CN108330095B - Recombinant corynebacterium glutamicum for accumulating N-acetylneuraminic acid and application thereof - Google Patents

Recombinant corynebacterium glutamicum for accumulating N-acetylneuraminic acid and application thereof Download PDF

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CN108330095B
CN108330095B CN201810171868.6A CN201810171868A CN108330095B CN 108330095 B CN108330095 B CN 108330095B CN 201810171868 A CN201810171868 A CN 201810171868A CN 108330095 B CN108330095 B CN 108330095B
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陈坚
堵国成
王淼
刘延峰
董迅衍
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Abstract

The invention discloses a recombinant corynebacterium glutamicum for accumulating N-acetylneuraminic acid and application thereof, belonging to the field of genetic engineering. The invention takes corynebacterium glutamicum as an expression host, and strengthens the synthesis path of N-acetylneuraminic acid by over-expressing glucosamine-fructose-6-phosphate aminotransferase gene, glucosamine acetylase coding gene, phosphatase coding gene, acetylglucosamine isomerase coding gene and N-acetylneuraminic acid synthase coding gene; by knocking out the coding gene of N-acetylneuraminic acid transport protein in the corynebacterium glutamicum and the related gene on the intracellular N-acetylneuraminic acid decomposition and utilization metabolic pathway, the corynebacterium glutamicum genetic engineering strain with extracellular accumulation of N-acetylneuraminic acid is obtained, the yield reaches 110mg/L, and a foundation is laid for further metabolic engineering modification of corynebacterium glutamicum to produce N-acetylneuraminic acid.

Description

Recombinant corynebacterium glutamicum for accumulating N-acetylneuraminic acid and application thereof
Technical Field
The invention relates to a recombinant corynebacterium glutamicum for accumulating N-acetylneuraminic acid and application thereof, belonging to the field of genetic engineering.
Background
N-acetylneuraminic acid is an important food nutrition additive and a novel prodrug as the most important compound molecule in sialic acid. Its effects include promoting the development of infant brain, maintaining the health of old people's brain, and improving immunity. At present, the industrial production of N-acetylneuraminic acid mainly adopts a chemical extraction and whole cell transformation method for production. Because the total content of N-acetylneuraminic acid in natural raw materials (egg yolk, cubilose, bovine colostrum and the like) is low, the extraction and separation process is complex, and the traditional extraction method has low yield and high cost. Therefore, the production of N-acetylneuraminic acid by the whole-cell transformation method has become a hot point of research in recent years. However, the whole-cell transformation method requires the addition of N-acetylglucosamine as a synthesis precursor, and most methods still require the addition of pyruvic acid as a synthesis precursor. In view of the high price cost (6-8 ten thousand yuan/ton) of N-acetylglucosamine and pyruvic acid, the development of a method for producing N-acetylneuraminic acid by fermenting glucose with microorganisms has important research significance and application value.
The corynebacterium glutamicum is used as a common industrial strain, has the advantages of clear genetic background, mature metabolic modification tools, mature fermentation process, difficult phage pollution in the fermentation process and the like, and is widely used for fermentation production of important chemicals. Meanwhile, the corynebacterium glutamicum is an internationally recognized food safety-level strain and is an ideal strain for producing food, nutriments and medicines. Therefore, the construction of the recombinant corynebacterium glutamicum by using a metabolic engineering means is an effective way for producing food safety-grade N-acetylneuraminic acid. However, the metabolic pathway of N-acetylneuraminic acid in Corynebacterium glutamicum is tightly regulated and does not result in the accumulation of N-acetylneuraminic acid. How to modify the N-acetylneuraminic acid metabolic pathway in Corynebacterium glutamicum is a matter of considerable investigation.
Disclosure of Invention
The technical problem to be solved by the invention is to construct a recombinant corynebacterium glutamicum for accumulating N-acetylneuraminic acid.
