CN108330095B - A kind of recombinant Corynebacterium glutamicum accumulating N-acetylneuraminic acid and its application - Google Patents

Abstract

The invention discloses a recombinant Corynebacterium glutamicum that accumulates N-acetylneuraminic acid and an application thereof, belonging to the field of genetic engineering. The present invention uses Corynebacterium glutamicum as an expression host, and by overexpressing glucosamine-fructose-6 phosphate aminotransferase gene, glucosamine acetylase coding gene, phosphatase coding gene, acetylglucosamine isomerase coding gene and N The gene encoding acetylneuraminic acid synthase enhances the N-acetylneuraminic acid synthesis pathway; and by knocking out the gene encoding the N-acetylneuraminic acid transporter and intracellular N-acetylneuraminic acid in Corynebacterium glutamicum Decomposition and utilization of the relevant genes on the metabolic pathway, the Corynebacterium glutamicum genetically engineered bacteria that accumulates N-acetylneuraminic acid extracellularly is obtained, and the yield reaches 110 mg/L, which is used for further metabolic engineering of Corynebacterium glutamicum to produce N-acetyl Neuraminidine lays the foundation.

Description

A kind of recombinant Corynebacterium glutamicum accumulating N-acetylneuraminic acid and its application

technical field

The invention relates to a recombinant Corynebacterium glutamicum that accumulates N-acetylneuraminic acid and its application, and belongs to the field of genetic engineering.

Background technique

N-acetylneuraminic acid, as the most important compound molecule in sialic acid, is an important food nutritional additive and a new type of drug precursor. Its effects include promoting brain development in infants, maintaining healthy brain function in the elderly, and enhancing immunity. At present, the industrial production of N-acetylneuraminic acid is mainly produced by chemical extraction and whole-cell transformation methods. Because the total content of N-acetylneuraminic acid in natural raw materials (eg, egg yolk, bird's nest and bovine colostrum, etc.) is low, the extraction and separation process is complicated, resulting in low yield and high cost of traditional extraction methods. Therefore, the production of N-acetylneuraminic acid by whole cell transformation has become a research hotspot in recent years. However, the whole-cell transformation methods all require the addition of N-acetylglucosamine as a synthetic precursor, and most methods still require the addition of pyruvate as a synthetic precursor. In view of the high cost of N-acetylglucosamine and pyruvic acid (60,000-80,000 yuan/ton), developing a method for producing N-acetylneuraminic acid by microbial fermentation of glucose will have important research significance and application value. .

As a common industrial strain, Corynebacterium glutamicum has many advantages, such as clear genetic background, mature metabolic transformation tools, mature fermentation process, and the fermentation process is not susceptible to phage contamination. It is widely used in the fermentation production of important chemicals. At the same time, Corynebacterium glutamicum is an internationally recognized food-safe strain, and it is an ideal strain for the production of food, nutritional products and medicines. Therefore, the use of metabolic engineering methods to construct recombinant Corynebacterium glutamicum is an effective way to produce food-safe N-acetylneuraminic acid. However, the N-acetylneuraminic acid metabolic pathway in C. glutamicum is tightly regulated, and the accumulation of N-acetylneuraminic acid does not occur. How to modify the N-acetylneuraminic acid metabolic pathway in Corynebacterium glutamicum is a question worthy of further study.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to construct a recombinant Corynebacterium glutamicum that accumulates N-acetylneuraminic acid.

For solving the above-mentioned technical problems, the technical scheme of the present invention is:

