WO2017174039A1 - Procédé de production de la n-acétyl-d-glucosamine et/ou d'un sel de d-glucosamine par fermentation microbienne - Google Patents

Procédé de production de la n-acétyl-d-glucosamine et/ou d'un sel de d-glucosamine par fermentation microbienne Download PDF

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WO2017174039A1
WO2017174039A1 PCT/CN2017/080652 CN2017080652W WO2017174039A1 WO 2017174039 A1 WO2017174039 A1 WO 2017174039A1 CN 2017080652 W CN2017080652 W CN 2017080652W WO 2017174039 A1 WO2017174039 A1 WO 2017174039A1
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glucosamine
acetyl
microorganism
phosphate
promoter
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PCT/CN2017/080652
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English (en)
Chinese (zh)
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孙镧
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孙镧
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Priority claimed from CN201710217322.5A external-priority patent/CN107267575B/zh
Application filed by 孙镧 filed Critical 孙镧
Priority to US16/091,714 priority Critical patent/US11118205B2/en
Publication of WO2017174039A1 publication Critical patent/WO2017174039A1/fr

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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 (MioT1) , Yamada-Okabe T, Arisawa M, Yamada-Okabe H: Saccharomyces cerevisiae GNA1, an essential gene encoding a novel acetyltransferase involved in UDP-N-acetylglucosamine synthesis, J Biol Chem., 1999 Jan 1;274(1):424-9 .).
  • the present invention is directed to the metabolic pathway of N-acetyl-D-glucosamine, using a novel genetic modification method to transform microorganisms, and using the microorganism to produce N-acetyl-D-glucosamine (GlcNAc) and/or with higher efficiency and higher yield. Or D-glucosamine salt.
  • the present invention enhances N-acetyl-D- in microorganisms by increasing the action of N-acetyl-mannosamine-6-phosphate epimerase (NanE) in microorganisms.
  • N-acetyl-mannosamine-6-phosphate epimerase NaE
  • GlcNAc-6-P N-acetyl-D-glucosamine-6-phosphate
  • GlcNAc-6-P excreted extracellularly to N-acetyl-D-glucosamine
  • GlcNAc allowing the microorganism to produce N-acetyl-D-glucosamine (GlcNAc) with higher efficiency and higher yield And / or
  • the present invention further relates to one or more of the following contents based on the above content:
  • D-Glucosamine-6-phosphate deaminase NagB
  • Glucosamine-6-phosphate synthase Glucosamine-6-phosphate synthase
  • GlmS also known as L-glutamine-6-phosphate fructose aminotransferase
  • -6-P is aminated to D-glucosamine-6-phosphate (GlcN-6-P).
  • Glucosamine-6-phosphatesynthase GlmS
  • L-glutamine-6-phosphate fructose aminotransferase L-glutamine: D-fructose-6-phosphate aminotransferase
  • 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.
  • N-acetyl-D-glucosamine-6-phosphate deacetylase N-acetylglucosamine-6-phosphate deacetylase, NagA
  • N-acetylglucosamine-6-phosphate deacetylase, NagA prevents the conversion of N-acetyl-D-glucosamine-6-phosphate (GlcNAc-6-P) in microorganisms It is D-glucosamine-6-phosphate (GlcN-6-P).
  • the invention relates to a method for the production of N-acetyl-D-glucosamine (GlcNAc) and/or D-glucosamine salt by microbial fermentation, the method comprising:
  • GlcNAc N-acetyl-D-glucosamine
  • the genetic modification for enhancing the action of N-acetyl-D-aminomannose-6-phosphate isomerase (NanE) in a microorganism is selected from the group consisting of a) N-acetyl-D-aminomannose-6-phosphate in a microorganism.
  • the enzymatic activity of the isomerase (NanE) is increased; and/or b) the N-acetyl-D-aminomannose-6-phosphate isomerase (NanE) is overexpressed in the microorganism.
  • N-acetyl-D-aminomannose-6-phosphate isomerase NaE
  • Phosphoric acid A gene mutant with increased enzyme activity of the enzyme NaE
  • Screening of NanE gene mutants can be accomplished by error-producing PCR techniques to obtain high frequency mutant genes.
  • N-acetyl-D-aminomannose-6-phosphate isomerase (NanE) in microorganisms it is also possible to increase the number of copies of the gene and replace the promoter with higher expression level than the native promoter.
  • N-acetyl-D-aminomannose-6-phosphate isomerase (NanE).
  • the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification that enhances the action of N-acetyl-D-aminomannose-6-phosphate isomerase (NanE) in the microorganism.
  • the microorganism is transformed with at least one recombinant nucleic acid molecule comprising a nucleic acid sequence encoding N-acetyl-D-aminomannose-6-phosphate isomerase (NanE).
  • NeE N-acetyl-D-aminomannose-6-phosphate isomerase
  • the nucleic acid sequence encoding N-acetyl-D-aminomannose-6-phosphate isomerase contains at least one increase in N-acetyl-D-aminomannose-6-phosphate isomerase (NanE) Genetic modification of the enzyme activity).
  • the genetic modification comprises one or two of substitutions at positions corresponding to the amino acid sequence of SEQ ID NO: 17: 133th cysteine is replaced by arginine and 187th tyrosine The acid is replaced by histidine.
  • nucleic acid sequence encoding the N-acetyl-D-aminomannose-6-phosphate isomerase is SEQ ID NO: 26; the N-acetyl-D-aminomannose-6-phosphate The amino acid sequence of the isomerase (NanE) is SEQ ID NO:27.
  • the N-acetyl-D-aminomannose-6-phosphate isomerase has at least about 30% identical, preferably at least about 50% identical to the amino acid sequence of SEQ ID NO:17, Further preferably at least about 70% identical, further preferably at least about 80% identical, still more preferably at least about 90% identical, most preferably at least about 95% identical amino acid sequence, wherein said N-acetyl-D-aminomannose- 6-phosphate isomerase (NanE) has enzymatic activity.
  • N-acetyl-D-aminomannose-6-phosphate isomerase has the amino acid sequence of SEQ ID NO:17.
  • the gene copy number encoding the N-acetyl-D-aminomannose-6-phosphate isomerase is increased in the recombinant nucleic acid molecule.
  • the recombinant nucleic acid molecule comprises an endogenous native promoter, a promoter having a higher expression level than the endogenous native promoter, an enhancer, a fusion sequence, and the like.
  • the recombinant nucleic acid molecule comprises a promoter having a higher expression level than the endogenous natural promoter, such as an HCE promoter, a gap promoter, a trc promoter, a T7 promoter, etc.; more preferably, the recombinant nucleic acid molecule comprises a trc promoter. child.
  • the trc promoter is a split promoter of the trp promoter and the lac promoter, which has higher transcription efficiency than trp And strong promoter properties regulated by lacI repressor.
  • the recombinant nucleic acid molecule is transformed into a microorganism selected from the group consisting of a free form (i.e., a recombinant nucleic acid molecule is loaded into a plasmid) and an integrated type (i.e., a recombinant nucleic acid molecule is integrated into the genome of the microorganism).
  • a free form i.e., a recombinant nucleic acid molecule is loaded into a plasmid
  • an integrated type i.e., a recombinant nucleic acid molecule is integrated into the genome of the microorganism.
  • the recombinant nucleic acid molecule is integrated into the genome of the microorganism.
  • the microorganism comprises at least one genetic modification of an endogenous native promoter of a gene encoding N-acetyl-D-aminomannose-6-phosphate isomerase (NanE).
  • an endogenous native promoter of the gene encoding N-acetyl-D-aminomannose-6-phosphate isomerase (NanE) is replaced by a promoter with a higher expression level, such as the HCE promoter, the gap promoter.
  • NeE N-acetyl-D-aminomannose-6-phosphate isomerase
  • the microorganism further comprises one or more of the following genetic modifications:
  • (1) 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 one molecule capable of reducing glucosamine-6-phosphate synthase (GlmS) Genetic modification
  • the genetic modification for enhancing the action of D-glucosamine-6-phosphate deaminase (NagB) in the microorganism is selected from the group consisting of a) D-glucosamine-6-phosphate deaminase (NagB) in the microorganism.
  • the enzyme activity is increased; and/or b) D-glucosamine-6-phosphate deaminase (NagB) is overexpressed in the microorganism.
  • D-glucosamine-6-phosphate deaminase (NagB) in microorganisms
  • the enzyme activity encoding D-glucosamine-6-phosphate deaminase (NagB) can be screened. Increased gene mutants are achieved. Screening for NagB gene mutants can be accomplished by error-producing PCR techniques to obtain high frequency mutant genes.
  • D-glucosamine-6-phosphate deaminase (NagB) in microorganisms it is also possible to overexpress D-amino by increasing the number of copies of the gene and replacing a promoter with a higher expression level than the native promoter.
  • Glucose-6-phosphate deaminase (NagB) is achieved.
  • the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification that enhances the action of D-glucosamine-6-phosphate deaminase (NagB) in the microorganism.
  • the microorganism is transformed with at least one recombinant nucleic acid molecule comprising a nucleic acid sequence encoding D-glucosamine-6-phosphate deaminase (NagB).
  • the nucleic acid sequence encoding D-glucosamine-6-phosphate deaminase contains at least one genetic modification that increases the enzymatic activity of D-glucosamine-6-phosphate deaminase (NagB).
  • the gene copy number encoding D-glucosamine-6-phosphate deaminase is increased in the recombinant nucleic acid molecule.
  • the recombinant nucleic acid molecule comprises an endogenous native promoter, a promoter having a higher expression level than the endogenous native promoter, an enhancer, a fusion sequence, and the like.
  • the recombinant nucleic acid molecule 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 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 comprises at least one of at least one capable of reducing ammonia in the microorganism Transformation of a genetically modified recombinant nucleic acid molecule that acts on a glucose-6-phosphate synthase (GlmS).
  • GlmS glucose-6-phosphate synthase
  • the genetic modification for increasing the action of glucosamine-6-phosphate synthase (GlmS) in the microorganism is selected from a) an increase in the enzymatic activity of glucosamine-6-phosphate synthase (GlmS) in the microorganism; And/or b) the glucosamine-6-phosphate synthase (GlmS) is overexpressed in the microorganism.
  • glucosamine-6-phosphate synthase in order to enhance the action of glucosamine-6-phosphate synthase (GlmS) in microorganisms, it is possible to screen for a gene mutant encoding an increase in enzymatic activity of glucosamine-6-phosphate synthase (GlmS). achieve. Screening of GlmS gene mutants can be accomplished by error-producing PCR techniques to obtain high frequency mutant genes. In order to enhance the action of glucosamine-6-phosphate synthase (GlmS) in microorganisms, it is also possible to overexpress glucosamine-6-phosphate by increasing its gene copy number and replacing a promoter with a higher expression level than the native promoter.
  • the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification that enhances the action of glucosamine-6-phosphate synthase (GlmS) in the microorganism.
  • GlmS glucosamine-6-phosphate synthase
  • the microorganism is transformed with at least one recombinant nucleic acid molecule comprising a nucleic acid sequence encoding glucosamine-6-phosphate synthase (GlmS).
  • GlmS glucosamine-6-phosphate synthase
  • the nucleic acid sequence encoding glucosamine-6-phosphate synthase contains at least one genetic modification that increases the enzymatic activity of glucosamine-6-phosphate synthase (GlmS).
