CN108330095A - It is a kind of accumulation N-acetyl-neuraminate recombination Corynebacterium glutamicum and its application - Google Patents

It is a kind of accumulation N-acetyl-neuraminate recombination Corynebacterium glutamicum and its application Download PDF

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CN108330095A
CN108330095A CN201810171868.6A CN201810171868A CN108330095A CN 108330095 A CN108330095 A CN 108330095A CN 201810171868 A CN201810171868 A CN 201810171868A CN 108330095 A CN108330095 A CN 108330095A
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corynebacterium glutamicum
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neuraminate
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陈坚
堵国成
王淼
刘延峰
董迅衍
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Jiangnan University
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Abstract

The invention discloses a kind of recombination Corynebacterium glutamicum of accumulation N n acetylneuraminic acid ns and its applications, belong to field of genetic engineering.The present invention is using Corynebacterium glutamicum as expressive host; by 6 phosphorylated amino transferase gene of overexpression ammonia sugar,confectioner's, Glucosamine acetylase encoding gene, phosphoric acid enzyme coding gene, acetylglucosamine isomery enzyme coding gene and N n acetylneuraminic acid n synthasee code genes, N n acetylneuraminic acid n route of synthesis is strengthened；And decompose the related gene utilized in metabolic pathway by knocking out N n acetylneuraminic acid n transport protein encoding genes and intracellular N n acetylneuraminic acid ns in Corynebacterium glutamicum, the Corynebacterium glutamicum gene engineering bacteria of extracellular accumulation N n acetylneuraminic acid ns is obtained, yield reaches 110mg/L, and producing N n acetylneuraminic acid ns for further metabolic engineering Corynebacterium glutamicum lays a good foundation.

Description

It is a kind of accumulation N-acetyl-neuraminate recombination Corynebacterium glutamicum and its application
Technical field
The present invention relates to a kind of recombination Corynebacterium glutamicum of accumulation N-acetyl-neuraminate and its applications, belong to hereditary work Journey field.
Background technology
N-acetyl-neuraminate as most important compound molecule in sialic acid, be important nutrient additive for food and Newtype drug precursor.Its effect includes promoting developing infant brain, maintaining elderly brain function health and enhancing immunity of organisms Deng.Currently, the industrial production of N-acetyl-neuraminate mainly uses chemical extraction and resting cell method to produce.Due to N- second Acyl neuraminic acid total content in natural material (yolk, bird's nest and colostrum etc.) is low, and extraction separation process is complicated, leads to tradition Extraction method yield is low, of high cost.Therefore, research in recent years hot spot is become using resting cell method production N-acetyl-neuraminate. But resting cell method is required to addition N-acetylglucosamine as synthesis precursor, and there is still a need for add for most of method Add pyruvic acid as synthesis precursor.It is higher (ten thousand yuan/ton of 6-8) in view of the Costco Wholesale of N-acetylglucosamine and pyruvic acid, Develop a kind of method using microbial fermentation glucose production N-acetyl-neuraminate by with important research significance and Application value.
Corynebacterium glutamicum is as industrial strain is commonly used, clear with genetic background, Metabolically engineered tool maturation, fermentation work Skill is ripe and fermentation process is not easy to be widely used in the fermentation method life of important chemical by many advantages such as Pollution of Phage Production.Meanwhile Corynebacterium glutamicum is aliment security level bacterial strain generally acknowledged in the world, is the reason for producing food, nutriment and drug Think bacterial strain.Therefore, it is production aliment security level N- acetyl nerve ammonia to build recombination Corynebacterium glutamicum with metabolic engineering means The effective way of acid.However, the regulation and control of N-acetyl-neuraminate metabolic pathway are tight in Corynebacterium glutamicum, N- second can't be formed The accumulation of acyl neuraminic acid.It is a worth deeply spy that N-acetyl-neuraminate metabolic pathway in Corynebacterium glutamicum, which how is transformed, The problem of begging for.
Invention content
The technical problem to be solved by the present invention is to build a kind of recombination glutamic acid rod of accumulation N-acetyl-neuraminate Bacterium.
In order to solve the above technical problems, the technical scheme is that：
By 5 genes of N-acetyl-neuraminate route of synthesis：Homologous ammonia sugar-fructose-6-phosphate aminopherase encodes base GlmS (the ammonia sugar-of coding of cause, especially Corynebacterium glutamicum (Corynebacterium glutamicum ATCC13869) The amino acid sequence of fructose-6-phosphate aminopherase such as SEQ ID NO:1)；Outer source glucosamine acetylase encodes base GNA1 (the Glucosamine acetyl of coding of cause, especially saccharomyces cerevisiae (Saccharomyces cerevisiae S288C) Change the amino acid sequence such as SEQ ID NO of enzyme:2)；External source phosphoric acid enzyme coding gene, especially Escherichia coli (Escherichia Coli K-12) the yqaB (amino acid sequence of the phosphatase of coding such as SEQ ID NO:3)；External source acetylglucosamine is different The age of structure enzyme coding gene, especially Anabaena (Anabaena sp.CH1) be (the acetylglucosamine isomerase of coding Amino acid sequence such as SEQ ID NO:4)；And external source N-acetyl-neuraminate synthasee code gene, especially Escherichia coli NeuB (the amino acid sequence of the N-acetyl-neuraminate synthase of the coding such as SEQ ID NO of (Escherichia coli K-1): 5)：It is cloned into expression vector pDXW-9, then converts Corynebacterium glutamicum (Corynebacterium glutamicum ATCC13869), by the way that metabolic pathway is transformed, the accumulation of N-acetyl-neuraminate is realized.
In one embodiment of the invention, expression vector pDXW-9 expression ammonia sugar-fructose-6-phosphate amino is selected to turn Enzyme coding gene, Glucosamine acetylase encoding gene, phosphoric acid enzyme coding gene, acetylglucosamine isomerase is moved to compile Code gene and N-acetyl-neuraminate synthasee code gene.
In one embodiment of the invention, age, neuB gene be connected to EcoRI on pDXW-9 expression vectors and Between HindIII restriction enzyme sites.
In one embodiment of the invention, glmS, GNA1, yqaB gene are connected on pDXW-9 expression vectors Between NheI and HindIII restriction enzyme sites.
In one embodiment of the invention, the N-acetyl-neuraminate transport protein of the Corynebacterium glutamicum is knocked out Encoding gene cg2937, specifically by build containing N-acetyl-neuraminate transport protein encoding gene knock out frame from Plasmid is killed, will knock out frame through homologous recombination substitutes N-acetyl-neuraminate transport protein coding base on Corynebacterium glutamicum chromosome Because of cg2937, to block N-acetyl-neuraminate from the extracellular transhipment to intracellular.
