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Escherichia coli engineering bacterium for producing 2'-fucosyllactose

A technology of fucosyllactose and Escherichia coli, which is applied in the direction of glycosyltransferase, bacteria, biochemical equipment and methods, etc., can solve the problem of high price of L-fucose and low yield of 2'-fucosyllactose Low, difficult fermentation production and other problems, to achieve the effect of increasing yield, increasing yield, and relieving metabolic pressure

Active Publication Date: 2020-02-18
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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  • Escherichia coli engineering bacterium for producing 2'-fucosyllactose
  • Escherichia coli engineering bacterium for producing 2'-fucosyllactose
  • Escherichia coli engineering bacterium for producing 2'-fucosyllactose

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0031] Using the CRISPR-Cas9 gene knockout system to knock out wacJ, lacZ, and fucIK in Escherichia coli BL21, 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, using 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 were amplified by PCR to amplify the upstream and downstream fragments of wacJ, lacZ, and fucIK, respectively, and the gel was recovered. Then use wacJ, lacZ, fucIK upstream and downstream fragments as templates respectively, using wcaJ-up-F / wcaJ-down-R, lacZ-up-F / lacZ-down-R and fucIK-up-F / fucIK-down-R The primers were used to obtain complete wacJ, lacZ, and fucIK templates by inversePCR, and the DNA fragments were recovered from the gel.

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

Embodiment 2

[0041] The construction of embodiment 2 recombinant expression vector

[0042] The specific steps for constructing the recombinant expression vector are as follows (see Table 2 for the primer sequences involved):

[0043] (1) Obtaining of manC-manB and gmd-wacG gene cluster fragments: using the genome of Escherichia coli K-12 (Escherichiacoli) as a template, using manCB-F / R (NcoI) and GW-F / R (NdeI) as primers , the manC-manB and gmd-wacG gene cluster fragments were amplified by PCR, and the DNA fragments were recovered from the gel;

[0044] (2) Obtaining the fkp gene fragment: using the Bacteroides fragilis 9343 genome as a template and using Fkp-F / R (NdeI) as a primer, the fkp gene fragment was amplified by PCR, and the DNA fragment was recovered from the gel

[0045] (4) Obtaining the fucT2 gene fragment: using Helicobacter pylori (Helicobacter pylori) genome as a template, using FucT2-F / R (NcoI) as a primer, amplify the fucT2 gene fragment by PCR, and recover the DNA frag...

Embodiment 3

[0050] The construction of embodiment 3 escherichia coli engineering strain

[0051] Cultivate wacJ, lacZ, fucIK gene knockout strain BWLF and prepare competent cells, use the chemical transformation method to introduce the extracted plasmids pET-BCGW and pCD-FF into the strain, and put them on the double-antibody LB plate (ampicillin and streptomycin) 37 Cultivate overnight at ℃ to obtain genetically engineered bacteria producing 2'-fucosyllactose. 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.

[0052] Table 3 Details of plasmids and engineered bacteria

[0053]

[0054]

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Abstract

The invention provides an escherichia coli engineering bacterium for producing 2'-fucosyllactose. Through a CRISPR / Cas9 gene editing system, related genes of an original strain 2'-fucosyllactose in ananabolic pathway can be knocked out; and a modular metabolic pathway is constructed, the expression level of phosphomannomutase (ManB), mannose-1-phosphateguanylyltransferase (ManC), GDP-mannose-6-dehydrogenase (Gmd), GDP-fucose-synthase (WcaG), L-fucose 1-kinase / GDP-fucose pyrophosphorylase (Fkp) and 2'-fucosyllactose synthetase (FucT2) in the metabolic pathway can be regulated and controlled through different plasmid combinations, so that 2'-fucosyllactose with higher concentration can be accumulated in cells. A method for efficiently producing the 2'-fucosyllactose is also provided.

Description

technical field [0001] The invention relates to an Escherichia coli engineering strain 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 pathogenic 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, has attracted widespread attention and ha...

Claims

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Application Information

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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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