Five genes in the N-acetylneuraminic acid synthesis pathway: the homologous glucosamine-fructose-6 phosphate aminotransferase encoding gene, especially the glmS (encoding glucosamine-fructose-) of Corynebacterium glutamicum ATCC13869 The amino acid sequence of 6-phosphate aminotransferase is such as SEQ ID NO: 1); the exogenous glucosamine acetylase coding gene, especially the GNA1 of Saccharomyces cerevisiae S288C (the amino acid sequence of the coded glucosamine acetylase such as SEQ ID NO: 1) ID NO: 2); exogenous phosphatase encoding gene, especially yqaB of Escherichia coli (Escherichiacoli K-12) (the amino acid sequence of the encoded phosphatase is as SEQ ID NO: 3); exogenous acetylglucosamine isomerase encoding Genes, especially the age of Anabaena sp. CH1 (the amino acid sequence of the acetylglucosamine isomerase encoded as SEQ ID NO: 4); and exogenous N-acetylneuraminic acid synthase encoding genes, especially neuB of Escherichia coli K-1 (the amino acid sequence of the encoded N-acetylneuraminic acid synthase is shown in SEQ ID NO: 5): cloned into the expression vector pDXW-9, and then transformed into Corynebacterium glutamicum glutamicumATCC13869), by modifying the metabolic pathway to achieve the accumulation of N-acetylneuraminic acid.

In one embodiment of the present invention, the expression vector pDXW-9 is selected to express the gene encoding glucosamine-fructose-6 phosphate aminotransferase, the gene encoding glucosamine acetylase, the gene encoding phosphatase, the gene encoding acetylglucosamine isomerase Gene and the gene encoding N-acetylneuraminic acid synthase.

In one embodiment of the present invention, the age and neuB genes are linked between the EcoRI and HindIII restriction sites on the pDXW-9 expression vector.

In one embodiment of the present invention, the glmS, GNA1, and yqaB genes are linked between the NheI and HindIII restriction sites on the pDXW-9 expression vector.

In one embodiment of the present invention, the N-acetylneuraminic acid transporter-encoding gene cg2937 of the Corynebacterium glutamicum is knocked out, specifically, by constructing a knockout gene containing the N-acetylneuraminic acid transporter-encoding gene An out-of-frame suicide plasmid, the knockout box was replaced by homologous recombination to replace the gene cg2937 encoding N-acetylneuraminic acid transporter on the chromosome of Corynebacterium glutamicum to block the transport of N-acetylneuraminic acid from extracellular to intracellular .

In one embodiment of the present invention, the N-acetylaminomannose kinase encoding gene nanK, the N-acetylaminomannose-6-phosphate isomerase encoding gene nanE, acetylamino Glucose-6-phosphate deacetylase-encoding gene nagA and glucosamine-6-phosphate deaminase-encoding gene nagB were constructed by constructing a gene containing N-acetylneuraminic acid operon 5'-nagB-nagA-nanA-nanK-nanE -Suicide plasmid with 3' knockout box, the knockout box is replaced by homologous recombination of the N-acetylneuraminic acid operon 5'-nagB-nagA-nanA-nanK-nanE-3' on the chromosome of Corynebacterium glutamicum, Then insert the nanA gene in the 5'-nagB-nagA-nanA-nanK-nanE-3' knockout box, and restore the nanA gene on the chromosome by homologous recombination to block the intracellular transfer of N-acetylneuraminic acid. Catabolism.

Another technical problem to be solved by the present invention is to provide a kind of construction method of constructing the above-mentioned recombinant Corynebacterium glutamicum, which is characterized in that comprising the steps:

1) Construction of recombinant plasmid

Cloning of the glucosamine-fructose-6 phosphate aminotransferase encoding gene (glmS) of Corynebacterium glutamicum, the glucosamine acetylase encoding gene (GNA1) of Saccharomyces cerevisiae, the phosphatase encoding gene (yqaB) of Escherichia coli, and the algae The gene encoding acetylglucosamine isomerase (age) and the gene encoding N-acetylneuraminic acid synthase (neuB) of Escherichia coli are connected to the recombinant expression plasmid;

2) Construction of N-acetylneuraminic acid-producing recombinant Corynebacterium glutamicum

The above recombinant expression vector is transformed into Corynebacterium glutamicum to obtain N-acetylneuraminic acid-producing recombinant Corynebacterium glutamicum.

The Corynebacterium glutamicum is C.glutamicum ATCC13869 or Corynebacterium glutamicumATCC13869△nanK△nanE△nagA△nagB or Corynebacterium glutamicum ATCC13869△cg2937△nanK△nanE△nagA△nagB; the recombinant expression vector is pDXW-9.