In one embodiment of the invention, the N- acetylamino mannokinases for knocking out the Corynebacterium glutamicum are compiled Code gene nanK, N- acetylamino mannose-6-phosphate isomerase encoding gene nanE, acetylglucosamine -6- phosphoric acid are de- Acetyl group enzyme coding gene nagA and Glucosamine -6- phosphate deaminase encoding gene nagB is to contain N- by structure 5 '-nagB-nagA-nanA-nanK-nanE-3 ' of n acetylneuraminic acid n operon knocks out the suicide plasmid of frame, will through homologous recombination It knocks out frame and substitutes 5 '-nagB-nagA-nanA-nanK- of N-acetyl-neuraminate operon on Corynebacterium glutamicum chromosome NanE-3 ', then nanA genes are inserted into 5 '-nagB-nagA-nanA-nanK-nanE-3 ' knock out frame, it will be contaminated through homologous recombination NanA genes on colour solid restore, to block N-acetyl-neuraminate in the catabolism of intracellular.
The invention solves another technical problem be to provide the structure of the above-mentioned recombination Corynebacterium glutamicum of structure a kind of Method, it is characterised in that include the following steps：
1) construction recombination plasmid
Clone ammonia sugar-fructose-6-phosphate transamination enzyme coding gene (glmS) of Corynebacterium glutamicum, saccharomyces cerevisiae The acetyl of Glucosamine acetylase encoding gene (GNA1), the phosphoric acid enzyme coding gene (yqaB) of Escherichia coli, Anabaena The N-acetyl-neuraminate synthasee code gene (neuB) of Glucosamine isomery enzyme coding gene (age) and Escherichia coli, even It is connected on recombinant expression plasmid；
2) structure production N-acetyl-neuraminate recombinates Corynebacterium glutamicum
Above-mentioned recombinant expression carrier is converted into Corynebacterium glutamicum, obtains production N-acetyl-neuraminate recombination glutamic acid rod Bacterium.
The Corynebacterium glutamicum is C.glutamicum ATCC13869 or Corynebacterium glutamicum ATCC13869 △ nanK △ nanE △ nagA △ nagB or Corynebacterium glutamicum ATCC13869 △ cg2937△nanK△nanE△nagA△nagB；The recombinant expression carrier is pDXW-9.
The present invention also provides a kind of sides using above-mentioned recombination Corynebacterium glutamicum fermenting and producing N-acetyl-neuraminate The seed that 12-24h is cultivated under 30-32 DEG C, 180-220rpm is transferred to fermentation medium by method with the inoculum concentration of 5%-10%, in 30-32 DEG C, cultivate 30-50h under the conditions of 180-220rpm.
Recombinate Corynebacterium glutamicum seed culture and fermentation：
Seed culture medium (g/L)：Glucose 10, urea 1.25, corn steep liquor 20, potassium dihydrogen phosphate 1, magnesium sulfate 0.5.
Fermentation medium (g/L)：Glucose 80, ammonium sulfate 35, corn steep liquor 20, potassium dihydrogen phosphate 1, magnesium sulfate 1, calcium carbonate 30。
Condition of culture：The seed that 12-24h is cultivated under 30-32 DEG C, 180-220rpm is transferred to the inoculum concentration of 5%-10% Fermentation medium cultivates 30-50h under the conditions of 30-32 DEG C, 180-220rpm.
The assay method of N-acetyl-neuraminate：
High performance liquid chromatography (HPLC) detection method：Agilent 1200, RID detector, NH2Column (250 × 4.6mm, 5 μ M), mobile phase：70% acetonitrile, flow velocity 0.6mL/min, 30 DEG C of column temperature, sampling volume are 10 μ L.
The present invention also provides a kind of recombination paddy ammonia that can be used glucose as fermenting substrate and produce N-acetyl-neuraminate Sour bar bacterium.The recombination Corynebacterium glutamicum is Corynebacterium glutamicum ATCC13869 △ cg2937 △ nanK△nanE△nagA△nagB(p9-age-neuB-Ptac- glmS-GNA1-yqaB), it is with Corynebacterium Glutamicum ATCC13869 △ cg2937 △ nanK △ nanE △ nagA △ nagB are host, with pDXW-9 pairs of expression vector GlmS, GNA1, yqaB, age and neuB are expressed.
Recombination Corynebacterium glutamicum provided by the invention can realize N-acetyl-neuraminate in extracellular accumulation, and concentration is reachable To 110mg/L, produces N-acetyl-neuraminate for further metabolic engineering Corynebacterium glutamicum and lay a good foundation.The present invention The recombination Corynebacterium glutamicum construction method of offer is simple, is easy to use, belongs to food-borne safe bacterial strain, has and applies well Foreground.
Description of the drawings
Fig. 1 recombinates Corynebacterium glutamicum C.glutamicum ATCC13869 △ cg2937 △ nanK △ nanE △ nagA △ nagB(p9-age-neuB-Ptac- glmS-GNA1-yqaB) it is metabolized the approach of synthesis N-acetyl-neuraminate.
Specific implementation mode
The structure of 1 recombinant plasmid of embodiment
Base is encoded according to the acetylglucosamine isomerase in the Anabaena (Anabaena sp.CH1) announced on NCBI Because of (age) sequence, age genes, and design primer age-F are synthesized by the bio tech ltd Jin Sirui:5’- TTCACACAGGAAACAGAATTCGAAGGAGTCTTCACATGGGCAAAAACTTACAAGCT CT-3 ', age-R:5’- CATTCTACTCTGACTTATGAAAGTGCTTCAAACTGTTGC-3’.Age gene pieces using above-mentioned primer to synthesize Section is template, amplification acetylglucosamine isomery enzyme coding gene (age).
According to the N-acetyl-neuraminate synthase in the Escherichia coli (Escherichia coli K-1) announced on NCBI Encoding gene (neuB) sequence synthesizes neuB genes, and design primer neuB-F by the bio tech ltd Jin Sirui: 5 '-TCATAAGTCAGAGTAGAATGAGAAGGAGTAGATTCATGTCTAACATCTACATCGTG GC-3 ', neuB-R:5’- CATCCGCCAAAACAGAAGCTTGTTAACTTTATTCTCCCTGGTTTTTAAATTCGC-3’.Using above-mentioned primer to synthesize Obtained neuB genetic fragments are template, amplification N-acetyl-neuraminate synthasee code gene (neuB).
Through one-step cloning method, (used one-step cloning kit is purchased from Nanjing to 2 genetic fragments that above-mentioned amplification is obtained Nuo Weizan Bioisystech Co., Ltd) it is connected to pDXW-9 expression vectors (Xu D, Tan Y, Shi F, Wang X.An improved shuttle vector constructed for metabolic engineering research in Corynebacteriun glutamicum.Plasmid.2010；64:EcoRI and HindIII restriction enzyme sites on 85-91) it Between.Digestion verification is simultaneously sequenced, and confirms that recombinant plasmid p9-age-neuB is built successfully.
