Construction method for engineered Escherichia coli strain capable of producing sialyllactose

A technology of sialyllactose and Escherichia coli, which is applied in the field of cell-catalyzed synthesis of sialyllactose, can solve the problems of large disturbance of chassis cells and achieve low cost, simplified production methods and high efficiency

Inactive Publication Date: 2018-04-13
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, Escherichia coli cannot automatically produce sialic acid in this method, and a recombinant plasmid is required to express the gene for synthesizing sialic acid in Escherichia coli, which greatly disturbs the chassis cells

Method used

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  • Construction method for engineered Escherichia coli strain capable of producing sialyllactose
  • Construction method for engineered Escherichia coli strain capable of producing sialyllactose
  • Construction method for engineered Escherichia coli strain capable of producing sialyllactose

Examples

Experimental program
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Effect test

Embodiment Construction

[0048] Strain: Escherichia coli K1 (CMCC number 44277, O2:K1:H4).

[0049] 1 polysialyltransferase gene (neuS) knockout strain E.coli K1-ΔneuS

[0050] The neuS gene knockout process and verification results are as follows:

[0051]The Escherichia coli K1 strain containing plasmid pKD46 expresses three recombinant proteins of bacteriophage γ after arabinose induction, thus having the ability of homologous recombination; using pKD3 as a template, the designed primers (neuS-H1-P1 and neuS-H2-P2 ) has a neuS gene homology arm of about 50 bp at the 5′ end, and an amplification primer at the 3′ end to amplify the chloramphenicol gene containing FRT sites on both sides, and then electrotransform the linear fragment into E.coli K1 after purification (pKD46) Competent cells, positive transformants were screened by chloramphenicol plate and colony PCR. Use high temperature to eliminate the temperature-sensitive plasmid pKD46, electrotransform the plasmid pCP20 expressing Flp recombin...

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Abstract

The invention discloses a method for constructing an engineering strain of sialyllactose-producing Escherichia coli, which includes: knocking out the polysialyltransferase gene (neuS) and knocking out the β-galactosidase gene (lacZ) in Escherichia coli to obtain ΔneuSΔlacZ double Defective strain; knock out the gene cluster related to the decomposition of N-acetylneuraminic acid (nanKETA) to obtain the ΔneuSΔlacZΔnanKETA-deficient strain; transform the recombinant plasmid pXC1k-Nst into it to obtain the strain K1-KETA-pNst. The Escherichia coli strain K1-KETA-pNst provided by the present invention has been deposited in the General Microbiology Center of the China Microbial Culture Collection Committee (CGMCC for short) on October 16, 2017, and the deposit number is: CGMCC No. 14825, the depository address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The advantage of the present invention is that it does not need to introduce genes for synthesizing and activating sialic acid, and directly utilizes the cells' own resources, which is simple and feasible, has low cost and high yield.

Description

Technical field [0001] The present invention relates to the technical field of cell catalytic synthesis of sialyllactose. Background technique [0002] Human milk oligosaccharides (HMOs) are mainly composed of sialic acid oligosaccharides, fuco oligosaccharides, oligosaccharides and their analogs. Because it is safe, reliable, does not produce drug resistance, and has partial immune function, the development of new drugs using human milk oligosaccharides as raw materials has become a hot topic in the current medical field. [0003] Sialyl oligosaccharides are an important component of human milk oligosaccharides, which are abundant in human milk and have important physiological functions. These oligosaccharides are located on gangliosides and glycoprotein molecules on the surface of human brain cells, which can enhance human immunity and memory, and play an important role in many biological processes such as cell recognition, cell adhesion and signal transduction. Sialylac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N1/21C12P19/12C12R1/19
CPCC12N15/70C12N9/1081C12N9/2465C12P19/12C12Y302/01022
Inventor 王梦楠杨静华陶勇金城
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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