  • the gene copy number encoding the glucosamine-6-phosphate synthase is increased in the recombinant nucleic acid molecule.
  • the recombinant nucleic acid molecule comprises an endogenous native promoter, a promoter having a higher expression level than the endogenous native promoter, an enhancer, a fusion sequence, and the like.
  • the recombinant nucleic acid molecule comprises a promoter having a higher expression level than the endogenous natural promoter, such as an HCE promoter, a gap promoter, a trc promoter, a T7 promoter, etc.; more preferably, the recombinant nucleic acid molecule comprises a trc promoter. child.
  • the recombinant nucleic acid molecule is transformed into a microorganism selected from the group consisting of a free form (i.e., a recombinant nucleic acid molecule is loaded into a plasmid) and an integrated type (i.e., a recombinant nucleic acid molecule is integrated into the genome of the microorganism).
  • a free form i.e., a recombinant nucleic acid molecule is loaded into a plasmid
  • an integrated type i.e., a recombinant nucleic acid molecule is integrated into the genome of the microorganism.
  • the recombinant nucleic acid molecule is integrated into the genome of the microorganism.
  • the microorganism comprises at least one genetic modification of an endogenous native promoter of a gene encoding glucosamine-6-phosphate synthase (GlmS).
  • the endogenous native promoter of the gene encoding glucosamine-6-phosphate synthase (GlmS) is replaced by a promoter with a higher expression level, such as the HCE promoter, the gap promoter, the trc promoter, the T7 promoter.
  • the endogenous natural promoter of the gene encoding glucosamine-6-phosphate synthase (GlmS) is initiated by trc Sub replacement.
  • the genetic modification for reducing the action of D-glucosamine-6-phosphate deaminase (NagB) in microorganisms is selected from a) the reduction of the enzyme activity of D-glucosamine-6-phosphate deaminase (NagB) in the microorganism.
  • D-glucosamine-6-phosphate deaminase (NagB) in the microorganism, including but not limited to: encoding D-glucosamine-6-phosphate deaminase (NagB) in the microorganism Partial or complete deletion, or partial or complete inactivation of the gene, and/or partial or complete deletion of the endogenous natural promoter of the D-glucosamine-6-phosphate deaminase (NagB) gene encoding the microorganism, Partially or completely inactivated.
  • the genetic modification to reduce the action of D-glucosamine-6-phosphate deaminase (NagB) in the microorganism is to completely encode the endogenous natural promoter of the D-glucosamine-6-phosphate deaminase (NagB) gene in the microorganism. Missing, that is, deleted.
  • the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification that reduces the action of D-glucosamine-6-phosphate deaminase (NagB) in the microorganism.
  • agB D-glucosamine-6-phosphate deaminase
  • the genetic modification for increasing the action of UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) in the microorganism is selected from the group consisting of a) UDP-N-acetyl-D- in the microorganism.
  • the enzymatic activity of glucosamine-2-isomerase (WecB) is increased; and/or b) UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) is overexpressed in the microorganism.
  • the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification that enhances the action of UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) in the microorganism.
  • the microorganism is transformed with at least one recombinant nucleic acid molecule comprising a nucleic acid sequence encoding UDP-N-acetyl-D-glucosamine-2-isomerase (WecB).
  • WecB UDP-N-acetyl-D-glucosamine-2-isomerase
  • the nucleic acid sequence encoding UDP-N-acetyl-D-glucosamine-2-isomerase contains at least one increase in UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) Genetic modification of the enzymatic activity; further preferably, the genetic modification is included in the amino acid sequence corresponding to SEQ ID NO: One or more of the substitutions at position 43: the cysteine at position 34 is replaced by serine, the histidine at position 145 is replaced by aspartic acid, and the cysteine at position 226 is replaced by phenylpropanoid More preferably, the nucleic acid sequence encoding the UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) is SEQ ID NO: 49; The amino acid sequence of UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) is SEQ ID NO:50.
  • the UDP-N-acetyl-D-glucosamine-2-isomerase has at least about 30% identical, preferably at least about 50% identical to the amino acid sequence of SEQ ID NO:43, Further preferably at least about 70% identical, further preferably at least about 80% identical, still more preferably at least about 90% identical, most preferably at least about 95% identical amino acid sequence, wherein said UDP-N-acetyl-D-glucosamine -2-isomerase (WecB) has enzymatic activity.
  • the UDP-N-acetyl-D-glucosamine-2-isomerase has the amino acid sequence of SEQ ID NO:43.
  • the gene copy number encoding the UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) in the recombinant nucleic acid molecule is increased.
  • the recombinant nucleic acid molecule comprises an endogenous native promoter, a promoter having a higher expression level than the endogenous native promoter, an enhancer, a fusion sequence, and the like.
  • the recombinant nucleic acid molecule comprises a promoter having a higher expression level than the endogenous natural promoter, such as an HCE promoter, a gap promoter, a trc promoter, a T7 promoter, etc.; more preferably, the recombinant nucleic acid molecule comprises a trc promoter. child.
  • the recombinant nucleic acid molecule is transformed into a microorganism selected from the group consisting of a free form (i.e., a recombinant nucleic acid molecule is loaded into a plasmid) and an integrated type (i.e., a recombinant nucleic acid molecule is integrated into the genome of the microorganism).
  • a free form i.e., a recombinant nucleic acid molecule is loaded into a plasmid
  • an integrated type i.e., a recombinant nucleic acid molecule is integrated into the genome of the microorganism.
  • the recombinant nucleic acid molecule is integrated into the genome of the microorganism.
  • the microorganism comprises at least one genetic modification of an endogenous native promoter of a gene encoding UDP-N-acetyl-D-glucosamine-2-isomerase (WecB).
  • the endogenous native promoter of the gene encoding UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) is replaced by a promoter with a higher expression level, such as the HCE promoter, the gap promoter.
  • WecB UDP-N-acetyl-D-glucosamine-2-isomerase
  • the microorganism further comprises one or more of the following genetic modifications:
  • (1) comprising at least one genetic modification capable of reducing the action of the mannose transporter EIIM, P/III man (ManXYZ) in the microorganism;
  • the genetic modification of reducing the action of the mannose transporter EIIM, P/III man (ManXYZ) in the microorganism includes, but is not limited to, encoding the mannose transporter EIIM, P/III man (ManXYZ) Partial or complete deletion, or partial or complete inactivation of the endogenous gene, and/or partial or complete encoding of the endogenous natural promoter of the mannose transporter EIIM, P/III man (ManXYZ) gene in the microorganism Missing, or partially or completely inactivated.
  • mannose transporter EIIM reducing microorganisms genetic P / III man (ManXYZ) acting microorganism modified to encode the mannose transporter EIIM, the complete absence of P / III man (ManXYZ) an endogenous gene, is deleted .
  • the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification that reduces the action of the mannose transporter EIIM, P/III man (ManXYZ) in the microorganism.
  • the genetic modification for reducing the action of N-acetylneuraminic acid lyase (NanA) in the microorganism includes, but is not limited to, encoding the endogenous source of N-acetylneuraminic acid lyase (NanA) in the microorganism. Partial or complete deletion, or partial or complete inactivation of a sex gene, and/or partial or complete deletion, or partial or complete, of an endogenous natural promoter of the N-acetylneuraminic acid lyase (NanA) gene in a microorganism Inactivated.
  • the genetic modification to reduce the action of N-acetylneuraminic acid lyase (NanA) in the microorganism is that the endogenous gene encoding the N-acetylneuraminic acid lyase (NanA) in the microorganism is completely deleted, ie, deleted.
  • the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification that reduces the action of N-acetylneuraminic acid lyase (NanA) in the microorganism.
  • N-acetyl-D-glucosamine-6-phosphate in the microorganism Genetic modifications of the action of acylase (NagA) include, but are not limited to, partial or complete deletion, or partial or complete, of an endogenous gene encoding N-acetyl-D-glucosamine-6-phosphate deacetylase (NagA) in a microorganism. Inactivated, and/or partially or completely deleted, or partially or completely inactivated, of the endogenous natural promoter of the N-acetyl-D-glucosamine-6-phosphate deacetylase (NagA) gene in the microorganism.
  • 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 comprises at least one glucosamine mutase encoding a phosphate Transformation of a recombinant nucleic acid molecule of a nucleic acid sequence of (GlmM).
  • 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 genetic modification of an endogenous native promoter of a gene encoding a phosphoglucosamine mutase (GlmM).
  • the endogenous native promoter encoding the gene for phosphoglucosamine mutase (GlmM) is replaced by a promoter with a higher expression level, such as the HCE promoter, gap promoter, trc promoter, T7 promoter, etc.
  • the endogenous native promoter of the gene encoding the phosphoglucosamine mutase (GlmM) is replaced by the trc promoter.
  • the genetic modification for enhancing the action of the bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU) in the microorganism is selected from a) the bifunctional enzyme N in the microorganism - an increase in the enzymatic activity of acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU); and/or b) bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine transfer in microorganisms
  • the enzyme (GlmU) is overexpressed.
  • N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU) in microorganisms
  • N-acetyl-D glucosamine-1-phosphate uridine acyltransferase
  • Screening for the GlmU gene mutant can be accomplished by error-producing PCR techniques to obtain high frequency mutant genes.
  • N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase GlmU
  • the promoter is expressed by overexpressing the bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU).
  • the microorganism comprises at least one of at least one capable of enhancing the bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU) in the microorganism Transformation of a genetically modified recombinant nucleic acid molecule.
  • 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 a free form (i.e., a recombinant nucleic acid molecule is loaded into a plasmid) and an integrated type (i.e., a recombinant nucleic acid molecule is integrated into the genome of the microorganism).
  • a free form i.e., a recombinant nucleic acid molecule is loaded into a plasmid
  • an integrated type i.e., a recombinant nucleic acid molecule is integrated into the genome of the microorganism.
  • the recombinant nucleic acid molecule is integrated into the genome of the microorganism.
  • the microorganism comprises at least one inheritance of an endogenous natural promoter of a gene encoding the bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU) Modification.
  • the endogenous native promoter of the gene encoding the bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU) is replaced by a promoter with a higher expression level, such as the HCE promoter.
  • a gap promoter a trc promoter, a T7 promoter, etc.; more preferably, an endogenous natural promoter encoding a gene of the bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU) Replaced by the trc promoter.
  • GlmU N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase
  • the invention relates to a process for the production of N-acetyl-D-glucosamine (GlcNAc) and/or D-glucosamine salt by microbial fermentation, the process comprising:
  • the microorganism further comprises at least one genetic modification that reduces the action of glucosamine-6-phosphate synthase (GlmS).
