。冰浴冷却。用超纯水洗一次，10％甘油洗两次，最后用10％甘油重悬，甘油用量以使菌体被浓缩500-1000倍的终浓度为宜。
②电击转化：将2mm电转杯从70％乙醇中取出，用灭菌超纯水洗2次，紫外灯照射30分钟。4℃预冷30分钟。取90μl最终重悬的细胞，移至预冷的离心管，加入5μl(100ng以上)步骤(1)得到的全长PCR片段(线性DNA)，用枪轻轻吸打混匀，冰浴30分钟。电转参数：2500V，200Ω，25μF。
③复苏与筛选阳性克隆：加入1ml的LB液体培养基，37℃，100rpm，1小时。然后每200μl涂布一个卡拉霉素(Kan)平板，一共5个。均匀、涂干。 30℃培养24个小时。挑在卡拉霉素抗性下生长的克隆，作PCR鉴定，筛选阳性克隆。
所获得菌种编号：AT-002-01(AT-001，△manXYZ::fKanrf)。
(3)抗性基因的消除
为便于后续工作，可消除所获得菌种(阳性克隆)中的抗性基因。消除抗性基因可借助pCP20质粒完成。pCP20是带有氨苄青霉素和氯霉素抗性基因的质粒，热诱导后可表达FLP重组酶，该酶可特异性识别FRT位点，通过重组可将FRT位点间的序列删除，只保留一个FRT位点。
将pCP20转入上述卡拉霉素抗性克隆，30℃培养8h，后提高到42℃过夜，热诱导FLP重组酶表达，质粒逐渐丢失。用接种环蘸菌液在无抗生素培养基上划板，挑长出的单克隆点到卡拉霉素抗性平板上，未生长的为卡拉霉素抗性基因已被FLP重组酶删除的克隆。用鉴定引物作PCR对卡拉霉素抗性消失的克隆进行鉴定。
所获得菌种编号：AT-002-02(AT-001，△manXYZ)。
2、删除nanA基因序列
N-乙酰神经氨酸裂解酶(N-acetylneuraminate lyase，NanA)能够将微生物中的N-乙酰-D-氨基甘露糖(ManNAc)降解成N-乙酰-D-神经氨酸(Neu5Ac)。将nanKETA操纵子中的nanA基因序列删除，可以阻止N-乙酰-D-氨基甘露糖(ManNAc)降解成N-乙酰-D-神经氨酸(Neu5Ac)。
(1)制备Red重组打靶用线性DNA全长PCR片段
设计同源臂引物：根据nanE-nanK前段nanA序列SEQ ID No.7，设计nanA序列删除的同源臂引物：正向引物(nanAKO-F)SEQ ID No.8，反向引物(nanAKO-R)SEQ ID No.9。
模板：扩增的fKanrf PCR片段。
PCR反应条件：第一步：94℃变性1min；第二步：94℃30s，55℃30s，72℃40s，循环30次；第三步：72℃延伸10min。
扩增产物：同源臂+fKanrf+同源臂。
将PCR产物琼脂糖凝胶电泳分离、纯化回收，得到100ng/μl的线性DNA全长PCR片段用于Red重组打靶。
(2)Red重组操作
首先，将pKD46载体转入大肠杆菌AT-002-02菌株中。然后，电转化制备好的打靶用线性DNA片段，筛选阳性克隆。最后，消除抗性基因。
1)转化pKD46质粒
①感受态制备：首先，将保存于-20℃的Escherichia coli AT-002-02(AT-001，△manXYZ)菌液，按1:50-100接种于10ml LB液体培养基中，37℃，225rpm，振荡培养2-3小时。再将培养液加入到10ml离心管中，4000g×5min，弃去上清，用冰浴的0.1M CaCl2 5ml悬浮5min。最后，4000g×5min离心，弃去上清，用冰浴的0.1M CaCl2 5ml悬浮。-4℃静置12小时，自然沉降。
②质粒转化：取自然沉降的菌体250μl，加入5μl pKD46质粒，-4℃，30min。然后，42℃水浴1.5min，加入SOC培养基0.7ml，30℃摇2小时。取0.2ml菌液，涂青霉素平板。30℃过夜(12-16小时)培养。挑单克隆，加入5ml LB液体培养基中培养，抽质粒鉴定。保存阳性菌种备用。
2)电转化制备好的打靶用线性DNA片段，筛选阳性克隆
①电转感受态的制备：将含pKD46Escherichia coli菌种AT-002-02接种于含有氨苄青霉素(Amp)LB培养基的试管，250rpm摇床过夜，第二天以1％的量接种至含有Amp的LB培养基中，30℃培养，待OD600达到0.2左右后，加入0.2％的L-阿拉伯糖，30℃诱导35分钟，直至OD600达到0.4左右。冰浴冷却。用超纯水洗一次，10％甘油洗两次，最后用10％甘油重悬，甘油用量以使菌体被浓缩500-1000倍的终浓度为宜。
②电击转化：将2mm电转杯从70％乙醇中取出，用灭菌超纯水洗2次，紫外灯照射30分钟。4℃预冷30分钟。取90μl最终重悬的细胞，移至预冷的离心管，加入5μl(100ng以上)步骤(1)得到的全长PCR片段(线性DNA)，用枪轻轻吸打混匀，冰浴30分钟。电转参数：2500V，200Ω，25μF。
③复苏与筛选阳性克隆：加入1ml的LB液体培养基，37℃，100rpm，1小时。然后每200μl涂布一个卡拉霉素(Kan)平板，一共5个。均匀、涂干。30℃培养24个小时。挑在卡拉霉素抗性下生长的克隆，作PCR鉴定，筛选阳性克隆。
所获得菌种编号：AT-003-01(AT-002-02，△nanA::fKanrf)。
(3)抗性基因的消除
将pCP20转入上述卡拉霉素抗性克隆，30℃培养8h，后提高到42℃过夜，热诱导FLP重组酶表达，质粒逐渐丢失。用接种环蘸菌液在无抗生素培养基上划板，挑长出的单克隆点到卡拉霉素抗性平板上，未生长的为卡拉霉素抗性基因已被FLP重组酶删除的克隆。用鉴定引物作PCR对卡拉霉素抗性消失的克隆进行鉴定。
所获得菌种编号：AT-003-02(AT-002-02，△nanA)。
3、删除nagA基因序列
N-乙酰-D-氨基葡萄糖-6-磷酸脱乙酰酶(N-acetylglucosamine-6-phosphate deacetylase，NagA)能将微生物中N-乙酰-D-氨基葡萄糖-6-磷酸(GlcNAc-6-P)转变为D-氨基葡萄糖-6-磷酸(GlcN-6-P)。将nag操纵子(nagE-nagBACD)中的nagA基因序列删除，可以阻止N-乙酰-D-氨基葡萄糖-6-磷酸(GlcNAc-6-P)转变为D-氨基葡萄糖-6-磷酸(GlcN-6-P)。
(1)制备Red重组打靶用线性DNA全长PCR片段
设计同源臂引物：根据NCBI查找NC_000913，Escherichia coli str.K-12N-乙酰-D-氨基葡萄糖-6-磷酸脱乙酰酶基因nagA序列SEQ ID No.10，设计nagA序列删除的同源臂引物：正向引物(nagAKO-F)SEQ ID No.11，反向引物(nagAKO-R)SEQ ID No.12。
模板：扩增的fKanrf PCR片段。
PCR反应条件：第一步：94℃变性1min；第二步：94℃30s，55℃30s，72℃40s，循环30次；第三步：72℃延伸10min。
扩增产物：同源臂+fKanf+同源臂。
将PCR产物琼脂糖凝胶电泳分离、纯化回收，得到100ng/μl的线性DNA全长PCR片段用于Red重组打靶。
(2)Red重组操作
首先，将pKD46载体转入大肠杆菌AT-003-02菌株中。然后，电转化制备好的打靶用线性DNA片段，筛选阳性克隆。最后，消除抗性基因。
1)转化pKD46质粒
①感受态制备：首先，将保存于-20℃的Escherichia coli AT-003-02(AT-002-02，△nanA)菌液，按1:50-100接种于10ml LB液体培养基中，37℃， 225rpm，振荡培养2-3小时。再将培养液加入到10ml离心管中，4000g×5min，弃去上清，用冰浴的0.1M CaCl2 5ml悬浮5min。最后，4000g×5min离心，弃去上清，用冰浴的0.1M CaCl2 5ml悬浮。-4℃静置12小时，自然沉降。
②质粒转化：取自然沉降的菌体250μl，加入5μl pKD46质粒，-4℃，30min。然后，42℃水浴1.5min，加入SOC培养基0.7ml，30℃摇2小时。取0.2ml菌液，涂青霉素平板。30℃过夜(12-16小时)培养。挑单克隆，加入5ml LB液体培养基中培养，抽质粒鉴定。保存阳性菌种备用。
2)电转化制备好的打靶用线性DNA片段，筛选阳性克隆
①电转感受态的制备：将含pKD46Escherichia coli菌种AT-003-02接种于含有氨苄青霉素(Amp)LB培养基的试管，250rpm摇床过夜，第二天以1％的量接种至含有Amp的LB培养基中，30℃培养，待OD600达到0.2左右后，加入0.2％的L-阿拉伯糖，30℃诱导35分钟，直至OD600达到0.4左右。冰浴冷却。用超纯水洗一次，10％甘油洗两次，最后用10％甘油重悬，甘油用量以使菌体被浓缩500-1000倍的终浓度为宜。
②电击转化：将2mm电转杯从70％乙醇中取出，用灭菌超纯水洗2次，紫外灯照射30分钟。4℃预冷30分钟。取90μl最终重悬的细胞，移至预冷的离心管，加入5μl(100ng以上)步骤(1)得到的全长PCR片段(线性DNA)，用枪轻轻吸打混匀，冰浴30分钟。电转参数：2500V，200Ω，25μF。
③复苏与筛选阳性克隆：加入1ml的LB液体培养基，37℃，100rpm，1小时。然后每200μl涂布一个卡拉霉素(Kan)平板，一共5个。均匀、涂干。30℃培养24个小时。挑在卡拉霉素抗性下生长的克隆，作PCR鉴定，筛选阳性克隆。
所获得菌种编号：AT-004-01(AT-003-02，△nagA::fKanrf)。
(3)抗性基因的消除
将pCP20转入上述卡拉霉素抗性克隆，30℃培养8h，后提高到42℃过夜，热诱导FLP重组酶表达，质粒逐渐丢失。用接种环蘸菌液在无抗生素培养基上划板，挑长出的单克隆点到卡拉霉素抗性平板上，未生长的为卡拉霉素抗性基因已被FLP重组酶删除的克隆。用鉴定引物作PCR对卡拉霉素抗性消失的克隆进行鉴定。
所获得菌种编号：AT-004-02(AT-003-02，△nagA)。
4、删除nagE基因序列
将N-乙酰-D-氨基葡萄糖特异酶IINag(N-acetyl-glucosamine-specific enzyme IINag，NagE)的基因序列nagE删除，可阻止细胞外GlcNAc被转运回细胞内降解。
(1)制备Red重组打靶用线性DNA全长PCR片段
设计同源臂引物：根据NCBI查找NC_000913，Escherichia coli str.K-12nagB启动子和nagE基因序列SEQ ID No.13，设计删除nagE基因序列的同源臂正向引物(nagEKO-F1)SEQ ID No.14，反向引物(nagEKO-R1)SEQ ID No.15。
模板：扩增的fKanrf PCR片段。
PCR反应条件：第一步：94℃变性1min；第二步：94℃30s，55℃30s，72℃40s，循环30次；第三步：72℃延伸10min。
扩增产物：同源臂+fKanrf+同源臂。
将PCR产物琼脂糖凝胶电泳分离、纯化回收，得到100ng/μl的线性DNA全长PCR片段用于Red重组打靶。
(2)Red重组操作
首先，将pKD46载体转入大肠杆菌AT-004-02菌株中。然后，电转化制备好的打靶用线性DNA片段，筛选阳性克隆。最后，消除抗性基因。
1)转化pKD46质粒
①感受态制备：首先，将保存于-20℃的Escherichia coli AT-004-02(AT-003-02，△nagA)菌液，按1:50-100接种于10ml LB液体培养基中，37℃，225rpm，振荡培养2-3小时。再将培养液加入到10ml离心管中，4000g×5min，弃去上清，用冰浴的0.1M CaCl2 5ml悬浮5min。最后，4000g×5min离心，弃去上清，用冰浴的0.1M CaCl2 5ml悬浮。-4℃静置12小时，自然沉降。
②质粒转化：取自然沉降的菌体250μl，加入5μl pKD46质粒，-4℃，30min。然后，42℃水浴1.5min，加入SOC培养基0.7ml，30℃摇2小时。取0.2ml菌液，涂青霉素平板。30℃过夜(12-16小时)培养。挑单克隆，加入5ml LB液体培养基中培养，抽质粒鉴定。保存阳性菌种备用。
2)电转化制备好的打靶用线性DNA片段，筛选阳性克隆
①电转感受态的制备：将含pKD46Escherichia coli菌种AT-004-02接种于 含有氨苄青霉素(Amp)LB培养基的试管，250rpm摇床过夜，第二天以1％的量接种至含有Amp的LB培养基中，30℃培养，待OD600达到0.2左右后，加入0.2％的L-阿拉伯糖，30℃诱导35分钟，直至OD600达到0.4左右。冰浴冷却。用超纯水洗一次，10％甘油洗两次，最后用10％甘油重悬，甘油用量以使菌体被浓缩500-1000倍的终浓度为宜。
②电击转化：将2mm电转杯从70％乙醇中取出，用灭菌超纯水洗2次，紫外灯照射30分钟。4℃预冷30分钟。取90μl最终重悬的细胞，移至预冷的离心管，加入5μl(100ng以上)步骤(1)得到的全长PCR片段(线性DNA)，用枪轻轻吸打混匀，冰浴30分钟。电转参数：2500V，200Ω，25μF。
③复苏与筛选阳性克隆：加入1ml的LB液体培养基，37℃，100rpm，1小时。然后每200μl涂布一个卡拉霉素(Kan)平板，一共5个。均匀、涂干。30℃培养24个小时。挑在卡拉霉素抗性下生长的克隆，作PCR鉴定，筛选阳性克隆。
所获得菌种编号：AT-005-01(AT-004-02，△nagE::fKanrf)。
(3)抗性基因的消除
将pCP20转入上述卡拉霉素抗性克隆，30℃培养8h，后提高到42℃过夜，热诱导FLP重组酶表达，质粒逐渐丢失。用接种环蘸菌液在无抗生素培养基上划板，挑长出的单克隆点到卡拉霉素抗性平板上，未生长的为卡拉霉素抗性基因已被FLP重组酶删除的克隆。用鉴定引物作PCR对卡拉霉素抗性消失的克隆进行鉴定。
所获得菌种编号：AT-005-02(AT-004-02，△nagE)。
实施例2
本实施例描述了N-乙酰-D-氨基甘露糖激酶(NanK)的基因nanK的克隆和大肠杆菌中转化nanK/pTrc99A质粒，以及ptrc-nanK基因盒向大肠杆菌染色体中的整合。
1、nanK基因的克隆、在大肠杆菌中转化nanK/pTrc99A质粒及其对N乙酰-D-氨基葡萄糖产量的影响
扩增Escherichia coli NanK(N-acetylmannosamine kinase，N-乙酰-D-氨基甘 露糖激酶)的基因nanK，置于Trc启动子控制下转化菌种，使之过量表达，能够加强ManNAc(N-Acetyl-D-mannosamine，N-乙酰-D-氨基甘露糖或N-乙酰-D-甘露糖胺)磷酸化为ManNAc-6-P(N-Acetyl-D-mannosamine-6-P，N-乙酰-D-氨基甘露糖-6-磷酸)。
1)大肠杆菌nanK基因的克隆
根据NCBI查找U00096，获得Escherichia coli nanK基因核苷酸序列SEQ ID No.16，其氨基酸序列SEQ ID No.17。
设计引物：正向引物(nanK-F)SEQ ID No.18，反向引物(nanK-R)SEQ ID No.19。
模板：Escherichia coli AT-001。
PCR反应条件：第一步：94℃变性1min；第二步：94℃30s，55℃30s，72℃40s，循环30次；第三步：72℃延伸10min。
扩增产物大小：0.9kb。
PCR产物经1％琼脂糖凝胶电泳分离、纯化回收片段。
将所获得的PCR扩增片段和pUC57-T载体连接并测序鉴定，获得nanK/pUC57。
2)将nanK基因置于Trc启动子控制下的质粒构建及转化
①质粒构建：扩增质粒nanK/pUC57，分别用Nco I和HindIII酶切质粒nanK/pUC57和载体pTrc99A，琼脂糖凝胶电泳分离、纯化回收nanK片段和pTrc99A片段，用T4DNA连接酶，16℃将连接过夜，并鉴定，得到nanK/pTrc99A质粒。
②感受态制备：首先，将保存于-20℃的AT-005-02菌液，按1:50-100接种于10ml LB液体培养基中，37℃，225rpm，振荡培养2-3小时。再将培养液加入到10ml离心管中，4000g×5min，弃去上清，用冰浴的0.1M CaCl2 5ml悬浮5min。最后，4000g×5min离心，弃去上清，用冰浴的0.1M CaCl2 5ml悬浮。-4℃静置12小时，自然沉降。
③质粒转化：取自然沉降的菌体250μl，加入5μl nanK/pTrc99A质粒，-4℃，30min。然后，42℃水浴1.5min，加入SOC培养基0.7ml，30℃摇2小时。取0.2ml菌液，涂青霉素平板。30℃过夜(12-16小时)培养。挑单克隆，加入5ml LB液体培养基中培养，抽质粒鉴定。保存阳性菌种备用。得到重组菌 nanK/pTrc99A(AT-005-02)
3)nanK/pTrc99A质粒转化对N-乙酰-D-氨基葡萄糖产量的影响
将重组菌nanK/pTrc99A(AT-005-02)和对照菌种，做摇瓶发酵试验。取新鲜培养的LB平板培养基上单克隆菌株，接种于3ml的LB液体培养基试管(13×150mm)中，30℃，225rpm，培养约8小时。LB液体培养基成分：5g/l酵母粉,10g/l蛋白胨,10g/l NaCl。然后取种子培养液，3％接种于含50ml的发酵培养液(M9培养液)250ml摇瓶中。起始OD600约0.5，37℃下，225rpm培养，发酵周期72小时。在第24小时、48小时，用10M NaOH调节发酵液pH至7.0。根据发酵液糖耗情况，分次加入65％葡萄糖液维持葡萄糖浓度在20g/L。发酵结束，取1ml发酵液，离心。用HPLC法测定N-乙酰-D-氨基葡萄糖含量。
①N-乙酰-D-氨基葡萄糖含量的HPLC法测定
缓冲液：将3.5g磷酸氢二钾加入1L容量瓶中，加水足以溶解，加0.25mL氨水，再加水稀释混匀，用磷酸调pH到7.5，以水定容。
流动相：乙腈：缓冲液(75:25)。
稀释液：乙腈和水(50:50)。
标准溶液：1.0mg/mL USP N-乙酰-D-氨基葡萄糖标准品(RS)溶于稀释液中。
样品溶液：1.0mg/mL N-乙酰-D-氨基葡萄糖样品溶于稀释液中。
液相条件：
型号：LC
检测器：UV 195nm
色谱柱：4.6-mm×15-cm；3-μm packing L8
流速：1.5ml/min
柱温：35℃
进样体积：10μL
②M9培养液的配制
先配制5×M9培养基：将在约800ml双蒸水(ddH2O)中加入64g Na2HPO4·7H2O、15g KH2PO4、2.5g NaCl、5.0g NH4Cl，溶解后，加水至1000ml。121℃灭菌30分钟。再分别配制1M MgSO4、1M CaCl2、20％葡萄糖，并单独灭菌。然后按表1配制M9培养液，其中，1000×微量元素溶液按表2配制。
表1. M9培养液成分
Figure PCTCN2017080653-appb-000003
表2. 1000×微量元素溶液成分
成分 用量(g/L)
CoCl2·6H2O 0.01
CuSO4·5H2O 0.01
MnSO4·H2O 0.033
Fe SO4·7H2O 0.50
ZnSO4·7H2O 0.38
H3BO3 0.01
NaMoO4·2H2O 0.01
pH 3
③nanK/pTrc99A质粒转化对摇瓶发酵N-乙酰-D-氨基葡萄糖产量的影响
摇瓶发酵产量情况见表3。结果表明：对照菌种AT-005-02产量很低，未检出，而nanK基因在Trc启动子控制下过量表达的重组菌nanK/pTrc99A(AT-005-02)产量明显提高。
表3.重组菌nanK/pTrc99A(AT-005-02)摇瓶发酵产量
Figure PCTCN2017080653-appb-000004
