WO2017174037A1 - 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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WO2017174037A1
WO2017174037A1 PCT/CN2017/080650 CN2017080650W WO2017174037A1 WO 2017174037 A1 WO2017174037 A1 WO 2017174037A1 CN 2017080650 W CN2017080650 W CN 2017080650W WO 2017174037 A1 WO2017174037 A1 WO 2017174037A1
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microorganism
glucosamine
promoter
phosphate
acetyl
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PCT/CN2017/080650
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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 CN201710217589.4A external-priority patent/CN107267577A/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
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • 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
    • 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 preferably simultaneously reduces glucosamine-6-phosphate synthase by increasing the action of D-Glucosamine-6-phosphate deaminase (NagB) in a microorganism.
  • D-Glucosamine-6-phosphate deaminase NagB
  • GlmS also known as L-glutamine-6-phosphate fructosamine aminotransferase
  • Glucose-6-phosphate, Glc-6-P strengthens glucose-6-phosphate in microorganisms
  • Glc-6-P is aminated to D-glucosamine-6-phosphate (GlcN-6-P).
  • the present invention enhances the glucosamine-6-phosphate synthase (GlmS, also known as L-glutamine-6-phosphate fructose aminotransferase, L-glutamine: D-fructose) -6-phosphate aminotransferase), and simultaneously reduce the role of D-Glucosamine-6-phosphate deaminase (NagB), strengthen the glucose-6-phosphate (Glucose-6) -phosphate, Glc-6-P) is aminated to D-glucosamine-6-phosphate (GlcN-6-P).
  • GlmS also known as L-glutamine-6-phosphate fructose aminotransferase, L-glutamine: D-fructose
  • GlcN-6-P Decreasing the effect of NagB, preventing the NagB catalytic reaction from proceeding to the Glc-6-P production by GlcN-6-P, and simultaneously overexpressing GlmS, accelerating GlmS-catalyzed Glc-6-P amination to GlcN-6-P,
  • the purpose of GlcN-6-P is increased to allow the microorganism to produce N-acetyl-D-glucosamine (GlcNAc) and/or D-glucosamine salt with higher efficiency and higher yield.
  • the present invention further enhances the sugar conversion rate and the N-acetyl-D-amino group in the microorganism by reducing the action of an enzyme or a protein in the microorganism which is associated with the degradation of the target product into the cell or the degradation of the beneficial intermediate product.
  • Glucose production thereby allowing the microorganism to produce N-acetyl-D-glucosamine (GlcNAc) and/or D-glucosamine salt with higher efficiency and higher yield.
  • GlcNAc N-acetyl-D-glucosamine
  • D-glucosamine salt Including but not limited to one or more of the following:
  • 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
  • N-acetyl-D-glucosamine-1-phosphate uridine transferase bifunctional N-acetyl glucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyl transferase, GlmU
  • GlcN-1-P D-glucosamine-1-phosphate
  • GlcNAc-1-P UDP-N-acetyl-D-glucosamine
  • 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 D-glucosamine-6-phosphate deaminase (NagB) in a microorganism is selected from a) an increase in the enzyme activity of D-glucosamine-6-phosphate deaminase (NagB) in the microorganism. 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 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 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 present invention relates to a method for producing 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 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) 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 natural promoter of the gene encoding glucosamine-6-phosphate synthase (GlmS) is of higher expression level Promoter substitution, such as HCE promoter, gap promoter, trc promoter, T7 promoter, etc.; more preferably, the endogenous natural promoter of the gene encoding glucosamine-6-phosphate synthase (GlmS) is trc promoter replace.
  • 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 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 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 of the glucosamine phosphatidamine Genetic modification of the endogenous natural promoter of the enzyme (GlmM) gene.
  • 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. More preferably, 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 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 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 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 comprises 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); another according to the present invention
  • the sugar-supplementing liquid comprises glucose, ribose and gluconate, preferably, the glucose concentration is 10%-85% (w/v), the ribose concentration is 0.5%-15% (w/v), and the gluconate concentration is 0.
  • 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. In one aspect, 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 comprises the step of contacting the fermentation broth with an ion exchange resin, including but 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 D-glucosamine-6-phosphate deaminase (NagB) in a microorganism, preferably comprising at least A genetic modification that reduces the action of glucosamine-6-phosphate synthase (GlmS).
  • This genetic modification has been described in detail above.
  • the present invention relates to a microorganism comprising at least one genetic modification capable of enhancing the action of glucosamine-6-phosphate synthase (GlmS) in a microorganism, and comprising at least one capable Genetic modification to reduce the effects of D-glucosamine-6-phosphate deaminase (NagB).
  • GlmS glucosamine-6-phosphate synthase
  • NagB D-glucosamine-6-phosphate deaminase
  • 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 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 the species of Streptomyces lividans bacteria.
  • 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 decreased and/or the expression of the enzyme is The reduction, 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 NagB, GlmS, or a combination thereof, and may further include ManXYZ, NanA, NagA, NagE, GlmM, GlmU or Combination modification.