  • GlmS glucosamine-6-phosphate synthase
  • the present invention relates to a method for producing N-acetyl-D-glucosamine (GlcNAc) and/or D-glucosamine salt by microbial fermentation, the method comprising:
  • A) cultivating a microorganism in a fermentation medium comprising: at least one genetic modification capable of increasing the action of N-acetyl-D-aminomannose-6-phosphate isomerase (NanE) in the microorganism; at least one capable a genetic modification that enhances the action of glucosamine-6-phosphate synthase (GlmS) in the microorganism; and at least one genetic modification that reduces the action of D-glucosamine-6-phosphate deaminase (NagB);
  • the present invention relates to a method for producing N-acetyl-D-glucosamine (GlcNAc) and/or D-glucosamine salt by microbial fermentation, the method comprising:
  • NaE N-acetyl-D-aminomannose-6-phosphate isomerase
  • WecB UDP-N-acetyl-D-glucosamine-2-isomerase
  • the present invention relates to a method for producing N-acetyl-D-glucosamine (GlcNAc) and/or D-glucosamine salt by microbial fermentation, the method comprising:
  • A) cultivating a microorganism in a fermentation medium comprising: at least one genetic modification capable of increasing the action of N-acetyl-D-aminomannose-6-phosphate isomerase (NanE) in the microorganism; at least one capable Genetic modification of the action of D-glucosamine-6-phosphate deaminase (NagB) in microorganisms; and at least one of which enhances the action of UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) in microorganisms Genetic modification; and
  • the microorganism further comprises at least one genetic modification that reduces the action of glucosamine-6-phosphate synthase (GlmS).
  • GlmS glucosamine-6-phosphate synthase
  • the present invention relates to a method for producing N-acetyl-D-glucosamine (GlcNAc) and/or D-glucosamine salt by microbial fermentation, the method comprising:
  • A) cultivating a microorganism in a fermentation medium comprising: at least one genetic modification capable of increasing the action of N-acetyl-D-aminomannose-6-phosphate isomerase (NanE) in the microorganism; at least one capable Genetic modification to increase the action of glucosamine-6-phosphate synthase (GlmS) in microorganisms; at least one genetic modification that reduces the action of D-glucosamine-6-phosphate deaminase (NagB); and at least one capable of enhancing microorganisms Genetic modification of the action of UDP-N-acetyl-D-glucosamine-2-isomerase (WecB);
  • the microorganism further comprises: at least one genetic modification capable of reducing the action of the mannose transporter EIIM, P/III man (ManXYZ) in the microorganism; at least one capable of reducing the N-acetyl nerve in the microorganism Genetic modification of the action of lyase (NanA); at least one genetic modification that reduces the action of N-acetyl-D-glucosamine-6-phosphate deacetylase (NagA) in microorganisms; and at least one that reduces microbial activity Genetic modification of the action of N-acetyl-D-glucosamine specific enzyme II Nag (NagE).
  • the 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-filling liquid comprises grapes Sugar and ribose, preferably, the glucose concentration is 10%-85% (w/v), the ribose concentration is 0.5%-15% (w/v), and further preferably, the glucose concentration is 55%-75% (w/v).
  • the ribose concentration is 5%-7% (w/v);
  • the sugar-comprising solution comprises glucose and gluconate, preferably, the glucose concentration is 10%-85% (w/v), glucose The acid salt concentration is from 0.5% to 15% (w/v), further preferably, the glucose concentration is from 55% to 75% (w/v), and the gluconate concentration is from 2% to 3% (w/v);
  • the sugar-retaining liquid comprises glucose, ribose and gluconate, preferably, the glucose concentration is from 10% to 85% (w/v), and the ribose concentration is from 0.5% to 15% (w/v), glucose.
  • the acid salt concentration is from 0.5% to 15% (w/v), further preferably, the glucose concentration is from 55% to 75% (w/v), the ribose concentration is from 5% to 7% (w/v), and the gluconate concentration It is 2%-3% (w/v).
  • the gluconate is sodium gluconate.
  • the culturing step is carried out at a temperature of from about 20 ° C to about 45 ° C, and more preferably, the culturing step is carried out at a temperature of from about 33 ° C to about 37 ° C.
  • the culturing step is carried out at a pH of from about 4.5 to about pH 8.5. 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 N-acetyl-D-aminomannose-6-phosphate isomerase (NanE) in a microorganism .
  • This genetic modification has been described in detail above.
  • the microorganism further comprises one or more of the following genetic modifications:
  • (1) 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 one molecule capable of reducing glucosamine-6-phosphate synthase (GlmS) Genetic modification
  • 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 invention relates to a microorganism comprising: at least one capable of enhancing the action of N-acetyl-D-aminomannose-6-phosphate isomerase (NanE) in a microorganism Genetic modification; and at least one genetic modification that enhances the action of D-glucosamine-6-phosphate deaminase (NagB) in the microorganism.
  • NaE N-acetyl-D-aminomannose-6-phosphate isomerase
  • CagB D-glucosamine-6-phosphate deaminase
  • the microorganism further comprises at least one genetic modification that reduces the action of glucosamine-6-phosphate synthase (GlmS).
  • GlmS glucosamine-6-phosphate synthase
  • the present invention relates to a microorganism comprising: at least one capable of enhancing N-acetyl-D-aminomannose-6-phosphate isomerase (NanE) in a microorganism Genetic modification of action; at least one genetic modification that increases the action of glucosamine-6-phosphate synthase (GlmS) in the microorganism; and at least one that reduces the action of D-glucosamine-6-phosphate deaminase (NagB) Genetic modification.
  • NaE N-acetyl-D-aminomannose-6-phosphate isomerase
  • GaM glucosamine-6-phosphate synthase
  • NagB D-glucosamine-6-phosphate deaminase
  • the invention relates to a microorganism comprising: at least one capable of enhancing the action of N-acetyl-D-aminomannose-6-phosphate isomerase (NanE) in a microorganism Genetic modification; and at least one genetic modification that enhances the action of UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) in microorganisms.
  • NaE N-acetyl-D-aminomannose-6-phosphate isomerase
  • WecB UDP-N-acetyl-D-glucosamine-2-isomerase
  • the present invention relates to a microorganism comprising: at least one capable of increasing N-acetyl-D-aminomannose-6-phosphate isomerase (NanE) in a microorganism Genetic modification of action; at least one genetic modification that enhances the action of D-glucosamine-6-phosphate deaminase (NagB) in microorganisms; and at least one that increases UDP-N-acetyl-D-glucosamine in microorganisms Genetic modification of the action of 2-isomerase (WecB).
  • NaE N-acetyl-D-aminomannose-6-phosphate isomerase
  • NagB D-glucosamine-6-phosphate deaminase
  • WecB 2-isomerase
  • the microorganism further comprises at least one genetic modification that reduces the action of glucosamine-6-phosphate synthase (GlmS).
  • GlmS glucosamine-6-phosphate synthase
  • the present invention relates to a microorganism comprising: at least one capable of enhancing N-acetyl-D-aminomannose-6-phosphate isomerase (NanE) in a microorganism Genetic modification of action; at least one genetic modification that enhances the action of glucosamine-6-phosphate synthase (GlmS) in microorganisms; at least one inheritance that reduces the action of D-glucosamine-6-phosphate deaminase (NagB) Modification; and at least one can increase UDP-N-acetyl-D-glucosamine-2-isomerase in microorganisms Genetic modification of the role of (WecB).
  • NagB D-glucosamine-6-phosphate deaminase
  • the microorganism further comprises: at least one genetic modification capable of reducing the action of the mannose transporter EIIM, P/III man (ManXYZ) in the microorganism; at least one capable of reducing the N-acetyl nerve in the microorganism Genetic modification of the action of lyase (NanA); at least one genetic modification that reduces the action of N-acetyl-D-glucosamine-6-phosphate deacetylase (NagA) in microorganisms; and at least one that reduces microbial activity Genetic modification of the action of N-acetyl-D-glucosamine specific enzyme II Nag (NagE).
  • the present invention relates to N-acetyl-D-aminomannose-6-phosphate isomerase (NanE) having a higher enzymatic activity, which has the SEQ ID NO: 27 Amino acid sequence.
  • the present invention further relates to a nucleic acid molecule encoding the above N-acetyl-D-aminomannose-6-phosphate isomerase (NanE) having the nucleic acid sequence of SEQ ID NO:26.
  • the invention further relates to a vector comprising the above nucleic acid molecule.
  • the invention further relates to a microorganism comprising the above vector.
  • the invention further relates to a microorganism comprising the above nucleic acid molecule in the genome.
  • the microorganism may be any microorganism (for example, a bacterium, a protist, an alga, a fungus, or other microorganisms).
  • the microorganism includes, but is not limited to, bacteria, yeast or fungi.
  • the microorganism is selected from the group consisting of bacteria or yeast.
  • the bacterium includes, but is not limited to, a genus selected from the group consisting of Escherichia, Bacillus, Lactobacillus, Pseudomonas, or Streptomyces.
  • the bacteria include, but are not limited to, selected from the group consisting of Escherichia coli, Bacillus subtilis, Bacillus licheniformis, Lactobacillus brevis, Pseudomonas aeruginosa ( Pseudomonas aeruginosa) or a species of Streptomyces lividans.
  • the yeast includes, but is not limited to, selected from the group consisting of Saccharomyces, Schizosaccharomyces, Candida, Hansenula, Pichia, and gram.
  • yeast includes, but is not limited to, Saccharomyce scerevisiae, Schizosaccharo mycespombe, Candida. Albicans), Hansenula polymorpha, Pichia pastoris, Pichia Canadensis), Kluyveromyces marxianus or Phaffia rohodozyma.
  • the microorganism is a fungus; further preferably, the fungus includes, but is not limited to, selected from the group consisting of Aspergillus, Absidia, Rhizopus, Chrysosporium, and Neurospora a fungus belonging to the genus Neurospora or Trichoderma; more preferably, the fungus includes, but is not limited to, selected from the group consisting of Aspergillus niger, Aspergillus nidulans, and Absidia coerulea. ), Rhizopus oryzae, Chrysosporium lucknowense, Neurospora crassa, Neurospora intermedia or Trichoderma reesei . Particularly preferred E.
  • coli strains include K-12, B and W, most preferably K-12.
  • E. coli is a preferred microorganism and is used as an example of various embodiments of the present invention, it is understood that N-acetyl-D-glucosamine can be used in the method of the invention and can be genetically modified to increase N-acetyl-D. - any other microorganism that produces glucosamine.
  • the microorganism used in the present invention may also be referred to as a production organism.
  • N-acetyl-D-glucosamine may be referred to as 2-acetamido-2-deoxy-D-glucose.
  • the terms N-acetyl-D-glucosamine, N-acetyl-D-glucosamine-6-phosphate and N-acetyl-D-glucosamine-1-phosphate can be abbreviated as GlcNAc, GlcNAc-6-P and GlcNAc-1, respectively.
  • N-acetyl-D-glucosamine is also abbreviated as NAG.
  • D-glucosamine, D-glucosamine-6-phosphate and D-glucosamine-1-phosphate can be abbreviated as GlcN, GlcN-6-P and GlcN, respectively.
  • -1-P the terms N-acetyl-D-aminomannose, N-acetyl-D-aminomannose-6-phosphate, glucose, glucose-6-phosphate, fructose-6-phosphate can be abbreviated as ManNAc, ManNAc-6, respectively.
  • increasing the action of an enzyme in a microorganism means that the activity of the enzyme in the microorganism is increased and/or the enzyme is overexpressed, thereby increasing the amount of substrate-producing product catalyzed by the enzyme in the microorganism.
  • reducing the action of an enzyme in a microorganism means that the activity of the enzyme in the microorganism is reduced and/or the expression of the enzyme is reduced, thereby reducing the amount of substrate-producing product catalyzed by the enzyme in the microorganism.
  • the term 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 increases the affinity of the substrate to mean that the enzyme is catalyzed by The affinity of the substrate increases.