2、pTrc-nanK基因盒向大肠杆菌染色体中整合
以nagE基因位点为pTrc-nanK基因盒在染色体上的整合位点。为达到pTrc-nanK基因盒向大肠杆菌染色体中的整合，首先，扩增带Trc启动子的nanK片段pTrc-nanK，以及两侧带有FLP重组酶识别位点(FRT位点)的卡拉霉素抗性基因片段：FRT-Kanr-FRT(fKanrf)，并拼接。然后，再一次设计删除nagE基因序列同源臂的引物，并以pTrc-nanK和fKanrf拼接的片段为模板，扩增Red重组打靶用线性DNA全长片段。
具体操作过程如下：
(1)PCR扩增pTrc-nanK片段
模板：nanK/pTrc99A。
设计引物：正向引物(Trcff-F)SEQ ID No.20，反向引物(Trcff-R)SEQ ID No.21。
PCR反应条件：第一步：94℃变性1min；第二步：94℃30s，55℃30s，72℃40s，循环30次；第三步：72℃延伸10min。
产物大小：1.05kb。
PCR产物经1％琼脂糖凝胶电泳分离、纯化回收片段。
(2)PCR扩增fKanrf片段
设计引物：正向引物(mfKanf-F)SEQ ID No.1，反向引物(mfKanf-R)SEQ ID No.2。
模板：pPic9K。
PCR反应条件：第一步：94℃变性1min；第二步：94℃30s，55℃30s，72℃40s，循环30次；第三步：72℃延伸10min。
fKanrf大小：1.28kb。其核苷酸序列SEQ ID No.3。
PCR产物经1％琼脂糖凝胶电泳分离、纯化回收片段。
(3)扩增与pTrc-nanK对接的fKanrf
设计引物：正向引物(fKanf-F)SEQ ID No.22，反向引物(fKanf-R)SEQ ID No.23。
模板：fKanrf。
PCR反应条件：第一步：94℃变性1min；第二步：94℃30s，55℃30s，72℃40s，循环30次；第三步：72℃延伸10min。
二次扩增的fKanrf大小：1.3kb。
PCR产物经1％琼脂糖凝胶电泳分离、纯化回收片段。
(4)制备Red重组打靶用线性DNA全长PCR片段
设计同源臂引物：再一次设计删除nagE基因序列同源臂的正向引物(nagEKO-F2)SEQ ID No.24，反向引物(nagEKO-R2)SEQ ID No.25。
模板：pTrc-nanK PCR片段和二次扩增的fKanrf PCR片段，1：1混合。
PCR反应条件：第一步：94℃变性1min；第二步：94℃30s，55℃30s，72℃40s，循环30次；第三步：72℃延伸10min。
扩增产物：同源臂+pTrc-nanK-fKanrf+同源臂。
将PCR产物琼脂糖凝胶电泳分离、纯化回收，得到100ng/μl的线性DNA全长PCR片段用于Red重组打靶。
(5)Red重组操作
首先，将pKD46载体转入大肠杆菌AT-004-02菌株中。然后，电转化制备好的打靶用线性DNA片段，筛选阳性克隆。最后，消除抗性基因。
1)转化pKD46质粒
①感受态制备：首先，将保存于-20℃的Escherichia coli AT-004-02菌液，按1:50-100接种于10ml LB液体培养基中，37℃，225rpm，振荡培养2-3小时。再将培养液加入到10ml离心管中，4000g×5min，弃去上清，用冰浴的0.1M CaCl25ml悬浮5min。最后，4000g×5min离心，弃去上清，用冰浴的0.1M CaCl2 5ml悬浮。-4℃静置12小时，自然沉降。
②质粒转化：取自然沉降的菌体250μl，加入5μl pKD46质粒，-4℃，30min。然后，42℃水浴1.5min，加入SOC培养基0.7ml，30℃摇2小时。取0.2ml菌液，涂青霉素平板。30℃过夜(12-16小时)培养。挑单克隆，加入5ml LB液体培养基中培养，抽质粒鉴定。保存阳性菌种备用。
2)电转化制备好的打靶用线性DNA片段，筛选阳性克隆
①电转感受态的制备：将含pKD46Escherichia coli菌种AT-004-02接种于含有氨苄青霉素(Amp)LB培养基的试管，250rpm摇床过夜，第二天以1％的量接种至含有Amp的LB培养基中，30℃培养，待OD600达到0.2左右后，加入0.2％的L-阿拉伯糖，30℃诱导35分钟，直至OD600达到0.4左右。冰浴冷却。用超纯水洗一次，10％甘油洗两次，最后用10％甘油重悬，甘油用量以使菌体被浓缩500-1000倍的终浓度为宜。
②电击转化：将2mm电转杯从70％乙醇中取出，用灭菌超纯水洗2次，紫外灯照射30分钟。4℃预冷30分钟。取90μl最终重悬的细胞，移至预冷的离心管，加入5μl(100ng以上)步骤(4)得到的全长PCR片段(线性DNA)，用枪轻轻吸打混匀，冰浴30分钟。电转参数：2500V，200Ω，25μF。
③复苏与筛选阳性克隆：加入1ml的LB液体培养基，37℃，100rpm，1小时。然后每200μl涂布一个卡拉霉素(Kan)平板，一共5个。均匀、涂干。30℃培养24个小时。挑在卡拉霉素抗性下生长的克隆，作PCR鉴定，筛选阳性克隆。
所获得菌种编号：AT-006-01(AT-004-02，△nagE::pTrc-nanK-fKanrf)。
(6)抗性基因的消除
将pCP20转入上述卡拉霉素抗性克隆，30℃培养8h，后提高到42℃过夜，热诱导FLP重组酶表达，质粒逐渐丢失。用接种环蘸菌液在无抗生素培养基上划板，挑长出的单克隆点到卡拉霉素抗性平板上，未生长的为卡拉霉素抗性基因已被FLP重组酶删除的克隆。用鉴定引物作PCR对卡拉霉素抗性消失的克隆进行鉴定。
所获得菌种编号：AT-006-02(AT-004-02，△nagE::pTrc-nanK)。
3、pTrc-nanK基因盒整合对N-乙酰-D-氨基葡萄糖产量的影响
将在染色体nagE基因位点上整合有pTrc-nanK基因盒的重组菌AT-006-02和对照菌种，做摇瓶发酵试验。取新鲜培养的LB平板培养基上单克隆菌株，接种于3ml的LB液体培养基试管(13×150mm)中，30℃，225rpm，培养约8小时。然后取种子培养液，3％接种于含50ml的发酵培养液(M9培养液)250ml摇瓶中。起始OD600约0.5，37℃下，225rpm培养，发酵周期72小时。在第 24小时、48小时，用10M NaOH调节发酵液pH至7.0。根据发酵液糖耗情况，分次加入65％葡萄糖液维持葡萄糖浓度在20g/L。发酵结束，取1ml发酵液，离心。用HPLC法测定N-乙酰-D-氨基葡萄糖含量。
摇瓶发酵产量情况见表4。结果表明：对照菌种AT-001、AT-005-02产量很低，未检出，而pTrc-nanK基因盒整合重组菌产量明显提高，且较未整合的重组菌nanK/pTrc99A(AT-005-02)产量亦有明显提高。
表4.pTrc-nanK基因盒整合重组菌摇瓶发酵产量
Figure PCTCN2017080653-appb-000005
实施例3
本实施例描述筛选突变的N-乙酰-D-氨基甘露糖激酶(NanK)的基因，所述基因编码酶活性增加的N-乙酰-D-氨基甘露糖激酶(NanK)。
为了进一步提高生产菌株中的N-乙酰-D-氨基葡萄糖合成量，筛选编码具有酶活性增加的N-乙酰-D-氨基甘露糖激酶(NanK)的基因突变体。为了达到该目的，用易错PCR技术扩增克隆的基因，通过用于扩增的DNA聚合酶，在导致高频错配的条件下扩增所述基因，以便在PCR产物中得到高频突变。
具体操作过程如下：
1、易错PCR扩增大肠杆菌N-乙酰-D-氨基甘露糖激酶基因nanK
利用Taq DNA聚合酶不具有3'-5'校对功能的性质，在高镁离子浓度(8mmol/L)和不同浓度dNTP的浓度下(其中，dATP和dGTP浓度为1.5mmol/L；dTTP和dCTP浓度为3.0mmol/L)，来控制随机突变的频率，向目的基因中引入随机突变，构建突变库；模板浓度A260值为1000ng/mL，酶浓度为5U/μL，引物浓度为100μM。
易错PCR反应体系(50μl)：10×PCR反应缓冲液5μl，dNTP(2.5mM)5μl，MgCl2(2.5mM)5μl，正向引物(nanK-F，SEQ ID No.18)1μl，反向引物(nanK-R，SEQ ID No.19)1μl，DNA模板(nanK/pUC57)0.1μl，Taq DNA聚合酶0.5μl，ddH2O 32.4μl。
PCR程序：96℃预变性4min；94℃变性1min，56℃退火1min，75℃延伸2min，45个循环；最后75℃延伸15min，采用胶回收方法回收PCR产物(产物大小：0.9kb)；取5μl产物1％琼脂糖凝胶电泳检验，-20℃保存备用。
2、构建N-乙酰-D-氨基甘露糖激酶的基因突变体库
将上述PCR产物经限制性内切酶Nco I和Hind III双酶切消化后，与用Nco I和Hind III内切酶消化的pTrc99A质粒进行连接反应，然后用连接产物混合物转化大肠杆菌AT-005-02，获得大量克隆转化子，构建转化菌体突变库。
3、筛选高酶活突变体
从转化菌体突变库中，随机挑取突变克隆300株，以野生型NanK/pTrc99A(AT-005-02)为对照，分别接种至含50μg/mL青霉素(Amp)的5ml LB培养基中，37℃、150rpm培养18h后，10000rpm，5mim离心收集菌体。弃上清后，在4℃下重悬于1ml PBS(pH值7.5，10mmol/L)溶液中，在冰浴条件下选取300V电压，超声3s间歇6s对其进行超声破碎10min，离心取上清作为酶粗提液，进行酶活测定。
N-乙酰-D-氨基甘露糖激酶(NanK)的活性检测：以N-乙酰-D-氨基甘露糖(ManNAc)被磷酸化多少为依据，也就是N-乙酰-D-氨基甘露糖减少为测定标记。酶活单位定义：在酶促反应条件下，每分钟减少相当于1μmol N-乙酰-D-氨基甘露糖的还原糖所需的酶量，定义为一个酶活力单位(IU)。具体操作如下：以5ml反应体系为酶活测定体系，其中含500mmol/L N-乙酰-D-氨基甘露糖、5mmol/L葡萄糖、100mmol/L Tris-HCl(pH8.0)及100μl粗酶液。酶活反应在37℃水浴中进行，保温4h，然后将酶解液在70℃下10min终止反应。3000rpm离心10min，取上清液。HPLC测定N-乙酰-D-氨基甘露糖含量。
结果表明：最高突变体菌株的酶活为77.5IU/ml，对照菌株的酶活为16.3IU/ml。通过易错PCR对NanK进行改造，获得酶活力提高约5倍的突变株。挑 选该酶活性最高的突变体菌株，提取质粒测序。结果表明：该N-乙酰-D-氨基甘露糖激酶突变体基因序列如SEQ ID No.26所示，对应的氨基酸序列如SEQ ID No.27所示。与野生型的N-乙酰-D-氨基甘露糖激酶基因序列比对，共发生了4处碱基点突变：107A/G，309T/G，669G/C，783A/G；致氨基酸3处错义突变，其突变点分别为：Q36R(第36位赖氨酸变为精氨酸)，I103M(第103位异亮氨酸变为蛋氨酸)，R223S(第223位精氨酸变为丝氨酸)。将该突变基因命名为nanKM。
4、pTrc-nanKM基因盒向大肠杆菌染色体nagE基因位点上整合
以nagE基因位点为pTrc-nanKM基因盒在染色体上的整合位点。为达到pTrc-nanKM基因盒向大肠杆菌染色体中的整合，首先，扩增带Trc启动子的nanKM片段pTrc-nanKM，以及两侧带有FLP重组酶识别位点(FRT位点)的卡拉霉素抗性基因片段：FRT-Kanr-FRT(fKanrf)，并拼接。然后，再一次设计删除nagE基因序列同源臂的引物，并以pTrc-nanKM和fKanrf拼接的片段为模板，扩增Red重组打靶用线性DNA全长片段。
具体操作过程如下：
(1)PCR扩增pTrc-nanKM片段
模板：nanKM/pTrc99A。
设计引物：正向引物(Trcff-F)SEQ ID No.20，反向引物(Trcff-R)SEQ ID No.21。
PCR反应条件：第一步：94℃变性1min；第二步：94℃30s，55℃30s，72℃40s，循环30次；第三步：72℃延伸10min。
产物大小：1.05kb。
PCR产物经1％琼脂糖凝胶电泳分离、纯化回收片段。
(2)PCR扩增fKanrf片段
设计引物：正向引物(mfKanf-F)SEQ ID No.1，反向引物(mfKanf-R)SEQ ID No.2。
模板：pPic9K。
PCR反应条件：第一步：94℃变性1min；第二步：94℃30s，55℃30s，72℃40s，循环30次；第三步：72℃延伸10min。
fKanrf大小：1.28kb。其核苷酸序列SEQ ID No.3。
PCR产物经1％琼脂糖凝胶电泳分离、纯化回收片段。
(3)扩增与pTrc-nanKM对接的fKanrf
设计引物：正向引物(fKanf-F)SEQ ID No.22，反向引物(fKanf-R)SEQ ID No.23。
模板：fKanrf。
PCR反应条件：第一步：94℃变性1min；第二步：94℃30s，55℃30s，72℃40s，循环30次；第三步：72℃延伸10min。
二次扩增的fKanrf大小：1.3kb。
PCR产物经1％琼脂糖凝胶电泳分离、纯化回收片段。
(4)制备Red重组打靶用线性DNA全长PCR片段
设计同源臂引物：再一次设计删除nagE基因序列同源臂的正向引物(nagEKO-F2)SEQ ID No.24，反向引物(nagEKO-R2)SEQ ID No.25。
模板：pTrc-nanKM PCR片段和二次扩增的fKanrf PCR片段，1：1混合。
PCR反应条件：第一步：94℃变性1min；第二步：94℃30s，55℃30s，72℃40s，循环30次；第三步：72℃延伸10min。
扩增产物：同源臂+pTrc-nanKM-fKanrf+同源臂。
将PCR产物琼脂糖凝胶电泳分离、纯化回收，得到100ng/μl的线性DNA全长PCR片段用于Red重组打靶。
(5)Red重组操作
首先，将pKD46载体转入大肠杆菌AT-004-02菌株中。然后，电转化制备好的打靶用线性DNA片段，筛选阳性克隆。最后，消除抗性基因。
1)转化pKD46质粒
①感受态制备：首先，将保存于-20℃的Escherichia coli AT-004-02菌液，按1:50-100接种于10ml LB液体培养基中，37℃，225rpm，振荡培养2-3小时。再将培养液加入10ml离心管中，4000g×5min，弃去上清，用冰浴的0.1M CaCl25ml悬浮5min。最后，4000g×5min离心，弃去上清，用冰浴的0.1M CaCl2 5ml悬浮。-4℃静置12小时，自然沉降。
②质粒转化：取自然沉降的菌体250μl，加入5μl pKD46质粒，-4℃，30min。然后，42℃水浴1.5min，加入SOC培养基0.7ml，30℃摇2小时。取0.2ml菌 液，涂青霉素平板。30℃过夜(12-16小时)培养。挑单克隆，加入5ml LB液体培养基中培养，抽质粒鉴定。保存阳性菌种备用。
2)电转化制备好的打靶用线性DNA片段，筛选阳性克隆
①电转感受态的制备：将含pKD46Escherichia coli菌种AT-004-02接种于含有氨苄青霉素(Amp)LB培养基的试管，250rpm摇床过夜，第二天以1％的量接种至含有Amp的LB培养基中，30℃培养，待OD600达到0.2左右后，加入0.2％的L-阿拉伯糖，30℃诱导35分钟，直至OD600达到0.4左右。冰浴冷却。用超纯水洗一次，10％甘油洗两次，最后用10％甘油重悬，甘油用量以使菌体被浓缩500-1000倍的终浓度为宜。
②电击转化：将2mm电转杯从70％乙醇中取出，用灭菌超纯水洗2次，紫外灯照射30分钟。4℃预冷30分钟。取90μl最终重悬的细胞，移至预冷的离心管，加入5μl(100ng以上)步骤(4)得到的全长PCR片段(线性DNA)，用枪轻轻吸打混匀，冰浴30分钟。电转参数：2500V，200Ω，25μF。
③复苏与筛选阳性克隆：加入1ml的LB液体培养基，37℃，100rpm，1小时。然后每200μl涂布一个卡拉霉素(Kan)平板，一共5个。均匀、涂干。30℃培养24个小时。挑在卡拉霉素抗性下生长的克隆，作PCR鉴定，筛选阳性克隆。
所获得菌种编号：AT-007-01(AT-004-02，△nagE::pTrc-nanKM-fKanrf)。
(6)抗性基因的消除
将pCP20转入上述卡拉霉素抗性克隆，30℃培养8h，后提高到42℃过夜，热诱导FLP重组酶表达，质粒逐渐丢失。用接种环蘸菌液在无抗生素培养基上划板，挑长出的单克隆点到卡拉霉素抗性平板上，未生长的为卡拉霉素抗性基因已被FLP重组酶删除的克隆。用鉴定引物作PCR对卡拉霉素抗性消失的克隆进行鉴定。
所获得菌种编号：AT-007-02(AT-004-02，△nagE::pTrc-nanKM)。
5、pTrc-nanKM基因盒整合对N-乙酰-D-氨基葡萄糖产量的影响
将在染色体nagE基因位点上整合有pTrc-nanKM基因盒的重组菌AT-007-02和对照菌种，做摇瓶发酵试验。取新鲜培养的LB平板培养基上单克隆菌株，接种于3ml的LB液体培养基试管(13×150mm)中，30℃，225rpm， 培养约8小时。然后取种子培养液，3％接种于含50ml的发酵培养液(M9培养液)250ml摇瓶中。起始OD600约0.5，37℃下，225rpm培养，发酵周期72小时。在第24小时、48小时，用10M NaOH调节发酵液pH至7.0。根据发酵液糖耗情况，分次加入65％葡萄糖液维持葡萄糖浓度在20g/L。发酵结束，取1ml发酵液，离心。用HPLC法测定N-乙酰-D-氨基葡萄糖含量。
摇瓶发酵产量情况见表5。结果表明：对照菌种AT-005-02产量很低，未检出，突变的pTrc-nanKM基因盒整合重组菌AT-007-02产量明显提高，且较未突变的对照菌种AT-006-02产量亦有明显提高。
表5.pTrc-nanKM基因盒整合重组菌摇瓶发酵产量
Figure PCTCN2017080653-appb-000006
以上结果显示：不仅N-乙酰-D-氨基甘露糖激酶超表达可明显提高N-乙酰-D-氨基葡萄糖产量，通过易错PCR技术筛选突变体也可大大提高N-乙酰-D-氨基葡萄糖产量，这是由于所获得的该激酶突变体酶活性增加所致。
实施例4
本实施例描述整合有pTrc-nanKM盒的大肠杆菌菌株，其中过量表达N-乙酰-D-氨基甘露糖-6-P异构酶(NanE)的基因nanE及其对N-乙酰-D-氨基葡萄糖产量的影响。
扩增Escherichia coli NanE(N-acetylmannosamine-6-phosphate epimerase，N-乙酰-D-氨基甘露糖-6-P异构酶)的基因nanE，并插入pTrc99A，从而将nanE置于Trc启动子控制下转化菌种，或将nanE基因内源性天然启动子换成Trc启动子，使之过量表达，能够加强N-乙酰-D-氨基甘露糖-6-磷酸(ManNAc-6-P)转化为N-乙酰-D-氨基葡萄糖-6-磷酸(GlcNAc-6-P)。
1、扩增nanE基因，并插入pTrc99A
根据NCBI查找U00096，获得Escherichia coli nanE基因核苷酸序列SEQ ID No.28，其氨基酸序列SEQ ID No.29。
设计引物：正向引物(nanE-F)SEQ ID No.30，反向引物(nanE-R)SEQ ID No.31。
模板：AT-001(Escherichia coli ATCC 27325)基因组。
PCR反应条件：第一步：94℃变性1min；第二步：94℃30s，55℃30s，72℃40s，循环30次；第三步：72℃延伸10min。
扩增产物大小：690bp。
PCR产物经1％琼脂糖凝胶电泳分离、纯化回收片段。