In order to solve the technical problems, the technical scheme of the invention is as follows:
synthesizing N-acetylneuraminic acid into 5 genes: a gene encoding homologous glucosamine-fructose-6 phosphate aminotransferase, particularly glmS of Corynebacterium glutamicum (Corynebacterium glutamicum ATCC13869) (the amino acid sequence of the encoded glucosamine-fructose-6 phosphate aminotransferase is shown in SEQ ID NO: 1); an exogenous glucosamine acetylase encoding gene, in particular GNA1 (the amino acid sequence of the encoded glucosamine acetylase is shown in SEQ ID NO:2) of Saccharomyces cerevisiae (Saccharomyces cerevisiae S288C); exogenous phosphatase encoding gene, especially yqaB of Escherichia coli (Escherichia coli K-12) (amino acid sequence of encoded phosphatase is shown as SEQ ID NO: 3); an exogenous acetylglucosamine isomerase encoding gene, in particular the age of the necklace algae (Anabaena sp.CH1) (the amino acid sequence of the encoded acetylglucosamine isomerase is shown as SEQ ID NO: 4); and a foreign N-acetylneuraminic acid synthase-encoding gene, particularly neuB of Escherichia coli (Escherichia coli K-1) (the amino acid sequence of the encoded N-acetylneuraminic acid synthase is shown in SEQ ID NO: 5): cloning to an expression vector pDXW-9, then transforming Corynebacterium glutamicum (Corynebacterium glutamicum ATCC13869), and realizing the accumulation of N-acetylneuraminic acid by modifying a metabolic pathway.
In one embodiment of the present invention, the expression vector pDXW-9 is selected to express an glucosamine-fructose-6-phosphate aminotransferase encoding gene, a glucosamine acetylase encoding gene, a phosphatase encoding gene, an acetylglucosamine isomerase encoding gene, and an N-acetylneuraminic acid synthase encoding gene.
In one embodiment of the invention, the age, neuB gene is ligated into the pDXW-9 expression vector between the EcoRI and HindIII cleavage sites.
In one embodiment of the invention, the glmS, GNA1, yqaB genes are ligated into the pDXW-9 expression vector between the NheI and HindIII cleavage sites.
In one embodiment of the invention, the N-acetylneuraminic acid transporter coding gene cg2937 of Corynebacterium glutamicum is knocked out, specifically, a suicide plasmid containing a knock-out frame of the N-acetylneuraminic acid transporter coding gene is constructed, and the knock-out frame is replaced by the N-acetylneuraminic acid transporter coding gene cg2937 on the chromosome of Corynebacterium glutamicum through homologous recombination, so that the transport of N-acetylneuraminic acid from outside to inside of cells is blocked.
In one embodiment of the invention, the knockout of the N-acetamidomannnK-encoding gene nanK, N-acetamidomannnose-6-phosphate isomerase-encoding gene nanE, acetylglucosamine-6-phosphate deacetylase-encoding gene nagA and glucosamine-6-phosphate deaminase-encoding gene nagB of Corynebacterium glutamicum is carried out by constructing a suicide plasmid containing a knockout frame of the N-acetylneuraminic acid operon 5 '-nagA-nanA-nanK-nanE-3', inserting the nan gene into the knockout frame of the 5 '-nagB-nagA-nanA-nanK-nanE-3' on the chromosome of Corynebacterium glutamicum instead of the knockout frame by homologous recombination, the nanA gene on the chromosome is restored by homologous recombination to block the intracellular catabolism of N-acetylneuraminic acid.
Another technical problem to be solved by the present invention is to provide a method for constructing the recombinant Corynebacterium glutamicum, which comprises the following steps:
1) construction of recombinant plasmids
Cloning an glucosamine-fructose-6 phosphate aminotransferase encoding gene (glmS) from Corynebacterium glutamicum, a glucosamine acetylase encoding gene (GNA1) from Saccharomyces cerevisiae, a phosphatase encoding gene (yqaB) from Escherichia coli, an acetylglucosamine isomerase encoding gene (age) from Colletoceria and an N-acetylneuraminic acid synthase encoding gene (neuB) from Escherichia coli, and ligating them to a recombinant expression plasmid;
2) construction of recombinant Corynebacterium glutamicum producing N-acetylneuraminic acid
Transforming the recombinant expression vector into corynebacterium glutamicum to obtain the recombinant corynebacterium glutamicum for producing N-acetylneuraminic acid.
