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Escherichia coli for producing 2'-fucosyllactose and application thereof

A technology of fucosyllactose and Escherichia coli, which is applied in the field of microbial metabolic engineering, can solve the problems of poor pathways, accumulation of intermediate metabolites, and high production costs, and achieve the effect of improving efficiency.

Active Publication Date: 2021-03-16
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The salvage synthesis pathway of 2’-fucosyllactose needs to use high-value fucose as a substrate, the production cost is high, and it basically has no industrial application value
Although the construction of 2'-fucosyllactose de novo synthesis pathway can produce 2'-fucosyllactose, there are still problems such as the pathway is not smooth and intermediate metabolites accumulate, resulting in the production level of 2'-fucosyllactose lower

Method used

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  • Escherichia coli for producing 2'-fucosyllactose and application thereof
  • Escherichia coli for producing 2'-fucosyllactose and application thereof
  • Escherichia coli for producing 2'-fucosyllactose and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Strains E. coli K12 MG1655△ wxya ::Ptrc- wcaG-gmd-lacy build

[0047] Using Escherichia coli MG1655 as the original strain, knock out the Plac promoter sequence in the lac operon sequence and lacI, lacZ gene, and in lacI, lacZ Gene loci overexpressed using the Ptrc promoter wcaG, gmd, lacy Gene. The Ptrc promoter is a spliced ​​promoter of the Ptrp promoter and the Plac promoter, and has higher transcription efficiency than the Plac promoter.

[0048] The method used to construct this strain is λRed recombination. The main purpose is to construct a two-step homologous recombination fragment, and use pKD46 (GenBank: MF287367) as a homologous recombination plasmid to carry out gene knockout and integration. The first step homologous recombination fragment contains upstream and downstream homology arms, chloramphenicol resistance gene and sacB Gene. The second-step homologous recombination fragment includes upstream and downstream homology arms...

Embodiment 2

[0058] Example 2: Strain W1△ adhE ::Ptrc- manB-manA build

[0059] for overexpression manB, manA gene, based on the W1 strain manB, manA Genome integration of , the integration site is selected as adhe (alcohol dehydrogenase) gene location. The gene integration method is consistent with Example 1. Need to construct the first step homologous recombination fragment adhe-cat-sacB . The required primers are adhe-up / adhe-2, adhe-down-FF / adhe-down, and Escherichia coli MG1655 is used as a template to obtain the upstream and downstream homology arms of homologous recombination by PCR. Construct cat-sacB gene fragments, of which cat is the chloramphenicol resistance gene, sacB Derived from pEX18Gm plasmid. Take three fragments, cat-sacB as well as adhe The upper and lower homologous arms of the gene are used as templates, and the first-step homologous recombination fragments are obtained by overlapping PCR. Construction of second-step homologous recombination ...

Embodiment 3

[0063] Example 3: Plasmid pTrc99a- futC-manC Construct

[0064] high copy overexpression futC, manC gene, need to construct plasmid pTrc99a- futC-manC . which was overexpressed using the Ptrc promoter f Gene, overexpressed using the arabinose-inducible promoter Para manC gene, so pTrc99a- f Plasmid and pKD46 -manC Plasmid, based on which pTrc99a- futC-manC plasmid.

[0065] pTrc99a- f Plasmid construction: Using the pTrc99a plasmid as a template, using primers ptrc99a-BA-R / ptrc99a-ty-F, PCR amplified to obtain a linear pTrc99a vector fragment containing a Ptrc promoter. Selection of fucosyltransferase genes from Helicobacter pylori f , using primers futc-11 / futc-12 to carry the total gene synthesis f The pUC57 plasmid DNA was used as a template, and PCR amplification was obtained f gene fragments, of which f Partial codon optimization of the gene, using seamless cloning enzyme to convert f Ligated with the linear vector pTrc99a fragment, the ligated...

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Abstract

The invention relates to recombinant escherichia coli for producing 2'-fucosyllactose and application thereof, and belongs to the field of microbial metabolic engineering. According to a regulation and control strategy for the expression level of 2'-fucosyllactose de novo synthesis route key enzyme, different enzymes are subjected to regulation and control of genome overexpression or plasmid overexpression, the efficiency of a metabolic pathway can be greatly improved, compared with the prior art, the efficiency of a genetic engineering strain constructed through related strategies is improvedby 13 times or above, and a foundation is laid for industrial production of the 2'-fucosyllactose.

Description

technical field [0001] The invention relates to a recombinant Escherichia coli producing 2'-fucosyllactose and an application thereof, belonging to the field of microbial metabolism engineering. Background technique [0002] One of the most important bioactive components in breast milk is human milk oligosaccharides (HMOs, Human Milk Oligosaccharides). The structure of HMOs is very complex. According to mass spectrometry analysis, it is speculated that there are thousands of types of HMOs. HMOs can shape the intestinal microbial flora by selectively stimulating the growth of specific microorganisms, especially probiotics such as bifidobacteria, effectively safeguarding the development and health of the gastrointestinal tract and immune system. In addition, with the deepening of the research on human intestinal microbiota, it has been fully proved that HMOs can not only promote the formation of beneficial microbial flora in infants, but also improve the intestinal microbial f...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P19/00C12R1/19
CPCC12N9/0006C12N9/1241C12N9/88C12N9/90C12N15/70C12P19/00C12Y101/01271C12Y207/07013C12Y402/01008C12Y402/01047C12Y504/02008
Inventor 李庆刚李玉卫鑫慧路福平王凤华秦慧民
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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