According in the Corynebacterium glutamicum (Corynebacterium glutamicumATCC 13032) announced on NCBI Ammonia sugar-fructose-6-phosphate transamination enzyme coding gene (glmS), design primer glmS-F:5’- CAGGAAACAGAATTCGCTAGCGAAGGAGTAATACGATGTGTGGAATTGTTGGATAT A-3 ', glmS-R：5’- TAGAGAGAGAGAGGTGGAAATTATTCGACGGTGACAGACTTTGC-3’.Using above-mentioned primer from Corynebacterium glutamicum It is expanded in (Corynebacterium glutamicum ATCC13869 are purchased from American Type Culture collection) genome Ammonia sugar-fructose-6-phosphate transamination enzyme coding gene (glmS).
According to saccharomyces cerevisiae (the Saccharomyces cerevisiae S288C, purchased from U.S. typical case announced on NCBI Organism Depositary, number ATCC 204508) in Glucosamine acetylase encoding gene (GNA1), design primer GNA1-F：5 '-GGGGTACCATTATAGGTAAGAGAGGAATGTACACATGAGCTTACCCGATGGATTTT ATA-3 ', GNA1-R：5’-CCCAAGCTTCTATTTTCTAATTTGCATTTCCACG-3’.Using above-mentioned primer from saccharomyces cerevisiae Glucosamine acetylase encoding gene (GNA1) is expanded in (Saccharomyces cerevisiaeS288C) genome.
According to the phosphoric acid enzyme coding gene for the Escherichia coli (Escherichia coli K-12) announced on NCBI (yqaB), design primer yqaB-F:5’- TAAGTGCTCCATGAAGTCGTGAAGGAGTGTCTACATGTACGAGCGTTATGCAGGTT TA-3 ', yqaB-R:5’- CATCCGCCAAAACAGAAGCTTTCACAGCAAGCGAACATCCA-3’.Using above-mentioned primer from Escherichia coli Phosphoric acid enzyme coding gene (yqaB) is expanded in (Escherichia coli K-12) genome.
3 genetic fragments glmS, GNA1, yqaB that above-mentioned amplification is obtained are through one-step cloning method (used one-step cloning Kit be purchased from Nanjing Nuo Weizan Bioisystech Co., Ltd) be connected to pDXW-9 expression vectors (Xu D, TanY, Shi F, Wang X.An improved shuttle vector constructed for metabolic engineering research in Corynebacteriun glutamicum.Plasmid.2010；64:NheI on 85-91) and Between HindIII restriction enzyme sites.Digestion verification is simultaneously sequenced, and confirms that recombinant plasmid p9-glmS-GNA1-yqaB is built successfully.
According to tac promoter sequence design primers Ptac-F：5’-ATTACCCGGGAAGCTGGCGATGTGGTGATT-3’； According to the phosphoric acid enzyme coding gene (yqaB) for the Escherichia coli (Escherichia coli K-12) announced on NCBI, design is drawn Object yqaB-R2:5’-ATTACCCGGGTCACAGCAAGCGAACATCCA-3’.Using above-mentioned primer from recombinant plasmid p9-glmS- Amplification is connected with the P of tac promoters on GNA1-yqaBtac- glmS-GNA1-yqaB segments are used in combination SmaI digestions to handle.It will recombination Plasmid p9-age-neuB is behind HindIII digestions, filling-in end, with PTac- glmS-GNA1-yqaB segments T4 ligase phases Connection.Digestion verification is simultaneously sequenced, and confirms recombinant plasmid p9-age-neuB-Ptac- glmS-GNA1-yqaB is built successfully.
The knockout of 2 N-acetyl-neuraminate transport protein encoding gene cg2937 of embodiment
According in the Corynebacterium glutamicum (Corynebacterium glutamicum ATCC 13032) announced on NCBI N-acetyl-neuraminate transport protein encoding gene (cg2937) upstream and downstream sequence, design primer cg2937-U-F:5’- CTATGACATGATTACGAATTCGCTGTGAGCTTTGATGGTTTC-3’；cg2937-U-R:5’- ACATTGATCTCTACTCTGACTGCCGGTGTTGTCTGGTGCA-3’；cg2937-D-F:5’- GTCAGAGTAGAGATCAATGTCGAATCCTACGACCAGGTACA-3’；cg2937-D-R:5’- TGCCTGCAGGTCGACTCTAGAGGTGATTGGGGTGATCAGC-3’.With Corynebacterium glutamicum (Corynebacterium Glutamicum ATCC13869, be purchased from American Type Culture collection) genome be pcr template, use primer Cg2937-U-F and cg2937-U-R amplifies the upstream N-acetyl-neuraminate transport protein encoding gene (cg2937) homology arm Sequence △ cg2937-Up；N-acetyl-neuraminate transport protein is amplified using primer cg2937-D-F and cg2937-D-R to compile The code downstream gene (cg2937) homology arm sequence △ cg2937-Down.By △ cg2937-Up and △ cg2937-Down through a step PCR cloning PCR (used one-step cloning kit is purchased from Nanjing Nuo Weizan Bioisystech Co., Ltd) is connected to knockout carrier pk18mobsacB( A,Tauch A, W,Kalinowski J,Thierbach G,PühlerA.Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18and pK19:selection of defined deletions in the chromosome of Corynebacterium glutamicum.Gene.1994；145(1):On 69-73.) between the sites EcoRI and XbaI to get To the suicide plasmid pk18mobsacB △ cg2937 for knocking out frame containing N-acetyl-neuraminate transport protein encoding gene.It will Pk18mobsacB △ cg2937 convert Corynebacterium glutamicum Corynebacterium glutamicum ATCC13869, pass through Kalamycin resistance plate screening selects plate transformation inoculation LB liquid medium and cultivates 12h in 30 DEG C, 200rpm, by bacterium Suspension dilution, LB tablet of the coating containing 10% sucrose, filter out without the tablet single bacterium colony for blocking that resistance, carry out bacterium colony PCR is verified, and confirms that N-acetyl-neuraminate transport protein encoding gene (cg2937) knocks out successfully, obtains recombination glutamic acid rod 13869 △ cg2937 of bacterium.
The blocking of 3 N-acetyl-neuraminate intracellular catabolic pathway of embodiment
By knocking out N- acetylamino mannokinase encoding gene nanK, N- acetylamino mannoses -6- on chromosome Phosphate isomerase enzyme coding gene nanE, acetylglucosamine -6- phosphoric acid deacetylation enzyme coding gene nagA and aminoglucose Sugar -6- phosphate deaminase encoding gene nagB, to block N-acetyl-neuraminate in the catabolism of intracellular.
