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A kind of recombinant Escherichia coli and the method for biosynthesizing 3'-sialyllactose

A technology for recombining Escherichia coli and sialyllactose, applied in the field of metabolic engineering, can solve the problems of complex by-products, too many synthesis steps, too many products, etc.

Active Publication Date: 2019-05-14
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the production method of 3'-sialyllactose is mainly chemical method. There are many disadvantages in the production of chemical method, such as too many synthesis steps, many products generated, complex by-products, and the reaction solution pollutes the environment. Biological method to prepare 3'-sialyllactose has attracted more and more attention of researchers

Method used

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  • A kind of recombinant Escherichia coli and the method for biosynthesizing 3'-sialyllactose
  • A kind of recombinant Escherichia coli and the method for biosynthesizing 3'-sialyllactose
  • A kind of recombinant Escherichia coli and the method for biosynthesizing 3'-sialyllactose

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Gene acquisition:

[0049] In this example, the CMP-acetylneuraminic acid synthetase gene derived from Escherichia coli was obtained neuA (Gene accession number GI:7152208), Acetylneuraminic acid synthetase gene neuB (Gene accession number GI:7152206), N-acetylglucosamine isomerase gene neuC (Gene accession number GI:7152210), β-galactoside permease gene lac Y (Gene accession number GI:949083) gene sequence. Get from Neisseria meningitidis of lst Gene (Gene accession number GI: 325207958)

Embodiment 2

[0051] Preparation of recombinant plasmids

[0052] Using the designed primer F 1 , R 1 The CMP-acetylneuraminic acid synthetase gene derived from Escherichia coli obtained in Example 1 neuA , acetylneuraminic acid synthetase gene neuB , N-acetylglucosamine isomerase gene neuC PCR amplification was carried out, and the amplified fragment was gel-cut and purified, and double-digested with NcoI and BamHI, and the digested fragment was ligated with the plasmid pCOLADuet-1, which was also double-digested with NcoI and BamHI, and the vector: The target fragments were mixed at a molar ratio of 1:3, added with T4 DNA Ligase, and then ligated for 5 hours at 22°C, and the ligated product was transformed E. coli DH5α, and screened on the kanamycin plate to obtain the recombinant plasmid pCOLADuet-1- neuBAC .

[0053] Using the designed primer F 2 , R 2 To the source that obtains in embodiment 1 Neisseria meningitidis of lst The gene was amplified by PCR, and the amplifie...

Embodiment 3

[0056] gene knockout

[0057] This example uses the λRed recombination system to knock out E. coli For multiple genes of BL21(DE3), this method eliminates resistance for each gene knocked out. Below to lacZ Taking a gene as an example, the steps of gene knockout are described in detail, and the knockout of the remaining 6 genes is the same. Find in NCBI E. coli BL21 lacZ Nucleotide sequence of gene, design lacZ Gene deletion primers and identification primers. lacZ The nucleotide sequences of gene deletion primers and identification primers are shown in SEQ ID NO.9-SEQ ID NO.12;

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Abstract

The invention discloses constructed recombinant escherichia coli and a method for biosynthesis of 3'-sialyllactose. The recombinant escherichia coli is named as E.coli-XYY which has a synthesis pathway of the 3'-sialyllactose. Meanwhile, the invention also discloses a constructing method. At the same time, the invention solves the problems of the prior art and provides an efficient gene knockout scheme; an original strain, after undergoing genetic engineering transformation, can produce the 3'-sialyllactose only, without changing other properties of the strain, so that influence on fermentation production is avoided; since mature escherichia coli plasmid is adopted by the strain, bacterium growth and normal metabolism do not affected in a metabolic process. The recombinant escherichia coli constructed by the invention has a good application prospect; therefore, a new thought is provided for the production of the 3'-sialyllactose by virtue of a biological method.

Description

technical field [0001] The invention relates to a method for synthesizing 3'-sialyllactose by using recombinant Escherichia coli, belonging to the field of metabolic engineering. Background technique [0002] Breast milk contains essential nutrients for infant growth and development, but it also contains substances that are not found in traditional nutrients and are beneficial to the body. Some of these substances are human milk oligosaccharides (HMOs). Sialyllactose is a common human milk oligosaccharide, which has the functions of anti-adhesion, maintenance of intestinal microbial composition and glycome modification. It is also a promising oligosaccharide in terms of nutrition and medicine. [0003] Sialyllactose, such as 3'-sialyllactose, plays an important role in glycome modification, regulates the expression of polysaccharides on the surface of intestinal epithelial cells, and regulates the adhesion sites of most pathogenic and commensal bacteria. CaCo-2 cells can a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/70C12P19/00C12R1/19
CPCC07K14/245C12N9/1085C12N9/1241C12N9/2402C12N9/2471C12N9/78C12N9/88C12N9/90C12N15/70C12N2800/101C12P19/00C12Y204/99C12Y205/01056C12Y207/0106C12Y207/07043C12Y302/01023C12Y305/99006C12Y501/03014
Inventor 王磊黄笛许莹莹王茹
Owner NANKAI UNIV
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