The present invention also provides a method for producing N-acetylneuraminic acid by using the above-mentioned recombinant Corynebacterium glutamicum by fermentation. The amount was transferred to the fermentation medium, and cultured at 30-32°C and 180-220rpm for 30-50h.

Recombinant Corynebacterium glutamicum seed culture and fermentation:

Seed 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.

Cultivation conditions: The seeds cultivated at 30-32°C and 180-220rpm for 12-24h were transferred to the fermentation medium with an inoculum of 5%-10%, and cultured at 30-32°C and 180-220rpm for 30-50h.

Determination method of N-acetylneuraminic acid:

High performance liquid chromatography (HPLC) detection method: Agilent 1200, RID detector, _NH2 column (250×4.6mm, 5μm), mobile phase: 70% acetonitrile, flow rate 0.6mL/min, column temperature 30℃, injection volume to 10 μL.

The invention also provides a recombinant Corynebacterium glutamicum capable of producing N-acetylneuraminic acid by fermentation using glucose as a substrate. The recombinant Corynebacterium glutamicum is Corynebacterium glutamicum ATCC13869△cg2937△nanK△nanE△nagA△nagB(p9-age-neuB-P _tac -glmS-GNA1-yqaB), which is Corynebacterium glutamicum ATCC13869△cg2937△nanK△nanE△ nagA△nagB was the host, and expression vector pDXW-9 was used to express glmS, GNA1, yqaB, age and neuB.

The recombinant Corynebacterium glutamicum provided by the invention can realize the extracellular accumulation of N-acetylneuraminic acid, and its concentration can reach 110 mg/L, which lays a solid foundation for the further metabolic engineering of Corynebacterium glutamicum to produce N-acetylneuraminic acid. Base. The recombinant Corynebacterium glutamicum provided by the invention has a simple construction method, is easy to use, belongs to a food-derived safe strain, and has a good application prospect.

Description of drawings

Fig. 1 The pathway of recombinant C. glutamicum ATCC13869△cg2937△nanK△nanE△nagA△nagB(p9-age-neuB-P _tac -glmS-GNA1-yqaB) to synthesize N-acetylneuraminic acid.

Detailed ways

Example 1 Construction of recombinant plasmid

According to the sequence of the acetylglucosamine isomerase encoding gene (age) in Anabaena sp. CH1 published on NCBI, the age gene was synthesized by GenScript Biotechnology Co., Ltd., and the primer age-F: 5' was designed. -TTCACACAGGAAACAGAATTCGAAGGAGTCTTCACATGGGCAAAAACTTACAAGCTCT-3',age-R:5'-CATTCTACTCTGACTTATGAAAGTGCTTCAAACTGTTGC-3'. The acetylglucosamine isomerase-encoding gene (age) was amplified using the above-mentioned primers with the synthetically obtained age gene fragment as a template.

According to the sequence of the N-acetylneuraminic acid synthase encoding gene (neuB) in Escherichia coli K-1 published on NCBI, the neuB gene was synthesized by GenScript Biotechnology Co., Ltd., and the primer neuB-F was designed. :5'-TCATAAGTCAGAGTAGAATGAGAAGGAGTAGATTCATGTCTAACATCTACATCGTGGC-3',neuB-R:5'-CATCCGCCAAAACAGAAGCTTGTTAACTTTATTCTCCCTGGTTTTTAAATTCGC-3'. The gene encoding N-acetylneuraminic acid synthase (neuB) was amplified using the above primers with the synthesized neuB gene fragment as a template.

The 2 gene fragments obtained by the above amplification were connected to the pDXW-9 expression vector (Xu D, Tan Y, Shi F, Wang) through the one-step cloning method (the one-step cloning kit used was purchased from Nanjing Novizan Biotechnology Co., Ltd.). X. Animproved shuttle vector constructed for metabolic engineering research in Corynebacteriun glutamicum. Plasmid. 2010; 64:85-91) between the EcoRI and HindIII restriction sites. Enzyme digestion verification and sequencing confirmed that the recombinant plasmid p9-age-neuB was successfully constructed.