According in the Corynebacterium glutamicum (Corynebacterium glutamicum ATCC 13032) announced on NCBI N-acetyl-neuraminate operon (5 '-nagB-nagA-nanA-nanK-nanE-3 ') upstream and downstream sequence, design primer NEU-U- F:5’-CTATGACATGATTACGAATTCGATTTCGGGGAGACATTCACT-3’；NEU-U-R:5’- GTACCTGAGAATGTAGTTTTTTGGTGCCAACGCGATCATC-3’；NEU-D-F:5’- AAAACTACATTCTCAGGTACAAACGCTGATCACTACCGTCT-3’；NEU-D-R:5’- TGCCTGCAGGTCGACTCTAGAGCGTAGAATTCATGGCCGAAAT-3’.With Corynebacterium glutamicum (Corynebacterium Glutamicum ATCC13869, be purchased from American Type Culture collection) genome be pcr template, use primer NEU- It is same that U-F and NEU-U-R amplifies the upstream N-acetyl-neuraminate operon (5 '-nagB-nagA-nanA-nanK-nanE-3 ') Source arm sequence △ NEU-Up；N-acetyl-neuraminate operon (5 '-nagB- are amplified using primer NEU-D-F and NEU-D-R NagA-nanA-nanK-nanE-3 ') downstream homology arm sequence △ NEU-Down.By △ NEU-Up and △ NEU-Down through a step PCR cloning PCR (used one-step cloning kit is purchased from Nanjing Nuo Weizan Bioisystech Co., Ltd) is connected to knockout carrier pk18mobsacB( A,Tauch A, W,Kalinowski J,Thierbach G,Pühler A.Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18and pK19:selection of defined deletions in the chromosome of Corynebacterium glutamicum.Gene.1994；145(1):On 69-73.) between the sites EcoRI and XbaI to get To the suicide plasmid pk18mobsacB △ NEU for knocking out frame containing N-acetyl-neuraminate operon.By pk18mobsacB △ NEU Corynebacterium glutamicum Corynebacterium glutamicum ATCC13869 and 13869 △ cg2937 are converted, card is passed through That chloramphenicol resistance plate screening selects plate transformation inoculation LB liquid medium and cultivates 12h in 30 DEG C, 200rpm, bacterium is hanged Liquid dilution, LB tablet of the coating containing 10% sucrose, filter out without the tablet single bacterium colony for blocking that resistance, carry out bacterium colony PCR Verification confirms that N-acetyl-neuraminate operon knocks out successfully, obtains recombination Corynebacterium glutamicum 13869 △ NEU and 13869 △cg2937△NEU。
According in the Corynebacterium glutamicum (Corynebacterium glutamicum ATCC 13032) announced on NCBI N-acetylneuraminic acid lyase encoding gene (nanA) and its upstream sequence, design primer nanA-F:5’- GTACCTGAGAATGTAGTTTTCACTTCTGCCATCTTTCTG-3’；nanA-R:5’- CGGAGATCTGGTACTTTCGAGGTGTGGGCCTTAAGCGGTGT-3’；NEU-U-F:5’- CTATGACATGATTACGAATTCGATTTCGGGGAGACATTCACT-3’；NEU-U-R2:5’- GTACCTGAGAATGTAGTTTTTTGGTGCCAACGCGATCATC-3’；NEU-D-F2:5’- CTCGAAAGTACCAGATCTCCGAAACGCTGATCACTACCGTCT-3’；NEU-D-R:5’- TGCCTGCAGGTCGACTCTAGAGCGTAGAATTCATGGCCGAAAT-3’.With Corynebacterium glutamicum (Corynebacterium Glutamicum ATCC13869, be purchased from American Type Culture collection) genome be pcr template, use primer NEU- It is same that U-F and NEU-U-R2 amplifies the upstream N-acetyl-neuraminate operon (5 '-nagB-nagA-nanA-nanK-nanE-3 ') Source arm sequence △ NEU-Up2；N-acetylneuraminic acid lyase is amplified using primer nanA-F and nanA-R and its upstream is opened Promoter sequences nanA；N-acetyl-neuraminate operon (5 '-nagB- are amplified using primer NEU-D-F2 and NEU-D-R NagA-nanA-nanK-nanE-3 ') downstream homology arm sequence △ NEU-Down2.By △ NEU-Up2, nanA and △ NEU- Down2 is connected to load through one-step cloning method (used one-step cloning kit is purchased from Nanjing Nuo Weizan Bioisystech Co., Ltd) Body pk18mobsacB ( A,Tauch A, W,Kalinowski J,Thierbach G,PühlerA.Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18and pK19:selection of defined deletions in the chromosome of Corynebacterium glutamicum.Gene.1994；145(1):On 69-73.) between the sites EcoRI and XbaI to get To nanA covering plasmid pk18mobsacB △ NEU::nanA.By pk18mobsacB △ NEU::NanA converts Corynebacterium glutamicum 13869 △ NEU and 13869 △ cg2937 △ NEU select the inoculation of plate transformation by kalamycin resistance plate screening LB liquid medium cultivates 12-18h in 30 DEG C, 200rpm, by bacteria suspension dilution, LB tablet of the coating containing 10% sucrose, sieve It selects without the tablet single bacterium colony for blocking that resistance, carries out bacterium colony PCR verifications, confirm that nanA restores successfully on chromosome, both To recombination 13869 △ nanK △ nanE △ nagA △ nagB of Corynebacterium glutamicum and 13869 △ cg2937 △ nanK △ nanE △nagA△nagB。
Embodiment 4 accumulates the structure of the recombination Corynebacterium glutamicum of N-acetyl-neuraminate
The recombinant expression plasmid p9-age-neuB-P that will be builttac- glmS-GNA1-yqaB converts Corynebacterium glutamicum Corynebacterium glutamicum ATCC13869,13869 △ nanK △ nanE △ nagA △ nagB and 13869 △ cg2937△nanK△nanE△nagA△nagB.Transformant is selected using age-F and yqaB-R primer pairs and carries out bacterium colony PCR, There is 5400bp bands, verification recombination Corynebacterium glutamicum Corynebacterium glutamicum ATCC13869 (p9- age-neuB-Ptac-glmS-GNA1-yqaB)、13869△nanK△nanE△nagA△nagB(p9-age-neuB-Ptac- ) and 13869 △ cg2937 △ nanK △ nanE △ nagA △ nagB (p9-age-neuB-P glmS-GNA1-yqaBtac-glmS- GNA1-yqaB it) builds successfully.
5 fermenting and producing N-acetyl-neuraminate of embodiment
Seed culture medium (g/L)：Glucose 10, urea 1.25, corn steep liquor 20, potassium dihydrogen phosphate 1, magnesium sulfate 0.5.
