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Recombinant microbe capable of yielding N-acetylneuraminic acid and application of recombinant microbe

A technology of acetylneuraminic acid and recombinant microorganisms, applied in the direction of microorganisms, microorganism-based methods, recombinant DNA technology, etc., can solve the problems of harsh chemical synthesis reaction conditions, environmental pollution, and difficult product separation, and achieve important industrial application value , reduce production costs, broad application prospects

Active Publication Date: 2021-07-16
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The content of N-acetylneuraminic acid in natural products is low, and the yield is low; the chemical synthesis reaction conditions are harsh, and it is easy to cause environmental pollution; whole-cell biological enzymes use pyruvate and acetylglucosamine as raw materials, which are expensive and neuraminic acid aldehyde Condensation enzyme catalyzes reversible decomposition, and a large amount of pyruvate remains after the reaction, which makes product separation difficult, resulting in low conversion rate and high production cost

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] The construction of embodiment 1 departure bacterial strain

[0059] In this example, Escherichia coli BL21 was taken as an example to construct a starting strain capable of synthesizing N-acetylneuraminic acid. Specifically, the nanATEK gene cluster was knocked out in Escherichia coli BL21, and the overexpression plasmid pTrc99A-neuBC-glmS* was introduced at the same time to obtain The starting strain BL21△nanATEK / pTrc99A-neuBC-glmS* capable of producing N-acetylneuraminic acid.

[0060] 1. The knockout method of nanATEK

[0061] 利用Red重组方法敲除nanATEK(基因簇序列如SEQ ID NO.8所示),具体方法如下:以nan-F(ggtataacaggtataaaggtatatcgtttatcagacaagcatcacttcagaggtatttgtgtaggctggagctgcttc,SEQ ID NO.9)和nan-R(tcataatttttctccctgggccaacagcgcagccccaagtaaacctgcatcatggcggtaatgcgccgccctgtcaaacatgagaattaa,SEQ ID NO.10 ) as a primer, using the plasmid pKD13 (purchased from Addgene) as a template to amplify the PCR fragment of 1.3Kb, the fragment was electroporated into Escherichia coli BL21 containing the ...

Embodiment 2

[0066] Example 2 Construction of Gene Knockout Recombinant Bacteria for Inactivation of Pyruvate Kinase

[0067] On the basis of BL21△nanATEK constructed in Example 1, the coding gene pyruvate kinase pykFA was further knocked out, and the overexpression plasmid pTrc99A-neuBC-glmS* was introduced at the same time to obtain a genetically engineered strain BL21△ capable of producing N-acetylneuraminic acid nanATEK-pykFA / pTrc99A-neuBC-glmS*.

[0068] 1. The knockout method of pykFA

[0069] 利用Red重组方法敲除pykF(编码基因序列如SEQ ID NO.1所示),具体方法如下:以pykf-F(gactcttgaatggtttcagcactttggactgtagaactcaacgactcaaagtgtaggctggagctgcttcgaa,SEQ ID NO.11)和pykf-R(atccccggaattaattctcatgtttgacagtattgcttttgtgaattaatttgtatatcgaagcgccctgatgggcgctt,SEQ ID NO.12 ) as a primer, using the plasmid pKD13 (purchased from Addgene) as a template to amplify a 1.3Kb PCR fragment, which was transferred into Escherichia coli BL21△nanATEK containing the plasmid pSIJ8 (purchased from Addgene) by electroporation. 50mg / L kanamy...

Embodiment 3

[0073] The fermentation verification of embodiment 3 genetically engineered bacterial strains

[0074] The starting strain constructed in Example 1 and the genetically engineered strain constructed in Example 2 were subjected to shake flask fermentation experiments to verify their ability to produce N-acetylneurarginine.

[0075] The medium formula used in fermentation is as follows:

[0076] M9Y-glucose medium: glucose 30g / L, dipotassium hydrogen phosphate 16g / L, potassium dihydrogen phosphate 14g / L, sodium citrate dihydrate 1g / L, ammonium sulfate 7.5g / L, magnesium sulfate heptahydrate 0.25g / L L, calcium chloride 15mg / L, yeast powder 5g / L, chloramphenicol 25mg / L.

[0077] 1. Shake flask fermentation

[0078]Escherichia coli BL21△nanATEK-pykFA / pTrc99A-neuBC-glmS* and the starting strain BL21△nanATEK / pTrc99A-neuBC-glmS* were cultured in a 500mL shake flask, the medium was M9Y-glucose, and the culture temperature was 37°C. The rotation speed is 200rpm, when the cells grow to ...

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Abstract

The invention relates to the technical field of microbial fermentation and particularly discloses a recombinant microbe capable of yielding N-acetylneuraminic acid and application of the recombinant microbe. According to the recombinant microbe provided by the invention, compared with an original strain, the recombinant microbe has lowered expression of pyruvate kinase and / or enzymatic activity; the pyruvate kinase is pyruvate kinase I and / or pyruvate kinase II; and the original strain is a microbe capable of synthesizing the N-acetylneuraminic acid. Compared with the original strain capable of synthesizing the N-acetylneuraminic acid, the recombinant microbe provided by the invention has lowered expression of the pyruvate kinase and / or enzymatic activity. The recombinant microbe is especially adapted for generating the N-acetylneuraminic acid through carrying out fermentation culture by using a cheap low-carbon raw material, i.e., glucose. According to the recombinant microbe provided by the invention, the fermentation level of synthesizing the N-acetylneuraminic acid is high, the production cost of the N-acetylneuraminic acid is reduced remarkably, and thus, the recombinant microbe has industrialized potentials.

Description

technical field [0001] The invention relates to the technical field of microbial fermentation, in particular to a recombinant microorganism producing N-acetylneuraminic acid and its application. Background technique [0002] N-acetylneuraminic acid is a nine-carbon sugar derivative that constitutes an important component of cell-adhesive glycoproteins, oligosaccharides, and brain ganglioside sugar chains in animals. The main natural sources of N-acetylneuraminic acid are breast milk, milk, eggs, cheese and bird's nest. Among them, the content of neuraminic acid in bird's nest is relatively high, reaching 7-12%, so it is also called "bird's nest acid". The content of N-acetylneuraminic acid in general food is very low, and the utilization rate of the hydrolyzed neuraminic acid monomer to reach human organs is quite low. Therefore, in order to meet the needs of functional metabolism, it is necessary for the human body, especially infants, to carry out exogenous supplementatio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P19/26C12R1/19
CPCC12N9/1205C12N15/80C12Y207/0104C12P19/26
Inventor 陈振吴佳鸿刘德华
Owner TSINGHUA UNIV
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