According to the gene encoding glucosamine-fructose-6 phosphate aminotransferase (glmS) in Corynebacterium glutamicumATCC 13032 published on NCBI, the primers glmS-F: 5'-CAGGAAACAGAATTCGCTAGCGAAGGAGTAATACGATGTGTGGAATTGTTGGATATA-3', glmS-R were designed: 5'-TAGAGAGAGAGAGGTGGAAATTATTCGACGGTGACAGACTTTGC-3'. The gene encoding glucosamine-fructose-6 phosphate aminotransferase (glmS) was amplified from the genome of Corynebacterium glutamicum ATCC13869 (purchased from American Type Collection) using the above primers.

According to the glucosamine acetylase encoding gene (GNA1) in Saccharomyces cerevisiae S288C published on NCBI (purchased from the American Collection of Types of Microorganisms, No. ATCC 204508), the primer GNA1-F was designed: 5'-GGGGTACCATTATAGGTAAGAGAGGAATGTACACATGAGCTTACCCGATGGATTTTATA-3', GNA1-R: 5'-CCCAAGCTTCTATTTTCTAATTTGCATTTCCACG-3'. The gene encoding glucosamine acetylase (GNA1) was amplified from the genome of Saccharomyces cerevisiae S288C using the above primers.

According to the phosphatase encoding gene (yqaB) of Escherichia coli K-12 published on NCBI, primers yqaB-F: 5'-TAAGTGCTCCATGAAGTCGTGAAGGAGTGTCTACATGTACGAGCGTTATGCAGGTTTA-3', yqaB-R: 5'-CATCCGCCAAAACAGAAGCTTTCAGCAAGCGAACATCCA-3' were designed. The phosphatase-encoding gene (yqaB) was amplified from the Escherichia coli K-12 genome using the primers described above.

The 3 gene fragments glmS, GNA1, yqaB obtained by the above amplification were connected to the pDXW-9 expression vector (Xu D, TanY, Shi F, Wang X. An improved shuttle vector constructed for metabolic engineering research in Corynebacteriun glutamicum. Plasmid. 2010;64:85-91) between NheI and HindIII cleavage sites. Enzyme digestion verification and sequencing confirmed that the recombinant plasmid p9-glmS-GNA1-yqaB was successfully constructed.

The primer P _tac -F was designed according to the tac promoter sequence: 5'-ATTACCCGGGAAGCTGGCGATGTGGTGATT-3'; the primer yqaB-R2:5 was designed according to the phosphatase encoding gene (yqaB) of Escherichia coli K-12 published on NCBI '-ATTACCCGGGTCACAGCAAGCGAACATCCA-3'. The Ptac-glmS-GNA1-yqaB fragment linked with the _tac promoter was amplified from the recombinant plasmid p9-glmS-GNA1-yqaB using the above primers, and digested with SmaI. The recombinant plasmid p9-age-neuB was digested with HindIII, blunt-ended, and then ligated with the P _Tac -glmS-GNA1-yqaB fragment using T4 ligase. Enzyme digestion and sequencing confirmed that the recombinant plasmid p9-age-neuB-P _tac -glmS-GNA1-yqaB was successfully constructed.

