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Recombinant escherichia coli with glucosamine accumulation and application thereof

A technology of recombinant Escherichia coli and glucosamine, applied in the field of genetic engineering, can solve the problems of allergic reaction, long reaction time and high efficiency, and achieve the effects of strengthening the synthesis pathway, reducing the feedback inhibition and improving the yield.

Inactive Publication Date: 2019-06-25
YANGZHOU RIXING BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The chitin hydrolysis method has limited sources of raw materials in production, is easy to cause environmental pollution and allergic reactions, and is difficult to realize industrial production; compared with the chitin hydrolysis method, the enzymatic biotransformation method has less environmental pollution, but has long reaction time and high efficiency. low problem; the microbial transformation method avoids the determination of the existence of the first two methods, and has high efficiency, and has become the main method of producing GlcN now

Method used

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  • Recombinant escherichia coli with glucosamine accumulation and application thereof
  • Recombinant escherichia coli with glucosamine accumulation and application thereof
  • Recombinant escherichia coli with glucosamine accumulation and application thereof

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Experimental program
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Effect test

Embodiment 1

[0023] Metabolic engineering based on wild-type E. coli strains

[0024] (1) Knockout of nagE gene

[0025] In order to block nagE to achieve partial inactivation of the glucosamine input line and reduce the uptake of glucosamine, the nagE gene in the wild-type strain was knocked out.

[0026] Edit wild type by CRISPR-Cas9 system (Jiang Y, Chen B, Duan C, et al. Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System [J]. Applied & Environmental Microbiology, 2015, 81(7): 2506.) The nagE gene encoding the L-methionine transport system in the genome of Escherichia coli Escherichiacoli W3110 (purchased from Escherichia coli strain collection center CGSC). The primers of SEQ ID NO.1 and SEQ ID NO.2 were used by PCR, and the pTargetF vector (Jiang Y, Chen B, Duan C, et al.Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System[J]. Applied & Environmental Microbiology, 2015, 81 (7): 2506.) as a template to construct a pTarget-AnagE mutation...

Embodiment 2

[0115] Enhanced expression of GNA1 gene based on metabolic modification of W3110ΔnagEΔmanXYZΔnagAΔnagB-GNA1(trc) strain

[0116] The original promoter sequence in the GNA1 gene was replaced with the trc promoter sequence to achieve the purpose of enhancing the expression of the GNA1 gene.

[0117] Editing W3110 ΔnagEΔmanXYZΔnagA by CRISPR-Cas9 system (Jiang Y, Chen B, Duan C, et al. Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System[J]·Applied & Environmental Microbiology, 2015, 81(7):2506.) The promoter sequence of the GNA1 gene encoding homoserine dehydrogenase in the strain genome. The primers of SEQ ID NO.33 and SEQ ID NO.34 were used by PCR, and the pTargetF vector (Jiang Y, Chen B, Duan C, et al.MultigeneEditing in the Escherichia coli Genome via the CRISPR-Cas9 System[J].Applied&Environmental Microbiology, 2015, 81(7): 2506.) was used as a template to construct a pTarget-ΔPGNA1::Ptrc mutation vector capable of expressing sgRNA targeting the pro...

Embodiment 3

[0139] Acquisition of the GlmS gene with resistance to feedback inhibition

[0140]Using the genome of wild-type Escherichia coli W3110 (purchased from Escherichia coli strain collection center CGSC) as a template, PCR amplification was performed together with the primers of SEQ ID NO.49 and SEQ ID NO.50. PCR reaction conditions: pre-denaturation at 95°C for 5min, 95°C for 30s, 60°C for 30s, 72°C for 1min, a total of 30 cycles, and finally extension at 72°C for 10min. The PCR product was detected by 1.0% agarose gel electrophoresis and the fragment was recovered and purified by cutting the gel, and base A was introduced into the 5' end of the fragment by using Taq DNA polymerase. The fragment was ligated with the pGEM-T vector under the action of T4 DNA ligase to obtain the cloning recombinant plasmid pGEM-T-GlmS. On the pGEM-T-GlmS plasmid, the GlmS gene was subjected to site-directed mutation, and the amino acid at position 64-glutamic acid-was replaced with glutamine (E64Q...

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Abstract

The invention discloses recombinant escherichia coli with glucosamine accumulation and application thereof, and belongs to the technical field of genetic engineering. The escherichia coli is used as ahost, the recombinant escherichia coli is obtained by knocking out four genes of an acetylglucosamine transporter encoding gene nagE, a mannose phosphate transporter encoding gene manXYZ, a deacetylase gene nagA and a deaminase gene nagB gene in the glucosamine synthesis pathway of the escherichia coli, and the recombinant escherichia coli is applied to the production of the glucosamine to achieve the purpose of blocking the transport pathway of glucosamine from the extracellular to the intracellular. By means of overexpression of a glucosamine synthetase encoding gene and a glucosamine acetylase encoding gene, the synthesis pathway of the glucosamine is enhanced, and the yield of the glucosamine is increased accordingly.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a recombinant Escherichia coli accumulating glucosamine and an application thereof. Background technique [0002] The sugar industry is the basic industry of the food industry, and it is also the raw material industry for various products such as papermaking, chemical industry, fermentation, medicine, and building materials. It occupies a very important position in the national economy. Functional sugars and their derivatives are an important part of the health industry and a sunrise industry in the field of food biotechnology. The industry has developed rapidly in recent years. Glucosamine (Glucosamine, 2-amino-2-deoxy-D-glucose, GlcN), also known as glucosamine, glucosamine or glucosamine, is a compound in which a hydroxyl group of glucose is replaced by an amino group. It is an important It is a functional monosaccharide and the first amino monosacchar...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P19/26C12R1/19
Inventor 张博丁振中张超王士刚平立凤李会高小燕张万宏徐俊山
Owner YANGZHOU RIXING BIO TECH
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