Genetically engineered bacterium for producing N-acetylglucosamine and application of genetically engineered bacterium

A technology of genetically engineered bacteria and acetylamino, applied in the field of genetic engineering, can solve problems such as limiting the conversion rate of N-acetylglucosamine, and achieve the effects of strengthening industrial production potential, improving conversion rate, and improving synthesis efficiency.

Active Publication Date: 2021-03-02
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The biosynthesis of N-acetylglucosamine requires fructose-6-phosphate in the glycolytic pathway as a precursor, so

Method used

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  • Genetically engineered bacterium for producing N-acetylglucosamine and application of genetically engineered bacterium
  • Genetically engineered bacterium for producing N-acetylglucosamine and application of genetically engineered bacterium
  • Genetically engineered bacterium for producing N-acetylglucosamine and application of genetically engineered bacterium

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1 produces the construction of N-acetylglucosamine bacterial strain:

[0048] (1) Integration of T7 RNA polymerase gene (T7-RNAP)

[0049] The integration of T7 RNA polymerase gene (T7-RNAP) was carried out by Red recombination technology.

[0050] ①Using PCR technology to use the E.coli W3110 genome as a template, according to the xylose promoter P xylF Gene sequence design a pair of primers (PxylF-F / R) to amplify xylose promoter P xylF ;

[0051] ②Using PCR technology to use the E.coli BL21 genome as a template, design a pair of primers (T7-F / R) according to the T7 RNA polymerase gene sequence, and amplify the T7 RNA polymerase gene;

[0052] ③Using PCR technology to use the pKD3 plasmid as a template, design primers (Cm r -F / R) amplifying the chloramphenicol resistance gene fragment;

[0053] ④Using PCR technology to use the E.coli W3110 genome as a template, according to the LacIZ gene sequence, design upstream homology arm primers (T7-UF / UR) and dow...

Embodiment 2

[0073] Example 2 Construction of N-acetylglucosamine-producing strains by complex carbon source metabolism division of labor

[0074] (1) Using CRISPR / Cas9 gene editing technology to knock out the pfkA gene

[0075] ①Using PCR technology to use the E.coli W3110 genome as a template, according to the pfkA gene sequence, design upstream homology arm primers (pfkA-UF / UR) and downstream homology arm primers (pfkA-DF / DR) at both ends of the gene, PCR Amplify and obtain the upstream and downstream homology arms of the pfkA gene;

[0076] ②Using overlapping PCR technology to use the upstream and downstream homology arms of the pfkA gene as templates, PCR amplification was used to obtain overlapping fragments of the upstream and downstream homology arms of the pfkA gene;

[0077]③ Construct a gRNA plasmid containing the Cas9 cleavage recognition sequence. The DNA fragment containing the target sequence is obtained by annealing the primers pfkA-F' and pfkA-R'. The constructed gRNA pla...

Embodiment 3

[0089] Embodiment 3 shaking flask fermentation experiment

[0090] Produce N-acetylglucosamine with the bacterial strain constructed in the above-mentioned embodiment 2 as a production strain:

[0091] (1) Seed culture: Use an inoculation loop to scrape a ring of strains into a 500 mL shaker flask with a liquid volume of 30 mL, and culture at 37° C. and 220 rpm for 12 hours.

[0092] (2) Shake flask fermentation: inoculate the fermentation medium with 10% inoculation amount, cultivate for 48 hours at 37° C. at 220 rpm; maintain the pH at 7.0-7.2, and maintain the fermentation with 60% (m / v) glycerol glucose composite carbon source (using phenol red As an indicator, if the color of the fermentation broth remains unchanged, it is considered to be short of sugar. During the lack of sugar, 60% (m / v) glycerol glucose composite carbon source) is added, and a xylose solution with a final concentration of 10g / L is added to the initial fermentation to induce the expression of the targe...

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Abstract

The invention relates to the technical field of genetic engineering, in particular to a genetically engineered bacterium for producing N-acetylglucosamine based on metabolic division of a composite carbon source and application of the genetically engineered bacterium. By constructing an enhanced N-acetylglucosamine synthesis pathway, knocking out a 6-phosphofructokinase gene and directionally mutating a glycerokinase expression gene, metabolic division is carried out on the utilization of a glycerol-glucose composite carbon source, so that glucose is mainly used for producing a target productN-acetylglucosamine, glycerol is mainly used for cell growth, and the yield of the Nacetylglucosamine is increased. Therefore, the synthesis efficiency of the N-acetylglucosamine is improved. After the mixed carbon source culture medium is subjected to shake-flask fermentation for 48 hours, the yield of the Nacetylglucosamine reaches 107.8 g/L at most, the conversion rate of the composite carbon source reaches 49% at most, compared with a glucose utilization strain, the yield is increased by 151.7%, the conversion rate is increased by 60.2%, and the method has high industrial production potential.

Description

Technical field: [0001] The invention relates to the technical field of genetic engineering, in particular to a genetically engineered bacterium for producing N-acetylglucosamine based on complex carbon source metabolism and division of labor and its application. Background technique: [0002] N-acetylglucosamine is a functional monosaccharide that plays an important role in the repair and health care of bones and joints, and relieves osteoarthritis. Therefore, it has a broad market application demand as health care products and medicines. The traditional production method is mainly to extract from shrimp shells and crab shells through acid hydrolysis, which has problems such as environmental pollution and easy allergies. In recent years, with the rapid development of metabolic engineering, reports on the production of N-acetylglucosamine by microbial fermentation are emerging. At present, the fermentation production of Escherichia coli, Bacillus subtilis, Corynebacterium g...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/54C12P19/26C12R1/19
CPCC12N9/1205C12N9/1247C12N9/1288C12N9/1096C12N1/20C12P19/26C12Y207/0103C12Y207/07006C12Y207/08C12Y206/01016
Inventor 马倩孙全伟谭淼夏利张颖杨蒙雅谢希贤陈宁徐庆阳张成林李燕军范晓光
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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