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A method for constructing a recombinant bacterium that efficiently produces 2'-fucosyllactose and its application

A technology of fucosyllactose and fucose, applied in biochemical equipment and methods, glycosyltransferase, recombinant DNA technology, etc., can solve difficult fermentation production and low yield of 2'-fucosyllactose , L-fucose is expensive and other problems, to achieve the effect of increasing production, increasing production and relieving metabolic pressure

Active Publication Date: 2021-05-28
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the yield of 2'-fucosyllactose has been significantly improved, the L-fucose used for its production is expensive, and the yield of 2'-fucosyllactose is also low (67.7%), making it difficult to Applied to large-scale fermentation production

Method used

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  • A method for constructing a recombinant bacterium that efficiently produces 2'-fucosyllactose and its application
  • A method for constructing a recombinant bacterium that efficiently produces 2'-fucosyllactose and its application
  • A method for constructing a recombinant bacterium that efficiently produces 2'-fucosyllactose and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Escherichia coli BL21 wxya , lacZ , wxya gene knockout

[0031] Knockout of Escherichia coli BL21 using CRISPR-Cas9 gene knockout system wxya , lacZ , wxya , the specific steps are as follows (see Table 1 for the primer sequences involved):

[0032] (1) Using the Escherichia coli BL21 genome as a template, use wcaJ-up-F / R and wcaJ-down-F / R, lacZ-up- F / R and lacZ-down-F / R, fucIK-up-F / R and fucIK-down-F / R Amplified by PCR wxya , lacZ , wxya The upstream and downstream fragments are recovered from the gel. and then separately wxya , lacZ , wxya The upstream and downstream fragments are used as templates, using wcaJ-up- F / wcaJ-down-R, lacZ-up-F / lacZ-down-R and fucIK-up-F / fucIK-down-R Primers were obtained by inversePCR complete wxya , lacZ , wxya Template, gel to recover DNA fragments.

[0033] (2) Using the original pTargetF plasmid as a template, wcaJ-sg-F / R, lacZ-sg-F / R and fucIK-sg-F / R As a primer, PCR amplificat...

Embodiment 2

[0040] Example 2 Construction of recombinant expression vector

[0041] The specific steps for constructing the recombinant expression vector are as follows (see Table 2 for the primer sequences involved):

[0042] (1) manC-manB and gmd-wacG Obtaining gene cluster fragments: using Escherichia coli K-12 ( Escherichia coli ) genome as a template, with manCB - F / R (NcoI) and GW - F / R (NdeI) As primers, PCR amplifies the manC- man B and gmd-wacG Gene cluster fragments, gel recovery DNA fragments;

[0043] (2) fkp Obtaining gene fragments: using Bacteroides fragilis ( Bacteroides fragilis 9343 ) genome as a template, with Fkp - F / R (NdeI) As primers, PCR amplified fkp Gene Fragments, Gel Recovery DNA Fragments

[0044] (4) fucT2 Acquisition of gene fragments: with Helicobacter pylori ( Helicobacter pylori ) genome as a template, with FucT2 - F / R (NcoI) As primers, PCR amplified fucT2 Gene Fragments, Gel Recovery DNA Fragments

[0045] ...

Embodiment 3

[0049] Example 3 Construction of Escherichia coli engineering strains

[0050] nourish wxya , lacZ , wxya Gene knockout strain BWLF was prepared and competent cells were prepared, and the extracted plasmids pET-BCGW and pCD-FF were introduced into the strain by chemical transformation method, and cultured overnight at 37°C on double-antibody LB plate (ampicillin and streptomycin) to obtain the product 2'-fucosyllactose genetically engineered bacteria. The construction of other recombinant genetically engineered bacteria is as above, and the specific recombinant plasmids, engineered bacteria and their detailed information are shown in Table 3.

[0051] Table 3 Details of plasmids and engineered bacteria

[0052] strain or plasmid nature plasmid pETDuet-1 double T7 promoters, two MCS, pBR322 origin, Ampr pCDFDuet-1 double T7 promoters, two MCS, CDF origin, Smr pACYCDuet-1 double T7 promoters, two MCS, P15A origin, Cmr pET-BCGW ...

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Abstract

The invention provides an engineering strain of Escherichia coli that produces 2'-fucosyllactose, through the CRISPR / Cas9 gene editing system, the relevant genes in the synthetic and metabolic pathway of the original strain 2'-fucosyllactose are knocked out; Modular metabolic pathway, phosphomannose mutase (ManB), mannose‑1‑phosphate guanyltransferase (ManC), GDP‑mannose‑6‑dehydrogenase ( Gmd), GDP-fucose synthase (WcaG), L-fucose 1-kinase / GDP-fucose pyrophosphorylase (Fkp) and 2'-fucosyllactose synthase (FucT2) expression level, so that a higher concentration of 2'-fucosyllactose can be accumulated in the cell. The present invention also provides a method for efficiently producing 2'-fucosyllactose.

Description

technical field [0001] The invention relates to a construction method and application of a recombinant bacterium for efficiently producing 2'-fucosyllactose, belonging to the field of microbial genetic engineering. Background technique [0002] Breast milk is generally considered the most important source of nutrition for infants. As the third solid component in breast milk, the synthesis of human milk oligosaccharides plays an important role in the development of intestinal flora in infants and in preventing the adhesion of pathogenic bacteria to epithelial cells. Fucosylated lactose, including 2'-fucosyllactose, 3-fucosyllactose, lacto-N-fucopentaose, etc., can selectively stimulate the growth of bifidobacteria and form pathogenic Analogues of bacterial receptors, thereby protecting infants against infection with enteric pathogens, such as Escherichia coli, Vibrio cholerae and Salmonella. 2’-fucosyllactose, as the most abundant component of human milk oligosaccharides, h...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/70C12P19/18C12P19/12
CPCC12N9/0006C12N9/1051C12N9/90C12N15/70C12P19/12C12P19/18C12Y101/01132C12Y101/01271C12Y204/01069C12Y504/02008
Inventor 沐万孟张文立李雯朱莺莺万李
Owner JIANGNAN UNIV
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