The Corynebacterium glutamicum is C.glutamcum ATCC13869 or Corynebacterium glutamcum ATCC13869 delta nanK delta nanE delta nagA delta nagB or Corynebacterium glutamcum ATCC13869 delta cg2937 delta nanK delta nanE delta nagA delta nagB; the recombinant expression vector is pDXW-9.
The invention also provides a method for producing N-acetylneuraminic acid by using the recombinant corynebacterium glutamicum, wherein seeds cultured for 12-24h at 30-32 ℃ and 180-220rpm are transferred into a fermentation medium by 5-10% of inoculum size, and cultured for 30-50h at 30-32 ℃ and 180-220 rpm.
Culturing and fermenting recombinant corynebacterium glutamicum seeds:
seed medium (g/L): 10 parts of glucose, 1.25 parts of urea, 20 parts of corn steep liquor, 1 part of monopotassium phosphate and 0.5 part of magnesium sulfate.
Fermentation medium (g/L): 80 parts of glucose, 35 parts of ammonium sulfate, 20 parts of corn steep liquor, 1 part of monopotassium phosphate, 1 part of magnesium sulfate and 30 parts of calcium carbonate.
The culture conditions are as follows: seeds cultured at the temperature of 30-32 ℃ and the rotational speed of 180-.
The method for measuring the N-acetylneuraminic acid comprises the following steps:
high Performance Liquid Chromatography (HPLC) detection: agilent 1200, RID Detector, NH2Column (250X 4.6mm, 5 μm), mobile phase: 70% acetonitrile, flow rate of 0.6mL/min, column temperature of 30 ℃, injection volume of 10 μ L.
The invention also provides a recombinant corynebacterium glutamicum capable of producing N-acetylneuraminic acid by fermentation by using glucose as a substrate. The recombinant Corynebacterium glutamicum is Corynebacterium glutamicum ATCC13869 delta cg2937 delta nanK delta nanE delta nagA delta nagB (P9-age-neuB-P)tacglmS-GNA1-yqaB), with Corynebacterium glutamicum ATCC 13869. delta. cg 2937. delta. nanK. delta. nanE. delta. nagA. delta. nagB as host, glmS, GNA1, yqaB, age and neuB were expressed using expression vector pDXW-9.
The recombinant corynebacterium glutamicum provided by the invention can realize extracellular accumulation of N-acetylneuraminic acid, the concentration of the recombinant corynebacterium glutamicum can reach 110mg/L, and a foundation is laid for further metabolic engineering modification of corynebacterium glutamicum to produce N-acetylneuraminic acid. The recombinant corynebacterium glutamicum provided by the invention is simple in construction method, convenient to use, belongs to a foodborne safe strain, and has a good application prospect.
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FIG. 1 recombinant Corynebacterium glutamicum C.glutamicum ATCC 13869. DELTA. cg 2937. DELTA. nanK. DELTA. nanE. DELTA. nagA. DELTA. nagB (P9-age-neuB-P)tacglmS-GNA1-yqaB) to synthesize N-acetylneuraminic acid.
Detailed Description
EXAMPLE 1 construction of recombinant plasmid
Age gene was synthesized by Kinsymond Biotechnology, Inc. based on the sequence of the acetylglucosamine isomerase-encoding gene (age) in the necklace algae published at NCBI (Anabaena sp. CH1), and primers age-F: 5'-TTCACACAGGAAACAGAATTCGAAGGAGTCTTCACATGGGCAAAAACTTACAAGCTCT-3' and age-R: 5'-CATTCTACTCTGACTTATGAAAGTGCTTCAAACTGTTGC-3' were designed. The acetylglucosamine isomerase-encoding gene (age) was amplified using the synthesized age gene fragment as a template using the above primers.
The neuB gene was synthesized by Kinsrui Biotechnology, Inc. based on the N-acetylneuraminic acid synthase coding gene (neuB) sequence in Escherichia coli (Escherichia coli K-1) published at NCBI, and primers neuB-F: 5'-TCATAAGTCAGAGTAGAATGAGAAGGAGTAGATTCATGTCTAACATCTACATCGTGGC-3' and neuB-R: 5'-CATCCGCCAAAACAGAAGCTTGTTAACTTTATTCTCCCTGGTTTTTAAATTCGC-3' were designed. The N-acetylneuraminic acid synthase encoding gene (neuB) was amplified using the synthesized neuB gene fragment as a template using the above primers.