Fermentation medium (g/L)：Glucose 80, ammonium sulfate 35, corn steep liquor 20, potassium dihydrogen phosphate 1, magnesium sulfate 1, calcium carbonate 30。
The seed that 12-24h is cultivated under 30-32 DEG C, 180-220rpm is transferred to fermented and cultured with the inoculum concentration of 5%-10% Base cultivates 30-50h under the conditions of 30-32 DEG C, 180-220rpm.Final Corynebacterium glutamicum 13869 △cg2937△nanK△nanE△nagA△nagB(p9-age-neuB-Ptac- glmS-GNA1-yqaB) in fermented supernatant fluid N-acetyl-neuraminate content has reached 110mg/L.Overexpression ammonia sugar-fructose-6-phosphate transamination enzyme coding gene (glmS), Glucosamine acetylase encoding gene (GNA1), phosphoric acid enzyme coding gene (yqaB), acetylglucosamine are different Structure enzyme coding gene (age) and N-acetyl-neuraminate synthasee code gene (neuB), and knock out N- acetyl nerve on chromosome Propylhomoserin transport protein encoding gene (cg2937) and intracellular N-acetyl-neuraminate, which decompose, utilizes the N- acetyl ammonia in metabolic pathway Base mannokinase encoding gene (nanK), N- acetylamino mannose-6-phosphate isomerase encoding genes (nanE), acetyl ammonia Base G-6-P deacetylation enzyme coding gene (nagA), Glucosamine -6- phosphate deaminase encoding genes (nagB), the N-acetyl-neuraminate accumulation extracellular in recombination Corynebacterium glutamicum is realized.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not limited to the present invention, any to be familiar with this skill The people of art can do various change and modification, therefore the protection model of the present invention without departing from the spirit and scope of the present invention Enclosing be subject to what claims were defined.
SEQUENCE LISTING
<110>Southern Yangtze University
<120>It is a kind of accumulation N-acetyl-neuraminate recombination Corynebacterium glutamicum and its application
<160> 31
<170> PatentIn version 3.3
<210> 1
<211> 623
<212> PRT
<213> Corynebacterium glutamicum ATCC13869
<400> 1
Met Cys Gly Ile Val Gly Tyr Ile Gly Gln Ala Gly Asp Ser Arg Asp
1 5 10 15
Tyr Phe Ala Leu Asp Val Val Leu Glu Gly Leu Arg Arg Leu Glu Tyr
20 25 30
Arg Gly Tyr Asp Ser Ala Gly Val Ala Val His Ala Asn Gly Glu Ile
35 40 45
Ser Tyr Arg Lys Lys Ala Gly Lys Val Ala Ala Leu Asp Ala Glu Ile
50 55 60
Ala Arg Ala Pro Leu Ala Asp Ser Ile Leu Ala Ile Gly His Thr Arg
65 70 75 80
Trp Ala Thr His Gly Gly Pro Thr Asp Ala Asn Ala His Pro His Val
85 90 95
Val Asp Gly Gly Lys Leu Ala Val Val His Asn Gly Ile Ile Glu Asn
100 105 110
Phe Ala Glu Leu Arg Ala Glu Leu Ser Ala Lys Gly Tyr Asn Phe Val
115 120 125
Ser Val Thr Asp Thr Glu Val Ala Ala Thr Leu Leu Ala Glu Ile Tyr
130 135 140
Asn Thr Gln Ala Asn Gly Asp Leu Thr Lys Ala Met Gln Leu Thr Gly
145 150 155 160
Gln Arg Leu Glu Gly Ala Phe Thr Leu Leu Ala Ile His Ala Asp His
165 170 175
Asp Asp Arg Ile Val Ala Ala Arg Arg Asn Ser Pro Leu Val Ile Gly
180 185 190
Leu Gly Glu Gly Glu Asn Phe Leu Gly Ser Asp Val Ser Gly Phe Ile
195 200 205
Asp Tyr Thr Arg Lys Ala Val Glu Met Gly Asn Asp Gln Ile Val Thr
210 215 220
Ile Thr Ala Asn Asp Tyr Gln Ile Thr Asn Phe Asp Gly Ser Glu Ala
225 230 235 240
Thr Gly Lys Pro Phe Asp Val Glu Trp Asp Ala Ala Ala Ala Glu Lys
245 250 255
Gly Gly Phe Asp Ser Phe Met Asp Lys Glu Ile His Asp Gln Pro Ala
260 265 270
Ala Val Arg Asp Thr Leu Leu Gly Arg Leu Asp Glu Asp Gly Lys Leu
275 280 285
Val Leu Asp Glu Leu Arg Ile Asp Glu Ala Thr Leu Arg Ser Val Asn
290 295 300
Lys Ile Ile Val Val Ala Cys Gly Thr Ala Ala Tyr Ala Gly Gln Val
305 310 315 320
Ala Arg Tyr Ala Ile Glu His Trp Cys Arg Ile Pro Thr Glu Val Glu
325 330 335
Leu Ala His Glu Phe Arg Tyr Arg Asp Pro Ile Val Asn Glu Lys Thr
340 345 350
Leu Val Val Ala Leu Ser Gln Ser Gly Glu Thr Met Asp Thr Leu Met
355 360 365
Ala Val Arg His Ala Arg Glu Gln Gly Ala Lys Val Ile Ala Ile Cys
370 375 380
Asn Thr Val Gly Ser Thr Leu Pro Arg Glu Ala Asp Ala Ser Leu Tyr
385 390 395 400
Thr Tyr Ala Gly Pro Glu Ile Ala Val Ala Ser Thr Lys Ala Phe Leu
405 410 415
Ala Gln Ile Thr Ala Ser Tyr Leu Leu Gly Leu Tyr Leu Ala Gln Leu
420 425 430
Arg Gly Asn Lys Phe Ala Asp Glu Val Ser Ser Ile Leu Asp Ser Leu
435 440 445
Arg Glu Met Pro Glu Lys Ile Gln Gln Val Ile Asp Ala Glu Glu Gln
450 455 460
Ile Lys Lys Leu Gly Gln Asp Met Ser Asp Ala Lys Ser Val Leu Phe
465 470 475 480
Leu Gly Arg His Val Gly Phe Pro Val Ala Leu Glu Gly Ala Leu Lys
485 490 495
Leu Lys Glu Ile Ala Tyr Leu His Ala Glu Gly Phe Ala Ala Gly Glu
500 505 510
Leu Lys His Gly Pro Ile Ala Leu Val Glu Glu Gly Gln Pro Val Phe
515 520 525
Val Ile Val Pro Ser Pro Arg Gly Arg Asp Ser Leu His Ser Lys Val