Example 2 Knockout of N-acetylneuraminic acid transporter-encoding gene cg2937

A,Tauch A,

W,Kalinowski J,Thierbach G,PühlerA.Small mobilizable multi-purpose cloning vectors derived from theEscherichia coli plasmids pK18and pK19:selection of defined deletions in thechromosome of Corynebacterium glutamicum.Gene.1994；145(1):69-73.)上EcoRI和XbaI位点之间，即得到含有N-乙酰神经氨酸转运蛋白编码基因敲除框的自杀质粒pk18mobsacB△cg2937。将pk18mobsacB△cg2937转化谷氨酸棒杆菌Corynebacteriumglutamicum ATCC13869，通过卡那霉素抗性平板筛选，挑选平板转化子接种LB液体培养基于30℃、200rpm培养12h，将菌悬液稀释、涂布含有10％蔗糖的LB平板，筛选出不具有卡那抗性的平板单菌落，进行菌落PCR验证，确认N-乙酰神经氨酸转运蛋白编码基因(cg2937)敲除成功，得到重组谷氨酸棒杆菌13869△cg2937。According to the upstream and downstream sequences of the N-acetylneuraminic acid transporter gene (cg2937) in Corynebacterium glutamicum ATCC 13032 published on NCBI, the primer cg2937-UF: 5'-CTATGACATGATTACGAATTCGCTGTGAGCTTTGATGGTTTC-3'; cg2937- UR: 5'-ACATTGATCTCTACTCTGACTGCCGGTGTTGTCTGGTGCA-3'; cg2937-DF: 5'-GTCAGAGTAGAGATCAATGTCGAATCCTACGACCAGGTACA-3'; cg2937-DR: 5'-TGCCTGCAGGTCGACTCTAGAGGTGATTGGGGTGATCAGC-3'. Using the genome of Corynebacterium glutamicum ATCC13869 (purchased from the American Collection of Types of Microorganisms) as a PCR template, the upstream of the N-acetylneuraminic acid transporter-encoding gene (cg2937) was amplified using primers cg2937-UF and cg2937-UR The homology arm sequence Δcg2937-Up; the homology arm sequence Δcg2937-Down downstream of the N-acetylneuraminic acid transporter encoding gene (cg2937) was amplified by primers cg2937-DF and cg2937-DR. △cg2937-Up and △cg2937-Down were ligated into the knockout vector pk18mobsacB (

A,Tauch A,

W, Kalinowski J, Thierbach G, Pühler A. Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in thechromosome of Corynebacterium glutamicum. Gene. 1994;145(1):69-73.) on Between the EcoRI and XbaI sites, the suicide plasmid pk18mobsacB△cg2937 containing the knockout box of the N-acetylneuraminic acid transporter-encoding gene was obtained. Transform pk18mobsacB△cg2937 into Corynebacterium glutamicum ATCC13869, screen by kanamycin resistance plate, select the plate transformants to inoculate LB liquid culture based on 30 ℃, 200rpm culture for 12h, dilute the bacterial suspension, spread containing 10% The LB plate of sucrose was used to screen out the single colony of the plate without kana resistance, and the colony PCR was carried out to confirm that the gene encoding the N-acetylneuraminic acid transporter (cg2937) was successfully knocked out, and the recombinant Corynebacterium glutamicum 13869△ was obtained. cg2937.

Example 3 Blockade of intracellular catabolic pathway of N-acetylneuraminic acid

By knocking out the genes encoding N-acetylaminomannose kinase nanK, N-acetylmannose-6-phosphate isomerase encoding genes nanE, acetylglucosamine-6-phosphate deacetylase encoding genes nagA and glucosamine on chromosomes -6-Phosphate deaminase encodes the gene nagB to block the intracellular catabolism of N-acetylneuraminic acid.

A,Tauch A,

W,Kalinowski J,ThierbachG,Pühler A.Small mobilizable multi-purpose cloning vectors derived from theEscherichia coli plasmids pK18and pK19:selection of defined deletions in thechromosome of Corynebacterium glutamicum.Gene.1994；145(1):69-73.)上EcoRI和XbaI位点之间，即得到含有N-乙酰神经氨酸操纵子敲除框的自杀质粒pk18mobsacB△NEU。将pk18mobsacB△NEU转化谷氨酸棒杆菌Corynebacterium glutamicum ATCC13869以及13869△cg2937，通过卡那霉素抗性平板筛选，挑选平板转化子接种LB液体培养基于30℃、200rpm培养12h，将菌悬液稀释、涂布含有10％蔗糖的LB平板，筛选出不具有卡那抗性的平板单菌落，进行菌落PCR验证，确认N-乙酰神经氨酸操纵子敲除成功，得到重组谷氨酸棒杆菌13869△NEU以及13869△cg2937△NEU。The primer NEU- UF: 5'-CTATGACATGATTACGAATTCGATTTCGGGGAGACATTCACT-3'; NEU-UR: 5'-GTACCTGAGAATGTAGTTTTTTGGTGCCAACGCGATCATC-3'; NEU-DF: 5'-AAAACTACATTCTCAGGTACAAACGCTGATCACTACCGTCT-3'; NEU-DR: 5'-TGCCTGCAGGTCGACTCTAGAGCGTAGAATTCATGGCCGAAAT-3'. The N-acetylneuraminic acid operon (5'-nagB- nagA-nanA-nanK-nanE-3') upstream homology arm sequence △NEU-Up; using primers NEU-DF and NEU-DR to amplify the N-acetylneuraminic acid operon (5'-nagB-nagA-nanA -nanK-nanE-3') downstream homology arm sequence ΔNEU-Down. △NEU-Up and △NEU-Down were ligated into the knockout vector pk18mobsacB (

