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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
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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

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Experimental program
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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 construction method for an engineered Escherichia coli strain capable of producing sialyllactose. The construction method comprises the following steps: knocking out a polysialytransferase gene (neuS) and beta-galactosidase gene (lacZ) in Escherichia coli so as to obtain a Delta neuS-Delta lacZ double-defective strain; knocking out a gene cluster (nanKETA) related to decomposition of N-acetylneuraminic acid so as to obtain a Delta neuS-Delta lacZ-Delta nanKETA defective strain; and transferring a recombinant plasmid pXC1k-Nst into the Delta neuS-Delta lacZ-Delta nanKETA defective strain so as to obtain a K1-KETA-pNst strain. The Escherichia coli strain K1-KETA-pNst provided by the invention is preserved in China General Microbiological Culture Collection Center, No. 3, Yard 1, West Beichen Road, Chaoyang District, Beijing City, China, on October 16, 2017, with an accession number of CGMCC No. 14825. The construction method has the advantages that introductionof genes for synthesis and activation of sialic acid is not needed; the resources of cells are directly used; and the method is simple and practical to implement, low in cost and high in 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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