The 2 gene fragments obtained by the above amplification were ligated by one-step cloning (the one-step cloning kit used was purchased from Nanjing Hookeonly Biotechnology Co., Ltd.) to EcoRI and HindIII cleavage sites on a pDXW-9 expression vector (Xu D, Tan Y, Shi F, Wang X. an engineered cut vector constructed for metabolic engineering research in Corynebacterium glutamicum. plasmid. 2010; 64: 85-91). Enzyme digestion verification and sequencing confirm that the recombinant plasmid p9-age-neuB is successfully constructed.
Based on the glucosamine-fructose-6 phosphate aminotransferase encoding gene (glmS) in Corynebacterium glutamicum (Corynebacterium glutamicum ATCC 13032) published on NCBI, primers glmS-F: 5'-CAGGAAACAGAATTCGCTAGCGAAGGAGTAATACGATGTGTGGAATTGTTGGATATA-3', glmS-R: 5'-TAGAGAGAGAGAGGTGGAAATTATTCGACGGTGACAGACTTTGC-3' are provided. The glucosamine-fructose-6 phosphate aminotransferase encoding gene (glmS) was amplified from the genome of Corynebacterium glutamicum (Corynebacterium glutamicum ATCC13869, available from the American type culture Collection) using the above primers.
According to the glucosamine acetylase-encoding gene (GNA1) in Saccharomyces cerevisiae (Saccharomyces cerevisiae S288C, available from the american type culture collection, accession No. ATCC 204508) published at NCBI, primers GNA1-F were designed: 5'-GGGGTACCATTATAGGTAAGAGAGGAATGTACACATGAGCTTACCCGATGGATTTTATA-3', GNA 1-R: 5'-CCCAAGCTTCTATTTTCTAATTTGCATTTCCACG-3' are provided. The glucosamine acetylase-encoding gene (GNA1) was amplified from the genome of Saccharomyces cerevisiae (Saccharomyces cerevisiae S288C) using the above primers.
Primers yqaB-F: 5'-TAAGTGCTCCATGAAGTCGTGAAGGAGTGTCTACATGTACGAGCGTTATGCAGGTTTA-3' and yqaB-R: 5'-CATCCGCCAAAACAGAAGCTTTCACAGCAAGCGAACATCCA-3' were designed based on the phosphatase-encoding gene (yqaB) of Escherichia coli (Escherichia coli K-12) published at NCBI. The phosphatase-encoding gene (yqaB) was amplified from the genome of E.coli (Escherichia coli K-12) using the above primers.
The 3 gene fragments glmS, GNA1 and yqaB obtained by the above amplification were ligated between NheI and dIHinII cleavage sites on a pDXW-9 expression vector (Xu D, TanY, Shi F, Wang X.an amplified cut vector construction for metabolic engineering research in Corynebacterium glutamicum plasmid 2010; 64:85-91) by a one-step cloning method (the one-step cloning kit used was purchased from Nanjing Hookenzan Biotechnology Co., Ltd.). Enzyme digestion verification and sequencing confirm that the recombinant plasmid p9-glmS-GNA1-yqaB is successfully constructed.
Design of primer P based on tac promoter sequencetac-F: 5'-ATTACCCGGGAAGCTGGCGATGTGGTGATT-3', respectively; based on the phosphatase-encoding gene (yqaB) of Escherichia coli (Escherichia coli K-12) published at NCBI, primers yqaB-R2: 5'-ATTACCCGGGTCACAGCAAGCGAACATCCA-3' were designed. Amplification of tac promoter-ligated P from recombinant plasmid P9-glmS-GNA1-yqaB Using the primers described abovetac-glmS-GNA1-yqaB fragment and treated with SmaI cleavage. The recombinant plasmid p9-age-neuB is cut by HindIII enzymeAfter the end is filled in, with PTacthe-glmS-GNA 1-yqaB fragment was ligated with T4 ligase. Enzyme digestion verification and sequencing are carried out, and the recombinant plasmid P9-age-neuB-P is confirmedtacThe construction of glmS-GNA1-yqaB was successful.