A,Tauch A,

W, Kalinowski J, Thierbach G, Pühler A. Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in thechromosome of Corynebacterium glutamicum. Gene. 1994;145(1):69-73.) on Between the EcoRI and XbaI sites, the suicide plasmid pk18mobsacBΔNEU containing the N-acetylneuraminic acid operon knockout box was obtained. The pk18mobsacB△NEU was transformed into Corynebacterium glutamicum ATCC13869 and 13869△cg2937, screened by kanamycin resistance plate, and the plate transformants were selected to inoculate LB liquid culture based on 30 ℃, 200rpm culture for 12h, the bacterial suspension was diluted, Coat the LB plate containing 10% sucrose, screen out the single colony on the plate without kana resistance, and perform colony PCR verification to confirm the successful knockout of the N-acetylneuraminic acid operon, and obtain the recombinant Corynebacterium glutamicum 13869△ NEU and 13869Δcg2937ΔNEU.

A,Tauch A,

W,Kalinowski J,Thierbach G,PühlerA.Small mobilizable multi-purpose cloningvectors derived from the Escherichia coli plasmids pK18and pK19:selection ofdefined deletions in the chromosome of Corynebacterium glutamicum.Gene.1994；145(1):69-73.)上EcoRI和XbaI位点之间，即得到nanA回补质粒pk18mobsacB△NEU::nanA。将pk18mobsacB△NEU::nanA转化谷氨酸棒杆菌13869△NEU以及13869△cg2937△NEU，通过卡那霉素抗性平板筛选，挑选平板转化子接种LB液体培养基于30℃、200rpm培养12-18h，将菌悬液稀释、涂布含有10％蔗糖的LB平板，筛选出不具有卡那抗性的平板单菌落，进行菌落PCR验证，确认染色体上nanA复原成功，既得到重组谷氨酸棒杆菌13869△nanK△nanE△nagA△nagB以及13869△cg2937△nanK△nanE△nagA△nagB。According to the N-acetylneuraminic acid lyase encoding gene (nanA) and its upstream sequence in Corynebacterium glutamicum ATCC 13032 published on NCBI, the primer nanA-F: 5'-GTACCTGAGAATGTAGTTTTCACTTCTGCCATCTTTTCG-3' was designed; nanA-R: 5'-CGGAGATCTGGTACTTTCGAGGTGTGGGCCTTAAGCGGTGT-3';NEU-UF:5'-CTATGACATGATTACGAATTCGATTTCGGGGAGACATTCACT-3';NEU-U-R2:5'-GTACCTGAGAATGTAGTTTTTTGGTGCCAACGCGATCATC-3';NEU-D-F2:5'-CTCGAAAGTACCAGATCTCCGAAACGCTGATCACTACC ';NEU-DR:5'-TGCCTGCAGGTCGACTCTAGAGCGTAGAATTCATGGCCGAAT-3'. The N-acetylneuraminic acid operon (5'- nagB-nagA-nanA-nanK-nanE-3') upstream homology arm sequence △NEU-Up2; use primers nanA-F and nanA-R to amplify N-acetylneuraminic acid lyase and its upstream promoter sequence nanA; using primers NEU-D-F2 and NEU-DR to amplify the downstream homology arm sequence of the N-acetylneuraminic acid operon (5'-nagB-nagA-nanA-nanK-nanE-3') △NEU-Down2 . △NEU-Up2, nanA and △NEU-Down2 were ligated into the vector pk18mobsacB (