  • 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 NanE, which may further include modifications to NagB, GlmS, WecB, or a combination thereof, and may further include ManXYZ, NanA Modification of NagA, NagE, GlmM, GlmU or a combination thereof.
  • Enzymes having the same biological activity are known in the art to have different names depending on which microorganism the enzyme is derived from. The following are 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.
  • N-acetyl-D-aminomannose-6-P isomerase from Escherichia coli has the amino acid sequence encoded by the nucleic acid sequence represented by SEQ ID NO: 16, represented by SEQ ID NO: 17.
  • UDP-N-acetyl-D-glucosamine-2-isomerase catalyzes the conversion of UDP-N-acetyl-D-glucosamine to N-acetyl-D-aminomannose.
  • UDP-N-acetyl-D-glucosamine-2-isomerase from E. coli is generally referred to as WecB.
  • UDP-N-acetyl-D-glucosamine-2-isomerases from various organisms are well known in the art and can be used in the genetic engineering strategies of the present invention.
  • UDP-N-acetyl-D-glucosamine-2-isomerase from Escherichia coli has the amino acid sequence encoded by the nucleic acid sequence represented by SEQ ID NO: 42 and represented by SEQ ID NO:43.
  • D-glucosamine-6-phosphate deaminase catalyzes the reversible reaction of D-glucosamine-6-phosphate with water to form glucose-6-phosphate and ammonium.
  • the enzyme is also known as D-glucosamine-6-phosphate isomerase, GlcN6P deaminase, D-glucosamine isomerase, D-glucosamine isomerase, D-glucosamine phosphate deaminase and 2-amino-2-deoxy-D-glucose-6-phosphate ethyl ketone alcohol isomerase (deamination).
  • D-glucosamine-6-phosphate deaminase from various organisms is well known in the art and can be used in the genetic engineering strategies of the present invention. In E. coli and other bacteria, the enzyme is generally referred to as NagB.
  • D-glucosamine-6-phosphate synthase catalyzes the formation of D-glucosamine-6-phosphate and glutamic acid from glucose-6-phosphate and glutamine.
  • the enzyme is also called D-glucosamine-fructose-6-phosphate aminotransferase (isomerization), hexose phosphate aminotransferase, D-fructose-6-phosphate transamidase, D-glucosamine-6-phosphate Isomerase (formation of glutamine), L-glutamine-fructose-6-phosphate transamidase and GlcN6P synthase.
  • D-glucosamine-6-phosphate synthase from various organisms is well known in the art and can be used in the genetic engineering strategies of the present invention.
  • D-glucosamine-6-phosphate synthase from E. coli and other bacteria is generally referred to as GlmS.
  • N-acetyl-D-glucosamine-6-phosphate deacetylase hydrolyzes N-acetyl-D-glucosamine-6-phosphate to D-glucosamine-6-phosphate and acetate .
  • N-acetyl-D-glucosamine-6-phosphate deacetylases from various organisms are well known in the art and can be used in the genetic engineering strategies of the present invention. For example, a method from E. coli called NagA is described herein.
  • N-acetylneuraminic lyase catalyzes the degradation of N-acetyl-D-aminomannose to N-acetylneuraminic acid.
  • N-acetylneuraminic lyases from various organisms are well known in the art and can be used in the genetic engineering strategies of the present invention.
  • an N-acetylneuraminic lyase from E. coli is described herein as NanoA.
  • phosphoglucosamine mutase catalyzes the conversion of D-glucosamine-6-phosphate to D-glucosamine-1-phosphate.
  • Phospho D-glucosamine mutases from various organisms are well known in the art and can be used in the genetic engineering strategies of the present invention.
  • the phosphoglucosamine mutase of this enzyme in Escherichia coli and other bacteria is generally referred to as GlmM.
  • D-glucosamine-1-phosphate N-acetyltransferase converts D-glucosamine-1-phosphate and acetyl-CoA to N-acetyl-D-glucosamine-1-phosphate, and Release the CoA.
  • N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase also known as UDP-N-acetyl-D-glucosamine pyrophosphorylase, UDP- N-acetyl-D-glucosamine diphosphatase, further converting N-acetyl-D-glucosamine-1-phosphate to UDP-N-acetyl-D-amino grape sugar.
  • 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:28.
  • 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.
  • UDP-N-acetyl-D-glucosamine is catalyzed by D-glucosamine-6-P (GlcN-6-P) in GlmM and GlmU in a cell, in UDP-N -Acetyl-glucosamine-2-isomerase (wecB) catalyzes to become N-acetyl-D-aminomannose (ManNAc), which is further converted to N-acetyl-D-glucosamine by overexpression of NanE Phosphorus 6-phosphate (GlcNAc-6-P) is phosphorylated by phosphatase and excreted as extracellular N-acetyl-D-glucosamine (GlcNAc).
  • the method of the present invention avoids the formation of D-glucosamine, thereby avoiding the toxic effects of D-glucosamine and/or its degradation products on the strain.
  • the present invention has the beneficial effects that the present invention proves that the completely natural N-acetyl-D-glucosamine can be directly produced by the microbial fermentation method; the new production method has no risk of heavy metal pollution, no antibiotics, drug residue risk, and production is not affected. Influence of raw material supply, long-term stable production, high yield and low cost; N-acetyl-D-glucosamine and D-glucosamine products produced are non-animal, chitin without shrimp shell, glucose, etc. Carbon source fermentation, is a vegetarian product, and an allergen of anhydrous products.
  • This example describes the construction of an E. coli mutant that blocks the metabolic pathway associated with uptake of N-acetyl-D-glucosamine and degradation of beneficial intermediates.
  • the parent strain of the production strain was AT-001 (Escherichia coli ATCC 27325) belonging to the E. coli K-12 derivative from the American Type Culture Collection.
  • Blocking the N-acetyl-D-glucosamine uptake and degradation of intermediate metabolites can reduce the loss in the metabolic process and increase the accumulation of the target product (N-acetyl-D-glucosamine).
  • Construction of such a mutant host strain can cause N-acetyl-D-glucosamine accumulation by completely or partially deleting the humanXYZ, nanA, nagA and nagE gene sequences on its chromosomal genome to disable its function.
  • Red recombination is a DNA homologous recombination technique mediated by the lambda phage Red operon and the Rac phage RecE/RecT system. By this technique, it is possible to easily and rapidly perform various modifications such as insertion, knockout, and mutation of any large DNA molecule.
  • Red Recombination Technology simply states that the pKD46 plasmid carrying the recombinase gene is first transferred into the cells, and then the linear DNA fragment for targeting is prepared by electroporation, and the positive clones are screened. Finally, the resistance in the recombinant strain is determined. Gene elimination.
  • the mannose transporter EIIM, P/III man can be used as the second transporter of N-acetyl-D-glucosamine, which can be used for N-acetyl-D-glucosamine, etc.
  • the hexose is transported into the cell, thereby transporting the extracellular and accumulated target product back into intracellular degradation. Deletion of the manXYZ gene sequence prevents extracellular N-acetyl-D-glucosamine from being transported back into the cell for degradation.
  • the fKanrf fragment that is, the FRT-Kanr-FRT fragment, refers to a base sequence of the FRT site specifically recognized by the FLP recombinase at both ends of the kanamycin resistance gene (Kanr).
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • fKanrf size 1.28kb. Its nucleotide sequence is SEQ ID No. 3.
  • the PCR product was separated and purified by 1% agarose gel electrophoresis.
  • Designing a homology arm primer Designing a homologous arm forward primer (manXYZKO-F) SEQ ID No. 5, reverse primer (manXYZKO-R) SEQ ID No. 6 to delete the manXYZ sequence according to the humanXYZ sequence SEQ ID No. 4. .
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • Amplification product homology arm + fKanrf + homology arm.
  • the PCR product was separated by agarose gel electrophoresis, purified and recovered, and 100 ng/ ⁇ l of linear DNA full-length PCR fragment was obtained for Red recombinant targeting.
  • the pKD46 vector was transferred into the E. coli AT-001 strain. Then, 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, which expresses the three gene segments of Exo, Bet and Gam.
  • the three genes are placed under the arabinose promoter and can be expressed by L-arabinose. Da.
  • Competency preparation First, Escherichia coli ATCC 27325 stock solution stored at -20 ° C was inoculated in 10 ml of LB liquid medium at 1:50-100, and cultured at 37 ° C, 225 rpm, shaking for 2-3 hours. The culture solution was further added to a 10 ml centrifuge tube, 4000 g ⁇ 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl 2 5 ml for 5 min. Finally, it was centrifuged at 4000 g ⁇ 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl 2 5 ml.
  • Plasmid transformation 250 ⁇ l of naturally-precipitated cells were taken, and 5 ⁇ l of pKD46 plasmid was added at -4 ° C for 30 min. Then, in a 42 ° C water bath for 1.5 min, 0.7 ml of SOC medium was added, and the mixture was shaken at 30 ° C for 2 hours. Take 0.2 ml of bacterial solution and apply penicillin plate. Incubate overnight (12-16 hours) at 30 °C. Monoclones were picked, cultured in 5 ml of LB liquid medium, and plasmids were identified. Preserve positive strains for use.
  • pCP20 is a plasmid with ampicillin and chloramphenicol resistance gene, which can express FLP recombinase after heat induction.
  • the enzyme can specifically recognize the FRT site, and the sequence between FRT sites can be deleted by recombination, leaving only one FRT site.
  • pCP20 was transferred into the above-mentioned caramycin resistant clone, cultured at 30 ° C for 8 h, and then increased to 42 ° C overnight, heat-induced FLP recombinase expression, and the plasmid was gradually lost.
  • the antimony inoculum was plated on the antibiotic-free medium, and the grown monoclonal spot was picked onto the caramycin resistant plate, and the undeveloped clone in which the caramycin resistance gene had been deleted by the FLP recombinase. Clones with the disappearance of resistance to caramycin were identified by PCR using the identified primers.
  • N-acetylneuraminate lyase is capable of degrading N-acetyl-D-aminomannose (ManNAc) in microorganisms to N-acetyl-D-neuraminic acid (Neu5Ac). Deletion of the nanA gene sequence in the nanKETA operon prevents the degradation of N-acetyl-D-aminomannose (ManNAc) to N-acetyl-D-neuraminic acid (Neu5Ac).
  • Designing the homology arm primer Designing the homologous arm primer for deletion of the nanA sequence according to the nanE sequence of nanE-nanK SEQ ID No. 7, forward primer (nanAKO-F) SEQ ID No. 8, reverse primer (nanAKO-R) ) SEQ ID No. 9.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • Amplification product homology arm + fKanrf + homology arm.
  • the PCR product was separated by agarose gel electrophoresis, purified and recovered, and 100 ng/ ⁇ l of linear DNA full-length PCR fragment was obtained for Red recombinant targeting.
  • the pKD46 vector was transferred into the E. coli AT-002-02 strain. Then, a linear DNA fragment for targeting was prepared by electroporation, and positive clones were selected. Finally, the resistance gene is eliminated.