530 535 540
Val Ser Asn Ile Gln Glu Ile Arg Ala Arg Gly Ala Val Thr Ile Val
545 550 555 560
Ile Ala Glu Glu Gly Asp Glu Ala Val Asn Asp Tyr Ala Asn Phe Ile
565 570 575
Ile Arg Ile Pro Gln Ala Pro Thr Leu Met Gln Pro Leu Leu Ser Thr
580 585 590
Val Pro Leu Gln Ile Phe Ala Cys Ala Val Ala Thr Ala Lys Gly Tyr
595 600 605
Asn Val Asp Gln Pro Arg Asn Leu Ala Lys Ser Val Thr Val Glu
610 615 620
<210> 2
<211> 159
<212> PRT
<213> Saccharomyces cerevisiae S288C
<400> 2
Met Ser Leu Pro Asp Gly Phe Tyr Ile Arg Arg Met Glu Glu Gly Asp
1 5 10 15
Leu Glu Gln Val Thr Glu Thr Leu Lys Val Leu Thr Thr Val Gly Thr
20 25 30
Ile Thr Pro Glu Ser Phe Ser Lys Leu Ile Lys Tyr Trp Asn Glu Ala
35 40 45
Thr Val Trp Asn Asp Asn Glu Asp Lys Lys Ile Met Gln Tyr Asn Pro
50 55 60
Met Val Ile Val Asp Lys Arg Thr Glu Thr Val Ala Ala Thr Gly Asn
65 70 75 80
Ile Ile Ile Glu Arg Lys Ile Ile His Glu Leu Gly Leu Cys Gly His
85 90 95
Ile Glu Asp Ile Ala Val Asn Ser Lys Tyr Gln Gly Gln Gly Leu Gly
100 105 110
Lys Leu Leu Ile Asp Gln Leu Val Thr Ile Gly Phe Asp Tyr Gly Cys
115 120 125
Tyr Lys Ile Ile Leu Asp Cys Asp Glu Lys Asn Val Lys Phe Tyr Glu
130 135 140
Lys Cys Gly Phe Ser Asn Ala Gly Val Glu Met Gln Ile Arg Lys
145 150 155
<210> 3
<211> 188
<212> PRT
<213> Escherichia coli K-12
<400> 3
Met Tyr Glu Arg Tyr Ala Gly Leu Ile Phe Asp Met Asp Gly Thr Ile
1 5 10 15
Leu Asp Thr Glu Pro Thr His Arg Lys Ala Trp Arg Glu Val Leu Gly
20 25 30
His Tyr Gly Leu Gln Tyr Asp Ile Gln Ala Met Ile Ala Leu Asn Gly
35 40 45
Ser Pro Thr Trp Arg Ile Ala Gln Ala Ile Ile Glu Leu Asn Gln Ala
50 55 60
Asp Leu Asp Pro His Ala Leu Ala Arg Glu Lys Thr Glu Ala Val Arg
65 70 75 80
Ser Met Leu Leu Asp Ser Val Glu Pro Leu Pro Leu Val Asp Val Val
85 90 95
Lys Ser Trp His Gly Arg Arg Pro Met Ala Val Gly Thr Gly Ser Glu
100 105 110
Ser Ala Ile Ala Glu Ala Leu Leu Ala His Leu Gly Leu Arg His Tyr
115 120 125
Phe Asp Ala Val Val Ala Ala Asp His Val Lys His His Lys Pro Ala
130 135 140
Pro Asp Thr Phe Leu Leu Cys Ala Gln Arg Met Gly Val Gln Pro Thr
145 150 155 160
Gln Cys Val Val Phe Glu Asp Ala Asp Phe Gly Ile Gln Ala Ala Arg
165 170 175
Ala Ala Gly Met Asp Ala Val Asp Val Arg Leu Leu
180 185
<210> 4
<211> 388
<212> PRT
<213> Anabaena sp. CH1
<400> 4
Met Gly Lys Asn Leu Gln Ala Leu Ala Gln Leu Tyr Lys Asn Ala Leu
1 5 10 15
Leu Asn Asp Val Leu Pro Phe Trp Glu Asn His Ser Leu Asp Ser Glu
20 25 30
Gly Gly Tyr Phe Thr Cys Leu Asp Arg Gln Gly Lys Val Tyr Asp Thr
35 40 45
Asp Lys Phe Ile Trp Leu Gln Asn Arg Gln Val Trp Thr Phe Ser Met
50 55 60
Leu Cys Asn Gln Leu Glu Lys Arg Glu Asn Trp Leu Lys Ile Ala Arg
65 70 75 80
Asn Gly Ala Lys Phe Leu Ala Gln His Gly Arg Asp Asp Glu Gly Asn
85 90 95
Trp Tyr Phe Ala Leu Thr Arg Gly Gly Glu Pro Leu Val Gln Pro Tyr
100 105 110
Asn Ile Phe Ser Asp Cys Phe Ala Ala Met Ala Phe Ser Gln Tyr Ala
115 120 125
Leu Ala Ser Gly Glu Glu Trp Ala Lys Asp Val Ala Met Gln Ala Tyr
130 135 140
Asn Asn Val Leu Arg Arg Lys Asp Asn Pro Lys Gly Lys Tyr Thr Lys
145 150 155 160
Thr Tyr Pro Gly Thr Arg Pro Met Lys Ala Leu Ala Val Pro Met Ile
165 170 175
Leu Ala Asn Leu Thr Leu Glu Met Glu Trp Leu Leu Pro Gln Glu Thr
180 185 190
Leu Glu Asn Val Leu Ala Ala Thr Val Gln Glu Val Met Gly Asp Phe
195 200 205
Leu Asp Gln Glu Gln Gly Leu Met Tyr Glu Asn Val Ala Pro Asp Gly
210 215 220
Ser His Ile Asp Cys Phe Glu Gly Arg Leu Ile Asn Pro Gly His Gly
225 230 235 240
Ile Glu Ala Met Trp Phe Ile Met Asp Ile Ala Arg Arg Lys Asn Asp
245 250 255
Ser Lys Thr Ile Asn Gln Ala Val Asp Val Val Leu Asn Ile Leu Asn
260 265 270
Phe Ala Trp Asp Asn Glu Tyr Gly Gly Leu Tyr Tyr Phe Met Asp Ala
275 280 285
Ala Gly His Pro Pro Gln Gln Leu Glu Trp Asp Gln Lys Leu Trp Trp
290 295 300
Val His Leu Glu Ser Leu Val Ala Leu Ala Met Gly Tyr Arg Leu Thr
305 310 315 320
Gly Arg Asp Ala Cys Trp Ala Trp Tyr Gln Lys Met His Asp Tyr Ser
325 330 335
Trp Gln His Phe Ala Asp Pro Glu Tyr Gly Glu Trp Phe Gly Tyr Leu
340 345 350
Asn Arg Arg Gly Glu Val Leu Leu Asn Leu Lys Gly Gly Lys Trp Lys
355 360 365
Gly Cys Phe His Val Pro Arg Ala Met Tyr Leu Cys Trp Gln Gln Phe
370 375 380
Glu Ala Leu Ser
385
<210> 5
<211> 346
<212> PRT
<213> Escherichia coli K-1
<400> 5
Met Ser Asn Ile Tyr Ile Val Ala Glu Ile Gly Cys Asn His Asn Gly
1 5 10 15
Ser Val Asp Ile Ala Arg Glu Met Ile Leu Lys Ala Lys Glu Ala Gly
20 25 30
Val Asn Ala Val Lys Phe Gln Thr Phe Lys Ala Asp Lys Leu Ile Ser
35 40 45
Ala Ile Ala Pro Lys Ala Glu Tyr Gln Ile Lys Asn Thr Gly Glu Leu
50 55 60
Glu Ser Gln Leu Glu Met Thr Lys Lys Leu Glu Met Lys Tyr Asp Asp
65 70 75 80
Tyr Leu His Leu Met Glu Tyr Ala Val Ser Leu Asn Leu Asp Val Phe
85 90 95
Ser Thr Pro Phe Asp Glu Asp Ser Ile Asp Phe Leu Ala Ser Leu Lys
