Method for biosynthesizing 2'-fucosyllactose by constructing recombinant colibacillus

A technology of Escherichia coli and construction methods, applied in the field of metabolic engineering, can solve problems such as many products, environmental pollution of reaction solution, complex by-products, etc.

Active Publication Date: 2016-12-07
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 2'-fucollactose is mainly chemical method, which has many disadvantages, such as too many synthesis steps, many products generate

Method used

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  • Method for biosynthesizing 2'-fucosyllactose by constructing recombinant colibacillus
  • Method for biosynthesizing 2'-fucosyllactose by constructing recombinant colibacillus
  • Method for biosynthesizing 2'-fucosyllactose by constructing recombinant colibacillus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Gene acquisition:

[0052] In this embodiment, the phosphomannosidase gene derived from Escherichia coli MG1655 was obtained man B (Gene Accession No. GI:946574), Phosphate Guanine Transferase Gene manC (Gene accession number GI:946580), GDP-mannose-4,6-dehydratase gene gmd (Gene accession number GI:946562), GDP-fucose synthase gene fcl (Gene accession number GI:946563), Mannose-6-phosphate isomerase gene man A (Gene accession number GI:944840), a positive regulator of GDP-fucose synthesis rcsA (Gene accession number GI:946467), obtained the β-galactoside permease gene from Escherichia coli BL21 lac Y (Gene accession number GI:949083), access to the α-1,2-fucosyltransferase gene from Helicobacter pylori f (Gene accession number GI: CP010436).

Embodiment 2

[0054] Preparation of recombinant plasmids

[0055] Using the designed primer F 1 ,R 1 The phosphomannosidase gene derived from Escherichia coli MG1655 obtained in Example 1 man B , phosphoguanine transferase gene manC Carry out PCR amplification, and the amplified fragments are gel-cut and purified, and double-digested with NcoI and HindIII, and the digested fragments are connected with the plasmid pETDuet-1 that has also been double-digested with NcoI and HindIII, 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 ampicillin plate to obtain the recombinant plasmid pETDuet-1-man B-man C .

[0056] Using the designed primer F 2 ,R 2 The GDP-mannose-4,6-dehydratase gene derived from Escherichia coli MG1655 obtained in Example 1 gmd , GDP-fucose synthetase gene fcl PCR amplification was carried out, and the...

Embodiment 3

[0060] gene knockout

[0061] 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. The following takes the wcaj gene as an example to describe the gene knockout steps in detail, and the knockout of the other two genes is the same.

[0062] Find in NCBI E. coli BL21(DE3) Nucleotide sequence of wcaj gene, design primers for deletion and identification of wcaj gene. The nucleotide sequences of the deletion primer and identification primer of wcaj gene are shown in SEQ ID NO.5-SEQ ID NO.8.

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Abstract

The invention discloses a method for biosynthesizing 2'-fucosyllactose by constructing recombinant colibacillus. The name of colibacillus genetic engineering bacteria is E.coli-XYY-1, a synthetic route of 2'-fucosyllactose is achieved, and meanwhile a construction method is disclosed. According to the method for biosynthesizing 2'-fucosyllactose by constructing recombinant colibacillus, existing technical problems are solved, and an efficient gene knockout scheme is obtained; after original strains are subjected to genetic reconstruction, 2'-fucosyllactose is produced, other characteristics of the strains are not changed, and fermentation production is not affected; plasmid adopted by the strains is mature colibacillus plasmid, and therefore growth and normal metabolism of bacteria are not affected in the metabolic process. The constructed recombinant colibacillus has a very large application prospect, and a new idea is provided for generating 2'-fucosyllactose through a biological method.

Description

technical field [0001] The invention relates to a method for synthesizing 2'-fucogalactose by using recombinant Escherichia coli, and belongs to the field of metabolic engineering. More specifically, it is a method for constructing recombinant Escherichia coli to biosynthesize 2'-fucogalactose. 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] Fucooligosaccharides, such as 2'-fucollactose, lacto-N-fucopentaose and lacto-N-fucohexaose, are common human milk oligosaccharides. Fucoidated oligosacchari...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P19/00C12R1/19
CPCC12N9/0006C12N9/1051C12N9/1077C12N9/2471C12N9/2488C12N9/50C12N9/88C12N9/90C12P19/00C12Y101/01271C12Y204/01069C12Y204/02008C12Y302/01023C12Y402/01047C12Y503/01008
Inventor 王磊黄笛许莹莹王茹
Owner NANKAI UNIV
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