Example 2 knock-out of the N-acetylneuraminic acid transporter encoding Gene cg2937
Designing primers cg2937-U-F: 5'-CTATGACATGATTACGAATTCGCTGTGAGCTTTGATGGTTTC-3' according to the upstream and downstream sequences of an N-acetylneuraminic acid transporter coding gene (cg2937) in Corynebacterium glutamicum (Corynebacterium glutamicum ATCC 13032) published on NCBI; cg2937-U-R: 5'-ACATTGATCTCTACTCTGACTGCCGGTGTTGTCTGGTGCA-3'; cg 2937-D-F5'-GTCAGAGTAGAGATCAATGTCGAATCCTACGACCAGGTACA-3'; cg 2937-D-R5'-TGCCTGCAGGTCGACTCTAGAGGTGATTGGGGTGATCAGC-3'. Amplifying an upstream homologous arm sequence delta cg2937-Up of an N-acetylneuraminic acid transporter coding gene (cg2937) by using primers cg2937-U-F and cg2937-U-R by using a Corynebacterium glutamicum (Corynebacterium glutamicum ATCC13869, purchased from American type culture Collection) genome as a PCR template; the primers cg2937-D-F and cg2937-D-R are used to amplify the sequence of the downstream homology arm of the N-acetylneuraminic acid transporter coding gene (cg 2937). DELTA.cg 2937-Down. Δ cg2937-Up and Δ cg2937-Down were ligated to knock-out vector pk18mobsacB (by one-step cloning using a one-step cloning kit from Nanjing Novozam Biotechnology Ltd.)
Figure BDA0001586079880000051
A,Tauch A,
Figure BDA0001586079880000052
W, Kalinowski J, Thierbach G, P ü hlera A. small mobile 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) 69-73.) between EcoRI and XbaI sites, thus obtaining the suicide plasmid pk18mobsacB delta cg2937 containing the N-acetylneuraminic acid transporter coding gene knockout frame. Transformation of pk18 mobsacB. DELTA. cg2937 into Corynebacterium glutamicum ATCC13869 by means of a cardAnd (3) screening natamycin resistant plates, selecting plate transformants, inoculating LB liquid culture for 12h based on 30 ℃ and 200rpm, diluting bacterial suspension, coating LB plates containing 10% of cane sugar, screening plate single colonies without kanamycin resistance, performing colony PCR verification, and confirming that the N-acetylneuraminic acid transporter coding gene (cg2937) is successfully knocked out to obtain recombinant corynebacterium glutamicum 13869 delta cg 2937.
Example 3 blocking of the intracellular catabolic pathway of N-acetylneuraminic acid
The catabolism of N-acetylneuraminic acid in cells is blocked by knocking out an N-acetylglucosamine kinase coding gene nanK, an N-acetylglucosamine-6-phosphate isomerase coding gene nanE, an acetylglucosamine-6-phosphate deacetylase coding gene nagA and a glucosamine-6-phosphate deaminase coding gene nagB on chromosomes.
