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Novel N-acetylneuraminic acid-producing escherichia coli engineering bacteria as well as construction method and application thereof

A technology of acetylneuraminic acid and Escherichia coli, applied in the field of bioengineering, can solve the problems of high cost, high production cost of sialic acid, and limitation of large-scale popularization and application.

Active Publication Date: 2014-02-26
武汉中科光谷绿色生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the natural raw material extraction method can obtain high-quality and pollution-free sialic acid products, but due to the extremely low content of sialic acid in natural raw materials, the production cost of sialic acid in this method remains high, which limits its large-scale application.
Chemical synthesis and enzyme catalysis require the addition of expensive raw materials and harmful organic solvents during the production process, resulting in the production of sialic acid that can only be used for the synthesis of drugs, but not for food and health products

Method used

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  • Novel N-acetylneuraminic acid-producing escherichia coli engineering bacteria as well as construction method and application thereof
  • Novel N-acetylneuraminic acid-producing escherichia coli engineering bacteria as well as construction method and application thereof
  • Novel N-acetylneuraminic acid-producing escherichia coli engineering bacteria as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1: Construction of high-yield N-acetylneuraminic acid genetically engineered bacteria

[0041] 1. Using the RED recombination method to inactivate the nagA gene and nanATEK gene cluster in the strain, the specific steps are as follows:

[0042] 1. Knockout of nagA gene

[0043] 1) According to the sequence of Escherichia coli BL21 (DE3) (Invitrogen Company) genome (Genbank No.CP001509), design primers: upstream primer F-KO-nagA:

[0044] TATGCATTAACCCAGGGCCGGATCTTTACCGGCCACGAAATTCCGGGGATCCGTCGACC (shown in SEQ ID NO.1) and downstream primer R-KO-nagA:

[0045] TTGAGTTACGACCTCGTTACCGTTAACGATGGTCCTGGTTGTAGGCTGGAGCTGCTTCG (shown in SEQ ID NO. 2).

[0046] Using the primers F-KO-nagA and R-KO-nagA, using the plasmid pIJ773 as a template, and using commercial PCR reagents, the DNA fragments were amplified by PCR and purified for future use.

[0047] 2) The RED recombinase expression plasmid vector pKD46 was transformed into Escherichia coli BL21(DE3) by electri...

Embodiment 2

[0111] Example 2 Glucose is used as carbon source to ferment and produce N-acetylneuraminic acid

[0112] 1. Seed and fermentation medium (1L):

[0113] (NH 4 ) 2 SO 4 4g / L; KH 2 PO 4 6g;K 2 HPO 4 ·3H 2 O8g; MgSO 4 ·7H 2 O0.25g; glucose 5g, trace elements 10ml.

[0114] Among them, trace elements are: manganese sulfate 100mg / L; zinc chloride 70mg / L; sodium molybdate 35mg / L; boric acid 60mg / L; cobalt chloride 200mg / L; copper sulfate 29.28mg / L; L; concentrated hydrochloric acid (37%) 0.9ml / L.

[0115] Feed solution: 500g / L glucose.

[0116] 2. Fermentation process:

[0117] 1) Pick a single colony and place it in a 4ml LB test tube, and incubate at 37°C for 8 hours.

[0118] 2) Inoculate 2ml of first-grade seeds into 200ml of seed medium and incubate at 37°C for 8-10 hours.

[0119] 3) Secondary seeds are inoculated in a fermenter with a liquid volume of 3.5L, at 37°C, with a stirring speed of 300-800 rpm, dissolved oxygen is kept above 30%, and the pH is controll...

Embodiment 3

[0123] Example 3 Glycerol is used as carbon source to ferment and produce N-acetylneuraminic acid

[0124] Replace glucose with glycerol of 20g / L, other components of fermentation medium are the same as embodiment 2, and feeding medium is 600g / L glycerol;

[0125] The fermentation process is the same as in Example 2.

[0126] Finally, after about 80 hours of fermentation, the concentration of N-acetylneuraminic acid produced by CASOV-1 can reach 30g / L; the concentration of N-acetylneuraminic acid produced by CASOV-2 can reach 5g / L.

[0127] The above culture results show that the genetically engineered bacteria constructed by metabolic engineering technology, especially CASOV-1, has the ability to produce N-acetylneuraminic acid and has the potential for industrialization.

[0128]

[0129]

[0130]

[0131]

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Abstract

The invention discloses novel N-acetylneuraminic acid-producing escherichia coli engineering bacteria as well as a construction method and application thereof. The engineering bacterial is constructed by introducing an encoded 6-glucosamine phosphate acetylase gene, an N-acetyl glucosamine-2-isomerase gene and an N-acetylneuraminic acid synthetase gene into escherichia coli to express, carrying out strengthened expression on 6-glucosamine phosphate deaminase gene contained in the escherichia coli per se and knocking off genes, for decomposing and utilizing enzyme in metabolic pathways, of the N-acetylneuraminic acid in the engineering bacteria. The engineering bacteria disclosed by the invention can be used for fermentation culture to synthesize the N-acetylneuraminic acid by using glucose or glycerinum as a substrate.

Description

technical field [0001] The invention relates to a new N-acetylneuraminic acid-producing Escherichia coli engineering bacterium and its construction method and application, belonging to the technical field of bioengineering. Background technique [0002] Sialic acid refers to a series of acidic amino sugars containing 9 carbon atoms and a pyranose structure, named 5-amino-3,5-dideoxy-D-glycerol-D-galactononulose. Sialic acid is widely distributed in nature. It has been found that many organisms have sialic acid. There are more than 50 kinds of sialic acid found so far. N-acetyl-D-neuraminic acid (Neu5Ac) is the main type of sialic acid. Its content accounts for more than 99% of the entire sialic acid family, and it is usually located at the end of non-reducing oligosaccharides such as glycoproteins and glycolipids in the form of α-glycosides. [0003] In recent years, sialic acid has attracted more and more people's attention. Sialic acid has been successfully applied in the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P19/26C12R1/19
Inventor 柳鹏福孙立洁王纪袁丽霞刘洋吴金勇陈祥松史吉平姚建铭
Owner 武汉中科光谷绿色生物技术有限公司
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