100 105 110
Gln Lys Ile Trp Lys Ile Pro Ser Gly Glu Leu Leu Asn Leu Pro Tyr
115 120 125
Leu Glu Lys Ile Ala Lys Leu Pro Ile Pro Asp Lys Lys Ile Ile Ile
130 135 140
Ser Thr Gly Met Ala Thr Ile Asp Glu Ile Lys Gln Ser Val Ser Ile
145 150 155 160
Phe Ile Asn Asn Lys Val Pro Val Gly Asn Ile Thr Ile Leu His Cys
165 170 175
Asn Thr Glu Tyr Pro Thr Pro Phe Glu Asp Val Asn Leu Asn Ala Ile
180 185 190
Asn Asp Leu Lys Lys His Phe Pro Lys Asn Asn Ile Gly Phe Ser Asp
195 200 205
His Ser Ser Gly Phe Tyr Ala Ala Ile Ala Ala Val Pro Tyr Gly Ile
210 215 220
Thr Phe Ile Glu Lys His Phe Thr Leu Asp Lys Ser Met Ser Gly Pro
225 230 235 240
Asp His Leu Ala Ser Ile Glu Pro Asp Glu Leu Lys His Leu Cys Ile
245 250 255
Gly Val Arg Cys Val Glu Lys Ser Leu Gly Ser Asn Ser Lys Val Val
260 265 270
Thr Ala Ser Glu Arg Lys Asn Lys Ile Val Ala Arg Lys Ser Ile Ile
275 280 285
Ala Lys Thr Glu Ile Lys Lys Gly Glu Val Phe Ser Glu Lys Asn Ile
290 295 300
Thr Thr Lys Arg Pro Gly Asn Gly Ile Ser Pro Met Glu Trp Tyr Asn
305 310 315 320
Leu Leu Gly Lys Ile Ala Glu Gln Asp Phe Ile Pro Asp Glu Leu Ile
325 330 335
Ile His Ser Glu Phe Lys Asn Gln Gly Glu
340 345
<210> 6
<211> 58
<212> DNA
<213>Artificial sequence
<400> 6
ttcacacagg aaacagaatt cgaaggagtc ttcacatggg caaaaactta caagctct 58
<210> 7
<211> 39
<212> DNA
<213>Artificial sequence
<400> 7
cattctactc tgacttatga aagtgcttca aactgttgc 39
<210> 8
<211> 58
<212> DNA
<213>Artificial sequence
<400> 8
tcataagtca gagtagaatg agaaggagta gattcatgtc taacatctac atcgtggc 58
<210> 9
<211> 54
<212> DNA
<213>Artificial sequence
<400> 9
catccgccaa aacagaagct tgttaacttt attctccctg gtttttaaat tcgc 54
<210> 10
<211> 57
<212> DNA
<213>Artificial sequence
<400> 10
caggaaacag aattcgctag cgaaggagta atacgatgtg tggaattgtt ggatata 57
<210> 11
<211> 44
<212> DNA
<213>Artificial sequence
<400> 11
tagagagaga gaggtggaaa ttattcgacg gtgacagact ttgc 44
<210> 12
<211> 59
<212> DNA
<213>Artificial sequence
<400> 12
ggggtaccat tataggtaag agaggaatgt acacatgagc ttacccgatg gattttata 59
<210> 13
<211> 34
<212> DNA
<213>Artificial sequence
<400> 13
cccaagcttc tattttctaa tttgcatttc cacg 34
<210> 14
<211> 58
<212> DNA
<213>Artificial sequence
<400> 14
taagtgctcc atgaagtcgt gaaggagtgt ctacatgtac gagcgttatg caggttta 58
<210> 15
<211> 41
<212> DNA
<213>Artificial sequence
<400> 15
catccgccaa aacagaagct ttcacagcaa gcgaacatcc a 41
<210> 16
<211> 30
<212> DNA
<213>Artificial sequence
<400> 16
attacccggg aagctggcga tgtggtgatt 30
<210> 17
<211> 30
<212> DNA
<213>Artificial sequence
<400> 17
attacccggg tcacagcaag cgaacatcca 30
<210> 18
<211> 42
<212> DNA
<213>Artificial sequence
<400> 18
ctatgacatg attacgaatt cgctgtgagc tttgatggtt tc 42
<210> 19
<211> 40
<212> DNA
<213>Artificial sequence
<400> 19
acattgatct ctactctgac tgccggtgtt gtctggtgca 40
<210> 20
<211> 41
<212> DNA
<213>Artificial sequence
<400> 20
gtcagagtag agatcaatgt cgaatcctac gaccaggtac a 41
<210> 21
<211> 40
<212> DNA
<213>Artificial sequence
<400> 21
tgcctgcagg tcgactctag aggtgattgg ggtgatcagc 40
<210> 22
<211> 42
<212> DNA
<213>Artificial sequence
<400> 22
ctatgacatg attacgaatt cgatttcggg gagacattca ct 42
<210> 23
<211> 40
<212> DNA
<213>Artificial sequence
<400> 23
gtacctgaga atgtagtttt ttggtgccaa cgcgatcatc 40
<210> 24
<211> 41
<212> DNA
<213>Artificial sequence
<400> 24
aaaactacat tctcaggtac aaacgctgat cactaccgtc t 41
<210> 25
<211> 43
<212> DNA
<213>Artificial sequence
<400> 25
tgcctgcagg tcgactctag agcgtagaat tcatggccga aat 43
<210> 26
<211> 39
<212> DNA
<213>Artificial sequence
<400> 26
gtacctgaga atgtagtttt cacttctgcc atctttctg 39
<210> 27
<211> 41
<212> DNA
<213>Artificial sequence
<400> 27
cggagatctg gtactttcga ggtgtgggcc ttaagcggtg t 41
<210> 28
<211> 42
<212> DNA
<213>Artificial sequence
<400> 28
ctatgacatg attacgaatt cgatttcggg gagacattca ct 42
<210> 29
<211> 40
<212> DNA
<213>Artificial sequence
<400> 29
gtacctgaga atgtagtttt ttggtgccaa cgcgatcatc 40
<210> 30
<211> 42
<212> DNA
<213>Artificial sequence
<400> 30
ctcgaaagta ccagatctcc gaaacgctga tcactaccgt ct 42
<210> 31
<211> 43
<212> DNA
<213>Artificial sequence
<400> 31
tgcctgcagg tcgactctag agcgtagaat tcatggccga aat 43

Claims (10)

1. a kind of recombination Corynebacterium glutamicum of accumulation N-acetyl-neuraminate, which is characterized in that close N-acetyl-neuraminate At 5 genes of approach：Ammonia sugar-fructose-6-phosphate transamination enzyme coding gene, Glucosamine acetylase encoding gene, phosphorus Sour enzyme coding gene, acetylglucosamine isomery enzyme coding gene, external source N-acetyl-neuraminate synthasee code gene, in paddy It is expressed in propylhomoserin bar bacterium, realizes the accumulation of N-acetyl-neuraminate.