将所获得的PCR扩增片段和载体pTrc99A分别用Nco I和Hind III酶切，琼脂糖凝胶电泳分离、纯化回收nanE片段和pTrc99A片段，用T4DNA连接酶，16℃连接过夜，并鉴定，得到nanE/pTrc99A质粒。
2、用nanE/pTrc99A转化整合有pTrc-NanKM盒的大肠杆菌菌株
(1)感受态制备
①保存于-20℃的AT-007-02菌液，按1:50-100接种于10ml LB液体培养基中，37℃，225rpm，振荡培养2-3小时。
②将培养液加入到10ml离心管中，4000g×5min，弃去上清，用冰浴的0.1M CaCl2 5ml悬浮5min。
③4000g×5min离心，弃去上清，用冰浴的0.1M CaCl2 5ml悬浮。-4℃静置12小时，自然沉降。
(2)质粒转化
①取自然沉降的菌体250μl，加入5μl nanE/pTrc99A质粒，-4℃，30min。
②42℃水浴1.5min，加入SOC培养基0.7ml，30℃摇2小时。
③取0.2ml菌液，涂青霉素平板。
④30℃过夜(12-16小时)培养。
⑤挑单克隆，加入5ml LB液体培养基中培养，抽质粒鉴定。
⑥保存阳性克隆备用。
所获得菌种编号：AT-008(AT-007-02，nanE/pTrc99A)。
3、将整合有pTrc-nanKM盒的大肠杆菌菌株的nanE基因内源性天然启动子换成Trc启动子
首先，扩增Trc启动子序列片段与fKanrf片段，并拼接。然后，设计同源臂引物，扩增Red重组打靶用线性DNA全长片段。
(1)扩增Trc启动子序列
根据公开信息，查得Trc启动子序列：SEQ ID No.32。
设计引物：正向引物(KanTrcRed-F)SEQ ID No.33，反向引物(KanTrcRed-R)SEQ ID No.34。
模板：pTrc99A
PCR反应条件：第一步：94℃变性1min；第二步：94℃30s，55℃30s，72℃40s，循环30次；第三步：72℃延伸10min。
产物大小：166bp。
PCR产物经1％琼脂糖凝胶电泳分离、纯化回收片段。
(2)扩增两侧带有FLP重组酶识别位点(FRT位点)的卡拉霉素抗性基因：fKanrf
设计引物：正向引物(mfKanf-F)SEQ ID No.1，反向引物(mfKanf-R)SEQ ID No.2。
模板：pPic9K。
PCR反应条件：第一步：94℃变性1min；第二步：94℃30s，55℃30s，72℃40s，循环30次；第三步：72℃延伸10min。
fKanrf大小：1.28kb。其核苷酸序列SEQ ID No.3。
PCR产物经1％琼脂糖凝胶电泳分离、纯化回收片段。
(3)扩增与Trc启动子对接的fKanrf
设计引物：正向引物(fKanfRed-F1)SEQ ID No.35，反向引物(fKanfRed-R1)SEQ ID No.36。
模板：fKanrf。
PCR反应条件：第一步：94℃变性1min；第二步：94℃30s，55℃30s，72℃40s，循环30次；第三步：72℃延伸10min。
二次扩增的fKanrf大小：1.3kb。
PCR产物经1％琼脂糖凝胶电泳分离、纯化回收片段。
(4)制备Red重组打靶用线性DNA全长PCR片段
设计同源臂引物：根据nanE基因启动子序列SEQ ID No.37。设计更换为Trc启动子的同源臂正向引物(ProNanEpTrc-F)SEQ ID No.38，反向引物(ProNanEpTrc-R)SEQ ID No.39。
模板：Trc启动子PCR片段和二次扩增的fKanrf PCR片段，1：1混合。
PCR反应条件：第一步：94℃变性1min；第二步：94℃30s，55℃30s，72℃40s，循环30次；第三步：72℃延伸10min。
扩增产物：同源臂+fKanrf+Trc启动子+同源臂。
将PCR产物琼脂糖凝胶电泳分离、纯化回收，得到100ng/μl的线性DNA全长PCR片段用于Red重组打靶。
(5)Red重组操作
首先，将pKD46载体转入大肠杆菌AT-007-02菌株中。然后，电转化制备好的打靶用线性DNA片段，筛选阳性克隆。最后，消除抗性基因。
所获得菌种编号：AT-009(AT-007-02，△nanE promotor::Trc promoter)。
4、整合有pTrc-nanKM盒的重组菌，其中转化有nanE/pTrc99A和其中更换nanE启动子为Trc启动子对N-乙酰-D-氨基葡萄糖产量的影响。
对整合有pTrc-nanKM盒的菌株、其中过量表达NanE的菌株(包括其中转化有nanE/pTrc99A的菌株和其中更换NanE启动子为Trc启动子的菌株)做摇瓶发酵试验。取新鲜培养的LB平板培养基上单克隆菌株，接种于3ml的LB液体培养基试管(13×150mm)中，30℃，225rpm，培养约8小时。然后取种子培养液，3％接种于含50ml的M9培养液250ml摇瓶中。起始OD600约0.5，37℃下，225rpm培养，发酵周期72小时。在第24小时、48小时，用10M NaOH调节发酵液pH至7.0。根据发酵液糖耗情况，分次加入65％葡萄糖液维持葡萄糖浓度在20g/L。发酵结束，取1ml发酵液，离心。用HPLC法测定N-乙酰-D-氨基葡萄糖含量。
重组菌摇瓶发酵产量情况见表6。结果表明：过量表达NanE，无论是更换NanE启动子为Trc启动子，还是转化nanE/pTrc99A质粒，均能明显提高产量， 且更换NanE启动子为Trc启动子重组菌较转化nanE/pTrc99A质粒对产量的提高更明显。
表6.pTrc-nanKM基因盒整合重组菌过量表达NanE摇瓶发酵产量
Figure PCTCN2017080653-appb-000007
实施例5
本实施例描述整合有pTrc-nanKM盒的大肠杆菌菌株，其中将氨基葡萄糖-6-磷酸合成酶(GlmS)的基因glmS和/或D-氨基葡萄糖-6-磷酸脱氨酶(NagB)的基因nagB的内源性天然启动子更换和/或删除对N-乙酰-D-氨基葡萄糖产量的影响。
1、将nagB基因内源性天然启动子换成Trc启动子，进一步删除glmS基因内源性天然启动子，对整合有pTrc-NanKM盒的大肠杆菌菌株N-乙酰-D-氨基葡萄糖产量的影响。
(1)将nagB基因内源性天然启动子换成Trc启动子
将nag调节子(nagE-nagBACD)中D-氨基葡萄糖-6-磷酸脱氨酶(D-glucosamine-6-phosphate deaminase，NagB)基因启动子删除，更换成Trc启动子。D-氨基葡萄糖-6-磷酸脱氨酶(D-Glucosamine-6-phosphate deaminase，NagB)所催化的反应是可逆的，过量表达nagB，加快NagB的正向催化反应，达到增加D-氨基葡萄糖-6-磷酸(GlcN-6-P)目的。
首先，扩增Trc启动子片段与fKanrf片段，并拼接。然后，设计同源臂引物，扩增Red重组打靶用线性DNA全长片段。
1)制备Red重组打靶用线性DNA全长PCR片段
设计同源臂引物：根据NCBI查找NC_000913，Escherichia coli str.K-12nagB启动子序列和nagE基因序列SEQ ID No.13，设计删除nagB启动子同源臂 的正向引物(nagBKO-F1)SEQ ID No.40，反向引物(nagBKO-R1)SEQ ID No.41。
模板：Trc启动子PCR片段和二次扩增的fKanrf PCR片段，1：1混合。
PCR反应条件：第一步：94℃变性1min；第二步：94℃30s，55℃30s，72℃40s，循环30次；第三步：72℃延伸10min。
扩增产物：同源臂+fKanrf+Trc启动子+同源臂。
将PCR产物琼脂糖凝胶电泳分离、纯化回收，得到100ng/μl的线性DNA全长PCR片段用于Red重组打靶。
2)Red重组操作
首先，将pKD46载体转入大肠杆菌AT-007-02菌株中。然后，电转化制备好的打靶用线性DNA片段，筛选阳性克隆。最后，消除抗性基因。
所获得菌种编号：AT-010(AT-007-02，△nagB promotor::Trc promoter)。
(2)删除glmS基因内源性天然启动子
删除氨基葡萄糖-6-磷酸合成酶(Glucosamine-6-phosphate synthase，glmS)基因启动子序列。氨基葡萄糖-6-磷酸合成酶(GlmS)又称L-谷氨酰胺-6-磷酸果糖转氨酶(L-glutamine：D-fructose-6-phosphate aminotransferase)，能够催化葡萄糖-6-磷酸(Glc-6-P)氨基化为D-氨基葡萄糖-6-磷酸(GlcN-6-P)，但有严重的产物抑制问题，将其启动子序列删除，使该酶丧失表达，可解除GlcN-6-P的产物抑制。
首先，扩增fKanrf片段，然后，设计同源臂引物，扩增Red重组打靶用线性DNA全长片段
1)扩增两侧带有FLP重组酶识别位点(FRT位点)的卡拉霉素抗性基因：fKanrf
设计引物：正向引物(mfKanf-F)SEQ ID No.1，反向引物(mfKanf-R)SEQ ID No.2。
模板：pPic9K。
PCR反应条件：第一步：94℃变性1min；第二步：94℃30s，55℃30s，72℃40s，循环30次；第三步：72℃延伸10min。
fKanrf大小：1.28kb。其核苷酸序列SEQ ID No.3。
PCR产物经1％琼脂糖凝胶电泳分离、纯化回收片段。
2)制备Red重组打靶用线性DNA全长PCR片段
设计同源臂引物：根据NCBI查找NC_000913，Escherichia coli str.K-12L-谷氨酰胺-6-磷酸果糖转氨酶(GlmS)基因启动子序列SEQ ID No.42，设计glmS基因启动子序列删除的同源臂正向引物(ProglmsKO-F)SEQ ID No.43，反向引物(ProglmsKO-R)SEQ ID No.44。
模板：fKanrf PCR片段。
PCR反应条件：第一步：94℃变性1min；第二步：94℃30s，55℃30s，72℃40s，循环30次；第三步：72℃延伸10min。
扩增产物：同源臂+fKanf+同源臂。
将PCR产物琼脂糖凝胶电泳分离、纯化回收，得到100ng/μl的线性DNA全长PCR片段用于Red重组打靶。
3)Red重组操作
首先，将pKD46载体转入大肠杆菌AT-010菌株中。然后，电转化制备好的打靶用线性DNA片段，筛选阳性克隆。最后，消除抗性基因。
所获得菌种编号：AT-011(AT-010，△glmS promotor)。
(3)将nagB启动子更换为更高表达水平的启动子及进一步删除glmS启动子对N-乙酰-D-氨基葡萄糖产量的影响
对整合有pTrc-nanKM盒的菌株，将nagB启动子更换为更高表达水平的启动子，以及进一步删除glmS启动子的重组菌做摇瓶发酵试验。取新鲜培养的LB平板培养基上单克隆菌株，接种于3ml的LB液体培养基试管(13×150mm)中，30℃，225rpm，培养约8小时。然后取种子培养液，3％接种于含50ml的发酵培养液(M9培养液)250ml摇瓶中。起始OD600约0.5，37℃下，225rpm培养，发酵周期72小时。在第24小时、48小时，用10M NaOH调节发酵液pH至7.0。根据发酵液糖耗情况，分次加入65％葡萄糖液维持葡萄糖浓度在20g/L。发酵结束，取1ml发酵液，离心。用HPLC法测定N-乙酰-D-氨基葡萄糖含量。
摇瓶发酵产量情况见表7。结果表明：将nagB启动子更换为Trc启动子的重组菌对N-乙酰-D-氨基葡萄糖产量明显增加，进一步删除glmS启动子后N-乙酰-D-氨基葡萄糖产量有更大提高。
表7.更换nagB启动子以及进一步删除glmS启动子的重组菌摇瓶发酵产量
Figure PCTCN2017080653-appb-000008
2、将glmS基因内源性天然启动子换成Trc启动子，进一步删除nagB基因内源性天然启动子，对整合有pTrc-NanKM盒的大肠杆菌菌株N-乙酰-D-氨基葡萄糖产量的影响。
(1)将glmS基因内源性天然启动子换成Trc启动子
将L-谷氨酰胺-6-磷酸果糖转氨酶(L-glutamine：D-fructose-6-phosphate aminotransferase)基因启动子序列更换为Trc启动子序列。L-谷氨酰胺-6-磷酸果糖转氨酶又称氨基葡萄糖-6-磷酸合成酶(Glucosamine-6-phosphate synthase，GlmS)，将其启动子序列更换为Trc启动子序列，可过量表达glmS，加快GlmS催化功能，达到增加D-氨基葡萄糖-6-磷酸(GlcN-6-P)目的。
首先，扩增Trc启动子序列片段与fKanrf片段，并拼接。然后，设计同源臂引物，扩增Red重组打靶用线性DNA全长片段。
1)扩增Red重组打靶用线性DNA全长PCR片段
设计同源臂引物：根据glmS基因启动子序列SEQ ID No.42，设计更换为Trc启动子的同源臂正向引物(ProglmspTrc-F)SEQ ID No.45，反向引物(ProglmspTrc-R)SEQ ID No.46。
模板：Trc启动子PCR片段和二次扩增的fKanrf PCR片段，1：1混合。
PCR反应条件：第一步：94℃变性1min；第二步：94℃30s，55℃30s，72℃40s，循环30次；第三步：72℃延伸10min。
扩增产物：同源臂+fKanrf+Trc启动子+同源臂。
将PCR产物琼脂糖凝胶电泳分离、纯化回收，得到100ng/μl的线性DNA全长PCR片段用于Red重组打靶。
2)Red重组操作
首先，将pKD46载体转入大肠杆菌AT-007-02菌株中。然后，电转化制备好的打靶用线性DNA片段，筛选阳性克隆。最后，消除抗性基因。
所获得菌种编号：AT-012(AT-007-02，△glmS promotor::Trc promoter)。
(2)删除NagB基因内源性天然启动子
将nag调节子(nagE-nagBACD)中D-氨基葡萄糖-6-磷酸脱氨酶(D-glucosamine-6-phosphate deaminase，NagB)基因启动子序列删除，使nagB失去功能，可消除NagB逆向催化功能，减少GlcN-6-P生成Glc-6-P。
首先，扩增fKanrf片段，然后，设计同源臂引物，制备Red重组打靶用线性DNA全长片段。
1)制备Red重组打靶用线性DNA全长PCR片段
设计同源臂引物：根据nagB启动子和nagE基因序列SEQ ID No.13，设计删除nagB启动子序列的同源臂正向引物(NagBKO-F2)SEQ ID No.47，反向引物(NagBKO-R2)SEQ ID No.48。
模板：fKanrf PCR片段
PCR反应条件：第一步：94℃变性1min；第二步：94℃30s，55℃30s，72℃40s，循环30次；第三步：72℃延伸10min。
扩增产物：同源臂+fKanrf+同源臂。
将PCR产物琼脂糖凝胶电泳分离、纯化回收，得到100ng/μl的线性DNA全长PCR片段用于Red重组打靶。
2)Red重组操作
首先，将pKD46载体转入大肠杆菌AT-012菌株中。然后，电转化制备好的打靶用线性DNA片段，筛选阳性克隆。最后，消除抗性基因。
所获得菌种编号：AT-013(AT-012，△nagB promotor)。
(3)将glmS启动子更换为更高表达水平的启动子及进一步删除nagB启动子对N-乙酰氨基葡萄糖产量的影响
对整合有pTrc-nanKM盒的菌株，将glmS启动子更换为更高表达水平的启动子，以及进一步删除nagB启动子的重组菌做摇瓶发酵试验。取新鲜培养的LB平板培养基上单克隆菌株，接种于3ml的LB液体培养基试管(13×150mm)中，30℃，225rpm，培养约8小时。然后取种子培养液，3％接种于含50ml的发酵培养液(M9培养液)250ml摇瓶中。起始OD600约0.5，37℃下，225rpm 培养，发酵周期72小时。在第24小时、48小时，用10M NaOH调节发酵液pH至7.0。根据发酵液糖耗情况，分次加入65％葡萄糖液维持葡萄糖浓度在20g/L。发酵结束，取1ml发酵液，离心。用HPLC法测定N-乙酰-D-氨基葡萄糖含量。
摇瓶发酵产量情况见表8。结果表明：将glmS启动子更换为Trc启动子的重组菌对N-乙酰-D-氨基葡萄糖产量增加效果不明显，但同时删除nagB启动子后N-乙酰-D-氨基葡萄糖产量较对照菌种有明显提高。
表8.更换glmS启动子以及进一步删除nagB启动子的重组菌摇瓶发酵产量
Figure PCTCN2017080653-appb-000009
实施例6
本实施例描述整合有pTrc-nanKM盒，并将D-氨基葡萄糖-6-磷酸脱氨酶(NagB)的基因nagB和氨基葡萄糖-6-磷酸合成酶(GlmS)的基因glmS的内源性天然启动子更换和/或删除的大肠杆菌菌株，转化nanE/pTrc99A质粒或将nanE基因内源性天然启动子换成Trc启动子，对N-乙酰-D-氨基葡萄糖产量的影响。
1、对整合有pTrc-nanKM盒，并将nagB基因内源性天然启动子换成Trc启动子和同时删除glmS基因内源性天然启动子的大肠杆菌菌株，转化nanE/pTrc99A质粒
(1)感受态制备：首先，将保存于-20℃重组菌AT-011菌液，按1:50-100接种于10ml LB液体培养基中，37℃，225rpm，振荡培养2-3小时。再将培养液加入10ml离心管中，4000g×5min，弃去上清，用冰浴的0.1M CaCl2 5ml悬浮5min。最后，4000g×5min离心，弃去上清，用冰浴的0.1M CaCl2 5ml悬浮。-4℃静置12小时，自然沉降。
(2)质粒转化：取自然沉降的菌体250μl，加入5μl nanE/pTrc99A质粒， -4℃，30min。然后，42℃水浴1.5min，加入SOC培养基0.7ml，30℃摇2小时。取0.2ml菌液，涂青霉素平板。30℃过夜(12-16小时)培养。挑单克隆，加入5ml LB液体培养基中培养，抽质粒鉴定。保存阳性克隆备用。
所获得菌种编号：AT-014(AT-011，nanE/pTrc99A)。
2、对整合有pTrc-nanKM盒，并将nagB基因内源性天然启动子换成Trc启动子和同时删除glmS基因内源性天然启动子的大肠杆菌菌株，将nanE基因内源性天然启动子换成Trc启动子
首先，扩增Trc启动子序列片段与fKanrf片段，并拼接。然后，设计同源臂引物，扩增Red重组打靶用线性DNA全长片段。
(1)制备Red重组打靶用线性DNA全长PCR片段
设计同源臂引物：根据nanE基因启动子序列SEQ ID No.37。设计更换为Trc启动子的同源臂正向引物(ProNanEpTrc-F)SEQ ID No.38，反向引物(ProNanEpTrc-R)SEQ ID No.39。
模板：Trc启动子PCR片段和二次扩增的fKanrf PCR片段，1：1混合。
PCR反应条件：第一步：94℃变性1min；第二步：94℃30s，55℃30s，72℃40s，循环30次；第三步：72℃延伸10min。
扩增产物：同源臂+fKanrf+Trc启动子+同源臂。
将PCR产物琼脂糖凝胶电泳分离、纯化回收，得到100ng/μl的线性DNA全长PCR片段用于Red重组打靶。
(2)Red重组操作
首先，将pKD46载体转入大肠杆菌AT-011菌株中。然后，电转化制备好的打靶用线性DNA片段，筛选阳性克隆。最后，消除抗性基因。
所获得菌种编号：AT-015(AT-011，△nanE promotor::Trc promoter)。
3、对整合有pTrc-nanKM盒，并将glmS基因内源性天然启动子换成Trc启动子和同时删除nagB基因内源性天然启动子的大肠杆菌菌株，转化nanE/pTrc99A质粒