  • Competent preparation First, Escherichia coli AT-002-02 (AT-001, ⁇ manXYZ) stock solution stored at -20 ° C, inoculated in 10 ml LB liquid medium at 1:50-100, 37 ° C, Incubate at 225 rpm for 2-3 hours with shaking. The culture solution was further added to a 10 ml centrifuge tube, 4000 g ⁇ 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl 2 5 ml for 5 min. Finally, it was centrifuged at 4000 g ⁇ 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl 2 5 ml. It was allowed to stand at -4 ° C for 12 hours and settled naturally.
  • Plasmid transformation 250 ⁇ l of naturally-precipitated cells were taken, and 5 ⁇ l of pKD46 plasmid was added at -4 ° C for 30 min. Then, in a 42 ° C water bath for 1.5 min, 0.7 ml of SOC medium was added, and the mixture was shaken at 30 ° C for 2 hours. Take 0.2 ml of bacterial solution and apply penicillin plate. Incubate overnight (12-16 hours) at 30 °C. Monoclones were picked, cultured in 5 ml of LB liquid medium, and plasmids were identified. Preserve positive strains for use.
  • pCP20 was transferred into the above-mentioned caramycin resistant clone, cultured at 30 ° C for 8 h, and then increased to 42 ° C overnight, heat-induced FLP recombinase expression, and the plasmid was gradually lost.
  • the antimony inoculum was plated on the antibiotic-free medium, and the grown monoclonal spot was picked onto the caramycin resistant plate, and the undeveloped clone in which the caramycin resistance gene had been deleted by the FLP recombinase. Identification of primers for PCR to eliminate the disappearance of resistance to carrageenin Line identification.
  • N-acetyl-D-glucosamine-6-phosphate deacetylase converts N-acetyl-D-glucosamine-6-phosphate (GlcNAc-6-P) in microorganisms It is D-glucosamine-6-phosphate (GlcN-6-P).
  • nagE-nagBACD 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 extracellular can prevent degradation of GlcNAc is transported back to the cell.
  • Designing the homology arm primer Designing the homologous arm forward primer (nagEKO-F1) SEQ ID No to delete the nagE gene sequence according to NCBI search NC_000913, Escherichia coli str. K-12nagB promoter and nagE gene sequence SEQ ID No. 13. .14, reverse primer (nagEKO-R1) SEQ ID No. 15.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • Amplification product homology arm + fKanrf + homology arm.
  • the PCR product was separated by agarose gel electrophoresis, purified and recovered, and 100 ng/ ⁇ l of linear DNA full-length PCR fragment was obtained for Red recombinant targeting.
  • the pKD46 vector was transferred into the E. coli AT-004-02 strain. Then, a linear DNA fragment for targeting was prepared by electroporation, and positive clones were selected. Finally, the resistance gene is eliminated.
  • Competent preparation First, the Escherichia coli AT-004-02 (AT-003-02, ⁇ nagA) stock solution stored at -20 ° C was inoculated in 10 ml LB liquid medium at 1:50-100, 37 Incubate for 2-3 hours at 225 rpm with °C. The culture solution was further added to a 10 ml centrifuge tube, 4000 g ⁇ 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl 2 5 ml for 5 min. Finally, it was centrifuged at 4000 g ⁇ 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl 2 5 ml. It was allowed to stand at -4 ° C for 12 hours and settled naturally.
  • Plasmid transformation 250 ⁇ l of naturally-precipitated cells were taken, and 5 ⁇ l of pKD46 plasmid was added at -4 ° C for 30 min. Then, in a 42 ° C water bath for 1.5 min, 0.7 ml of SOC medium was added, and the mixture was shaken at 30 ° C for 2 hours. Take 0.2 ml of bacterial solution and apply penicillin plate. Incubate overnight (12-16 hours) at 30 °C. Monoclones were picked, cultured in 5 ml of LB liquid medium, and plasmids were identified. Preserve positive strains for use.
  • pCP20 was transferred into the above-mentioned caramycin resistant clone, cultured at 30 ° C for 8 h, and then increased to 42 ° C overnight, heat-induced FLP recombinase expression, and the plasmid was gradually lost.
  • the antimony inoculum was plated on the antibiotic-free medium, and the grown monoclonal spot was picked onto the caramycin resistant plate, and the undeveloped clone in which the caramycin resistance gene had been deleted by the FLP recombinase. Clones with the disappearance of resistance to caramycin were identified by PCR using the identified primers.
  • This example describes the cloning of the gene nanE of N-acetyl-D-aminomannose-6-P isomerase (NanE) and the transformation of the nanE/pTrc99A plasmid in E. coli, and the ptrc-nanE gene cassette into the E. coli chromosome. Integration.
  • the Uscherichia coli nanE gene nucleotide sequence SEQ ID No. 16 was obtained according to NCBI, and the amino acid sequence of SEQ ID No. 17 was obtained.
  • AT-001 Erscherichia coli ATCC 27325 genome.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • Amplification product size 690 bp.
  • the PCR product was separated and purified by 1% agarose gel electrophoresis.
  • the obtained PCR amplified fragment and the pUC57-T vector were ligated and sequenced to obtain nanE/pUC57.
  • Plasmid construction Amplification of plasmid nanE/pUC57, digestion of plasmid nanE/pUC57 and vector pTrc99A with Nco I and HindIII, respectively, separation and purification of nanE fragment and pTrc99A fragment by agarose gel electrophoresis, using T4 DNA ligase, 16 ° C The mixture was ligated overnight and identified to give the nanE/pTrc99A plasmid.
  • Competency preparation First, the AT-005-02 bacterial solution stored at -20 ° C was inoculated into 10 ml of LB liquid medium at 1:50-100, and cultured at 37 ° C, 225 rpm, and shaken for 2-3 hours. The culture solution was further added to a 10 ml centrifuge tube, 4000 g ⁇ 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl 2 5 ml for 5 min. Finally, it was centrifuged at 4000 g ⁇ 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl 2 5 ml. It was allowed to stand at -4 ° C for 12 hours and settled naturally.
  • Plasmid transformation 250 ⁇ l of naturally-precipitated cells were taken, and 5 ⁇ l of nanE/pTrc99A plasmid was added at -4 ° C for 30 min. Then, in a 42 ° C water bath for 1.5 min, 0.7 ml of SOC medium was added, and the mixture was shaken at 30 ° C for 2 hours. Take 0.2 ml of bacterial solution and apply penicillin plate. Incubate overnight (12-16 hours) at 30 °C. Monoclones were picked, cultured in 5 ml of LB liquid medium, and plasmids were identified. Preserve positive strains for use. Obtained recombinant nanE/pTrc99A (AT-005-02)
  • the recombinant strain nanE/pTrc99A (AT-005-02) and the control strain were subjected to a shake flask fermentation test.
  • the monoclonal strain on the freshly cultured LB plate medium was inoculated into a 3 ml LB liquid medium test tube (13 x 150 mm), and cultured at 30 ° C, 225 rpm for about 8 hours.
  • LB liquid medium composition 5 g/l yeast powder, 10 g/l peptone, 10 g/l NaCl. Then, the seed culture solution was taken, and 3% was inoculated into a 250 ml shake flask containing 50 ml of the fermentation broth (M9 medium).
  • the initial OD 600 was about 0.5, cultured at 225 rpm at 37 ° C, and the fermentation cycle was 72 hours.
  • the pH of the fermentation broth was adjusted to 7.0 with 10 M NaOH.
  • 65% glucose solution was added in portions to maintain the glucose concentration at 20 g/L.
  • 1 ml of the fermentation broth was taken and centrifuged.
  • the N-acetyl-D-glucosamine content was determined by HPLC.
  • Buffer Add 3.5g of dipotassium hydrogen phosphate to a 1L volumetric flask, add enough water to dissolve, add 0.25mL of ammonia water, add water to dilute and mix, adjust the pH to 7.5 with phosphoric acid, and dilute to volume with water.
  • Standard solution 1.0 mg/mL USP N-acetyl-D-glucosamine standard (RS) was dissolved in the diluent.
  • Sample solution 1.0 mg/mL N-acetyl-D-glucosamine sample was dissolved in the diluent.
  • 5 ⁇ M9 medium was prepared: 64 g of Na 2 HPO 4 ⁇ 7H 2 O, 15 g of KH 2 PO 4 , 2.5 g of NaCl, 5.0 g of NH 4 Cl were added to about 800 ml of double distilled water (ddH 2 O), and dissolved. Add water to 1000ml. Sterilize at 121 ° C for 30 minutes. 1 M MgSO 4 , 1 M CaCl 2 , 20% glucose were separately prepared and sterilized separately. Then, M9 culture solution was prepared according to Table 1, wherein 1000 ⁇ trace element solution was prepared according to Table 2.
  • the yield of shake flask fermentation is shown in Table 3. The results showed that the yield of the control strain AT-005-02 was very low and not detected, and the yield of the recombinant nanE/pTrc99A (AT-005-02) overexpressed by the nanE gene under the control of the Trc promoter was significantly increased.
  • the pTrc-nanE gene cassette is integrated into the E. coli chromosome.
  • the nagE gene locus is the integration site of the pTrc-nanE gene cassette on the chromosome.
  • the nanE fragment pTrc-nanE with the Trc promoter and the carrageenin with the FLP recombinase recognition site (FRT site) on both sides were amplified. Resistance gene fragment: FRT-Kanr-FRT (fKanrf), and spliced.
  • the primers for deleting the homologous arm of the nagE gene sequence were designed again, and the full-length linear DNA fragment of Red recombinant targeting was amplified by using the fragment fused by pTrc-nanE and fKanrf as a template.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • the PCR product was separated and purified by 1% agarose gel electrophoresis.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • fKanrf size 1.28kb. Its nucleotide sequence is SEQ ID No. 3.
  • the PCR product was separated and purified by 1% agarose gel electrophoresis.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • the size of the second amplified fKanrf was 1.3 kb.
  • the PCR product was separated and purified by 1% agarose gel electrophoresis.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • Amplification product homology arm + pTrc-nanE-fKanrf + homology arm.
  • the PCR product was separated by agarose gel electrophoresis, purified and recovered, and 100 ng/ ⁇ l of linear DNA full-length PCR fragment was obtained for Red recombinant targeting.
  • the pKD46 vector was transferred into the E. coli AT-004-02 strain. Then, a linear DNA fragment for targeting was prepared by electroporation, and positive clones were selected. Finally, the resistance gene is eliminated.
  • Competent preparation First, the Escherichia coli AT-004-02 stock solution stored at -20 ° C was inoculated in 10 ml LB liquid medium at 1:50-100, and cultured at 37 ° C, 225 rpm, shaking for 2-3 hours. . The culture solution was further added to a 10 ml centrifuge tube, 4000 g ⁇ 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl 2 5 ml for 5 min. Finally, it was centrifuged at 4000 g ⁇ 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl 2 5 ml. It was allowed to stand at -4 ° C for 12 hours and settled naturally.
  • Plasmid transformation 250 ⁇ l of naturally-precipitated cells were taken, and 5 ⁇ l of pKD46 plasmid was added at -4 ° C for 30 min. Then, in a 42 ° C water bath for 1.5 min, 0.7 ml of SOC medium was added, and the mixture was shaken at 30 ° C for 2 hours. Take 0.2 ml of bacterial solution and apply penicillin plate. Incubate overnight (12-16 hours) at 30 °C. Pick a single, add 5ml LB solution The medium was cultured in the medium, and the plasmid was identified. Preserve positive strains for use.