A,Tauch A,

W, Kalinowski J, Thierbach G, Pühler A. Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene. 1994;145(1):69-73.) on Between the EcoRI and XbaI sites, the nanA complementing plasmid pk18mobsacB△NEU::nanA was obtained. pk18mobsacB△NEU::nanA was transformed into Corynebacterium glutamicum 13869△NEU and 13869△cg2937△NEU, screened by kanamycin resistance plate, and the plate transformants were selected to inoculate LB liquid culture based on 30℃, 200rpm culture for 12-18h , the bacterial suspension was diluted, coated with LB plate containing 10% sucrose, and the single colony on the plate without kana resistance was screened out, and the colony PCR was carried out to confirm the successful recovery of nanA on the chromosome, and the recombinant Corynebacterium glutamicum was obtained. 13869△nanK△nanE△nagA△nagB and 13869△cg2937△nanK△nanE△nagA△nagB.

Example 4 Construction of recombinant Corynebacterium glutamicum accumulating N-acetylneuraminic acid

The constructed recombinant expression plasmid p9-age-neuB-P _tac -glmS-GNA1-yqaB was transformed into Corynebacterium glutamicum ATCC13869, 13869△nanK△nanE△nagA△nagB and 13869△cg2937△nanK△nanE△nagA ΔnagB. Using age-F and yqaB-R primers to select transformants for colony PCR, a 5400bp band appeared, verifying that the recombinant Corynebacterium glutamicum ATCC13869 (p9-age-neuB-P _tac -glmS-GNA1-yqaB), 13869 △nanK△nanE△nagA△nagB(p9-age-neuB-P _tac -glmS-GNA1-yqaB) and 13869△cg2937△nanK△nanE△nagA△nagB(p9-age-neuB-P _tac -glmS-GNA1- yqaB) build successfully.

Example 5 Fermentation to produce N-acetylneuraminic acid

Seed 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 seeds cultured at 30-32°C and 180-220rpm for 12-24h were transferred to the fermentation medium at an inoculum of 5%-10%, and cultured at 30-32°C and 180-220rpm for 30-50h. Finally, the content of N-acetylneuraminic acid in the fermentation supernatant of Corynebacterium glutamicum 13869△cg2937△nanK△nanE△nagA△nagB(p9-age-neuB-P _tac -glmS-GNA1-yqaB) reached 110mg/L. Overexpression of the gene encoding glucosamine-fructose-6 phosphate aminotransferase (glmS), the gene encoding glucosamine acetylase (GNA1), the gene encoding phosphatase (yqaB), the gene encoding acetylglucosamine isomerase (age) and N - The gene encoding acetylneuraminic acid synthase (neuB), and the gene encoding the N-acetylneuraminic acid transporter (cg2937) on the chromosome and the N-acetylneuramidase on the metabolic pathway of intracellular N-acetylneuraminic acid catabolism are knocked out Mannokinase encoding gene (nanK), N-acetylaminomannose-6-phosphate isomerase encoding gene (nanE), acetylglucosamine-6-phosphate deacetylase encoding gene (nagA), glucosamine-6- Phosphate deaminase encoding gene (nagB) realizes the extracellular accumulation of N-acetylneuraminic acid in recombinant Corynebacterium glutamicum.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone who is familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, The protection scope of the present invention should be defined by the claims.