首先，制备重组大肠杆菌菌株AT-013感受态，然后，将NanE/pTrc99A质粒利用CaCl2转化法转化至AT-013中，挑单克隆培养，抽质粒鉴定阳性克隆。
所获得菌种编号：AT-016(AT-013，nanE/pTrc99A)。
4、对整合有pTrc-nanKM盒，并将glmS基因内源性天然启动子换成Trc启动子和同时删除nagB基因内源性天然启动子的大肠杆菌菌株，将nanE基因内源性天然启动子换成Trc启动子
(1)制备Red重组打靶用线性DNA全长PCR片段
首先，扩增Trc启动子序列片段与fKanrf片段，并拼接。然后，设计同源臂引物，扩增Red重组打靶用线性DNA全长片段。
(2)Red重组操作
首先，将pKD46载体转入大肠杆菌AT-013菌株中。然后，电转化制备好的打靶用线性DNA片段，筛选阳性克隆。最后，消除抗性基因。
所获得菌种编号：AT-017(AT-013，△nanE promotor::Trc promoter)。
5、整合有pTrc-nanKM盒，并将nagB基因和glmS基因的内源性天然启动子更换和/或删除的大肠杆菌菌株，转化nanE/pTrc99A质粒和将nanE基因内源性天然启动子换成Trc启动子对N-乙酰-D-氨基葡萄糖产量的影响
对整合有pTrc-nanKM盒的菌株，其中将glmS和nagB内源性天然启动子更换和/或删除，并转化nanE/pTrc99A质粒或将nanE基因内源性天然启动子更换为Trc启动子，所形成的不同基因型重组菌，做摇瓶发酵试验。取新鲜培养的LB平板培养基上单克隆菌株，接种于3ml的LB液体培养基试管(13×150mm)中，30℃，225rpm，培养约8小时。然后取种子培养液，3％接种于含50ml的发酵培养液(M9培养液)250ml摇瓶中。起始OD600约0.5，37℃下，225rpm培养，发酵周期72小时。在第24小时、48小时，用10M NaOH调节发酵液pH至7.0。根据发酵液糖耗情况，分次加入65％葡萄糖液维持葡萄糖浓度在20g/L。发酵结束，取1ml发酵液，离心。用HPLC法测定N-乙酰-D-氨基葡萄糖含量。
摇瓶发酵产量情况见表9。结果表明：过量表达NanE，无论是更换NanE启动子为Trc启动子，还是转化NanE/pTrc99A质粒，均对N-乙酰-D-氨基葡萄糖产量有实质改进，但更换NanE启动子为Trc启动子重组菌较转化nanE/pTrc99A质粒对N-乙酰-D-氨基葡萄糖的产量有更明显提高。
表9.转化NanE/pTrc99A质粒或更换NanE启动子为Trc启动子的重组菌摇瓶发酵产量
Figure PCTCN2017080653-appb-000010
实施例7
本实施例描述整合有pTrc-nanKM盒的大肠杆菌菌株，其中过量表达UDP-N-乙酰-D-氨基葡萄糖-2-异构酶(WecB)的基因wecB及其对N-乙酰-D-氨基葡萄糖产量的影响。
将大肠杆菌UDP-N-乙酰氨基葡萄糖-2-异构酶(UDP-N-acetyl-D-glucosamine-2-epimerase，WecB)基因wecB，置于Trc启动子控制下转化菌种，或将wecB基因内源性天然启动子换成Trc启动子，使之过量表达，可加强UDP-GlcNAc(UDP-N-acetyl glucosamine，UDP-N-乙酰-D-氨基葡萄糖)变成ManNAc(N-Acetyl-D-mannosamine，N-乙酰-D-氨基甘露糖或N-乙酰-D-甘露糖胺)。
1、用wecB/pTrc99A质粒转化整合有pTrc-NanKM盒的大肠杆菌株菌
(1)扩增大肠杆菌wecB基因，并插入pTrc99A
根据NCBI查找大肠杆菌wecB基因核苷酸序列SEQ ID No.49，其氨基酸序列SEQ ID No.50。
设计引物：正向引物(TrcwecB-F)SEQ ID No.51，反向引物(TrcwecB-R)SEQ ID No.52。
模板：AT-001(Escherichia coli ATCC 27325)基因组。
PCR反应条件：第一步：94℃变性1min；第二步：94℃30s，55℃30s，72℃40s，循环30次；第三步：72℃延伸10min。
扩增产物大小：1.13kb。
PCR产物经1％琼脂糖凝胶电泳分离、纯化回收片段。
将所获得的PCR扩增片段和载体pTrc99A分别用Nco I和Hind III酶切，琼脂糖凝胶电泳分离、纯化回收wecB片段和pTrc99A片段，用T4DNA连接酶，16℃连接过夜，并鉴定，得到wecB/pTrc99A质粒。
(2)用wecB/pTrc99A质粒转化整合有pTrc-NanKM盒的大肠杆菌菌株
(1)感受态制备：首先，将保存于-20℃重组菌AT-007-02菌液，按1:50-100接种于10ml LB液体培养基中，37℃，225rpm，振荡培养2-3小时。再将培养液加入10ml离心管中，4000g×5min，弃去上清，用冰浴的0.1M CaCl2 5ml悬浮5min。最后，4000g×5min离心，弃去上清，用冰浴的0.1M CaCl2 5ml悬浮。-4℃静置12小时，自然沉降。
(2)质粒转化：取自然沉降的菌体250μl，加入5μl wecB/pTrc99A质粒，-4℃，30min。然后，42℃水浴1.5min，加入SOC培养基0.7ml，30℃摇2小时。取0.2ml菌液，涂青霉素平板。30℃过夜(12-16小时)培养。挑单克隆，加入5ml LB液体培养基中培养，抽质粒鉴定。保存阳性克隆备用。
所获得菌种编号：AT-018(AT-007-02，wecB/pTrc99A)。
2、将整合有pTrc-nanKM盒的大肠杆菌中，wecB基因内源性天然启动子换成Trc启动子
首先，扩增Trc启动子序列片段与fKanrf片段，并拼接。然后，设计同源臂引物，扩增Red重组打靶用线性DNA全长片段。
(1)制备Red重组打靶用线性DNA全长PCR片段
设计同源臂引物：根据NCBI查找NC_000913，获得Escherichia coli UDP-N-乙酰氨基葡萄糖-2-异构酶(wecB)基因启动子的核苷酸序列SEQ ID No.53，设计更换为Trc启动子的同源臂正向引物(ProwecBpTrc-F)SEQ ID No.54，反向引物(Pro wecBpTrc-R)SEQ ID No.55。
模板：Trc启动子PCR片段和二次扩增的fKanrf PCR片段，1：1混合。
PCR反应条件：第一步：94℃变性1min；第二步：94℃30s，55℃30s，72℃40s，循环30次；第三步：72℃延伸10min。
扩增产物：同源臂+fKanrf+Trc启动子+同源臂。
将PCR产物琼脂糖凝胶电泳分离、纯化回收，得到100ng/μl的线性DNA全长PCR片段用于Red重组打靶。
(2)Red重组操作
首先，将pKD46载体转入大肠杆菌AT-007-02菌株中。然后，电转化制备好的打靶用线性DNA片段，筛选阳性克隆。最后，消除抗性基因。
所获得菌种编号：AT-019(AT-007-02，△wecB promotor::Trc promoter)。
3、整合有pTrc-NanKM盒的重组菌，转化有wecB/pTrc99A质粒和更换wecB启动子为Trc启动子对N-乙酰-D-氨基葡萄糖产量的影响。
对整合有pTrc-nanKM盒的菌株，过量表达wecB的菌株(包括转化有wecB/pTrc99A的菌株和更换wecB启动子为Trc启动子)产生的重组菌，做摇瓶发酵试验。取新鲜培养的LB平板培养基上单克隆菌株，接种于3ml的LB液体培养基试管(13×150mm)中，30℃，225rpm，培养约8小时。然后取种子培养液，3％接种于含50ml的M9培养液250ml摇瓶中。起始OD600约0.5，37℃下，225rpm培养，发酵周期72小时。在第24小时、48小时，用10M NaOH调节发酵液pH至7.0。根据发酵液糖耗情况，分次加入65％葡萄糖液维持葡萄糖浓度在20g/L。发酵结束，取1ml发酵液，离心。用HPLC法测定N-乙酰-D-氨基葡萄糖含量。
摇瓶发酵产量情况见表10。结果表明：与对照菌种AT-007-02相比，转化wecB/pTrc99A质粒对N-乙酰-D-氨基葡萄糖产量有明显提高，更换wecB启动子为Trc启动子的重组菌对N-乙酰-D-氨基葡萄糖产量有更大的提高。
表10.转化wecB/pTrc99A质粒或更换wecB启动子为Trc启动子的重组菌摇瓶发酵产量
Figure PCTCN2017080653-appb-000011
实施例8
本实施例描述整合有pTrc-nanKM盒且其中nanE基因内源性天然启动子换成Trc启动子，并转化wecB/pTrc99A质粒或将wecB基因内源性天然启动子换成Trc启动子，对N-乙酰-D-氨基葡萄糖产量的影响。
1、对整合有pTrc-nanKM盒且其中nanE基因内源性天然启动子换成Trc启动子的大肠杆菌菌株，转化wecB/pTrc99A质粒
制备感受态，将wecB/pTrc99A质粒用CaCl2转化法转化到整合有pTrc-NanKM盒且其中nanE基因内源性天然启动子换成Trc启动子的大肠杆菌菌株AT-009中，挑单克隆培养，抽质粒鉴定阳性克隆。
所获得菌种编号：AT-020(AT-009，wecB/pTrc99A)。
2、将整合有pTrc-nanKM盒且其中nanE基因内源性天然启动子换成Trc启动子的大肠杆菌菌株中，wecB基因内源性天然启动子换成Trc启动子
首先，将pKD46载体转入大肠杆菌AT-009菌株中。然后，电转化制备好的打靶用线性DNA片段，筛选阳性克隆。最后，消除抗性基因。
所获得菌种编号：AT-021(AT-009，△wecB promotor::Trc promoter)。
3、整合有pTrc-nanKM盒且其中nanE基因内源性天然启动子换成Trc启动子的重组菌，转化wecB/pTrc99A质粒和更换wecB启动子为Trc启动子对N-乙酰-D-氨基葡萄糖产量的影响
对整合有pTrc-nanKM盒且其中nanE基因内源性天然启动子换成Trc启动子的菌株，过量表达wecB(包括转化有wecB/pTrc99A的菌株和更换wecB启动子为Trc启动子)产生的重组菌，做摇瓶发酵试验。取新鲜培养的LB平板培养基上单克隆菌株，接种于3ml的LB液体培养基试管(13×150mm)中，30℃，225rpm，培养约8小时。然后取种子培养液，3％接种于含50ml的M9培养液250ml摇瓶中。起始OD600约0.5，37℃下，225rpm培养，发酵周期72小时。在第24小时、48小时，用10M NaOH调节发酵液pH至7.0。根据发酵液糖耗情况，分次加入65％葡萄糖液维持葡萄糖浓度在20g/L。发酵结束，取1ml发酵液，离心。用HPLC法测定N-乙酰-D-氨基葡萄糖含量。
摇瓶发酵产量情况见表11。结果表明：与对照菌种AT-009相比，转化wecB/pTrc99A质粒N-乙酰-D-氨基葡萄糖产量有明显提高，更换wecB启动子为Trc启动子的重组菌对N-乙酰-D-氨基葡萄糖产量有更大的提高。
表11.转化wecB/pTrc99A质粒或更换wecB启动子为Trc启动子的重组菌摇瓶发酵产量
Figure PCTCN2017080653-appb-000012
实施例9
本实施例描述整合有pTrc-nanKM盒，并将glmS基因和nagB基因的内源性天然启动子更换和/或删除的大肠杆菌菌株，转化wecB/pTrc99A质粒或将wecB基因内源性天然启动子换成Trc启动子，对N-乙酰-D-氨基葡萄糖产量的影响
1、对整合有pTrc-nanKM盒，并将nagB基因内源性天然启动子换成Trc启动子和同时删除glmS基因内源性天然启动子的大肠杆菌菌株，转化 wecB/pTrc99A质粒
制备感受态，将wecB/pTrc99A质粒用CaCl2转化法转化到整合有pTrc-NanKM盒，并将nagB基因内源性天然启动子换成Trc启动子和同时删除glmS基因内源性天然启动子的大肠杆菌菌株AT-011中，挑单克隆培养，抽质粒鉴定阳性克隆。
所获得菌种编号：AT-022(AT-011，wecB/pTrc99A)。
2、对整合有pTrc-nanKM盒，并将nagB基因内源性天然启动子换成Trc启动子和同时删除glmS基因内源性天然启动子的大肠杆菌菌株，将wecB基因内源性天然启动子换成Trc启动子
首先，将pKD46载体转入大肠杆菌AT-011菌株中。然后，电转化制备好的打靶用线性DNA片段，筛选阳性克隆。最后，消除抗性基因。
所获得菌种编号：AT-023(AT-011，△wecB promotor::Trc promoter)。
3、对整合有pTrc-nanKM盒，并将glmS基因内源性天然启动子换成Trc启动子和同时删除nagB基因内源性天然启动子的大肠杆菌菌株，转化wecB/pTrc99A质粒
制备感受态，将wecB/pTrc99A质粒用CaCl2转化法转化到整合有pTrc-nanKM盒，并将glmS基因内源性天然启动子换成Trc启动子和同时删除nagB基因内源性天然启动子的大肠杆菌菌株AT-013中，挑单克隆培养，抽质粒鉴定阳性克隆。
所获得菌种编号：AT-024(AT-013，wecB/pTrc99A)。
4、对整合有pTrc-nanKM盒并将glmS基因内源性天然启动子换成Trc启动子和同时删除nagB基因内源性天然启动子的大肠杆菌菌株，将wecB基因内源性天然启动子换成Trc启动子
首先，将pKD46载体转入大肠杆菌AT-013菌株中。然后，电转化制备好的打靶用线性DNA片段，筛选阳性克隆。最后，消除抗性基因。
所获得菌种编号：AT-025(AT-013，△wecB promotor::Trc promoter)。
5、整合有pTrc-nanKM盒，并将nagB基因和glmS基因的内源性天然启动子更换和/或删除的大肠杆菌菌株，转化wecB/pTrc99A质粒和将wecB基因内源性天然启动子换成Trc启动子对N-乙酰-D-氨基葡萄糖产量的影响
对整合有pTrc-nanKM盒，并将glmS基因和nagB基因的内源性天然启动子更换和/或删除后，并转化wecB/pTrc99A质粒或将wecB基因内源性天然启动子更换为Trc启动子，所形成的不同基因型重组菌，做摇瓶发酵试验。取新鲜培养的LB平板培养基上单克隆菌株，接种于3ml的LB液体培养基试管(13×150mm)中，30℃，225rpm，培养约8小时。然后取种子培养液，3％接种于含50ml的发酵培养液(M9培养液)250ml摇瓶中。起始OD600约0.5，37℃下，225rpm培养，发酵周期72小时。在第24小时、48小时，用10M NaOH调节发酵液pH至7.0。根据发酵液糖耗情况，分次加入65％葡萄糖液维持葡萄糖浓度在20g/L。发酵结束，取1ml发酵液，离心。用HPLC法测定N-乙酰-D-氨基葡萄糖含量。
摇瓶发酵产量情况见表12。结果表明：与对照菌种AT-011或AT-013相比，转化wecB/pTrc99A质粒使得N-乙酰-D-氨基葡萄糖产量有明显提高，更换wecB启动子为Trc启动子的重组菌对N-乙酰-D-氨基葡萄糖产量有更大的提高。
表12.转化wecB/pTrc99A质粒或更换wecB启动子为Trc启动子的重组菌摇瓶发酵产量
Figure PCTCN2017080653-appb-000013
实施例10
本实施例描述整合有pTrc-NanKM盒、将glmS基因和NagB基因的内源性天然启动子更换和/或删除并将nanE基因内源性天然启动子换成Trc启动子的大肠杆菌菌株，转化wecB/pTrc99A质粒或将wecB基因内源性天然启动子换成Trc启动子，对N-乙酰-D-氨基葡萄糖产量的影响
1、对整合有pTrc-nanKM盒、nagB基因内源性天然启动子换成Trc启动子、删除glmS基因内源性天然启动子、并将nanE基因内源性天然启动子换成Trc启动子的大肠杆菌菌株，转化wecB/pTrc99A质粒
制备感受态，将wecB/pTrc99A质粒用CaCl2转化法转化到整合有pTrc-nanKM盒、nagB基因内源性天然启动子换成Trc启动子、删除glmS基因内源性天然启动子、并将nanE基因内源性天然启动子换成Trc启动子的大肠杆菌菌株AT-015中，挑单克隆培养，抽质粒鉴定阳性克隆。
所获得菌种编号：AT-026(AT-015，wecB/pTrc99A)。
2、对整合有pTrc-nanKM盒、nagB基因内源性天然启动子换成Trc启动子、删除glmS基因内源性天然启动子、并将nanE基因内源性天然启动子换成Trc启动子的大肠杆菌菌株，将wecB基因内源性天然启动子换成Trc启动子
首先，将pKD46载体转入大肠杆菌AT-015菌株中。然后，电转化制备好的打靶用线性DNA片段，筛选阳性克隆。最后，消除抗性基因。
所获得菌种编号：AT-027(AT-015，△wecB promotor::Trc promoter)。
3、对整合有pTrc-nanKM盒、glmS基因内源性天然启动子换成Trc启动子、删除nagB基因内源性天然启动子、并将nanE基因内源性天然启动子换成Trc启动子的大肠杆菌菌株，转化wecB/pTrc99A质粒
制备感受态，将wecB/pTrc99A质粒用CaCl2法转化到整合有pTrc-nanKM盒、glmS基因内源性天然启动子换成Trc启动子、删除nagB基因内源性天然启动子、并将nanE基因内源性天然启动子换成Trc启动子的大肠杆菌菌株AT-017中，挑单克隆培养，抽质粒鉴定阳性克隆。
所获得菌种编号：AT-028(AT-017，wecB/pTrc99A)。
4、对整合有pTrc-nanKM盒、glmS基因内源性天然启动子换成Trc启动子、删除NnagB基因内源性天然启动子、并将nanE基因内源性天然启动子换成Trc启动子的大肠杆菌菌株，将wecB基因内源性天然启动子换成Trc启动子
首先，将pKD46载体转入大肠杆菌AT-017菌株中。然后，电转化制备好的打靶用线性DNA片段，筛选阳性克隆。最后，消除抗性基因。
所获得菌种编号：AT-029(AT-017，△wecB promotor::Trc promoter)。
5、整合有pTrc-nanKM盒、将NagB基因和glmS基因的内源性天然启动子更换和/或删除并将nanE基因内源性天然启动子换成Trc启动子的大肠杆菌菌株，转化wecB/pTrc99A质粒将wecB基因内源性天然启动子换成Trc启动子对N-乙酰-D-氨基葡萄糖产量的影响
对整合有pTrc-nanKM盒，并将glmS基因和nagB基因的内源性天然启动子更换和/或删除并将nanE基因内源性天然启动子换成Trc启动子的大肠杆菌菌株，转化wecB/pTrc99A质粒和更换wecB启动子为Trc启动子产生的重组菌，做摇瓶发酵试验。取新鲜培养的LB平板培养基上单克隆菌株，接种于3ml的LB液体培养基试管(13×150mm)中，30℃，225rpm，培养约8小时。然后取种子培养液，3％接种于含50ml的发酵培养液(M9培养液)250ml摇瓶中。起始OD600约0.5，37℃下，225rpm培养，发酵周期72小时。在第24小时、48小时，用10M NaOH调节发酵液pH至7.0。根据发酵液糖耗情况，分次加入65％葡萄糖液维持葡萄糖浓度在20g/L。发酵结束，取1ml发酵液，离心。用HPLC法测定N-乙酰-D-氨基葡萄糖含量。
摇瓶发酵产量情况见表13。结果表明：与对照菌种AT-015或AT-017相比，转化wecB/pTrc99A质粒使得N-乙酰-D-氨基葡萄糖产量有明显提高，更换wecB启动子为Trc启动子的重组菌对N-乙酰-D-氨基葡萄糖产量有更大的提高。
表13.转化wecB/pTrc99A质粒或更换wecB启动子为Trc启动子的重组菌摇瓶发酵产量
Figure PCTCN2017080653-appb-000014
实施例11
本实施例描述以10L发酵罐生产N-乙酰-D-氨基葡萄糖发酵试验