According to the sequences of the N-acetylneuraminic acid operon (5 '-nagB-nagA-nanA-nanK-nanE-3') and the upstream and downstream of the N-acetylneuraminic acid operon in Corynebacterium glutamicum (Corynebacterium glutamicum ATCC 13032) published on NCBI, a primer NEU-U-F: 5'-CTATGACATGATTACGAATTCGATTTCGGGGAGACATTCACT-3' is designed; 5'-GTACCTGAGAATGTAGTTTTTTGGTGCCAACGCGATCATC-3' for NEU-U-R; 5'-AAAACTACATTCTCAGGTACAAACGCTGATCACTACCGTCT-3' for NEU-D-F; 5'-TGCCTGCAGGTCGACTCTAGAGCGTAGAATTCATGGCCGAAAT-3' is NEU-D-R. Amplifying an upstream homologous arm sequence delta NEU-Up of an N-acetylneuraminic acid operon (5 '-nagB-nagA-nanA-nanK-nanE-3') by using primers NEU-U-F and NEU-U-R by taking a genome of Corynebacterium glutamicum (Corynebacterium glutamicum ATCC13869, purchased from American type culture Collection) as a PCR template; the primers NEU-D-F and NEU-D-R were used to amplify the sequence of the downstream homology arm of the N-acetylneuraminic acid operon (5 '-nagB-nagA-nanA-nanK-nanE-3'). DELTA.NEU-Down. Delta NEU-Up and Delta NEU-Down were ligated to the knock-out vector pk18mobsacB (by one-step cloning kit from Biotechnology Ltd of Nanjing Novozam) by one-step cloning
Figure BDA0001586079880000061
A,Tauch A,
Figure BDA0001586079880000062
W, Kalinowski J, Thierbach G, P ü hler A. Small mobile multi-phosphorous circulating from the Escherichia coli plasmids pK18and pK19 selection of defined definitions in the chromosome of Corynebacterium glutamicum, Gene.1994; 145, (1) 69-73.) between EcoRI and XbaI sites, the suicide plasmid pk18mobsacB delta NEU containing the knock-out frame of the N-acetylneuraminic acid operon is obtained. The method comprises the steps of converting pk18mobsacB delta NEU into Corynebacterium glutamicum ATCC13869 and 13869 delta cg2937, screening kanamycin-resistant plates, selecting plate transformants, inoculating LB liquid culture medium for 12 hours at 30 ℃ and 200rpm, diluting bacterial suspension, coating LB liquid culture medium with 10% of cane sugar, screening plate single colonies without kanamycin resistance, carrying out colony PCR verification, confirming that N-acetylneuraminic acid operon is successfully knocked out, and obtaining recombinant Corynebacterium glutamicum 13869 delta NEU and 13869 delta cg2937 delta NEU.
According to an N-acetylneuraminic acid lyase coding gene (nanA) in Corynebacterium glutamicum (Corynebacterium glutamicum ATCC 13032) published on NCBI and an upstream sequence thereof, primers nanA-F: 5'-GTACCTGAGAATGTAGTTTTCACTTCTGCCATCTTTCTG-3' are designed; nanA-R: 5'-CGGAGATCTGGTACTTTCGAGGTGTGGGCCTTAAGCGGTGT-3'; 5'-CTATGACATGATTACGAATTCGATTTCGGGGAGACATTCACT-3' is NEU-U-F; NEU-U-R2: 5'-GTACCTGAGAATGTAGTTTTTTGGTGCCAACGCGATCATC-3'; NEU-D-F2: 5'-CTCGAAAGTACCAGATCTCCGAAACGCTGATCACTACCGTCT-3'; 5'-TGCCTGCAGGTCGACTCTAGAGCGTAGAATTCATGGCCGAAAT-3' is NEU-D-R. The primer NEU-U-F and NEU-U-R2 are used for amplifying an upstream homologous arm sequence delta NEU-Up2 of an N-acetylneuraminic acid operon (5 '-nagB-nagA-nanA-nanK-nanE-3') by using a Corynebacterium glutamicum ATCC13869 (purchased from American type culture Collection) genome as a PCR template; amplifying N-acetylneuraminic acid lyase and an upstream promoter sequence nanA thereof by using primers nanA-F and nanA-R; the primers NEU-D-F2 and NEU-D-R were used to amplify the sequence of the downstream homology arm of the N-acetylneuraminic acid operon (5 '-nagB-nagA-nanA-nanK-nanE-3'). DELTA.NEU-Down 2. Delta NEU-Up2, nanA and Delta NEU-Down2 were subjected to a one-step cloning method (the one-step cloning kit used was purchased from Nanjing NovozamBiotechnology Ltd.) was ligated to vector pk18mobsacB (
Figure BDA0001586079880000071
A,Tauch A,
Figure BDA0001586079880000072
W, Kalinowski J, Thierbach G, P ü hlera A. small mobile 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) 69-73), and obtaining nanA anaplerotic plasmid pk18mobsacB delta NEU between EcoRI and XbaI sites. The recombinant Corynebacterium glutamicum 13869 delta 1NEU and 13869 delta 2cg2937 delta 3NEU are obtained by the steps of pnam 18mobsacB delta 0NEU, kanamycin resistance plate screening, plate transformant selection, LB liquid culture based on 30 ℃ and 200rpm for 12-18h, bacterial suspension dilution, LB plate culture based on 10% cane sugar, plate single colony without kanamycin resistance is screened, colony PCR verification is carried out, and the restoration success of the nanA on the chromosome is confirmed, thus obtaining the recombinant Corynebacterium glutamicum 13869 delta 4nanK delta nanE delta nagA delta nagB and 13869 delta cg2937 delta nanK delta nanE delta nagA nagB.