  • pCP20 was transferred into the above-mentioned caramycin resistant clone, cultured at 30 ° C for 8 h, and then increased to 42 ° C overnight, heat-induced FLP recombinase expression, and the plasmid was gradually lost.
  • the antimony inoculum was plated on the antibiotic-free medium, and the grown monoclonal spot was picked onto the caramycin resistant plate, and the undeveloped clone in which the caramycin resistance gene had been deleted by the FLP recombinase. Clones with the disappearance of resistance to caramycin were identified by PCR using the identified primers.
  • the recombinant strain AT-030-02 and the control strain in which the pTrc-nanE gene cassette was integrated at the chromosome nagE gene locus were subjected to a shake flask fermentation test.
  • the monoclonal strain on the freshly cultured LB plate medium was inoculated into a 3 ml LB liquid medium test tube (13 x 150 mm), and cultured at 30 ° C, 225 rpm for about 8 hours. Then, the seed culture solution was taken, and 3% was inoculated into a 250 ml shake flask containing 50 ml of the fermentation broth (M9 medium).
  • the initial OD 600 was about 0.5, cultured at 225 rpm at 37 ° C, and the fermentation cycle was 72 hours.
  • the pH of the fermentation broth was adjusted to 7.0 with 10 M NaOH.
  • 65% glucose solution was added in portions to maintain the glucose concentration at 20 g/L.
  • 1 ml of the fermentation broth was taken and centrifuged.
  • the N-acetyl-D-glucosamine content was determined by HPLC.
  • This example describes the screening of a gene for the mutated N-acetyl-D-aminomannose-6-P isomerase (NanE), which encodes an N-acetyl-D-aminomannose-6-P with increased enzymatic activity. Isomerase.
  • a gene mutant encoding N-acetyl-D-aminomannose-6-P isomerase having an increased enzyme activity was screened.
  • the cloned gene is amplified by error-prone PCR technology, and the gene is amplified by a DNA polymerase for amplification under conditions that cause high frequency mismatches to obtain high frequency mutations in the PCR product. .
  • Taq DNA polymerase does not have the 3'-5' proofreading property at high magnesium ion concentration (8mmol/L) and different concentrations of dNTP (where dATP and dGTP concentrations are 1.5mmol/L; dTTP and dCTP)
  • concentration of 3.0mmol/L was used to control the frequency of random mutations, and random mutations were introduced into the target gene to construct a mutant library.
  • the template concentration of A260 was 1000 ng/mL, and the enzyme concentration was 5 U/ ⁇ L. The concentration was 100 ⁇ M.
  • Error-prone PCR reaction system 10 ⁇ l of PCR reaction buffer, 5 ⁇ l of dNTP (2.5 mM), 5 ⁇ l of MgCl 2 (2.5 mM), 1 ⁇ l of forward primer (nanE-F, SEQ ID No. 18), reversed Primer (nanE-R, SEQ ID No. 19) 1 ⁇ l, DNA template (nanE/pUC57) 0.1 ⁇ l, Taq DNA polymerase 0.5 ⁇ l, ddH 2 O 32.4 ⁇ l.
  • PCR procedure pre-denaturation at 96 °C for 4 min; denaturation at 94 °C for 1 min, annealing at 56 °C for 1 min, extension at 75 °C for 2 min, 45 cycles; final extension at 75 °C for 15 min, recovery of PCR product by gel recovery method (product size: 0.7 kb); 5 ⁇ l of the product was examined by 1% agarose gel electrophoresis and stored at -20 ° C until use.
  • the above PCR product was digested with restriction endonucleases Nco I and Hind III, and ligated with the pTrc99A plasmid digested with Nco I and Hind III endonuclease, and then transformed into E. coli AT-005 with the ligation product mixture. -02, a large number of cloned transformants were obtained, and a transformed mutant library was constructed.
  • Activity assay of N-acetyl-D-aminomannose-6-P isomerase conversion to N-acetyl-D-amino group with N-acetyl-D-aminomannose-6-phosphate (ManNAc-6-P) Based on the amount of glucose-6-phosphate (GlcNAc-6-P), that is, N-acetyl-D-aminomannose-6-phosphate is reduced to the assay label.
  • Enzyme unit definition The amount of enzyme required to reduce the equivalent of 1 ⁇ mol of N-acetyl-D-aminomannose-6-phosphate per minute under enzymatic reaction conditions, defined as an enzyme activity unit (IU).
  • an isotope-labeled ManNAc-6-P is prepared as a substrate.
  • the reaction was terminated by the addition of 350 ul of ethanol.
  • the product was eluted with water and lyophilized.
  • reaction solution a total volume of 26.5 ul of the reaction solution was prepared as an enzyme activity assay system containing 1 mM isotope-labeled ManNAc-6-P, 37 mM Tris-HCl, pH 8.0 and 19 mM MgCl 2 .
  • the reaction was boiled for 3 min, then 0.1 volume of alkaline phosphatase buffer was added to adjust the pH and 20 units of alkaline phosphatase. After incubation at 37 ° C for 1 hour, samples were taken onto dry chromatography paper and pre-soaked with 1% sodium tetraborate.
  • the solvent system used was ethyl acetate: isopropanol: pyridine: water (50:22:14:14).
  • the radioactive compound was separated by chromatography on paper. The radioactivity was measured by a liquid scintillation counter, and the activity unit of N-acetyl-D-aminomannose-6-P isomerase was calculated from the amount of ManNAc-6-P converted to GlcNAc-6-P.
  • the NanE was modified by error-prone PCR to obtain a mutant strain with greatly improved enzyme activity.
  • the mutant strain with the highest activity of the enzyme was selected and plasmid sequencing was performed.
  • the pTrc-nanEM gene cassette integrates into the nagE gene locus of E. coli
  • the nagE gene locus is the integration site of the pTrc-nanEM gene cassette on the chromosome.
  • the nanEM fragment pTrc-nanEM with the Trc promoter and the claratinmycin flanked by the FLP recombinase recognition site (FRT site) were amplified. Resistance gene fragment: FRT-Kanr-FRT (fKanrf), and spliced.
  • the primers for deleting the homologous arm of the nagE gene sequence were designed again, and the full-length linear DNA fragment of Red recombinant targeting was amplified by using the fragment of pTrc-nanEM and fKanrf splicing as a template.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • the PCR product was separated and purified by 1% agarose gel electrophoresis.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • fKanrf size 1.28kb. Its nucleotide sequence is SEQ ID No. 3.
  • the PCR product was separated and purified by 1% agarose gel electrophoresis.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • the size of the second amplified fKanrf was 1.3 kb.
  • the PCR product was separated and purified by 1% agarose gel electrophoresis.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • Amplification product homology arm + pTrc-nanEM-fKanrf + homology arm.
  • the PCR product was separated by agarose gel electrophoresis, purified and recovered, and 100 ng/ ⁇ l of linear DNA full-length PCR fragment was obtained for Red recombinant targeting.
  • the pKD46 vector was transferred into the E. coli AT-004-02 strain. Then, a linear DNA fragment for targeting was prepared by electroporation, and positive clones were selected. Finally, the resistance gene is eliminated.
  • Competent preparation First, the Escherichia coli AT-004-02 stock solution stored at -20 ° C was inoculated in 10 ml LB liquid medium at 1:50-100, and cultured at 37 ° C, 225 rpm, shaking for 2-3 hours. . The culture solution was further added to a 10 ml centrifuge tube, 4000 g ⁇ 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl 2 5 ml for 5 min. Finally, it was centrifuged at 4000 g ⁇ 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl 2 5 ml. It was allowed to stand at -4 ° C for 12 hours and settled naturally.
  • Plasmid transformation 250 ⁇ l of naturally-precipitated cells were taken, and 5 ⁇ l of pKD46 plasmid was added at -4 ° C for 30 min. Then, in a 42 ° C water bath for 1.5 min, 0.7 ml of SOC medium was added, and the mixture was shaken at 30 ° C for 2 hours. Take 0.2 ml of bacterial solution and apply penicillin plate. Incubate overnight (12-16 hours) at 30 °C. Monoclones were picked, cultured in 5 ml of LB liquid medium, and plasmids were identified. Preserve positive strains for use.
  • pCP20 was transferred into the above-mentioned caramycin resistant clone, cultured at 30 ° C for 8 h, and then increased to 42 ° C overnight, heat-induced FLP recombinase expression, and the plasmid was gradually lost.
  • the ungrown caramycin resistant group A clone that has been deleted by FLP recombinase. Clones with the disappearance of resistance to caramycin were identified by PCR using the identified primers.
  • the recombinant strain AT-031-02 and the control strain in which the pTrc-nanEM gene cassette was integrated at the chromosome nagE gene locus were subjected to a shake flask fermentation test.
  • the monoclonal strain on the freshly cultured LB plate medium was inoculated into a 3 ml LB liquid medium test tube (13 x 150 mm), and cultured at 30 ° C, 225 rpm for about 8 hours. Then, the seed culture solution was taken, and 3% was inoculated into a 250 ml shake flask containing 50 ml of the fermentation broth (M9 medium).
  • the initial OD600 was about 0.5, cultured at 225 rpm at 37 ° C, and the fermentation cycle was 72 hours.
  • the pH of the fermentation broth was adjusted to 7.0 with 10 M NaOH.
  • 65% glucose solution was added in portions to maintain the glucose concentration at 20 g/L.
  • 1 ml of the fermentation broth was taken and centrifuged.
  • the N-acetyl-D-glucosamine content was determined by HPLC.
  • This example describes an E. coli strain integrated with a pTrc-nanEM cassette in which glucosamine-6- Phospho-synthase (GlmS) gene glmS and/or D-glucosamine-6-phosphate deaminase (NagB) gene nagB endogenous natural promoter replacement and / or deletion of N-acetyl-D-glucosamine The impact of production.
  • GlmS glucosamine-6- Phospho-synthase
  • NagB D-glucosamine-6-phosphate deaminase
  • D-glucosamine-6-phosphate deaminase (NagB) gene in the nagE-nagBACD was deleted and replaced with the Trc promoter.
  • the reaction catalyzed by D-Glucosamine-6-phosphate deaminase (NagB) is reversible, over-expressing nagB, accelerating the forward catalytic reaction of NagB, and increasing D-glucosamine- 6-phosphoric acid (GlcN-6-P) purpose.
  • Trc promoter fragment and the fKanrf fragment were amplified and spliced. Then, a homology arm primer was designed to amplify a full-length linear DNA fragment for Red recombinant targeting.
  • Trc promoter sequence was found: SEQ ID No. 28.
  • 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, Cycle 30 times
  • third step extend at 72 ° C for 10 min.
  • fKanrf size 1.28kb. Its nucleotide sequence is SEQ ID No. 3.
  • the PCR product was separated and purified by 1% agarose gel electrophoresis.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • the size of the second amplified fKanrf was 1.3 kb.
  • the PCR product was separated and purified by 1% agarose gel electrophoresis.
  • Trc promoter PCR fragment and secondary amplified fKanrf PCR fragment, 1:1 mixing.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • Amplification product homology arm + fKanrf + Trc promoter + homology arm.
  • the PCR product was separated by agarose gel electrophoresis, purified and recovered, and 100 ng/ ⁇ l of linear DNA full-length PCR fragment was obtained for Red recombinant targeting.