以重组工程菌株AT-029作为生产菌种，以10L发酵罐做生产N-乙酰-D-氨基葡萄糖发酵试验。
1、种子培养
(1)一级种子培养：挑取新鲜培养的LB平板培养基上单克隆菌株，接种于8ml的LB液体培养基中，37℃、225rpm培养8小时。
(2)二级种子培养：取6ml一级种子培养液，接种于含200ml的M9培养液的1000ml摇瓶中，37℃、225rpm培养16小时。至OD600值为6.0-10，约为对数生长中期。
(3)发酵培养基按表14配制，其中，微量元素溶液按表15配制，复合维生素溶液按表16配制。
表14.发酵培养基
成分 用量(/L)
K2HPO4 1.30g
KH2PO4 1.00g
MgSO4.7H2O 0.10g
NH4Cl 0.02g
(NH4)2SO4 0.20g
NaH2PO4 0.60g
聚醚类消泡剂 10ml
微量元素溶液 4ml
复合维生素溶液 4ml
葡萄糖 6.00g
注：
①微量元素溶液单独灭菌后加入，维生素溶液过滤后加入；
②葡萄糖：浓度65％(w/v)，单独灭菌，在接种前加入，葡萄糖加入量：6.0g/L；
③以上合并一起后，用10M NH4OH调pH至7.0；
④发酵培养基在加入葡萄糖之前为基础培养基，基础培养基的初始装液量
(培养基的初始体积占发酵罐总容量体积)：50％。
表15.微量元素溶液
成分 用量(g/L)
CaCl2·2H2O 10
FeCl3·6H2O 10
MnSO4·5H2O 2.5
AlCl3·6H2O 2.5
CoCl2·6H2O 1.75
ZnSO4·2H2O 0.5
NaMoO4·2H2O 0.5
CuSO4·5H2O 0.25
H3BO3 0.125
pH 3～4
表16.复合维生素溶液
2、接种
将二级种子液按40ml/L接种至发酵罐，接种量：2.5-5％(以体积计)，起始OD600为0.3-0.5。
3、工艺参数
利用10L自控发酵罐进行高密度发酵，以机带软件对其进行数据采集，实现计算机在线控制。控制参数为：空气流量按0.5-1vvm.；溶氧≥20％，以增加转速和通气调节；温度37℃；pH值7.0，自动流加饱和氨水以保持恒定。在基础培养基中葡萄糖消耗完，即溶氧回升时开始补糖。补糖速度以控制残糖浓度0.45g/L以下为标准。补糖液葡萄糖为浓度65％(w/v)，并加入2.5％葡萄糖酸钠或6％核糖。60-72h发酵中止。总装液量：75-80％。
4、实例(10L发酵罐)
(1)菌种编号：AT-029。批号：0072。
(2)种子液浓度：OD600为2.7。
(3)基料：4L。
(4)接种量200mL。
(5)补糖速度：控制残糖浓度0.45g/L以下。
(6)补糖液：葡萄糖为浓度65％(w/v)并加入2.5％葡萄糖酸钠。
(7)跟踪指标：检测OD600、残糖含量(发酵液中残存葡萄糖)。
(8)产物：N-乙酰-D-氨基葡萄糖。效价：72小时145.5g/L。
实施例12
本实施例描述N-乙酰-D-氨基葡萄糖和D-氨基葡萄糖盐酸盐分离纯化后处理工艺
1、N-乙酰-D-氨基葡萄糖精制
(1)灭活：发酵液80℃，30min。
(2)固液分离：4000-8000rpm离心，弃菌渣与蛋白，取发酵液。也可以用陶瓷膜过滤。
(3)脱色：产品：水：活性炭＝1：(1.5-3)：(0.01-0.1)，搅拌0.5-5h。
(4)除盐：电渗析除盐。浓室罐装入发酵液初始盐浓度：0.01-0.05mol/L。淡室发酵液流速：40-80L/h，浓室发酵液流速：40-80L/h，单膜对的电压0.5-1.4V。也可以采用阴、阳离子交换树脂除盐。
(5)浓缩：将除盐后发酵液真空条件下(0.095MPa)加热50-80℃，浓缩8-15h，至过饱和，约4-6倍。
(6)结晶：浓缩后的发酵液先25℃水降温至25-35℃，再用0℃水降温1-3h，至0-10℃。加无水乙醇(用量为产品重量5-20倍)，700-1500rpm搅拌15min-1h。
(7)洗涤：加无水乙醇(与产品等量)搅拌10-100rpm，0.5-2h。
(8)烘干：50-100℃，3-10h。纯度：99.93％。总产率为91.1％。
2、D-氨基葡萄糖盐酸盐精制
(1)灭活：发酵液80℃，30min。
(2)固液分离：4000-8000rpm离心，弃菌渣与蛋白，取发酵液。也可以用陶瓷膜过滤。
(3)脱色：产品：水：活性炭＝1：(1.5-3)：(0.01-0.1)，搅拌0.5-5h。
(4)除盐：电渗析除盐。浓室罐装入发酵液初始盐浓度：0.01-0.05mol/L。 淡室发酵液流速：40-80L/h，浓室发酵液流速：40-80L/h，单膜对的电压0.5-1.4V。也可以采用阴、阳离子交换树脂除盐。
(5)浓缩：将除盐后发酵液真空条件下(0.095MPa)加热50-80℃，浓缩8-15h，至过饱和，约4-6倍。
(6)水解：浓缩后的发酵液转入搪瓷或玻璃容器中，加入浓盐酸(37％)至终浓度为12-16％，搅匀，70℃保温90min。盐酸可循环套用。
(7)结晶：先25℃水降温至25-35℃，再用0℃水降温1-3h，至4℃。
(8)洗涤：加无水乙醇(与产品等量)搅拌10-100rpm，0.5-2h。离心700-1500rpm，15-60min，得氨基葡萄糖盐酸盐，转化率为90.2％。
(9)溶解：洗涤后的产品溶于水中，水用量约为原发酵液体积。
(10)脱色：加入活性炭(用量为1％)。混合30分钟后。离心700-1500rpm，15-60min。或过滤，得到无色滤液。
(11)重结晶：在50℃与55cmHg真空中对滤液进行真空蒸发至过饱和。加无水乙醇(用量为产品重量5-20倍)，700-1500rpm搅拌15min-1h。
(12)洗涤：加无水乙醇(与产品等量)搅拌10-100rpm，0.5-2h。离心700-1500rpm，15-60min。
(13)烘干：50-100℃，3-10h。纯度：99.91％。总产率为83.8％。
实施例13
本实施例描述筛选突变的N-乙酰-D-氨基甘露糖-6-P异构酶(NanE)的基因，所述基因编码酶活性增加的N-乙酰-D-氨基甘露糖-6-P异构酶。
为了进一步提高生产菌株中的N-乙酰-D-氨基葡萄糖合成量，筛选编码具有酶活性增加的基因突变体。为了达到该目的，用易错PCR技术扩增克隆的基因，通过用于扩增的DNA聚合酶，在导致高频错配的条件下扩增所述基因，以便在PCR产物中得到高频突变。
具体操作过程如下：
1、易错PCR扩增大肠杆菌N-乙酰-D-氨基甘露糖-6-P异构酶基因nanE
利用Taq DNA聚合酶不具有3'-5'校对功能的性质，在高镁离子浓度(8mmol/L)和不同浓度dNTP的浓度下(其中，dATP和dGTP浓度为1.5mmol/L；dTTP和dCTP浓度为3.0mmol/L)，来控制随机突变的频率，向目的基因中引入 随机突变，构建突变库；模板浓度A260值为1000ng/mL，酶浓度为5U/μL，引物浓度为100μM。
易错PCR反应体系(50μl)：10×PCR反应缓冲液5μl，dNTP(2.5mM)5μl，MgCl2(2.5mM)5μl，正向引物(nanE-F，SEQ ID No.30)1μl，反向引物(nanE-R，SEQ ID No.31)1μl，DNA模板(nanE/pUC57)0.1μl，Taq DNA聚合酶0.5μl，ddH2O 32.4μl。
PCR程序：96℃预变性4min；94℃变性1min，56℃退火1min，75℃延伸2min，45个循环；最后75℃延伸15min，采用胶回收方法回收PCR产物(产物大小：0.7kb)；取5μl产物1％琼脂糖凝胶电泳检验，-20℃保存备用。
2、构建N-乙酰-D-氨基甘露糖-6-P异构酶的基因突变体库
将上述PCR产物经限制性内切酶Nco I和Hind III双酶切消化后，与用Nco I和Hind III内切酶消化的pTrc99A质粒进行连接反应，然后用连接产物混合物转化大肠杆菌AT-005-02，获得大量克隆转化子，构建转化菌体突变库。
3、筛选高酶活突变体
从转化菌体突变库中，随机挑取突变克隆350株，以野生型NanE/pTrc99A(AT-005-02)为对照，分别接种至含50μg/mL青霉素(Amp)的5ml LB培养基中，37℃、150rpm培养18h后，10000rpm，5mim离心收集菌体。弃上清后，在4℃下重悬于1ml PBS(pH值7.5，10mmol/L)溶液中，在冰浴条件下选取300V电压，超声3s间歇6s对其进行超声破碎10min，离心取上清作为酶粗提液，进行酶活测定。
N-乙酰-D-氨基甘露糖-6-P异构酶的活性检测：以N-乙酰-D-氨基甘露糖-6-磷酸(ManNAc-6-P)转化为N-乙酰-D-氨基葡萄糖-6-磷酸(GlcNAc-6-P)多少为依据，也就是N-乙酰-D-氨基甘露糖-6-磷酸减少为测定标记。酶活单位定义：在酶促反应条件下，每分钟减少相当于1μmol N-乙酰-D-氨基甘露糖-6-磷酸所需的酶量，定义为一个酶活力单位(IU)。具体操作如下：首先，制备作为底物的同位素标记的ManNAc-6-P。配制总体积225ul反应液，含ManNAc激酶(NanK)粗酶液(含1-5mg蛋白)，20mM ATP二钠盐,60mM Tris-HCl,pH8.1，20mM MgCl2和5mM ManNAc，50nCi[14C]ManNAc。37℃下孵育30min。加350ul乙醇终止 反应。产物用水洗脱，冻干。其次，制备总体积26.5ul反应液为酶活测定体系，其中含1mM同位素标记的ManNAc-6-P，37mM Tris-HCl，pH 8.0和19mM MgCl2。37℃下，孵育30min后，反应液煮3min，然后加0.1体积的碱性磷酸酶缓冲液调节pH和20单位碱性磷酸酶。37℃下，孵育1小时后，取样品加入干层析纸上，用1％四硼酸钠预浸。所用溶剂体系为醋酸乙脂：异丙醇：吡啶：水(50：22：14：14)。带放射性化合物用纸色谱分离。用液相闪烁计数仪测定其放射性强度，根据ManNAc-6-P转化为GlcNAc-6-P的多少，计算N-乙酰-D-氨基甘露糖-6-P异构酶的活性单位。
结果表明：最高突变体菌株的酶活为72IU/ml，对照菌株的酶活为9.5IU/ml。通过易错PCR对NanE进行改造，获得酶活力大大提高的突变株。挑选该酶活性最高的突变体菌株，提取质粒测序。结果表明：该N-乙酰-D-氨基甘露糖-6-P异构酶突变体基因序列如SEQ ID No.56所示，对应的氨基酸序列如SEQ ID No.57所示。与野生型的N-乙酰-D-氨基甘露糖-6-P异构酶基因序列比对，共发生了3处碱基点突变：198C/T，397T/C，559T/C；致氨基酸2处错义突变，其突变点分别为：C133R(第133位半胱氨酸变为精氨酸)，Y187H(第187位酪氨酸变为组氨酸)。将该突变基因命名为nanEM。
4、pTrc-nanEM基因盒向大肠杆菌染色体nagE基因位点上整合
以nagE基因位点为pTrc-nanEM基因盒在染色体上的整合位点。为达到pTrc-nanEM基因盒向大肠杆菌染色体中的整合，首先，扩增带Trc启动子的nanEM片段pTrc-nanEM，以及两侧带有FLP重组酶识别位点(FRT位点)的卡拉霉素抗性基因片段：FRT-Kanr-FRT(fKanrf)，并拼接。然后，再一次设计删除nagE基因序列同源臂的引物，并以pTrc-nanEM和fKanrf拼接的片段为模板，扩增Red重组打靶用线性DNA全长片段。
具体操作过程如下：
(1)PCR扩增pTrc-nanEM片段
模板：nanEM/pTrc99A。
设计引物：正向引物(Trcff-F)SEQ ID No.20，反向引物(Trcff-R)SEQ ID No.21。
PCR反应条件：第一步：94℃变性1min；第二步：94℃30s，55℃30s，72℃40s， 循环30次；第三步：72℃延伸10min。
产物大小：0.86kb。
PCR产物经1％琼脂糖凝胶电泳分离、纯化回收片段。
(2)PCR扩增fKanrf片段
设计引物：正向引物(mfKanf-F)SEQ ID No.1，反向引物(mfKanf-R)SEQ ID No.2。
模板：pPic9K。
PCR反应条件：第一步：94℃变性1min；第二步：94℃30s，55℃30s，72℃40s，循环30次；第三步：72℃延伸10min。
fKanrf大小：1.28kb。其核苷酸序列SEQ ID No.3。
PCR产物经1％琼脂糖凝胶电泳分离、纯化回收片段。
(3)扩增与pTrc-nanEM对接的fKanrf
设计引物：正向引物(fKanf-F)SEQ ID No.22，反向引物(fKanf-R)SEQ ID No.23。
模板：fKanrf。
PCR反应条件：第一步：94℃变性1min；第二步：94℃30s，55℃30s，72℃40s，循环30次；第三步：72℃延伸10min。
二次扩增的fKanrf大小：1.3kb。
PCR产物经1％琼脂糖凝胶电泳分离、纯化回收片段。
(4)制备Red重组打靶用线性DNA全长PCR片段
设计同源臂引物：再一次设计删除nagE基因序列同源臂的正向引物(nagEKO-F2)SEQ ID No.24，反向引物(nagEKO-R2)SEQ ID No.25。
模板：pTrc-nanEM PCR片段和二次扩增的fKanrf PCR片段，1：1混合。
PCR反应条件：第一步：94℃变性1min；第二步：94℃30s，55℃30s，72℃40s，循环30次；第三步：72℃延伸10min。
扩增产物：同源臂+pTrc-nanEM-fKanrf+同源臂。
将PCR产物琼脂糖凝胶电泳分离、纯化回收，得到100ng/μl的线性DNA全长PCR片段用于Red重组打靶。
(5)Red重组操作
首先，将pKD46载体转入大肠杆菌AT-004-02菌株中。然后，电转化制备 好的打靶用线性DNA片段，筛选阳性克隆。最后，消除抗性基因。
1)转化pKD46质粒
①感受态制备：首先，将保存于-20℃的Escherichia coli AT-004-02菌液，按1:50-100接种于10ml LB液体培养基中，37℃，225rpm，振荡培养2-3小时。再将培养液加入10ml离心管中，4000g×5min，弃去上清，用冰浴的0.1M CaCl25ml悬浮5min。最后，4000g×5min离心，弃去上清，用冰浴的0.1M CaCl2 5ml悬浮。-4℃静置12小时，自然沉降。
②质粒转化：取自然沉降的菌体250μl，加入5μl pKD46质粒，-4℃，30min。然后，42℃水浴1.5min，加入SOC培养基0.7ml，30℃摇2小时。取0.2ml菌液，涂青霉素平板。30℃过夜(12-16小时)培养。挑单克隆，加入5ml LB液体培养基中培养，抽质粒鉴定。保存阳性菌种备用。
2)电转化制备好的打靶用线性DNA片段，筛选阳性克隆
①电转感受态的制备：将含pKD46Escherichia coli菌种AT-004-02接种于含有氨苄青霉素(Amp)LB培养基的试管，250rpm摇床过夜，第二天以1％的量接种至含有Amp的LB培养基中，30℃培养，待OD600达到0.2左右后，加入0.2％的L-阿拉伯糖，30℃诱导35分钟，直至OD600达到0.4左右。冰浴冷却。用超纯水洗一次，10％甘油洗两次，最后用10％甘油重悬，甘油用量以使菌体被浓缩500-1000倍的终浓度为宜。
②电击转化：将2mm电转杯从70％乙醇中取出，用灭菌超纯水洗2次，紫外灯照射30分钟。4℃预冷30分钟。取90μl最终重悬的细胞，移至预冷的离心管，加入5μl(100ng以上)步骤(4)得到的全长PCR片段(线性DNA)，用枪轻轻吸打混匀，冰浴30分钟。电转参数：2500V，200Ω，25μF。
③复苏与筛选阳性克隆：加入1ml的LB液体培养基，37℃，100rpm，1小时。然后每200μl涂布一个卡拉霉素(Kan)平板，一共5个。均匀、涂干。30℃培养24个小时。挑在卡拉霉素抗性下生长的克隆，作PCR鉴定，筛选阳性克隆。
所获得菌种编号：AT-031-01(AT-004-02，△nagE::pTrc-nanEM-fKanrf)。
按照上述相同的方法，制备菌种AT-030-01(AT-004-02，△nagE::pTrc-nanE-fKanrf)。
(6)抗性基因的消除
将pCP20转入上述卡拉霉素抗性克隆，30℃培养8h，后提高到42℃过夜，热诱导FLP重组酶表达，质粒逐渐丢失。用接种环蘸菌液在无抗生素培养基上划板，挑长出的单克隆点到卡拉霉素抗性平板上，未生长的为卡拉霉素抗性基因已被FLP重组酶删除的克隆。用鉴定引物作PCR对卡拉霉素抗性消失的克隆进行鉴定。
所获得菌种编号：AT-031-02(AT-004-02，△nagE::pTrc-nanEM)。
按照上述相同的方法，制备菌种AT-030-02(AT-004-02，△nagE::pTrc-nanE)。
5、pTrc-nanE、pTrc-nanEM基因盒整合对N-乙酰-D-氨基葡萄糖产量的影响
将在染色体nagE基因位点上整合有pTrc-nanE、pTrc-nanEM基因盒的重组菌AT-030-02、AT-031-02和对照菌种，做摇瓶发酵试验。取新鲜培养的LB平板培养基上单克隆菌株，接种于3ml的LB液体培养基试管(13×150mm)中，30℃，225rpm，培养约8小时。然后取种子培养液，3％接种于含50ml的发酵培养液(M9培养液)250ml摇瓶中。起始OD600约0.5，37℃下，225rpm培养，发酵周期72小时。在第24小时、48小时，用10M NaOH调节发酵液pH至7.0。根据发酵液糖耗情况，分次加入65％葡萄糖液维持葡萄糖浓度在20g/L。发酵结束，取1ml发酵液，离心。用HPLC法测定N-乙酰-D-氨基葡萄糖含量。
摇瓶发酵产量情况见表17。结果表明：对照菌种AT-005-02产量很低，未检出，突变的pTrc-nanEM基因盒整合重组菌AT-031-02产量明显提高，且较未突变的对照菌种AT-030-02产量亦有明显提高。
表17.pTrc-nanEM基因盒整合重组菌摇瓶发酵产量
Figure PCTCN2017080653-appb-000016
以上结果显示：不仅N-乙酰-D-氨基甘露糖-6-P异构酶超表达可明显提高N-乙酰-D-氨基葡萄糖产量，通过易错PCR技术筛选突变体也可大大提高N-乙 酰-D-氨基葡萄糖产量，这是由于所获得的该异构酶突变体酶活性增加所致。
实施例14
本实施例描述筛选突变的UDP-N-乙酰氨基葡萄糖-2-异构酶(WecB)的基因，所述基因编码酶活性增加的UDP-N-乙酰氨基葡萄糖-2-异构酶。
为了进一步提高生产菌株中的N-乙酰-D-氨基葡萄糖合成量，筛选编码具有酶活性增加的UDP-N-乙酰氨基葡萄糖-2-异构酶的基因突变体。为了达到该目的，用易错PCR技术扩增克隆的基因，通过用于扩增的DNA聚合酶，在导致高频错配的条件下扩增所述基因，以便在PCR产物中得到高频突变。
具体操作过程如下：
1、易错PCR扩增大肠杆菌UDP-N-乙酰氨基葡萄糖-2-异构酶基因wecB
利用Taq DNA聚合酶不具有3'-5'校对功能的性质，在高镁离子浓度(8mmol/L)和不同浓度dNTP的浓度下(其中，dATP和dGTP浓度为1.5mmol/L；dTTP和dCTP浓度为3.0mmol/L)，来控制随机突变的频率，向目的基因中引入随机突变，构建突变库；模板浓度A260值为1000ng/mL，酶浓度为5U/μL，引物浓度为100μM。
易错PCR反应体系(50μl)：10×PCR反应缓冲液5μl，dNTP(2.5mM)5μl，MgCl2(2.5mM)5μl，正向引物(TrcwecB-F，SEQ ID No.51)1μl，反向引物(TrcwecB-R，SEQ ID No.52)1μl，DNA模板(wecB/pUC57)0.1μl，Taq DNA聚合酶0.5μl，ddH2O 32.4μl。