EXAMPLE 4 construction of recombinant Corynebacterium glutamicum accumulating N-acetylneuraminic acid
The constructed recombinant expression plasmid P9-age-neuB-PtacTransformation of Corynebacterium glutamicum ATCC13869, 13869. delta. nanK. delta. nanE. delta. nagA. delta. nagB and 13869. delta. cg 2937. delta. nanK. delta. nanE. delta. nagA. delta. nagB by glmS-GNA 1-yqaB. Colony PCR is carried out on selected transformants by using age-F and yqaB-R primers, a 5400bp band appears, and the recombinant Corynebacterium glutamicum ATCC13869 (P9-age-neuB-P) is verifiedtac-glmS-GNA1-yqaB)、13869△nanK△nanE△nagA△nagB(p9-age-neuB-PtacglmS-GNA1-yqaB) and 13869 Δ cg2937 Δ nanK Δ nanE Δ nagA Δ nagB (P9-age-neuB-P)tacglmS-GNA1-yqaB) was successfully constructed.
EXAMPLE 5 fermentative production of N-acetylneuraminic acid
Seed medium (g/L): 10 parts of glucose, 1.25 parts of urea, 20 parts of corn steep liquor, 1 part of monopotassium phosphate and 0.5 part of magnesium sulfate.
Fermentation medium (g/L): 80 parts of glucose, 35 parts of ammonium sulfate, 20 parts of corn steep liquor, 1 part of monopotassium phosphate, 1 part of magnesium sulfate and 30 parts of calcium carbonate.
Seeds cultured at the temperature of 30-32 ℃ and the rotational speed of 180-. Finally Corynebacterium glutamicum 13869. delta. cg 2937. delta. nanK. delta. nanE. delta. nagA. delta. nagB (P9-age-neuB-P)tacThe content of N-acetylneuraminic acid in the fermentation supernatant of the glmS-GNA1-yqaB) reaches 110 mg/L. Overexpresses the glucosamine-fructose-6-phosphate aminotransferase encoding gene (glmS), the glucosamine acetylase encoding gene (GNA1), the phosphatase encoding gene (yqaB), the acetylglucosamine isomerase encoding gene (age), and the N-acetylneuraminic acid synthase encoding gene (neuB), and an N-acetylneuraminic acid transporter coding gene (cg2937) on the chromosome and an intracellular N-acetylneuraminic acid decomposition N-acetylmannokinase coding gene (nanK), an N-acetylmannosyl-6-phosphate isomerase coding gene (nanE), an acetylglucosamine-6-phosphate deacetylase coding gene (nagA) and a glucosamine-6-phosphate deaminase coding gene (nagB) on a metabolic pathway are knocked out, so that the accumulation of the N-acetylneuraminic acid outside the recombinant corynebacterium glutamicum cells is realized.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> recombinant corynebacterium glutamicum for accumulating N-acetylneuraminic acid and application thereof
<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 (8)

1. Recombinant corynebacterium glutamicum (C) for accumulating N-acetylneuraminic acidCorynebacterium glutamicum) Its special featureCharacterized in that the synthesis pathway of N-acetylneuraminic acid is divided into 5 genes: the coding gene of glucosamine-fructose-6 phosphate aminotransferase, the coding gene of glucosamine acetylase, the coding gene of phosphatase, the coding gene of acetylglucosamine isomerase and the coding gene of exogenous N-acetylneuraminic acid synthase are expressed in corynebacterium glutamicum to realize the accumulation of N-acetylneuraminic acid; the host bacterium is corynebacterium glutamicum ATCC13869 delta cg2937 delta nanK delta nanE delta nagB with the knock-out of an N-acetylneuraminic acid transporter coding gene cg2937, an N-acetylaminomannokinase coding gene nanK, an N-acetylaminomannose-6-phosphate isomerase coding gene nanE, an acetylglucosamine-6-phosphate deacetylase coding gene nagA and a glucosamine-6-phosphate deaminase coding gene nagB.