  • the pKD46 vector was transferred into the E. coli AT-031-02 strain. Then, a linear DNA fragment for targeting was prepared by electroporation, and positive clones were selected. Finally, the resistance gene is eliminated.
  • AT-032 AT-031-02, ⁇ nagB promotor::Trc promoter.
  • Glucosamine-6-phosphate synthase (glmS) gene promoter sequence was deleted.
  • Glucosamine-6-phosphate synthase (GlmS) also known as L-glutamine-6-phosphate aminotransferase, catalyzes glucose-6-phosphate (Glc-6) -P) amination to D-glucosamine-6-phosphate (GlcN-6-P), but severe
  • the product inhibits the problem, deleting its promoter sequence, and losing the expression of the enzyme, which can inhibit the inhibition of GlcN-6-P product.
  • 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-032 strain. Then, a linear DNA fragment for targeting was prepared by electroporation, and positive clones were selected. Finally, the resistance gene is eliminated.
  • AT-033 AT-032, ⁇ glmS promotor.
  • the nagB promoter was replaced with a promoter of a higher expression level, and the recombinant strain in which the glmS promoter was further deleted was subjected to a shake flask fermentation test.
  • the monoclonal strain on the freshly cultured LB plate medium was inoculated into a 3 ml LB liquid medium test tube (13 x 150 mm), and cultured at 30 ° C, 225 rpm for about 8 hours. Then, the seed culture solution was taken, and 3% was inoculated into a 250 ml shake flask containing 50 ml of the fermentation broth (M9 medium).
  • the initial OD600 was about 0.5, cultured at 225 rpm at 37 ° C, and the fermentation cycle was 72 hours.
  • the pH of the fermentation broth was adjusted to 7.0 with 10 M NaOH.
  • 65% glucose solution was added in portions to maintain the glucose concentration at 20 g/L.
  • 1 ml of the fermentation broth was taken and centrifuged.
  • the N-acetyl-D-glucosamine content was determined by HPLC.
  • L-glutamine-6-phosphate aminotransferase gene promoter sequence was replaced with a Trc promoter sequence.
  • L-Glutamine-6-phosphate fructose aminotransferase also known as Glucosamine-6-phosphate synthase (GlmS) replaces its promoter sequence with the Trc promoter sequence, overexpressing glmS, accelerating GlmS catalyzes the function of increasing D-glucosamine-6-phosphate (GlcN-6-P).
  • Trc promoter sequence fragment and the fKanrf fragment were amplified and spliced. Then, a homology arm primer was designed to amplify a full-length linear DNA fragment for Red recombinant targeting.
  • Designing the homology arm primer designing the homologous arm forward primer (ProglmspTrc-F) SEQ ID No. 38, reverse primer (ProglmspTrc-R), which was replaced with the Trc promoter according to the glmS gene promoter sequence SEQ ID No. SEQ ID No. 39.
  • 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-031-02 strain. Then, a linear DNA fragment for targeting was prepared by electroporation, and positive clones were selected. Finally, the resistance gene is eliminated.
  • AT-034 AT-031-02, ⁇ 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. 40, 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. 41.
  • 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 E. coli AT-034 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-035 (AT-034, ⁇ nagB promotor).
  • the glmS promoter was replaced with a promoter of a higher expression level, and the recombinant strain in which the nagB promoter was further deleted was subjected to a shake flask fermentation test.
  • the monoclonal strain on the freshly cultured LB plate medium was inoculated into a 3 ml LB liquid medium test tube (13 x 150 mm), and cultured at 30 ° C, 225 rpm for about 8 hours. Then, the seed culture solution was taken, and 3% was inoculated into a 250 ml shake flask containing 50 ml of the fermentation broth (M9 medium).
  • the initial OD600 was about 0.5, cultured at 225 rpm at 37 ° C, and the fermentation cycle was 72 hours.
  • the pH of the fermentation broth was adjusted to 7.0 with 10 M NaOH.
  • 65% glucose solution was added in portions to maintain the glucose concentration at 20 g/L.
  • 1 ml of the fermentation broth was taken and centrifuged.
  • the N-acetyl-D-glucosamine content was determined by HPLC.
  • This example describes an E. coli strain integrated with a pTrc-nanEM cassette in which the gene wecB overexpressing UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) and its N-acetyl-D-amino group The effect of glucose production.
  • WecB UDP-N-acetyl-D-glucosamine-2-isomerase
  • UDP-N-acetylglucosamine-2-isomerase UDP-N-acetyl-D-glucosamine -2-epimerase, WecB gene wecB, placed under the control of Trc promoter, or the endogenous natural promoter of wecB gene into Trc promoter, over-expression, can strengthen UDP-GlcNAc (UDP- N-acetyl glucosamine, UDP-N-acetyl-D-glucosamine) becomes ManNAc (N-Acetyl-D-mannosamine, N-acetyl-D-aminomannose or N-acetyl-D-mannosamine).
  • UDP-GlcNAc UDP- N-acetyl glucosamine
  • UDP-N-acetyl-D-glucosamine UDP-N-acetyl-D-glucosamine
  • ManNAc N-Acetyl-D-mannosamine, N-acetyl
  • the nucleotide sequence of the E. coli wecB gene SEQ ID No. 42 was found according to NCBI, and its amino acid sequence is SEQ ID No. 43.
  • AT-001 Erscherichia coli ATCC 27325 genome.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • Amplification product size 1.13 Kb.
  • the PCR product was separated and purified by 1% agarose gel electrophoresis.
  • the obtained PCR amplified fragment and the vector pTrc99A were digested with Nco I and Hind III respectively, and the wecB fragment and the pTrc99A fragment were separated and purified by agarose gel electrophoresis, and ligated with T4 DNA ligase at 16 ° C overnight, and identified. wecB/pTrc99A plasmid.
  • Competent preparation First, the recombinant strain AT-031-02 stored at -20 °C is inoculated into 10 ml of LB liquid medium at 1:50-100, shaken at 37 ° C, 225 rpm, 2-3 hour. The culture solution was further added to a 10 ml centrifuge tube, 4000 g ⁇ 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl 2 5 ml for 5 min. Finally, it was centrifuged at 4000 g ⁇ 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl 2 5 ml. It was allowed to stand at -4 ° C for 12 hours and settled naturally.
  • Plasmid transformation 250 ⁇ l of the naturally-precipitated cells were taken, and 5 ⁇ l of the wecB/pTrc99A plasmid was added at -4 ° C for 30 min. Then, in a 42 ° C water bath for 1.5 min, 0.7 ml of SOC medium was added, and the mixture was shaken at 30 ° C for 2 hours. Take 0.2 ml of bacterial solution and apply penicillin plate. Incubate overnight (12-16 hours) at 30 °C. Monoclones were picked, cultured in 5 ml of LB liquid medium, and plasmids were identified. Save positive clones for use.
  • strain number AT-036 (AT-031-02, wecB/pTrc99A).
  • Trc promoter sequence fragment and the fKanrf fragment were amplified and spliced. Then, a homology arm primer was designed to amplify a full-length linear DNA fragment for Red recombinant targeting.
  • Trc promoter PCR fragment and secondary amplified fKanrf PCR fragment, 1:1 mixing.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • Amplification product homology arm + fKanrf + Trc promoter + homology arm.
  • the PCR product was separated by agarose gel electrophoresis, purified and recovered, and 100 ng/ ⁇ l of linear DNA full-length PCR fragment was obtained for Red recombinant targeting.
  • the pKD46 vector was transferred into the E. coli AT-031-02 strain. Then, a linear DNA fragment for targeting was prepared by electroporation, and positive clones were selected. Finally, the resistance gene is eliminated.
  • the recombinant strain produced by the strain overexpressing the wecB (including the strain transformed with the wecB/pTrc99A and the replacement of the wecB promoter into the Trc promoter) was subjected to a shake flask fermentation test.
  • the monoclonal strain on the freshly cultured LB plate medium was inoculated into a 3 ml LB liquid medium test tube (13 x 150 mm), and cultured at 30 ° C, 225 rpm for about 8 hours. Then, the seed culture solution was taken, and 3% was inoculated into a 250 ml shake flask containing 50 ml of M9 medium.
  • the initial OD 600 was about 0.5, cultured at 225 rpm at 37 ° C, and the fermentation cycle was 72 hours.
  • the pH of the fermentation broth was adjusted to 7.0 with 10 M NaOH.
  • 65% glucose solution was added in portions to maintain the glucose concentration at 20 g/L.
  • 1 ml of the fermentation broth was taken and centrifuged.
  • the N-acetyl-D-glucosamine content was determined by HPLC.
  • This example describes an E. coli strain that incorporates a pTrc-nanEM cassette and replaces and/or deletes the endogenous native promoter of the glmS gene and the nagB gene, transforms the wecB/pTrc99A plasmid or the wecB gene endogenous natural promoter. Effect of switching to Trc promoter on N-acetyl-D-glucosamine production
  • the wecB/pTrc99A plasmid was transformed into the integrated pTrc-NanEM cassette by CaCl 2 transformation, and the endogenous natural promoter of nagB gene was replaced with the Trc promoter and the endogenous natural promoter of glmS gene was deleted at the same time.
  • the E. coli strain AT-033 monoclonal culture was picked and the positive clones were identified by plasmid extraction.
  • strain number AT-038 (AT-033, wecB/pTrc99A).
  • the pKD46 vector was transferred into E. coli AT-033 strain. Then, a linear DNA fragment for targeting was prepared by electroporation, and positive clones were selected. Finally, the resistance gene is eliminated.
  • AT-039 AT-033, ⁇ wecB promotor::Trc promoter.
  • the wecB/pTrc99A plasmid was transformed into the integrated pTrc-nanEM cassette by CaCl 2 transformation, and the endogenous natural promoter of the glmS gene was replaced with the Trc promoter and the endogenous natural promoter of the nagB gene was simultaneously deleted.
  • the monoclonal culture was picked and the positive clones were identified by plasmid extraction.
  • the obtained strain number AT-040 (AT-035, wecB/pTrc99A).
  • Trc promoter of the wecB gene was exchanged for the E. coli strain in which the pTrc-nanEM cassette was integrated and the endogenous natural promoter of the glmS gene was replaced with the Trc promoter and the endogenous natural promoter of the nagB gene was simultaneously deleted. Trc promoter
  • the pKD46 vector was transferred into the E. coli AT-035 strain. Then, a linear DNA fragment for targeting was prepared by electroporation, and positive clones were selected. Finally, the resistance gene is eliminated.
  • AT-041 AT-035, ⁇ wecB promotor::Trc promoter.
  • the E. coli strain in which the pTrc-nanEM cassette is integrated and the endogenous natural promoter of the nagB gene and the glmS gene are replaced and/or deleted is transformed into the wecB/pTrc99A plasmid and the endogenous natural promoter of the wecB gene is replaced with Effect of Trc promoter on N-acetyl-D-glucosamine production
  • the endogenous natural promoter of the glmS gene and the nagB gene is replaced and/or deleted, and the wecB/pTrc99A plasmid is transformed or the wecB gene endogenous natural promoter is replaced with the Trc promoter.
  • the different genotypes of recombinant bacteria formed were used for shake flask fermentation experiments.
  • the monoclonal strain on the freshly cultured LB plate medium was inoculated into a 3 ml LB liquid medium test tube (13 x 150 mm), and cultured at 30 ° C, 225 rpm for about 8 hours. Then take the seed culture solution and inoculate 3% in 50ml.