SEQUENCE LISTING

<110> Jiangnan University

<120> A kind of recombinant Corynebacterium glutamicum accumulating N-acetylneuraminic acid 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 sequences

<400> 6

ttcacacagg aaacagaatt cgaaggagtc ttcacatggg caaaaactta caagctct 58

<210> 7

<211> 39

<212> DNA

<213> Artificial sequences

<400> 7

cattctactc tgacttatga aagtgcttca aactgttgc 39

<210> 8

<211> 58

<212> DNA

<213> Artificial sequences

<400> 8

tcataagtca gagtagaatg agaaggagta gattcatgtc taacatctac atcgtggc 58

<210> 9

<211> 54

<212> DNA

<213> Artificial sequences

<400> 9

catccgccaa aacagaagct tgttaacttt attctccctg gtttttaaat tcgc 54

<210> 10

<211> 57

<212> DNA

<213> Artificial sequences

<400> 10

caggaaacag aattcgctag cgaaggagta atacgatgtg tggaattgtt ggatata 57

<210> 11

<211> 44

<212> DNA

<213> Artificial sequences

<400> 11

tagagagaga gaggtggaaa ttattcgacg gtgacagact ttgc 44

<210> 12

<211> 59

<212> DNA

<213> Artificial sequences

<400> 12

ggggtaccat tataggtaag agaggaatgt acacatgagc ttacccgatg gattttata 59

<210> 13

<211> 34

<212> DNA

<213> Artificial sequences

<400> 13

cccaagcttc tattttctaa tttgcatttc cacg 34

<210> 14

<211> 58

<212> DNA

<213> Artificial sequences

<400> 14

taagtgctcc atgaagtcgt gaaggagtgt ctacatgtac gagcgttatg caggttta 58

<210> 15

<211> 41

<212> DNA

<213> Artificial sequences

<400> 15

catccgccaa aacagaagct ttcacagcaa gcgaacatcc a 41

<210> 16

<211> 30

<212> DNA

<213> Artificial sequences

<400> 16

attacccggg aagctggcga tgtggtgatt 30

<210> 17

<211> 30

<212> DNA

<213> Artificial sequences

<400> 17

attacccggg tcacagcaag cgaacatcca 30

<210> 18

<211> 42

<212> DNA

<213> Artificial sequences

<400> 18

ctatgacatg attacgaatt cgctgtgagc tttgatggtt tc 42

<210> 19

<211> 40

<212> DNA

<213> Artificial sequences

<400> 19

acattgatct ctactctgac tgccggtgtt gtctggtgca 40

<210> 20

<211> 41

<212> DNA

<213> Artificial sequences

<400> 20

gtcagagtag agatcaatgt cgaatcctac gaccaggtac a 41

<210> 21

<211> 40

<212> DNA

<213> Artificial sequences

<400> 21

tgcctgcagg tcgactctag aggtgattgg ggtgatcagc 40

<210> 22

<211> 42

<212> DNA

<213> Artificial sequences

<400> 22

ctatgacatg attacgaatt cgatttcggg gagacattca ct 42

<210> 23

<211> 40

<212> DNA

<213> Artificial sequences

<400> 23

gtacctgaga atgtagtttt ttggtgccaa cgcgatcatc 40

<210> 24

<211> 41

<212> DNA

<213> Artificial sequences

<400> 24

aaaactacat tctcaggtac aaacgctgat cactaccgtc t 41

<210> 25

<211> 43

<212> DNA

<213> Artificial sequences

<400> 25

tgcctgcagg tcgactctag agcgtagaat tcatggccga aat 43

<210> 26

<211> 39

<212> DNA

<213> Artificial sequences

<400> 26

gtacctgaga atgtagtttt cacttctgcc atctttctg 39

<210> 27

<211> 41

<212> DNA

<213> Artificial sequences

<400> 27

cggagatctg gtactttcga ggtgtgggcc ttaagcggtg t 41

<210> 28

<211> 42

<212> DNA

<213> Artificial sequences

<400> 28

ctatgacatg attacgaatt cgatttcggg gagacattca ct 42

<210> 29

<211> 40

<212> DNA

<213> Artificial sequences

<400> 29

gtacctgaga atgtagtttt ttggtgccaa cgcgatcatc 40

<210> 30

<211> 42

<212> DNA

<213> Artificial sequences

<400> 30

ctcgaaagta ccagatctcc gaaacgctga tcactaccgt ct 42

<210> 31

<211> 43

<212> DNA

<213> Artificial sequences

<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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