PCR程序：96℃预变性4min；94℃变性1min，56℃退火1min，75℃延伸2min，45个循环；最后75℃延伸15min，采用胶回收方法回收PCR产物(产物大小：1.13kb)；取5μl产物1％琼脂糖凝胶电泳检验，-20℃保存备用。
2、构建UDP-N-乙酰氨基葡萄糖-2-异构酶的基因突变体库
将上述PCR产物经限制性内切酶Nco I和Hind III双酶切消化后，与用Nco I和Hind III内切酶消化的pTrc99A质粒进行连接反应，然后用连接产物混合物转化大肠杆菌AT-005-02，获得大量克隆转化子，构建转化菌体突变库。
3、筛选高酶活突变体
从转化菌体突变库中，随机挑取突变克隆640株，以野生型WecB/pTrc99A(AT-005-02)为对照，分别接种至含50μg/mL青霉素(Amp)的5ml LB培养基中，37℃、150rpm培养18h后，10000rpm，5mim离心收集菌体。弃上清后，在4℃下重悬于1ml PBS(pH值7.5，10mmol/L)溶液中，在冰浴条件下选取300V电压，超声3s间歇6s对其进行超声破碎10min，离心取上清作为酶粗提液，进行酶活测定。
UDP-N-乙酰氨基葡萄糖-2-异构酶的活性检测：以UDP-N-乙酰-D-氨基葡萄糖转化为N-乙酰-D-氨基甘露糖多少为依据。也就是UDP-N-乙酰-D-氨基葡萄糖减少为测定标记。酶活单位定义：在酶促反应条件下，每分钟减少相当于1μmol UDP-N-乙酰-D-氨基葡萄糖所需的酶量，定义为一个酶活力单位(IU)。具体操作如下：以20ml反应体系为酶活测定体系，其中含45mmol/L磷酸缓冲液(pH7.5)、10mM MgCl2and 100nCi of UDPGlcNAc及5mg粗酶液。酶活反应在37℃水浴中进行孵育30min。加乙醇终止反应。带放射性化合物用纸色谱分离。用液相闪烁计数仪测定其放射性强度。所用溶剂体系为正丙醇：1M醋酸钠，pH 5.0：水(7：1：2)。根据UDPGlcNAc转化为ManNAc的多少，计算UDP-N-乙酰氨基葡萄糖-2-异构酶的活性单位。
结果表明：最高突变体菌株的酶活为653IU/ml，对照菌株的酶活为21.0IU/ml。通过易错PCR对WecB进行改造，获得酶活力大大提高的突变株。挑选该酶活性最高的突变体菌株，提取质粒测序。结果表明：该UDP-N-乙酰氨基葡萄糖-2-异构酶突变体基因序列如SEQ ID No.58所示，对应的氨基酸序列如SEQ ID No.59所示。与野生型的UDP-N-乙酰氨基葡萄糖-2-异构酶基因序列比对，共发生了5处碱基点突变：101G/C，433C/G，677G/T，734T/G，1038T/C；致氨基酸4处错义突变，其突变点分别为：C34S(第34位半胱氨酸变为丝氨酸)，H145D(第145位组氨酸变为天冬氨酸)，C226F(第226位半胱氨酸变为苯丙氨酸)，V245G(第245位缬氨酸变为甘氨酸)。将该突变基因命名为wecBM。
4、pTrc-wecBM基因盒向大肠杆菌染色体nagE基因位点上整合
以nagE基因位点为pTrc-wecBM基因盒在染色体上的整合位点。为达到pTrc-wecBM基因盒向大肠杆菌染色体中的整合，首先，扩增带Trc启动子的 wecBM片段pTrc-wecBM，以及两侧带有FLP重组酶识别位点(FRT位点)的卡拉霉素抗性基因片段：FRT-Kanr-FRT(fKanrf)，并拼接。然后，再一次设计删除nagE基因序列同源臂的引物，并以pTrc-wecBM和fKanrf拼接的片段为模板，扩增Red重组打靶用线性DNA全长片段。
具体操作过程如下：
(1)PCR扩增pTrc-wecBM片段
模板：wecBM/pTrc99A。
设计引物：正向引物(Trcff-F)SEQ ID No.20，反向引物(Trcff-R)SEQ ID No.21。
PCR反应条件：第一步：94℃变性1min；第二步：94℃30s，55℃30s，72℃40s，循环30次；第三步：72℃延伸10min。
产物大小：1.3kb。
PCR产物经1％琼脂糖凝胶电泳分离、纯化回收片段。
(2)PCR扩增fKanrf片段
设计引物：正向引物(mfKanf-F)SEQ ID No.1，反向引物(mfKanf-R)SEQ ID No.2。
模板：pPic9K。
PCR反应条件：第一步：94℃变性1min；第二步：94℃30s，55℃30s，72℃40s，循环30次；第三步：72℃延伸10min。
fKanrf大小：1.28kb。其核苷酸序列SEQ ID No.3。
PCR产物经1％琼脂糖凝胶电泳分离、纯化回收片段。
(3)扩增与pTrc-wecBM对接的fKanrf
设计引物：正向引物(fKanf-F)SEQ ID No.22，反向引物(fKanf-R)SEQ ID No.23。
模板：fKanrf。
PCR反应条件：第一步：94℃变性1min；第二步：94℃30s，55℃30s，72℃40s，循环30次；第三步：72℃延伸10min。
二次扩增的fKanrf大小：1.3kb。
PCR产物经1％琼脂糖凝胶电泳分离、纯化回收片段。
(4)制备Red重组打靶用线性DNA全长PCR片段
设计同源臂引物：再一次设计删除nagE基因序列同源臂的正向引物(nagEKO-F2)SEQ ID No.24，反向引物(nagEKO-R2)SEQ ID No.25。
模板：pTrc-wecBM PCR片段和二次扩增的fKanrf PCR片段，1：1混合。
PCR反应条件：第一步：94℃变性1min；第二步：94℃30s，55℃30s，72℃40s，循环30次；第三步：72℃延伸10min。
扩增产物：同源臂+pTrc-wecBM-fKanrf+同源臂。
将PCR产物琼脂糖凝胶电泳分离、纯化回收，得到100ng/μl的线性DNA全长PCR片段用于Red重组打靶。
(5)Red重组操作
首先，将pKD46载体转入大肠杆菌AT-004-02菌株中。然后，电转化制备好的打靶用线性DNA片段，筛选阳性克隆。最后，消除抗性基因。
1)转化pKD46质粒
①感受态制备：首先，将保存于-20℃的Escherichia coli AT-004-02菌液，按1:50-100接种于10ml LB液体培养基中，37℃，225rpm，振荡培养2-3小时。再将培养液加入10ml离心管中，4000g×5min，弃去上清，用冰浴的0.1M CaCl25ml悬浮5min。最后，4000g×5min离心，弃去上清，用冰浴的0.1M CaCl2 5ml悬浮。-4℃静置12小时，自然沉降。
②质粒转化：取自然沉降的菌体250μl，加入5μl pKD46质粒，-4℃，30min。然后，42℃水浴1.5min，加入SOC培养基0.7ml，30℃摇2小时。取0.2ml菌液，涂青霉素平板。30℃过夜(12-16小时)培养。挑单克隆，加入5ml LB液体培养基中培养，抽质粒鉴定。保存阳性菌种备用。
2)电转化制备好的打靶用线性DNA片段，筛选阳性克隆
①电转感受态的制备：将含pKD46Escherichia coli菌种AT-004-02接种于含有氨苄青霉素(Amp)LB培养基的试管，250rpm摇床过夜，第二天以1％的量接种至含有Amp的LB培养基中，30℃培养，待OD600达到0.2左右后，加入0.2％的L-阿拉伯糖，30℃诱导35分钟，直至OD600达到0.4左右。冰浴冷却。用超纯水洗一次，10％甘油洗两次，最后用10％甘油重悬，甘油用量以使菌体被浓缩500-1000倍的终浓度为宜。
②电击转化：将2mm电转杯从70％乙醇中取出，用灭菌超纯水洗2次，紫外灯照射30分钟。4℃预冷30分钟。取90μl最终重悬的细胞，移至预冷的离 心管，加入5μl(100ng以上)步骤(4)得到的全长PCR片段(线性DNA)，用枪轻轻吸打混匀，冰浴30分钟。电转参数：2500V，200Ω，25μF。
③复苏与筛选阳性克隆：加入1ml的LB液体培养基，37℃，100rpm，1小时。然后每200μl涂布一个卡拉霉素(Kan)平板，一共5个。均匀、涂干。30℃培养24个小时。挑在卡拉霉素抗性下生长的克隆，作PCR鉴定，筛选阳性克隆。
所获得菌种编号：AT-043-01(AT-004-02，△nagE::pTrc-wecBM-fKanrf)。
按照上述相同的方法，制备菌种AT-042-01(AT-004-02，△nagE::pTrc-wecB-fKanrf)。
(6)抗性基因的消除
将pCP20转入上述卡拉霉素抗性克隆，30℃培养8h，后提高到42℃过夜，热诱导FLP重组酶表达，质粒逐渐丢失。用接种环蘸菌液在无抗生素培养基上划板，挑长出的单克隆点到卡拉霉素抗性平板上，未生长的为卡拉霉素抗性基因已被FLP重组酶删除的克隆。用鉴定引物作PCR对卡拉霉素抗性消失的克隆进行鉴定。
所获得菌种编号：AT-043-02(AT-004-02，△nagE::pTrc-wecBM)。
按照上述相同的方法，制备菌种AT-042-02(AT-004-02，△nagE::pTrc-wecB)。
5、pTrc-wecB、pTrc-wecBM基因盒整合对N-乙酰-D-氨基葡萄糖产量的影响
将在染色体nagE基因位点上整合有pTrc-wecB、pTrc-wecBM基因盒的重组菌AT-042-02、AT-043-02和对照菌种，做摇瓶发酵试验。取新鲜培养的LB平板培养基上单克隆菌株，接种于3ml的LB液体培养基试管(13×150mm)中，30℃，225rpm，培养约8小时。然后取种子培养液，3％接种于含50ml的发酵培养液(M9培养液)250ml摇瓶中。起始OD600约0.5，37℃下，225rpm培养，发酵周期72小时。在第24小时、48小时，用10M NaOH调节发酵液pH至7.0。根据发酵液糖耗情况，分次加入65％葡萄糖液维持葡萄糖浓度在20g/L。发酵结束，取1ml发酵液，离心。用HPLC法测定N-乙酰-D-氨基葡萄糖含量。
摇瓶发酵产量情况见表18。结果表明：对照菌种AT-005-02产量很低，未检出，突变的pTrc-wecBM基因盒整合重组菌AT-043-02产量明显提高，且较未突变的对照菌种AT-042-02产量亦有明显提高。
表18.pTrc-wecBM基因盒整合重组菌摇瓶发酵产量
Figure PCTCN2017080653-appb-000017
以上结果显示：不仅UDP-N-乙酰氨基葡萄糖-2-异构酶超表达可明显提高N-乙酰-D-氨基葡萄糖产量，通过易错PCR技术筛选突变体也可大大提高N-乙酰-D-氨基葡萄糖产量，这是由于所获得的该异构酶突变体酶活性增加所致。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。

Claims (73)

  1. 一种通过微生物发酵生产N-乙酰-D-氨基葡萄糖和/或D-氨基葡萄糖盐的方法，该方法包括：
    A)在发酵培养基中培养微生物，所述微生物包含至少一种能提高微生物中N-乙酰-D-氨基甘露糖激酶作用的遗传修饰；和
    B)收集从培养步骤A)中产生的N-乙酰-D-氨基葡萄糖。
    优选，进一步包括C)由N-乙酰-D-氨基葡萄糖脱乙酰化得到D-氨基葡萄糖盐。
    进一步优选，所述盐选自盐酸盐、硫酸盐、钠盐、磷酸盐和硫酸氢盐。
  2. 如权利要求1所述的方法，其中，提高微生物中N-乙酰-D-氨基甘露糖激酶作用的遗传修饰选自a)微生物中N-乙酰-D-氨基甘露糖激酶的酶活性增加；和/或b)微生物中N-乙酰-D-氨基甘露糖激酶被过量表达；
    优选，微生物用至少一种包含至少一种能提高微生物中N-乙酰-D-氨基甘露糖激酶作用的遗传修饰的重组核酸分子转化。
  3. 如权利要求2所述的方法，其中，微生物用至少一种包含编码N-乙酰-D-氨基甘露糖激酶的核酸序列的重组核酸分子转化。
    优选，编码N-乙酰-D-氨基甘露糖激酶的核酸序列含有至少一种增加N-乙酰-D-氨基甘露糖激酶的酶活性的遗传修饰；进一步优选，所述遗传修饰包括在对应于氨基酸序列SEQ ID NO：17的下述位置处的取代中的一种或多种：第36位赖氨酸被精氨酸取代、第103位异亮氨酸被蛋氨酸取代和第223位精氨酸被丝氨酸取代；更优选，编码N-乙酰-D-氨基甘露糖激酶的核酸序列为SEQ ID NO：26。
    优选，所述的N-乙酰-D-氨基甘露糖激酶具有与SEQ ID NO:17的氨基酸序列至少约30％相同，优选至少约50％相同，进一步优选至少约70％相同，进一步优选至少约80％相同，更进一步优选至少约90％相同，最优选至少约95％相同的氨基酸序列，其中所述的N-乙酰-D-氨基甘露糖激酶具有酶活性；进一步优选，所述的N-乙酰-D-氨基甘露糖激酶具有SEQ ID NO:17的氨基酸序列。
    进一步优选，重组核酸分子中编码N-乙酰-D-氨基甘露糖激酶的基因拷贝数 增加。
    进一步优选，重组核酸分子中包含内源性天然启动子或具有比内源性天然启动子更高表达水平的启动子；优选，具有比内源性天然启动子更高表达水平的启动子选自HCE启动子、gap启动子、trc启动子、T7启动子；进一步优选，具有比内源性天然启动子更高表达水平的启动子为trc启动子。
  4. 如权利要求2所述的方法，其中，微生物包括至少一种对编码N-乙酰-D-氨基甘露糖激酶的基因的内源性天然启动子的遗传修饰；优选，编码N-乙酰-D-氨基甘露糖激酶的基因的内源性天然启动子被具有更高表达水平的启动子替换；进一步优选，具有更高表达水平的启动子选自HCE启动子、gap启动子、trc启动子、T7启动子；最优选，具有更高表达水平的启动子为trc启动子。
  5. 如权利要求1-4中任一项所述的方法，其中，所述微生物进一步包含下述遗传修饰中的一种或多种：
    (1)包含至少一种能提高微生物中N-乙酰-D-氨基甘露糖-6-磷酸异构酶作用的遗传修饰；
    (2)包含至少一种能提高微生物中D-氨基葡萄糖-6-磷酸脱氨酶作用的遗传修饰，优选同时包含至少一种能降低氨基葡萄糖-6-磷酸合成酶作用的遗传修饰；
    (3)包含至少一种能提高微生物中氨基葡萄糖-6-磷酸合成酶作用的遗传修饰，并同时包含至少一种能降低D-氨基葡萄糖-6-磷酸脱氨酶作用的遗传修饰；
    (4)包含至少一种能提高微生物中UDP-N-乙酰-D-氨基葡萄糖-2-异构酶(WecB)作用的遗传修饰。
  6. 如权利要求5所述的方法，其中，提高微生物中N-乙酰-D-氨基甘露糖-6-磷酸异构酶作用的遗传修饰选自a)微生物中N-乙酰-D-氨基甘露糖-6-磷酸异构酶的酶活性增加；和/或b)微生物中N-乙酰-D-氨基甘露糖-6-磷酸异构酶被过量表达；
    优选，微生物用至少一种包含至少一种能提高微生物中N-乙酰-D-氨基甘露糖-6-磷酸异构酶作用的遗传修饰的重组核酸分子转化。
  7. 如权利要求6所述的方法，其中，微生物用至少一种包含编码N-乙酰-D-氨基甘露糖-6-磷酸异构酶的核酸序列的重组核酸分子转化。
    优选，编码N-乙酰-D-氨基甘露糖-6-磷酸异构酶的核酸序列含有至少一种增加N-乙酰-D-氨基甘露糖-6-磷酸异构酶的酶活性的遗传修饰。优选，所述遗传修饰包括在对应于氨基酸序列SEQ ID NO：29的下述位置处的取代中的一种或两种：第133位半胱氨酸被精氨酸取代和第187位酪氨酸被组氨酸取代。进一步优选，编码所述N-乙酰-D-氨基甘露糖-6-磷酸异构酶(NanE)的核酸序列为SEQ ID NO：56。
    优选，所述的N-乙酰-D-氨基甘露糖-6-磷酸异构酶具有与SEQ ID NO：29的氨基酸序列至少约30％相同，优选至少约50％相同，进一步优选至少约70％相同，进一步优选至少约80％相同，更进一步优选至少约90％相同，最优选至少约95％相同的氨基酸序列，其中所述的N-乙酰-D-氨基甘露糖-6-磷酸异构酶具有酶活性；进一步优选，所述的N-乙酰-D-氨基甘露糖-6-磷酸异构酶具有SEQ ID NO：29的氨基酸序列。
    进一步优选，重组核酸分子中编码N-乙酰-D-氨基甘露糖-6-磷酸异构酶的基因拷贝数增加。
    进一步优选，重组核酸分子中包含内源性天然启动子或具有比内源性天然启动子更高表达水平的启动子；优选，具有比内源性天然启动子更高表达水平的启动子选自HCE启动子、gap启动子、trc启动子、T7启动子；进一步优选，具有比内源性天然启动子更高表达水平的启动子为trc启动子。
  8. 如权利要求6所述的方法，其中，微生物包括至少一种对编码N-乙酰-D-氨基甘露糖-6-磷酸异构酶的基因的内源性天然启动子的遗传修饰；优选，编码N-乙酰-D-氨基甘露糖-6-磷酸异构酶的基因的内源性天然启动子被具有更高表达水平的启动子替换；进一步优选，具有更高表达水平的启动子选自HCE启动子、gap启动子、trc启动子、T7启动子；最优选，具有更高表达水平的启动子为trc启动子。
  9. 如权利要求5所述的方法，其中，提高微生物中D-氨基葡萄糖-6-磷酸脱 氨酶作用的遗传修饰选自a)微生物中D-氨基葡萄糖-6-磷酸脱氨酶的酶活性增加；和/或b)微生物中D-氨基葡萄糖-6-磷酸脱氨酶被过量表达；
    优选，微生物用至少一种包含至少一种能提高微生物中D-氨基葡萄糖-6-磷酸脱氨酶作用的遗传修饰的重组核酸分子转化。
  10. 如权利要求9所述的方法，其中，微生物用至少一种包含编码D-氨基葡萄糖-6-磷酸脱氨酶的核酸序列的重组核酸分子转化。
    优选，编码D-氨基葡萄糖-6-磷酸脱氨酶的核酸序列含有至少一种增加D-氨基葡萄糖-6-磷酸脱氨酶的酶活性的遗传修饰。
    进一步优选，重组核酸分子中编码D-氨基葡萄糖-6-磷酸脱氨酶的基因拷贝数增加。
    进一步优选，重组核酸分子中包含内源性天然启动子或具有比内源性天然启动子更高表达水平的启动子；优选，具有比内源性天然启动子更高表达水平的启动子选自HCE启动子、gap启动子、trc启动子、T7启动子；进一步优选，具有比内源性天然启动子更高表达水平的启动子为trc启动子。
  11. 如权利要求9所述的方法，其中，微生物包括至少一种对编码D-氨基葡萄糖-6-磷酸脱氨酶的基因的内源性天然启动子的遗传修饰。优选，编码D-氨基葡萄糖-6-磷酸脱氨酶的基因的内源性天然启动子被具有更高表达水平的启动子替换；进一步优选，具有更高表达水平的启动子选自HCE启动子、gap启动子、trc启动子、T7启动子；最优选，具有更高表达水平的启动子为trc启动子。
  12. 如权利要求5所述的方法，其中，降低微生物中氨基葡萄糖-6-磷酸合成酶作用的遗传修饰选自a)微生物中氨基葡萄糖-6-磷酸合成酶的酶活性降低；和/或b)微生物中氨基葡萄糖-6-磷酸合成酶的表达减少；
    优选，微生物用至少一种包含至少一种能降低微生物中氨基葡萄糖-6-磷酸合成酶作用的遗传修饰的重组核酸分子转化。
    进一步优选，降低微生物中氨基葡萄糖-6-磷酸合成酶作用的遗传修饰选自编码微生物中氨基葡萄糖-6-磷酸合成酶的内源性基因的部分或完全缺失、或部分或完全失活，和/或编码微生物中氨基葡萄糖-6-磷酸合成酶基因的内源性天然 启动子的部分或完全缺失、或部分或完全失活；更优选，降低微生物中氨基葡萄糖-6-磷酸合成酶作用的遗传修饰为编码微生物中氨基葡萄糖-6-磷酸合成酶基因的内源性天然启动子完全缺失，即被删除。
  13. 如权利要求5所述的方法，其中，提高微生物中氨基葡萄糖-6-磷酸合成酶作用的遗传修饰选自a)微生物中氨基葡萄糖-6-磷酸合成酶的酶活性增加；和/或b)微生物中氨基葡萄糖-6-磷酸合成酶被过量表达；
    优选，微生物用至少一种包含至少一种能提高微生物中氨基葡萄糖-6-磷酸合成酶作用的遗传修饰的重组核酸分子转化。
  14. 如权利要求13所述的方法，其中，微生物用至少一种包含编码氨基葡萄糖-6-磷酸合成酶的核酸序列的重组核酸分子转化。