  • the fermentation broth (M9 medium) was shaken in a 250 ml shake flask.
  • the initial OD600 was about 0.5, cultured at 225 rpm at 37 ° C, and the fermentation cycle was 72 hours.
  • the pH of the fermentation broth was adjusted to 7.0 with 10 M NaOH.
  • 65% glucose solution was added in portions to maintain the glucose concentration at 20 g/L.
  • 1 ml of the fermentation broth was taken and centrifuged.
  • the N-acetyl-D-glucosamine content was determined by HPLC.
  • the yield of shake flask fermentation is shown in Table 9.
  • Table 9 The results showed that compared with the control strains AT-033 or AT-035, the transformation of wecB/pTrc99A plasmid resulted in a significant increase in N-acetyl-D-glucosamine production, and the replacement of the wecB promoter was a recombinant strain of the Trc promoter against N- There is a greater increase in acetyl-D-glucosamine production.
  • This example describes the production of N-acetyl-D-glucosamine fermentation in a 10 L fermentor.
  • the recombinant engineering strain AT-039 was used as the production strain, and the N-acetyl-D-glucosamine fermentation test was carried out in a 10 L fermentor.
  • Secondary seed culture 6 ml of the primary seed culture solution was taken, inoculated into a 1000 ml shake flask containing 200 ml of M9 medium, and cultured at 37 ° C, 225 rpm for 16 hours.
  • the OD 600 value is 6.0-10, which is about the middle of logarithmic growth.
  • the fermentation medium was prepared according to Table 10, wherein the trace element solution was prepared according to Table 11, and the multivitamin solution was prepared according to Table 12.
  • 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.
  • This example describes the screening of a gene for the mutated UDP-N-acetylglucosamine-2-isomerase (WecB), which encodes a UDP-N-acetylglucosamine-2-isomerase with increased enzymatic activity.
  • WecB mutated UDP-N-acetylglucosamine-2-isomerase
  • a gene mutant encoding UDP-N-acetylglucosamine-2-isomerase having an increased enzyme activity was screened.
  • the cloned gene is amplified by error-prone PCR technology, and the gene is amplified by a DNA polymerase for amplification under conditions that cause high frequency mismatches to obtain high frequency mutations in the PCR product. .
  • Taq DNA polymerase does not have the 3'-5' proofreading property at high magnesium ion concentration (8mmol/L) and different concentrations of dNTP (where dATP and dGTP concentrations are 1.5mmol/L; dTTP and dCTP) The concentration was 3.0mmol/L) to control the frequency of random mutations, and random mutations were introduced into the target gene to construct a mutant library.
  • the template concentration of A260 was 1000 ng/mL, the enzyme concentration was 5 U/ ⁇ L, and the primer concentration was 100 ⁇ M.
  • Error-prone PCR reaction system 50 ⁇ l: 5 ⁇ l of 10 ⁇ PCR reaction buffer, 5 ⁇ l of dNTP (2.5 mM), 5 ⁇ l of MgCl 2 (2.5 mM), 1 ⁇ l of forward primer (Trcwec B-F, SEQ ID No. 44), reversed Primer (TrcwecB-R, SEQ ID No. 45) 1 ⁇ l, DNA template (wecB/pUC57) 0.1 ⁇ l, Taq DNA polymerase 0.5 ⁇ l, ddH 2 O 32.4 ⁇ l.
  • PCR procedure pre-denaturation at 96 °C for 4 min; denaturation at 94 °C for 1 min, annealing at 56 °C for 1 min, extension at 75 °C for 2 min, 45 cycles; final extension at 75 °C for 15 min, recovery of PCR product by gel recovery method (product size: 1.13 kb); 5 ⁇ l of the product was examined by 1% agarose gel electrophoresis and stored at -20 ° C until use.
  • the above PCR product was digested with restriction endonucleases Nco I and Hind III, and ligated with the pTrc99A plasmid digested with NcoI and Hind III endonuclease, and then transformed into E. coli AT-005 with a ligation product mixture. 02, a large number of cloned transformants were obtained, and a transformed mutant library was constructed.
  • 640 mutant clones were randomly picked and wild-type WecB/pTrc99A (AT-005-02) was used as control.
  • the cells were inoculated into 5 ml LB medium containing 50 ⁇ g/mL penicillin (Amp). After incubation at 37 ° C, 150 rpm for 18 h, the cells were collected by centrifugation at 10,000 rpm at 5 mM.
  • Activity detection of UDP-N-acetylglucosamine-2-isomerase based on the conversion of UDP-N-acetyl-D-glucosamine to N-acetyl-D-aminomannose. That is, UDP-N-acetyl-D-glucosamine is reduced to the assay marker.
  • Enzyme unit definition The amount of enzyme required to reduce the equivalent of 1 ⁇ mol of UDP-N-acetyl-D-glucosamine per minute under enzymatic reaction conditions, defined as an enzyme activity unit (IU).
  • the specific operation was as follows: a 20 ml reaction system was used as an enzyme activity assay system, which contained 45 mmol/L phosphate buffer (pH 7.5), 10 mM MgCl2 and 100 nCi of UDPGlcNAc, and 5 mg of crude enzyme solution. The enzyme was incubated for 30 min in a 37 ° C water bath. The reaction was terminated by the addition of ethanol. The radioactive compound was separated by chromatography on paper. The radioactivity was measured by a liquid scintillation counter. The solvent system used was n-propanol: 1 M sodium acetate, pH 5.0: water (7:1:2). The activity unit of UDP-N-acetylglucosamine-2-isomerase was calculated based on how much UDPGlcNAc was converted to ManNAc.
  • the pTrc-wecBM gene cassette integrates into the nagE gene locus of E. coli
  • the nagE gene locus is the integration site of the pTrc-wecBM gene cassette on the chromosome.
  • the wecBM fragment pTrc-wecBM with the Trc promoter and the carrageenin with the FLP recombinase recognition site (FRT site) on both sides were amplified. Resistance gene fragment: FRT-Kanr-FRT (fKanrf), and spliced.
  • the primer for deleting the homologous arm of the nagE gene sequence was designed again, and the full-length linear DNA fragment of Red recombinant targeting was amplified by using the fragment fused with pTrc-wecBM and fKanrf as a template.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • the PCR product was separated and purified by 1% agarose gel electrophoresis.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • fKanrf size 1.28kb. Its nucleotide sequence is SEQ ID No. 3.
  • the PCR product was separated and purified by 1% agarose gel electrophoresis.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles
  • third step 72 ° C for 10 min.
  • the size of the second amplified fKanrf was 1.3 kb.
  • the PCR product was separated and purified by 1% agarose gel electrophoresis.
  • first step denaturation at 94 ° C for 1 min
  • second step 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, Cycle 30 times
  • third step extend at 72 ° C for 10 min.
  • Amplification product homology arm + pTrc-wecBM-fKanrf + homology arm.
  • the PCR product was separated by agarose gel electrophoresis, purified and recovered, and 100 ng/ ⁇ l of linear DNA full-length PCR fragment was obtained for Red recombinant targeting.
  • the pKD46 vector was transferred into the E. coli AT-004-02 strain. Then, a linear DNA fragment for targeting was prepared by electroporation, and positive clones were selected. Finally, the resistance gene is eliminated.
  • Competent preparation First, the Escherichia coli AT-004-02 stock solution stored at -20 ° C was inoculated in 10 ml LB liquid medium at 1:50-100, and cultured at 37 ° C, 225 rpm, shaking for 2-3 hours. . The culture solution was further added to a 10 ml centrifuge tube, 4000 g ⁇ 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl 2 5 ml for 5 min. Finally, it was centrifuged at 4000 g ⁇ 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl 2 5 ml. It was allowed to stand at -4 ° C for 12 hours and settled naturally.
  • Plasmid transformation 250 ⁇ l of naturally-precipitated cells were taken, and 5 ⁇ l of pKD46 plasmid was added at -4 ° C for 30 min. Then, in a 42 ° C water bath for 1.5 min, 0.7 ml of SOC medium was added, and the mixture was shaken at 30 ° C for 2 hours. Take 0.2 ml of bacterial solution and apply penicillin plate. Incubate overnight (12-16 hours) at 30 °C. Monoclones were picked, cultured in 5 ml of LB liquid medium, and plasmids were identified. Preserve positive strains for use.
  • strain AT-042-01 (AT-004-02, ⁇ nagE::pTrc-wecB-fKanrf) was prepared in the same manner as above.
  • pCP20 was transferred into the above-mentioned caramycin resistant clone, cultured at 30 ° C for 8 h, and then increased to 42 ° C overnight, heat-induced FLP recombinase expression, and the plasmid was gradually lost.
  • the antimony inoculum was plated on the antibiotic-free medium, and the grown monoclonal spot was picked onto the caramycin resistant plate, and the undeveloped clone in which the caramycin resistance gene had been deleted by the FLP recombinase. Clones with the disappearance of resistance to caramycin were identified by PCR using the identified primers.
  • strain AT-042-02 (AT-004-02, ⁇ nagE::pTrc-wecB) was prepared in the same manner as above.
  • the recombinant strains AT-042-02, AT-043-02 and the control strains in which the pTrc-wecB and pTrc-wecBM gene cassettes were integrated at the chromosome nagE gene locus were subjected to a shake flask fermentation test.
  • the monoclonal strain on the freshly cultured LB plate medium was inoculated into a 3 ml LB liquid medium test tube (13 x 150 mm), and cultured at 30 ° C, 225 rpm for about 8 hours. Then, the seed culture solution was taken, and 3% was inoculated into a 250 ml shake flask containing 50 ml of the fermentation broth (M9 medium).
  • the initial OD600 was about 0.5, cultured at 225 rpm at 37 ° C, and the fermentation cycle was 72 hours.
  • the pH of the fermentation broth was adjusted to 7.0 with 10 M NaOH.
  • 65% glucose solution was added in portions to maintain the glucose concentration at 20 g/L.
  • 1 ml of the fermentation broth was taken and centrifuged.
  • the N-acetyl-D-glucosamine content was determined by HPLC.
  • the yield of shake flask fermentation is shown in Table 13. The results showed that the yield of the control strain AT-005-02 was very low and was not detected. The yield of the recombinant pTrc-wecBM gene cassette integrated recombinant strain AT-043-02 was significantly improved, and compared with the unmutated control strain AT-042- 02 production has also increased significantly.

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Abstract

La présente invention concerne un procédé de production de la N-acétyl-D-glucosamine et/ou d'un sel de D-glucosamine par fermentation microbienne. Le procédé produit la N-acétyl-D-glucosamine et/ou le sel de D-glucosamine en augmentant l'expression d'une N-acétyl-D-amino mannose-6-phosphate isomérase dans des microbes.
PCT/CN2017/080652 2016-04-05 2017-04-14 Procédé de production de la n-acétyl-d-glucosamine et/ou d'un sel de d-glucosamine par fermentation microbienne WO2017174039A1 (fr)

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CN114592020A (zh) * 2020-12-07 2022-06-07 上海医药工业研究院 一种发酵生产n-乙酰氨基葡萄糖的方法
CN114592020B (zh) * 2020-12-07 2023-07-14 上海医药工业研究院 一种发酵生产n-乙酰氨基葡萄糖的方法

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