  • 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 alternative names for many of the enzymes referred to herein and specific gene names from some organisms encoding such enzymes. 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-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.
  • 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-amino group from various organisms Glucose-2-isomerases are well known in the art and can be used in the genetic engineering strategies of the present invention.
  • 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 diphosphate
  • UDP-N-acetyl-D-glucosamine pyrophosphorylase UDP- N-acetyl-D-glucosamine diphosphate
  • the enzyme is further converted to 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: 16.
  • 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 in part due to D-glucosamine degradation products in the medium with a starting pH of 7.0. 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.
  • Glc-6-P becomes D-glucosamine-6-P (GlcN-6-P) catalyzed by NagB and GlmS in cells, and further catalyzes the formation of UDP-N-acetyl group in GlmM and GlmU.
  • UDP-GlcNAc N-acetyl-D-aminomannose
  • ManNAc N-acetyl-D-aminomannose
  • WecB UDP-N-acetyl-glucosamine-2-isomerase
  • GlcNAc-6-P N-acetyl-D-glucosamine-6-phosphate
  • GlcNAc-6-P N-acetyl-D-glucosamine-6-phosphate
  • 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 is AT-001 (Escherichia coli ATCC 27325), belonging to 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, a linear DNA fragment for targeting was prepared by electroporation, and positive clones were selected. 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 microstimulation N-acetyl-D-aminomannose (ManNAc) in the product is degraded 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, 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 (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. Inoculation with cyclosporine on antibiotic-free medium The plated, picked-up monoclonal spots were added to the caramycin resistant plates, and the ungrown clones 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 replacement of the endogenous natural promoter of nagB gene into the Trc promoter, and further deletion of the endogenous natural promoter of glmS gene, which has an effect on the yield of N-acetyl-D-glucosamine.
  • 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.
  • Trc promoter sequence was found: SEQ ID No. 16.
  • 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.
  • Designing the homology arm primer Designing the forward primer (nagBKO-F1) SEQ ID of the homologous arm of the nagB promoter according to NCBI search NC_000913, Escherichia coli str.K-12nagB promoter sequence and nagE gene sequence SEQ ID No.13 No. 21, reverse primer (nagBKO-R1) SEQ ID No. 22.
  • 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-005-02 strain. Then, a linear DNA fragment for targeting was prepared by electroporation, and positive clones were selected. Finally, the resistance gene is eliminated.
  • AT-048 (AT-005-02, ⁇ nagB promotor::Trc promoter).
  • Glucosamine-6-phosphate synthase 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 E. coli AT-048 strain. Then, a linear DNA fragment for targeting was prepared by electroporation, and positive clones were selected. Finally, the resistance gene is eliminated.
  • AT-049 AT-048, ⁇ glmS promotor
  • a shake flask fermentation test was performed on a strain in which the nagB promoter was replaced with a promoter of a higher expression level, and a recombinant strain in which the glmS promoter was further deleted.
  • 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.
  • 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.
  • This example describes the replacement of the endogenous native promoter of the glmS gene into the Trc promoter, and further deletion of the endogenous natural promoter of the nagB gene, which has an effect on the yield of N-acetyl-D-glucosamine.
  • 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. 26, reverse primer (ProglmspTrc-R), which was replaced with the Trc promoter according to the glmS gene promoter sequence SEQ ID No. 23. SEQ ID No. 27.
  • 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 to obtain 100 ng/ ⁇ l of linear DNA. Full length PCR fragments were used for Red recombination targeting.
  • the pKD46 vector was transferred into the E. coli AT-005-02 strain. Then, a linear DNA fragment for targeting was prepared by electroporation, and positive clones were selected. Finally, the resistance gene is eliminated.
  • AT-050 AT-005-02, ⁇ 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 the homology arm primer Designing the homologous arm forward primer (NagBKO-F2) SEQ ID No. 28, reverse primer (NagBKO-) to delete the nagB promoter sequence according to the nagB promoter and the nagE gene sequence SEQ ID No. R2) SEQ ID No. 29.
  • 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-050 strain. Then, a linear DNA fragment for targeting was prepared by electroporation, and positive clones were selected. Finally, the resistance gene is eliminated.
  • the obtained strain number AT-051 (AT-050, ⁇ nagB promotor).
  • 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. At 24 hours and 48 hours, the pH of the fermentation broth was adjusted to 7.0 with 10 M NaOH. According to the sugar consumption of the fermentation broth, 65% glucose solution was added in portions to maintain the glucose concentration at 20 g/L. At the end of the fermentation, 1 ml of the fermentation broth was taken and centrifuged. The N-acetyl-D-glucosamine content was determined by HPLC.
  • This example describes the production of N-acetyl-D-glucosamine fermentation in a 10 L fermentor.
  • the recombinant engineering strain AT-049 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 5, wherein the trace element solution was prepared according to Table 6, and the multivitamin solution was prepared according to Table 7.
  • 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;
  • the fermentation medium is the basal medium before the addition of glucose, and the initial liquid volume of the basal medium (the initial volume of the medium accounts for the total volume of the fermenter): 50%.
  • 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.