    优选，编码氨基葡萄糖-6-磷酸合成酶的核酸序列含有至少一种增加氨基葡萄糖-6-磷酸合成酶的酶活性的遗传修饰。
    进一步优选，重组核酸分子中编码氨基葡萄糖-6-磷酸合成酶的基因拷贝数增加。
    进一步优选，重组核酸分子中包含内源性天然启动子或具有比内源性天然启动子更高表达水平的启动子；优选，具有比内源性天然启动子更高表达水平的启动子选自HCE启动子、gap启动子、trc启动子、T7启动子；进一步优选，具有比内源性天然启动子更高表达水平的启动子为trc启动子。
  15. 如权利要求13所述的方法，其中，微生物包括至少一种对编码氨基葡萄糖-6-磷酸合成酶的基因的内源性天然启动子的遗传修饰。优选，编码氨基葡萄糖-6-磷酸合成酶的基因的内源性天然启动子被具有更高表达水平的启动子替换；进一步优选，具有更高表达水平的启动子选自HCE启动子、gap启动子、trc启动子、T7启动子；最优选，具有更高表达水平的启动子为trc启动子。
  16. 如权利要求5所述的方法，其中，降低微生物中D-氨基葡萄糖-6-磷酸脱氨酶作用的遗传修饰选自a)微生物中D-氨基葡萄糖-6-磷酸脱氨酶的酶活性降低；和/或b)微生物中D-氨基葡萄糖-6-磷酸脱氨酶的表达减少；
    优选，微生物用至少一种包含至少一种能降低微生物中D-氨基葡萄糖-6-磷酸脱氨酶作用的遗传修饰的重组核酸分子转化。
    进一步优选，降低微生物中D-氨基葡萄糖-6-磷酸脱氨酶作用的遗传修饰选自编码微生物中D-氨基葡萄糖-6-磷酸脱氨酶的内源性基因的部分或完全缺失、或部分或完全失活，和/或编码微生物中D-氨基葡萄糖-6-磷酸脱氨酶基因的内源性天然启动子的部分或完全缺失、或部分或完全失活；更优选，降低微生物中D-氨基葡萄糖-6-磷酸脱氨酶作用的遗传修饰为编码微生物中D-氨基葡萄糖-6-磷酸脱氨酶基因的内源性天然启动子完全缺失，即被删除。
  17. 如权利要求5所述的方法，其中，提高微生物中UDP-N-乙酰-D-氨基葡萄糖-2-异构酶作用的遗传修饰选自a)微生物中UDP-N-乙酰-D-氨基葡萄糖-2-异构酶的酶活性增加；和/或b)微生物中UDP-N-乙酰-D-氨基葡萄糖-2-异构酶被过量表达；
    优选，微生物用至少一种包含至少一种能提高微生物中UDP-N-乙酰-D-氨基葡萄糖-2-异构酶作用的遗传修饰的重组核酸分子转化。
  18. 如权利要求17所述的方法，其中，微生物用至少一种包含编码UDP-N-乙酰-D-氨基葡萄糖-2-异构酶的核酸序列的重组核酸分子转化。
    优选，编码UDP-N-乙酰-D-氨基葡萄糖-2-异构酶的核酸序列含有至少一种增加UDP-N-乙酰-D-氨基葡萄糖-2-异构酶的酶活性的遗传修饰。优选，所述遗传修饰包括在对应于氨基酸序列SEQ ID NO：50的下述位置处的取代中的一种或多种：第34位半胱氨酸被丝氨酸取代、第145位组氨酸被天冬氨酸取代、第226位半胱氨酸被苯丙氨酸取代和245位缬氨酸被甘氨酸取代；更优选，编码UDP-N-乙酰-D-氨基葡萄糖-2-异构酶(WecB)的核酸序列为SEQ ID NO：58。
    优选，所述的UDP-N-乙酰-D-氨基葡萄糖-2-异构酶具有与SEQ ID NO：50的氨基酸序列至少约30％相同，优选至少约50％相同，进一步优选至少约70％相同，进一步优选至少约80％相同，更进一步优选至少约90％相同，最优选至少约95％相同的氨基酸序列，其中所述的UDP-N-乙酰-D-氨基葡萄糖-2-异构酶(WecB)具有酶活性；进一步优选，所述的UDP-N-乙酰-D-氨基葡萄糖-2-异构酶具有SEQ ID NO：50的氨基酸序列。
    进一步优选，重组核酸分子中编码UDP-N-乙酰-D-氨基葡萄糖-2-异构酶的基因拷贝数增加。
    进一步优选，重组核酸分子中包含内源性天然启动子或具有比内源性天然启动子更高表达水平的启动子；优选，具有比内源性天然启动子更高表达水平的启动子选自HCE启动子、gap启动子、trc启动子、T7启动子；进一步优选，具有比内源性天然启动子更高表达水平的启动子为trc启动子。
  19. 如权利要求17所述的方法，其中，微生物包括至少一种对编码UDP-N-乙酰-D-氨基葡萄糖-2-异构酶的基因的内源性天然启动子的遗传修饰；优选，编码UDP-N-乙酰-D-氨基葡萄糖-2-异构酶的基因的内源性天然启动子被具有更高表达水平的启动子替换；进一步优选，具有更高表达水平的启动子选自HCE启动子、gap启动子、trc启动子、T7启动子；最优选，具有更高表达水平的启动子为trc启动子。
  20. 如权利要求1-19任一项所述的方法，其中，所述微生物进一步包含下述遗传修饰中的一种或多种：
    (1)包含至少一种能降低微生物中甘露糖转运蛋白EIIM，P/IIIman(ManXYZ)作用的遗传修饰；
    (2)包含至少一种能降低微生物中N-乙酰神经氨酸裂解酶(NanA)作用的遗传修饰；
    (3)包含至少一种能降低微生物中N-乙酰-D-氨基葡萄糖-6-磷酸脱乙酰酶(NagA)作用的遗传修饰；
    (4)包含至少一种能降低微生物中N-乙酰-D-氨基葡萄糖特异酶IINag(NagE)作用的遗传修饰；
    (5)包含至少一种能提高微生物中磷酸葡糖胺变位酶(GlmM)作用的遗传修饰；
    (6)包含至少一种能提高微生物中双功能酶N-乙酰-D-氨基葡萄糖-1-磷酸尿苷酰转移酶(GlmU)作用的遗传修饰。
  21. 如权利要求20所述的方法，其中，微生物用至少一种包含至少一种能 降低微生物中甘露糖转运蛋白EIIM，P/IIIman(ManXYZ)作用的遗传修饰的重组核酸分子转化。
    优选，降低微生物中甘露糖转运蛋白EIIM，P/IIIman(ManXYZ)作用的遗传修饰选自编码微生物中甘露糖转运蛋白EIIM，P/IIIman(ManXYZ)的内源性基因的部分或完全缺失、或部分或完全失活，和/或编码微生物中甘露糖转运蛋白EIIM，P/IIIman(ManXYZ)基因的内源性天然启动子的部分或完全缺失、或部分或完全失活；更优选，降低微生物中甘露糖转运蛋白EIIM，P/IIIman(ManXYZ)作用的遗传修饰为编码微生物中甘露糖转运蛋白EIIM，P/IIIman(ManXYZ)的内源性基因完全缺失，即被删除。
  22. 如权利要求20所述的方法，其中，微生物用至少一种包含至少一种能降低微生物中N-乙酰神经氨酸裂解酶(NanA)作用的遗传修饰的重组核酸分子转化。
    优选，降低微生物中N-乙酰神经氨酸裂解酶(NanA)作用的遗传修饰选自编码微生物中N-乙酰神经氨酸裂解酶(NanA)的内源性基因的部分或完全缺失、或部分或完全失活，和/或编码微生物中N-乙酰神经氨酸裂解酶(NanA)基因的内源性天然启动子的部分或完全缺失、或部分或完全失活；更优选，降低微生物中N-乙酰神经氨酸裂解酶(NanA)作用的遗传修饰为编码微生物中N-乙酰神经氨酸裂解酶(NanA)的内源性基因完全缺失，即被删除。
  23. 如权利要求20所述的方法，其中，微生物用至少一种包含至少一种能降低微生物中N-乙酰-D-氨基葡萄糖-6-磷酸脱乙酰酶(NagA)作用的遗传修饰的重组核酸分子转化。
    优选，降低微生物中N-乙酰-D-氨基葡萄糖-6-磷酸脱乙酰酶(NagA)作用的遗传修饰选自编码微生物中N-乙酰-D-氨基葡萄糖-6-磷酸脱乙酰酶(NagA)的内源性基因的部分或完全缺失、或部分或完全失活，和/或编码微生物中N-乙酰-D-氨基葡萄糖-6-磷酸脱乙酰酶(NagA)基因的内源性天然启动子的部分或完全缺失、或部分或完全失活；更优选，降低微生物中N-乙酰-D-氨基葡萄糖-6-磷酸脱乙酰酶(NagA)作用的遗传修饰为编码微生物中N-乙酰-D-氨基葡萄糖-6-磷酸脱乙酰酶(NagA)的内源性基因完全缺失，即被删除。
  24. 如权利要求20所述的方法，其中，微生物用至少一种包含至少一种能降低微生物中N-乙酰-D-氨基葡萄糖特异酶IINag(NagE)作用的遗传修饰的重组核酸分子转化。
    优选，降低微生物中N-乙酰-D-氨基葡萄糖特异酶IINag(NagE)作用的遗传修饰选自编码微生物中N-乙酰-D-氨基葡萄糖特异酶IINag(NagE)的内源性基因的部分或完全缺失、或部分或完全失活，和/或编码微生物中N-乙酰-D-氨基葡萄糖特异酶IINag(NagE)基因的内源性天然启动子的部分或完全缺失、或部分或完全失活；更优选，降低微生物中N-乙酰-D-氨基葡萄糖特异酶IINag(NagE)作用的遗传修饰为编码微生物中N-乙酰-D-氨基葡萄糖特异酶IINag(NagE)的内源性基因完全缺失，即被删除。
  25. 如权利要求20所述的方法，其中，提高微生物中磷酸葡糖胺变位酶(GlmM)作用的遗传修饰选自a)微生物中磷酸葡糖胺变位酶(GlmM)的酶活性增加；和/或b)微生物中磷酸葡糖胺变位酶(GlmM)被过量表达；
    优选，微生物用至少一种包含至少一种能提高微生物中磷酸葡糖胺变位酶(GlmM)作用的遗传修饰的重组核酸分子转化。
  26. 如权利要求25所述的方法，其中，微生物用至少一种包含编码磷酸葡糖胺变位酶(GlmM)的核酸序列的重组核酸分子转化。
    优选，编码磷酸葡糖胺变位酶(GlmM)的核酸序列含有至少一种增加磷酸葡糖胺变位酶(GlmM)的酶活性的遗传修饰。
    进一步优选，重组核酸分子中编码磷酸葡糖胺变位酶(GlmM)的基因拷贝数增加。
    进一步优选，重组核酸分子中包含内源性天然启动子或具有比内源性天然启动子更高表达水平的启动子；优选，具有比内源性天然启动子更高表达水平的启动子选自HCE启动子、gap启动子、trc启动子、T7启动子；进一步优选，具有比内源性天然启动子更高表达水平的启动子为trc启动子。
  27. 如权利要求25所述的方法，其中，微生物包括至少一种对编码磷酸葡 糖胺变位酶(GlmM)的基因的内源性天然启动子的遗传修饰；优选，编码磷酸葡糖胺变位酶(GlmM)的基因的内源性天然启动子被具有更高表达水平的启动子替换；进一步优选，具有更高表达水平的启动子选自HCE启动子、gap启动子、trc启动子、T7启动子；最优选，具有更高表达水平的启动子为trc启动子。
  28. 如权利要求20所述的方法，其中，提高微生物中双功能酶N-乙酰-D-氨基葡萄糖-1-磷酸尿苷酰转移酶(GlmU)作用的遗传修饰选自a)微生物中双功能酶N-乙酰-D-氨基葡萄糖-1-磷酸尿苷酰转移酶(GlmU)的酶活性增加；和/或b)微生物中双功能酶N-乙酰-D-氨基葡萄糖-1-磷酸尿苷酰转移酶(GlmU)被过量表达；
    优选，微生物用至少一种包含至少一种能提高微生物中双功能酶N-乙酰-D-氨基葡萄糖-1-磷酸尿苷酰转移酶(GlmU)作用的遗传修饰的重组核酸分子转化。
  29. 如权利要求28所述的方法，其中，微生物用至少一种包含编码双功能酶N-乙酰-D-氨基葡萄糖-1-磷酸尿苷酰转移酶(GlmU)的核酸序列的重组核酸分子转化。
    优选，编码双功能酶N-乙酰-D-氨基葡萄糖-1-磷酸尿苷酰转移酶(GlmU)的核酸序列含有至少一种增加双功能酶N-乙酰-D-氨基葡萄糖-1-磷酸尿苷酰转移酶(GlmU)的酶活性的遗传修饰。
    进一步优选，重组核酸分子中编码双功能酶N-乙酰-D-氨基葡萄糖-1-磷酸尿苷酰转移酶(GlmU)的基因拷贝数增加。
    进一步优选，重组核酸分子中包含内源性天然启动子或具有比内源性天然启动子更高表达水平的启动子；优选，具有比内源性天然启动子更高表达水平的启动子选自HCE启动子、gap启动子、trc启动子、T7启动子；进一步优选，具有比内源性天然启动子更高表达水平的启动子为trc启动子。
  30. 如权利要求28所述的方法，其中，微生物包括至少一种对编码双功能酶N-乙酰-D-氨基葡萄糖-1-磷酸尿苷酰转移酶(GlmU)的基因的内源性天然启动子的遗传修饰；优选，编码双功能酶N-乙酰-D-氨基葡萄糖-1-磷酸尿苷酰转移酶(GlmU)的基因的内源性天然启动子被具有更高表达水平的启动子替换；进 一步优选，具有更高表达水平的启动子选自HCE启动子、gap启动子、trc启动子、T7启动子；最优选，具有更高表达水平的启动子为trc启动子。
  31. 如权利要求1-30中任一项所述的方法，其中，重组核酸分子被装入质粒中或重组核酸分子被整合到微生物的基因组中。
  32. 如权利要求1-31中任一项所述的方法，其中，所述重组核酸分子的表达是可诱导的；优选，所述重组核酸分子的表达可由乳糖诱导。
  33. 如权利要求1-32中任一项所述的方法，其中，所述培养步骤A)在约20℃-约45℃进行；优选，在约33℃-约37℃进行。
    优选，所述培养步骤A)在约pH4.5-约pH8.5进行；优选在约pH6.7-约pH7.2进行。
    优选，所述培养步骤A)采用补料发酵法。
    进一步优选，补糖液包含葡萄糖和核糖，优选，葡萄糖浓度为10％-85％(w/v)，核糖浓度为0.5％-15％(w/v)，进一步优选，葡萄糖浓度为55％-75％(w/v)，核糖浓度为5％-7％(w/v)；
    进一步优选，补糖液包含葡萄糖和葡萄糖酸盐，优选，葡萄糖浓度为10％-85％(w/v)，葡萄糖酸盐浓度为0.5％-15％(w/v)，进一步优选，葡萄糖浓度为55％-75％(w/v)，葡萄糖酸盐浓度为2％-3％(w/v)；
    进一步优选，补糖液包含葡萄糖、核糖和葡糖酸盐，优选，葡萄糖浓度为10％-85％(w/v)，核糖浓度为0.5％-15％(w/v)，葡萄糖酸盐浓度为0.5％-15％(w/v)，进一步优选，葡萄糖浓度为55％-75％(w/v)，核糖浓度为5％-7％(w/v)，葡萄糖酸盐浓度为2％-3％(w/v)。
    更优选，葡萄糖酸盐为葡萄糖酸钠。
  34. 如权利要求1-33中任一项所述的方法，其中，所述收集步骤B)包括(a)从去除微生物的发酵液中沉淀N-乙酰-D-氨基葡萄糖；和/或(b)从去除微生物的发酵液中结晶N-乙酰-D-氨基葡萄糖。
    优选，所述收集步骤B)进一步包括将发酵液脱色的步骤；进一步优选，所 述脱色步骤在对发酵液进行沉淀或结晶之前、在对发酵液进行一次或多次沉淀或结晶重溶解之后进行；更优选，所述脱色步骤包括活性炭处理和/或色谱脱色。
  35. 如权利要求1-34中任一项所述的方法，其中，所述步骤C)在酸性和加热条件下进行或在酶催化下进行。
    优选，在30％-37％盐酸溶液中、60℃-90℃下脱乙酰化水解N-乙酰-D-氨基葡萄糖得到D-氨基葡萄糖盐酸盐。
    优选，在UDP-3-O-N-乙酰葡萄糖胺脱乙酰基酶作用下水解N-乙酰-D-氨基葡萄糖得到D-氨基葡萄糖，并进一步成盐。
  36. 一种微生物，该微生物包含至少一种能提高微生物中N-乙酰-D-氨基甘露糖激酶作用的遗传修饰。
  37. 如权利要求36所述的微生物，其中，提高微生物中N-乙酰-D-氨基甘露糖激酶作用的遗传修饰选自a)微生物中N-乙酰-D-氨基甘露糖激酶的酶活性增加；和/或b)微生物中N-乙酰-D-氨基甘露糖激酶被过量表达；
    优选，微生物用至少一种包含至少一种能提高微生物中N-乙酰-D-氨基甘露糖激酶作用的遗传修饰的重组核酸分子转化。
  38. 如权利要求37所述的微生物，其中，微生物用至少一种包含编码N-乙酰-D-氨基甘露糖激酶的核酸序列的重组核酸分子转化。
    优选，编码N-乙酰-D-氨基甘露糖激酶的核酸序列含有至少一种增加N-乙酰-D-氨基甘露糖激酶的酶活性的遗传修饰；进一步优选，所述遗传修饰包括在对应于氨基酸序列SEQ ID NO：17的下述位置处的取代中的一种或多种：第36位赖氨酸被精氨酸取代、第103位异亮氨酸被蛋氨酸取代和第223位精氨酸被丝氨酸取代；更优选，编码N-乙酰-D-氨基甘露糖激酶的核酸序列为SEQ ID NO：26。
    优选，所述的N-乙酰-D-氨基甘露糖激酶具有与SEQ ID NO:17的氨基酸序列至少约30％相同，优选至少约50％相同，进一步优选至少约70％相同，进一步优选至少约80％相同，更进一步优选至少约90％相同，最优选至少约95％相 同的氨基酸序列，其中所述的N-乙酰-D-氨基甘露糖激酶具有酶活性；进一步优选，所述的N-乙酰-D-氨基甘露糖激酶具有SEQ ID NO:17的氨基酸序列。
    进一步优选，重组核酸分子中编码N-乙酰-D-氨基甘露糖激酶的基因拷贝数增加。
    进一步优选，重组核酸分子中包含内源性天然启动子或具有比内源性天然启动子更高表达水平的启动子；优选，具有比内源性天然启动子更高表达水平的启动子选自HCE启动子、gap启动子、trc启动子、T7启动子；进一步优选，具有比内源性天然启动子更高表达水平的启动子为trc启动子。
  39. 如权利要求37所述的微生物，其中，微生物包括至少一种对编码N-乙酰-D-氨基甘露糖激酶的基因的内源性天然启动子的遗传修饰；优选，编码N-乙酰-D-氨基甘露糖激酶的基因的内源性天然启动子被具有更高表达水平的启动子替换；进一步优选，具有更高表达水平的启动子选自HCE启动子、gap启动子、trc启动子、T7启动子；最优选，具有更高表达水平的启动子为trc启动子。
  40. 如权利要求36-39中任一项所述的微生物，其中，所述微生物进一步包含下述遗传修饰中的一种或多种：
    (1)包含至少一种能提高微生物中N-乙酰-D-氨基甘露糖-6-磷酸异构酶作用的遗传修饰；
    (2)包含至少一种能提高微生物中D-氨基葡萄糖-6-磷酸脱氨酶作用的遗传修饰，优选同时包含至少一种能降低氨基葡萄糖-6-磷酸合成酶作用的遗传修饰；
    (3)包含至少一种能提高微生物中氨基葡萄糖-6-磷酸合成酶作用的遗传修饰，并同时包含至少一种能降低D-氨基葡萄糖-6-磷酸脱氨酶作用的遗传修饰；
    (4)包含至少一种能提高微生物中UDP-N-乙酰-D-氨基葡萄糖-2-异构酶(WecB)作用的遗传修饰。
  41. 如权利要求40所述的微生物，其中，提高微生物中N-乙酰-D-氨基甘露糖-6-磷酸异构酶作用的遗传修饰选自a)微生物中N-乙酰-D-氨基甘露糖-6-磷酸异构酶的酶活性增加；和/或b)微生物中N-乙酰-D-氨基甘露糖-6-磷酸异构 酶被过量表达；
    优选，微生物用至少一种包含至少一种能提高微生物中N-乙酰-D-氨基甘露糖-6-磷酸异构酶作用的遗传修饰的重组核酸分子转化。
  42. 如权利要求41所述的微生物，其中，微生物用至少一种包含编码N-乙酰-D-氨基甘露糖-6-磷酸异构酶的核酸序列的重组核酸分子转化。
    优选，编码N-乙酰-D-氨基甘露糖-6-磷酸异构酶的核酸序列含有至少一种增加N-乙酰-D-氨基甘露糖-6-磷酸异构酶的酶活性的遗传修饰。优选，所述遗传修饰包括在对应于氨基酸序列SEQ ID NO：29的下述位置处的取代中的一种或两种：第133位半胱氨酸被精氨酸取代和第187位酪氨酸被组氨酸取代。进一步优选，编码所述N-乙酰-D-氨基甘露糖-6-磷酸异构酶的核酸序列为SEQ ID NO：56。
    优选，所述的N-乙酰-D-氨基甘露糖-6-磷酸异构酶具有与SEQ ID NO：29的氨基酸序列至少约30％相同，优选至少约50％相同，进一步优选至少约70％相同，进一步优选至少约80％相同，更进一步优选至少约90％相同，最优选至少约95％相同的氨基酸序列，其中所述的N-乙酰-D-氨基甘露糖-6-磷酸异构酶具有酶活性；进一步优选，所述的N-乙酰-D-氨基甘露糖-6-磷酸异构酶具有SEQ ID NO：29的氨基酸序列。
    进一步优选，重组核酸分子中编码N-乙酰-D-氨基甘露糖-6-磷酸异构酶的基因拷贝数增加。
    进一步优选，重组核酸分子中包含内源性天然启动子或具有比内源性天然启动子更高表达水平的启动子；优选，具有比内源性天然启动子更高表达水平的启动子选自HCE启动子、gap启动子、trc启动子、T7启动子；进一步优选，具有比内源性天然启动子更高表达水平的启动子为trc启动子。