2. a kind of Corynebacterium glutamicum of accumulation N-acetyl-neuraminate according to claim 1, which is characterized in that as The Corynebacterium glutamicum of host is Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13869, or strikes In addition to N-acetyl-neuraminate transport protein encoding gene cg2937 Corynebacterium glutamicum ATCC13869, or N- second is knocked out Acyl neuraminic acid transport protein encoding gene cg2937 and N- acetylamino mannokinase encoding gene nanK, N- acetyl ammonia Base mannose-6-phosphate isomerase encoding gene nanE, acetylglucosamine -6- phosphoric acid deacetylation enzyme coding genes nagA With the Corynebacterium glutamicum ATCC13869 of Glucosamine -6- phosphate deaminase encoding genes nagB.
3. a kind of Corynebacterium glutamicum of accumulation N-acetyl-neuraminate according to claim 1 or 2, which is characterized in that Ammonia sugar-fructose-6-phosphate transamination enzyme coding gene, Glucosamine acetylase are carried with expression vector pDXW-9 structures Encoding gene, phosphoric acid enzyme coding gene, acetylglucosamine isomery enzyme coding gene, external source N-acetyl-neuraminate synthase are compiled The recombinant expression carrier of code gene.
4. according to a kind of Corynebacterium glutamicum of any accumulation N-acetyl-neuraminate of claims 1 to 3, feature exists In ammonia sugar-fructose-6-phosphate transamination enzyme coding gene derives from Corynebacterium glutamicum ATCC13869 and/or aminoglucose Sugared acetylase encoding gene is compiled from saccharomyces cerevisiae (Saccharomyces cerevisiae) S288C and/or phosphatase Code gene source is in Escherichia coli (Escherichia coli) K-12 and/or acetylglucosamine isomery enzyme coding gene Escherichia coli are derived from from Anabaena (Anabaena sp.) CH1 and/or N-acetyl-neuraminate synthasee code gene (Escherichia coli)K-1。
5. a kind of Corynebacterium glutamicum of accumulation N-acetyl-neuraminate according to any one of claims 1 to 4, feature exist In age, neuB gene are connected between EcoRI the and HindIII restriction enzyme sites on pDXW-9 expression vectors；glmS、GNA1、 YqaB genes are connected between NheI the and HindIII restriction enzyme sites on pDXW-9 expression vectors.
6. according to a kind of Corynebacterium glutamicum of any accumulation N-acetyl-neuraminate of claim 2~5, feature exists In, knock out the N-acetyl-neuraminate transport protein encoding gene cg2937 of the Corynebacterium glutamicum, be by structure contain N-acetyl-neuraminate transport protein encoding gene knocks out the suicide plasmid of frame, and will knock out frame through homologous recombination substitutes glutamic acid rod N-acetyl-neuraminate transport protein encoding gene cg2937 on bacillus chromosome, with block N-acetyl-neuraminate from it is extracellular to The transhipment of intracellular.
7. according to a kind of Corynebacterium glutamicum of any accumulation N-acetyl-neuraminate of claim 2~5, feature exists In knocking out N- acetylamino mannokinase encoding gene nanK, N- acetylamino mannoses -6- of the Corynebacterium glutamicum Phosphate isomerase enzyme coding gene nanE, acetylglucosamine -6- phosphoric acid deacetylation enzyme coding gene nagA and aminoglucose Sugar -6- phosphate deaminase encoding gene nagB are to contain 5 '-nagB-nagA- of N-acetyl-neuraminate operon by structure NanA-nanK-nanE-3 ' knocks out the suicide plasmid of frame, and will knock out frame through homologous recombination substitutes on Corynebacterium glutamicum chromosome 5 '-nagB-nagA-nanA-nanK-nanE-3 ' of N-acetyl-neuraminate operon, then in 5 '-nagB-nagA-nanA- NanK-nanE-3 ', which is knocked out, is inserted into nanA genes in frame, restore the nanA genes on chromosome through homologous recombination, to block N- Catabolism of the n acetylneuraminic acid n in intracellular.
8. a kind of method of structure recombination Corynebacterium glutamicum, which is characterized in that include the following steps：
1) construction recombination plasmid
Clone ammonia sugar-fructose-6-phosphate transamination enzyme coding gene (glmS) of Corynebacterium glutamicum, the amino of saccharomyces cerevisiae The acetylamino of glucose acetylase encoding gene (GNA1), the phosphoric acid enzyme coding gene (yqaB) of Escherichia coli, Anabaena The N-acetyl-neuraminate synthasee code gene (neuB) of glucose isomerase enzyme coding gene (age) and Escherichia coli, is connected to On recombinant expression plasmid；
2) structure production N-acetyl-neuraminate recombinates Corynebacterium glutamicum
Above-mentioned recombinant expression carrier is converted into Corynebacterium glutamicum, obtains production N-acetyl-neuraminate recombination Corynebacterium glutamicum；
The Corynebacterium glutamicum is C.glutamicum ATCC13869 or Corynebacterium glutamicum ATCC13869 △ nanK △ nanE △ nagA △ nagB or Corynebacterium glutamicum ATCC13869 △ cg2937△nanK△nanE△nagA△nagB。
9. a kind of side using any recombination Corynebacterium glutamicum fermenting and producing N-acetyl-neuraminate of claim 1~7 Method, which is characterized in that the seed that 12-24h is cultivated under 30-32 DEG C, 180-220rpm is transferred to hair with the inoculum concentration of 5%-10% Ferment culture medium cultivates 30-50h under the conditions of 30-32 DEG C, 180-220rpm.
10. according to the method described in claim 9, it is characterized in that,
Seed culture medium (g/L)：Glucose 10, urea 1.25, corn steep liquor 20, potassium dihydrogen phosphate 1, magnesium sulfate 0.5；
Fermentation medium (g/L)：Glucose 80, ammonium sulfate 35, corn steep liquor 20, potassium dihydrogen phosphate 1, magnesium sulfate 1, calcium carbonate 30.
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