2. The Corynebacterium glutamicum accumulating N-acetylneuraminic acid of claim 1, wherein the glucosamine-fructose-6-phosphate aminotransferase encoding gene is derived from Corynebacterium glutamicum ATCC13869, and/or the glucosamine acetylase encoding gene is derived from Saccharomyces cerevisiae S288C, and/or the phosphatase encoding gene is derived from Escherichia coli K-12, and/or the acetylglucosamine isomerase encoding gene is derived from Colletocerina sp CH1, and/or the N-acetylneuraminic acid synthase encoding gene is derived from Escherichia coli K-1.
3. The method for constructing Corynebacterium glutamicum accumulating N-acetylneuraminic acid of claim 1 or 2, wherein a recombinant expression vector carrying a glucosamine-fructose-6-phosphate aminotransferase encoding gene, a glucosamine acetylase encoding gene, a phosphatase encoding gene, an acetylglucosamine isomerase encoding gene, and an exogenous N-acetylneuraminic acid synthase encoding gene is constructed using expression vector pDXW-9.
4. The method for constructing Corynebacterium glutamicum accumulating N-acetylneuraminic acid of claim 3, wherein the age and neuB genes are ligated between EcoRI and HindIII cleavage sites on the pDXW-9 expression vector; the glmS, GNA1, yqaB genes were ligated into the pDXW-9 expression vector between the NheI and HindIII cleavage sites.
5. The method for constructing Corynebacterium glutamicum accumulating N-acetylneuraminic acid of claim 3 or 4, wherein the knockout of the N-acetylneuraminic acid transporter coding gene cg2937 of Corynebacterium glutamicum is performed by constructing a suicide plasmid containing a knockout frame of the N-acetylneuraminic acid transporter coding gene, and replacing the knockout frame with the N-acetylneuraminic acid transporter coding gene cg2937 on the chromosome of Corynebacterium glutamicum through homologous recombination, so as to block the transport of N-acetylneuraminic acid from the outside to the inside of the cell.
6. The method of claim 5, wherein the knockout of the N-acetylneuraminic acid-encoding gene nanK, the N-acetylmannosyl-6-phosphate isomerase-encoding gene nanE, the acetylglucosamine-6-phosphate deacetylase-encoding gene nagA and the glucosamine-6-phosphate deaminase-encoding gene nagB is performed by constructing a suicide plasmid containing a knockout frame of the N-acetylneuraminic acid operon 5 '-nagB-nagA-nanA-nanK-nanE-3', substituting the knockout frame for the N-acetylneuraminic acid operon 5 '-nagB-nagA-nanA-nanK-nanE-3' on the chromosome of Corynebacterium glutamicum by homologous recombination, then the nanA gene is inserted into the 5 '-nagB-nagA-nanA-nanK-nanE-3' knockout frame, and the nanA gene on the chromosome is restored through homologous recombination so as to block the catabolism of the N-acetylneuraminic acid in the cells.
7. A method for producing N-acetylneuraminic acid by fermentation of the recombinant Corynebacterium glutamicum as claimed in claim 1 or 2, which is characterized in that the seed liquid cultured at 30-32 ℃ and 180-220rpm for 12-24h is transferred into the fermentation medium with an inoculum size of 5% -10% and cultured at 30-32 ℃ and 180-220rpm for 30-50 h.
8. The method of claim 7,
seed medium (g/L): 10 parts of glucose, 1.25 parts of urea, 20 parts of corn steep liquor, 1 part of monopotassium phosphate and 0.5 part of magnesium sulfate; fermentation medium (g/L): 80 parts of glucose, 35 parts of ammonium sulfate, 20 parts of corn steep liquor, 1 part of monopotassium phosphate, 1 part of magnesium sulfate and 30 parts of calcium carbonate.
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