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Abstract

本发明提供了一种通过微生物发酵生产N-乙酰-D-氨基葡萄糖和D-氨基葡萄糖盐的方法。所述方法通过提高微生物中D-氨基葡萄糖-6-磷酸脱氨酶（NagB）的作用，同时降低氨基葡萄糖-6-磷酸合成酶（GlmS）的作用，或者提高微生物中GlmS的作用，同时降低NagB的作用，以加强N-乙酰-D-氨基葡萄糖和/或D-氨基葡萄糖盐的生产。

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，一方面，本发明通过提高微生物中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。
另一方面，本发明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。
本发明在上述内容的基础上还通过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；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。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。
根据本发明的另一个实施方案，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；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。
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。
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。在一个方面中，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。上文已经对这一遗传修饰进行了详细描述。
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。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。
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NagB、GlmS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。
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。
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。
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：16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Glc-6-P在NagB和GlmS催化下变成D-氨基葡萄糖-6-P(GlcN-6-P)，并进一步在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)，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 PCTCN2017080650-appb-000001
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)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.16。
设计引物：正向引物(KanTrcRed-F)SEQ ID No.17，反向引物(KanTrcRed-R)SEQ ID No.18。
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.19，反向引物(fKanfRed-R1)SEQ ID No.20。
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.21，反向引物(nagBKO-R1)SEQ ID No.22。
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-005-02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-048(AT-005-02，△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.23，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.24，反向引物(ProglmsKO-R)SEQ ID No.25。
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-048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-049(AT-048，△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
对将nagB启动子更换为更高表达水平的启动子的菌株，以及进一步删除glmS启动子的重组菌株，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。
(1)N-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
(2)M9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 PCTCN2017080650-appb-000002
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
(3)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：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3.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 PCTCN2017080650-appb-000003
实施例3
本实施例描述将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.23，设计更换为Trc启动子的同源臂正向引物(ProglmspTrc-F)SEQ ID No.26，反向引物(ProglmspTrc-R)SEQ ID No.27。
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-005-02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-050(AT-005-02，△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.28，反向引物(NagBKO-R2)SEQ ID No.29。
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-050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-051(AT-050，△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
对将glmS启动子更换为更高表达水平的启动子的菌株，以及进一步删除 nagB启动子的重组菌株，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：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，未检出产量。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4.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 PCTCN2017080650-appb-000004
实施例4
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-049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5配制，其中，微量元素溶液按表6配制，复合维生素溶液按表7配制。
表5.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6.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7.复合维生素溶液
Figure PCTCN2017080650-appb-000005
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-049。批号：0070-2。
(2)种子液浓度：OD600为2.9。
(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109.0g/L。
实施例5
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91.3％。
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89.9％。
(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：99.95％。总产率为84.2％。
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 (50)

  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；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。
  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。
  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。
  5. 如权利要求1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。
  6. 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；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。
  7. 如权利要求6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。
  8. 如权利要求7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。
  9. 如权利要求7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。
  10. 如权利要求6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。
  11. 如权利要求1-10一项所述的方法，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。
  12. 如权利要求11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。
  13. 如权利要求11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。
  14. 如权利要求11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。
  15. 如权利要求11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。
  16. 如权利要求11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。
  17. 如权利要求16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。
  18. 如权利要求16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。
  19. 如权利要求11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。
  20. 如权利要求19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。
  21. 如权利要求19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。
  22. 如权利要求1-21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。
  23. 如权利要求1-22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。
  24. 如权利要求1-23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。
  25. 如权利要求1-24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。
  26. 如权利要求1-25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。
  27. 一种微生物，该微生物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。
  28. 如权利要求27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。
  29. 如权利要求28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。
  30. 如权利要求28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。
  31. 如权利要求27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。
  32. 一种微生物，该微生物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。
  33. 如权利要求32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。
  34. 如权利要求33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。
  35. 如权利要求33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。
  36. 如权利要求32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。
  37. 如权利要求27-36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。
  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。
  39. 如权利要求37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。
  40. 如权利要求37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。
  41. 如权利要求37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。
  42. 如权利要求37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。
  43. 如权利要求42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。
  44. 如权利要求42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。
  45. 如权利要求37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。
  46. 如权利要求45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。
  47. 如权利要求45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。
  48. 如权利要求27-47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。
  49. 如权利要求27-48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。
  50. 如权利要求1-26中任一项所述的方法或者如权利要求27-49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)。
PCT/CN2017/080650 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 WO2017174037A1 (zh)

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