  43. 如权利要求41所述的微生物，其中，微生物包括至少一种对编码N-乙酰-D-氨基甘露糖-6-磷酸异构酶的基因的内源性天然启动子的遗传修饰；优选，编码N-乙酰-D-氨基甘露糖-6-磷酸异构酶的基因的内源性天然启动子被具有更高表达水平的启动子替换；进一步优选，具有更高表达水平的启动子选自HCE启动子、gap启动子、trc启动子、T7启动子；最优选，具有更高表达水平的启 动子为trc启动子。
  44. 如权利要求40所述的微生物，其中，提高微生物中D-氨基葡萄糖-6-磷酸脱氨酶作用的遗传修饰选自a)微生物中D-氨基葡萄糖-6-磷酸脱氨酶的酶活性增加；和/或b)微生物中D-氨基葡萄糖-6-磷酸脱氨酶被过量表达；
    优选，微生物用至少一种包含至少一种能提高微生物中D-氨基葡萄糖-6-磷酸脱氨酶作用的遗传修饰的重组核酸分子转化。
  45. 如权利要求44所述的微生物，其中，微生物用至少一种包含编码D-氨基葡萄糖-6-磷酸脱氨酶的核酸序列的重组核酸分子转化。
    优选，编码D-氨基葡萄糖-6-磷酸脱氨酶的核酸序列含有至少一种增加D-氨基葡萄糖-6-磷酸脱氨酶的酶活性的遗传修饰。
    进一步优选，重组核酸分子中编码D-氨基葡萄糖-6-磷酸脱氨酶的基因拷贝数增加。
    进一步优选，重组核酸分子中包含内源性天然启动子或具有比内源性天然启动子更高表达水平的启动子；优选，具有比内源性天然启动子更高表达水平的启动子选自HCE启动子、gap启动子、trc启动子、T7启动子；进一步优选，具有比内源性天然启动子更高表达水平的启动子为trc启动子。
  46. 如权利要求44所述的微生物，其中，微生物包括至少一种对编码D-氨基葡萄糖-6-磷酸脱氨酶的基因的内源性天然启动子的遗传修饰。优选，编码D-氨基葡萄糖-6-磷酸脱氨酶的基因的内源性天然启动子被具有更高表达水平的启动子替换；进一步优选，具有更高表达水平的启动子选自HCE启动子、gap启动子、trc启动子、T7启动子；最优选，具有更高表达水平的启动子为trc启动子。
  47. 如权利要求40所述的微生物，其中，降低微生物中氨基葡萄糖-6-磷酸合成酶作用的遗传修饰选自a)微生物中氨基葡萄糖-6-磷酸合成酶的酶活性降低；和/或b)微生物中氨基葡萄糖-6-磷酸合成酶的表达减少；
    优选，微生物用至少一种包含至少一种能降低微生物中氨基葡萄糖-6-磷酸 合成酶作用的遗传修饰的重组核酸分子转化。
    进一步优选，降低微生物中氨基葡萄糖-6-磷酸合成酶作用的遗传修饰选自编码微生物中氨基葡萄糖-6-磷酸合成酶的内源性基因的部分或完全缺失、或部分或完全失活，和/或编码微生物中氨基葡萄糖-6-磷酸合成酶基因的内源性天然启动子的部分或完全缺失、或部分或完全失活；更优选，降低微生物中氨基葡萄糖-6-磷酸合成酶作用的遗传修饰为编码微生物中氨基葡萄糖-6-磷酸合成酶基因的内源性天然启动子完全缺失，即被删除。
  48. 如权利要求40所述的微生物，其中，提高微生物中氨基葡萄糖-6-磷酸合成酶作用的遗传修饰选自a)微生物中氨基葡萄糖-6-磷酸合成酶的酶活性增加；和/或b)微生物中氨基葡萄糖-6-磷酸合成酶被过量表达；
    优选，微生物用至少一种包含至少一种能提高微生物中氨基葡萄糖-6-磷酸合成酶作用的遗传修饰的重组核酸分子转化。
  49. 如权利要求48所述的微生物，其中，微生物用至少一种包含编码氨基葡萄糖-6-磷酸合成酶的核酸序列的重组核酸分子转化。
    优选，编码氨基葡萄糖-6-磷酸合成酶的核酸序列含有至少一种增加氨基葡萄糖-6-磷酸合成酶的酶活性的遗传修饰。
    进一步优选，重组核酸分子中编码氨基葡萄糖-6-磷酸合成酶的基因拷贝数增加。
    进一步优选，重组核酸分子中包含内源性天然启动子或具有比内源性天然启动子更高表达水平的启动子；优选，具有比内源性天然启动子更高表达水平的启动子选自HCE启动子、gap启动子、trc启动子、T7启动子；进一步优选，具有比内源性天然启动子更高表达水平的启动子为trc启动子。
  50. 如权利要求48所述的微生物，其中，微生物包括至少一种对编码氨基葡萄糖-6-磷酸合成酶的基因的内源性天然启动子的遗传修饰。优选，编码氨基葡萄糖-6-磷酸合成酶的基因的内源性天然启动子被具有更高表达水平的启动子替换；进一步优选，具有更高表达水平的启动子选自HCE启动子、gap启动子、trc启动子、T7启动子；最优选，具有更高表达水平的启动子为trc启动子。
  51. 如权利要求40所述的微生物，其中，降低微生物中D-氨基葡萄糖-6-磷酸脱氨酶作用的遗传修饰选自a)微生物中D-氨基葡萄糖-6-磷酸脱氨酶的酶活性降低；和/或b)微生物中D-氨基葡萄糖-6-磷酸脱氨酶的表达减少；
    优选，微生物用至少一种包含至少一种能降低微生物中D-氨基葡萄糖-6-磷酸脱氨酶作用的遗传修饰的重组核酸分子转化。
    进一步优选，降低微生物中D-氨基葡萄糖-6-磷酸脱氨酶作用的遗传修饰选自编码微生物中D-氨基葡萄糖-6-磷酸脱氨酶的内源性基因的部分或完全缺失、或部分或完全失活，和/或编码微生物中D-氨基葡萄糖-6-磷酸脱氨酶基因的内源性天然启动子的部分或完全缺失、或部分或完全失活；更优选，降低微生物中D-氨基葡萄糖-6-磷酸脱氨酶作用的遗传修饰为编码微生物中D-氨基葡萄糖-6-磷酸脱氨酶基因的内源性天然启动子完全缺失，即被删除。
  52. 如权利要求40所述的微生物，其中，提高微生物中UDP-N-乙酰-D-氨基葡萄糖-2-异构酶作用的遗传修饰选自a)微生物中UDP-N-乙酰-D-氨基葡萄糖-2-异构酶的酶活性增加；和/或b)微生物中UDP-N-乙酰-D-氨基葡萄糖-2-异构酶被过量表达；
    优选，微生物用至少一种包含至少一种能提高微生物中UDP-N-乙酰-D-氨基葡萄糖-2-异构酶作用的遗传修饰的重组核酸分子转化。
  53. 如权利要求52所述的微生物，其中，微生物用至少一种包含编码UDP-N-乙酰-D-氨基葡萄糖-2-异构酶的核酸序列的重组核酸分子转化。
    优选，编码UDP-N-乙酰-D-氨基葡萄糖-2-异构酶的核酸序列含有至少一种增加UDP-N-乙酰-D-氨基葡萄糖-2-异构酶的酶活性的遗传修饰。优选，所述遗传修饰包括在对应于氨基酸序列SEQ ID NO：50的下述位置处的取代中的一种或多种：第34位半胱氨酸被丝氨酸取代、第145位组氨酸被天冬氨酸取代、第226位半胱氨酸被苯丙氨酸取代和245位缬氨酸被甘氨酸取代；更优选，编码UDP-N-乙酰-D-氨基葡萄糖-2-异构酶(WecB)的核酸序列为SEQ ID NO：58。
    优选，所述的UDP-N-乙酰-D-氨基葡萄糖-2-异构酶具有与SEQ ID NO：50的氨基酸序列至少约30％相同，优选至少约50％相同，进一步优选至少约70％ 相同，进一步优选至少约80％相同，更进一步优选至少约90％相同，最优选至少约95％相同的氨基酸序列，其中所述的UDP-N-乙酰-D-氨基葡萄糖-2-异构酶(WecB)具有酶活性；进一步优选，所述的UDP-N-乙酰-D-氨基葡萄糖-2-异构酶具有SEQ ID NO：50的氨基酸序列。
    进一步优选，重组核酸分子中编码UDP-N-乙酰-D-氨基葡萄糖-2-异构酶的基因拷贝数增加。
    进一步优选，重组核酸分子中包含内源性天然启动子或具有比内源性天然启动子更高表达水平的启动子；优选，具有比内源性天然启动子更高表达水平的启动子选自HCE启动子、gap启动子、trc启动子、T7启动子；进一步优选，具有比内源性天然启动子更高表达水平的启动子为trc启动子。
  54. 如权利要求52所述的微生物，其中，微生物包括至少一种对编码UDP-N-乙酰-D-氨基葡萄糖-2-异构酶的基因的内源性天然启动子的遗传修饰；优选，编码UDP-N-乙酰-D-氨基葡萄糖-2-异构酶的基因的内源性天然启动子被具有更高表达水平的启动子替换；进一步优选，具有更高表达水平的启动子选自HCE启动子、gap启动子、trc启动子、T7启动子；最优选，具有更高表达水平的启动子为trc启动子。
  55. 如权利要求36-54任一项所述的微生物，其中，所述微生物进一步包含下述遗传修饰中的一种或多种：
    (1)包含至少一种能降低微生物中甘露糖转运蛋白EIIM，P/IIIman(ManXYZ)作用的遗传修饰；
    (2)包含至少一种能降低微生物中N-乙酰神经氨酸裂解酶(NanA)作用的遗传修饰；
    (3)包含至少一种能降低微生物中N-乙酰-D-氨基葡萄糖-6-磷酸脱乙酰酶(NagA)作用的遗传修饰；
    (4)包含至少一种能降低微生物中N-乙酰-D-氨基葡萄糖特异酶IINag(NagE)作用的遗传修饰；
    (5)包含至少一种能提高微生物中磷酸葡糖胺变位酶(GlmM)作用的遗传修饰；
    (6)包含至少一种能提高微生物中双功能酶N-乙酰-D-氨基葡萄糖-1-磷酸尿苷酰转移酶(GlmU)作用的遗传修饰。
  56. 如权利要求55所述的微生物，其中，微生物用至少一种包含至少一种能降低微生物中甘露糖转运蛋白EIIM，P/IIIman(ManXYZ)作用的遗传修饰的重组核酸分子转化。
    优选，降低微生物中甘露糖转运蛋白EIIM，P/IIIman(ManXYZ)作用的遗传修饰选自编码微生物中甘露糖转运蛋白EIIM，P/IIIman(ManXYZ)的内源性基因的部分或完全缺失、或部分或完全失活，和/或编码微生物中甘露糖转运蛋白EIIM，P/IIIman(ManXYZ)基因的内源性天然启动子的部分或完全缺失、或部分或完全失活；更优选，降低微生物中甘露糖转运蛋白EIIM，P/IIIman(ManXYZ)作用的遗传修饰为编码微生物中甘露糖转运蛋白EIIM，P/IIIman(ManXYZ)的内源性基因完全缺失，即被删除。
  57. 如权利要求55所述的微生物，其中，微生物用至少一种包含至少一种能降低微生物中N-乙酰神经氨酸裂解酶(NanA)作用的遗传修饰的重组核酸分子转化。
    优选，降低微生物中N-乙酰神经氨酸裂解酶(NanA)作用的遗传修饰选自编码微生物中N-乙酰神经氨酸裂解酶(NanA)的内源性基因的部分或完全缺失、或部分或完全失活，和/或编码微生物中N-乙酰神经氨酸裂解酶(NanA)基因的内源性天然启动子的部分或完全缺失、或部分或完全失活；更优选，降低微生物中N-乙酰神经氨酸裂解酶(NanA)作用的遗传修饰为编码微生物中N-乙酰神经氨酸裂解酶(NanA)的内源性基因完全缺失，即被删除。
  58. 如权利要求55所述的微生物，其中，微生物用至少一种包含至少一种能降低微生物中N-乙酰-D-氨基葡萄糖-6-磷酸脱乙酰酶(NagA)作用的遗传修饰的重组核酸分子转化。
    优选，降低微生物中N-乙酰-D-氨基葡萄糖-6-磷酸脱乙酰酶(NagA)作用的遗传修饰选自编码微生物中N-乙酰-D-氨基葡萄糖-6-磷酸脱乙酰酶(NagA)的内源性基因的部分或完全缺失、或部分或完全失活，和/或编码微生物中N-乙 酰-D-氨基葡萄糖-6-磷酸脱乙酰酶(NagA)基因的内源性天然启动子的部分或完全缺失、或部分或完全失活；更优选，降低微生物中N-乙酰-D-氨基葡萄糖-6-磷酸脱乙酰酶(NagA)作用的遗传修饰为编码微生物中N-乙酰-D-氨基葡萄糖-6-磷酸脱乙酰酶(NagA)的内源性基因完全缺失，即被删除。
  59. 如权利要求55所述的微生物，其中，微生物用至少一种包含至少一种能降低微生物中N-乙酰-D-氨基葡萄糖特异酶IINag(NagE)作用的遗传修饰的重组核酸分子转化。
    优选，降低微生物中N-乙酰-D-氨基葡萄糖特异酶IINag(NagE)作用的遗传修饰选自编码微生物中N-乙酰-D-氨基葡萄糖特异酶IINag(NagE)的内源性基因的部分或完全缺失、或部分或完全失活，和/或编码微生物中N-乙酰-D-氨基葡萄糖特异酶IINag(NagE)基因的内源性天然启动子的部分或完全缺失、或部分或完全失活；更优选，降低微生物中N-乙酰-D-氨基葡萄糖特异酶IINag(NagE)作用的遗传修饰为编码微生物中N-乙酰-D-氨基葡萄糖特异酶IINag(NagE)的内源性基因完全缺失，即被删除。
  60. 如权利要求55所述的微生物，其中，提高微生物中磷酸葡糖胺变位酶(GlmM)作用的遗传修饰选自a)微生物中磷酸葡糖胺变位酶(GlmM)的酶活性增加；和/或b)微生物中磷酸葡糖胺变位酶(GlmM)被过量表达；
    优选，微生物用至少一种包含至少一种能提高微生物中磷酸葡糖胺变位酶(GlmM)作用的遗传修饰的重组核酸分子转化。
  61. 如权利要求60所述的微生物，其中，微生物用至少一种包含编码磷酸葡糖胺变位酶(GlmM)的核酸序列的重组核酸分子转化。
    优选，编码磷酸葡糖胺变位酶(GlmM)的核酸序列含有至少一种增加磷酸葡糖胺变位酶(GlmM)的酶活性的遗传修饰。
    进一步优选，重组核酸分子中编码磷酸葡糖胺变位酶(GlmM)的基因拷贝数增加。
    进一步优选，重组核酸分子中包含内源性天然启动子或具有比内源性天然启动子更高表达水平的启动子；优选，具有比内源性天然启动子更高表达水平 的启动子选自HCE启动子、gap启动子、trc启动子、T7启动子；进一步优选，具有比内源性天然启动子更高表达水平的启动子为trc启动子。
  62. 如权利要求60所述的微生物，其中，微生物包括至少一种对编码磷酸葡糖胺变位酶(GlmM)的基因的内源性天然启动子的遗传修饰；优选，编码磷酸葡糖胺变位酶(GlmM)的基因的内源性天然启动子被具有更高表达水平的启动子替换；进一步优选，具有更高表达水平的启动子选自HCE启动子、gap启动子、trc启动子、T7启动子；最优选，具有更高表达水平的启动子为trc启动子。
  63. 如权利要求55所述的微生物，其中，提高微生物中双功能酶N-乙酰-D-氨基葡萄糖-1-磷酸尿苷酰转移酶(GlmU)作用的遗传修饰选自a)微生物中双功能酶N-乙酰-D-氨基葡萄糖-1-磷酸尿苷酰转移酶(GlmU)的酶活性增加；和/或b)微生物中双功能酶N-乙酰-D-氨基葡萄糖-1-磷酸尿苷酰转移酶(GlmU)被过量表达；
    优选，微生物用至少一种包含至少一种能提高微生物中双功能酶N-乙酰-D-氨基葡萄糖-1-磷酸尿苷酰转移酶(GlmU)作用的遗传修饰的重组核酸分子转化。
  64. 如权利要求63所述的微生物，其中，微生物用至少一种包含编码双功能酶N-乙酰-D-氨基葡萄糖-1-磷酸尿苷酰转移酶(GlmU)的核酸序列的重组核酸分子转化。
    优选，编码双功能酶N-乙酰-D-氨基葡萄糖-1-磷酸尿苷酰转移酶(GlmU)的核酸序列含有至少一种增加双功能酶N-乙酰-D-氨基葡萄糖-1-磷酸尿苷酰转移酶(GlmU)的酶活性的遗传修饰。
    进一步优选，重组核酸分子中编码双功能酶N-乙酰-D-氨基葡萄糖-1-磷酸尿苷酰转移酶(GlmU)的基因拷贝数增加。
    进一步优选，重组核酸分子中包含内源性天然启动子或具有比内源性天然启动子更高表达水平的启动子；优选，具有比内源性天然启动子更高表达水平的启动子选自HCE启动子、gap启动子、trc启动子、T7启动子；进一步优选，具有比内源性天然启动子更高表达水平的启动子为trc启动子。
  65. 如权利要求63所述的微生物，其中，微生物包括至少一种对编码双功能酶N-乙酰-D-氨基葡萄糖-1-磷酸尿苷酰转移酶(GlmU)的基因的内源性天然启动子的遗传修饰；优选，编码双功能酶N-乙酰-D-氨基葡萄糖-1-磷酸尿苷酰转移酶(GlmU)的基因的内源性天然启动子被具有更高表达水平的启动子替换；进一步优选，具有更高表达水平的启动子选自HCE启动子、gap启动子、trc启动子、T7启动子；最优选，具有更高表达水平的启动子为trc启动子。
  66. 如权利要求36-65中任一项所述的微生物，其中，重组核酸分子被装入质粒中或重组核酸分子被整合到微生物的基因组中。
  67. 如权利要求36-66中任一项所述的微生物，其中，所述重组核酸分子的表达是可诱导的；优选，所述重组核酸分子的表达可由乳糖诱导。
  68. 如权利要求1-35中任一项所述的方法或者如权利要求36-67中任一项所述的微生物，其中，所述微生物微生物为细菌、酵母或真菌。
    优选，所述的微生物选自细菌或酵母。
    进一步优选，细菌选自埃希氏菌属(Escherichia)、芽孢杆菌属(Bacillus)、乳杆菌属(Lactobacillus)、假单胞菌属(Pseudomonas)或链霉菌属(Streptomyces)的属的细菌；进一步优选，细菌选自大肠杆菌(Escherichia coli)、枯草芽孢杆菌(Bacillus subtilis)、地衣芽孢杆菌(Bacillus licheniformis)、短乳杆菌(Lactobacillus brevis)、铜绿假单胞菌(Pseudomonas aeruginosa)或浅青紫链霉菌(Streptomyces lividans)的种的细菌；更优选，细菌为大肠杆菌；更进一步优选，大肠杆菌选自K-12、B和W菌株；最优选大肠杆菌为K-12菌株。
    进一步优选，酵母选自糖酵母属(Saccharomyces)、裂殖糖酵母属(Schizosaccharomyces)、念珠菌属(Candida)、汉逊酵母属(Hansenula)、毕赤酵母属(Pichia)、克鲁维酵母属(Kluveromyces)和红法夫属(Phaffia)的酵母；更优选，酵母包括但不限于选自酿酒酵母(Saccharomyce scerevisiae)、粟酒裂殖酵母(Schizosaccharo mycespombe)、白色念珠菌(Candida albicans)、多形汉逊酵母(Hansenulapolymorpha)、巴氏毕赤酵母(Pichia pastoris)、加拿大毕赤酵母(Pichia  canadensis)、马克斯克鲁维酵母(Kluyveromyces marxianus)或红法夫酵母(Phaffia rohodozyma)。
    优选，所述的微生物为真菌；进一步优选，真菌选自曲霉属(Aspergillus)、犁头霉属(Absidia)、根霉属(Rhizopus)、金孢子菌属(Chrysosporium)、脉孢霉属(Neurospora)或木霉属(Trichoderma)的属的真菌；更优选，真菌选自黑曲霉(Aspergillus niger)、构巢曲霉(Aspergillus nidulans)、蓝色犁头霉(Absidia coerulea)、米根霉(Rhizopus oryzae)、劳肯诺温斯金孢子菌(Chrysosporium lucknowense)、粗糙脉孢霉(Neurospora crassa)、间型脉孢霉(Neurospora intermedia)或里氏木霉(Trichoderma reesei)。
  69. 一种具有更高酶活性的N-乙酰-D-氨基甘露糖激酶(NanK)，所述酶具有SEQ ID NO：27所示的氨基酸序列。
  70. 一种编码如权利要求69所述N-乙酰-D-氨基甘露糖激酶(NanK)的核酸分子，所述核酸分子具有SEQ ID NO：26所示的核酸序列。
  71. 一种包含如权利要求70所述核酸分子的载体。
  72. 一种包含如权利要求71所述载体的微生物。
  73. 一种基因组中包含如权利要求70所述核酸分子的微生物。
PCT/CN2017/080653 2016-04-05 2017-04-14 微生物发酵生产n-乙酰-d-氨基葡萄糖和/或d-氨基葡萄糖盐的方法 WO2017174040A1 (zh)

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