Genetic engineering bacterium capable of producing uridine at high yield as well as building method and application thereof

A technology of genetically engineered bacteria and uridine, applied in the field of genetic engineering, can solve the problems of low production efficiency, unfavorable product separation and purification, etc., and achieve the effects of high production efficiency, favorable separation and purification, and high production intensity

Active Publication Date: 2018-06-08
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to accumulate a large amount of uridine, a large amount of organic nitrogen sources such as corn steep liquor need to be added to the fermentation medium, which is not conducive to the separation and purification of subsequent products. At the same time, the cycle of fermentation of uridine by Bacillus subtilis is as long as more than 70 hours, and the production efficiency is relatively low.

Method used

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  • Genetic engineering bacterium capable of producing uridine at high yield as well as building method and application thereof
  • Genetic engineering bacterium capable of producing uridine at high yield as well as building method and application thereof
  • Genetic engineering bacterium capable of producing uridine at high yield as well as building method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Construction of strain E.coli UR11:

[0049] 1 Methods of gene editing

[0050] The gene editing method used in the present invention is carried out with reference to the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli using CRISPR–Cas9 edited genome editing. Metabolic engineering, 2015, 31:13-21.), the method used The two plasmid maps of the attached figure 1 . Among them, pREDCas9 carries gRNA expression plasmid pGRB elimination system, bacteriophage λ Red recombination system and Cas9 protein expression system, spectinomycin resistance (working concentration: 100mg / L), cultured at 32°C; pGRB uses pUC18 as the backbone, including the promoter J23100, gRNA-Cas9 binding region sequence and terminator sequence, ampicillin resistance (working concentration: 100mg / L), cultured at 37°C.

[0051] The concrete steps of this method are as follows:

[0052] 1.1 pGRB plasmid construction

[0053] The purpose of constructing the plasmid pGR...

Embodiment 2

[0125] The method for producing uridine by fermentation of genetically engineered bacteria E.coli UR11 is as follows:

[0126] (1) Shake flask fermentation:

[0127] Slant culture: Streak inoculation of -80°C preserved strains on the activated slant, culture at 37°C for 12 hours, and passage once;

[0128] Shake flask seed culture: Scrape a ring of slant seeds with an inoculation loop and inoculate in a 500mL Erlenmeyer flask containing 30mL of seed medium, seal with nine layers of gauze, and incubate at 37°C and 200rpm for 7-10h;

[0129] Shake flask fermentation culture: Inoculate 10-15% inoculum into a 500mL Erlenmeyer flask with fermentation medium (final volume is 30mL), seal with nine layers of gauze, 37°C, 200r / min shaking culture, during the fermentation process by supplementing Add ammonia water to maintain the pH at 7.0-7.2; add 60% (m / v) glucose solution to maintain the fermentation; the fermentation period is 24-30h;

[0130] The composition of the slant medium i...

Embodiment 3

[0143] Fermentation experiment of strain E.coli UR11 on 5L fermenter:

[0144] Slope activation culture: Scrape a ring of strains from the bacteria preservation tube of the -80°C refrigerator, spread evenly on the activation slope, incubate at 37°C for 12 hours, transfer to an eggplant-shaped bottle and continue to cultivate for 12 hours;

[0145] Seed culture: Take an appropriate amount of sterile water in an eggplant-shaped bottle, insert the bacterial suspension into the seed medium, keep the pH at about 7.0, the temperature at 37°C, and the dissolved oxygen at 25-35%, and cultivate for 6 hours;

[0146] Fermentation culture: add fresh fermentation medium according to 15% inoculation amount, start fermentation, control pH to be stable at about 7.0 during fermentation, maintain temperature at 37°C, and dissolve oxygen between 25-35%; After the glucose is consumed, add 80% (m / v) glucose solution to maintain the glucose concentration in the fermentation medium at 0.1-5g / L; the...

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Abstract

The invention provides a genetic engineering bacterium capable of producing uridine at high yield as well as a building method and application thereof. The genetic engineering bacterium is characterized in that pyrimidine nucleoside operons pyrBCAKDFE with the nucleotide sequence shown as SEQ ID NO:1 is integrated on a genome of colon bacillus; the starting is realized by a strong promoter P[trc];a uridine synthesis path is reconstituted; the self PRPP synthetase coding gene prsA on the genome is subjected to dual copying, and the starting is realized by the strong promoter P[trc]; meanwhile,the activity of udk, udp, and rihA, rihB and rihC is lacked; the thrA activity is lacked; the argF activity is lacked. The genetic engineering bacterium is applied to fermentation production of uridine; 40 to 67g/L of uridine can be produced after the fermentation is performed for 40 to 70h in a 5L fermentation tank; the maximum production intensity can reach 1.5g/(L*h); the glucoside conversionrate is 15 to 25 percent; the genetic engineering bacterium belongs to the highest level for producing the uridine by the fermentation method reported in the prior art.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a genetically engineered bacterium with high uridine production and its construction method and application. Background technique [0002] Uridine is a pyrimidine nucleoside widely used in food, health products, cosmetics and pharmaceutical industries. Uridine is the precursor of uridine acid. Uridine acid can not only be used as seasoning and pharmaceutical raw materials, but also be added to milk powder as a food additive to greatly improve the immunity of infants. Uridine can be used as a pharmaceutical intermediate to produce some anti-viral and anti-tumor drugs, such as iodine, bromide, fluoroglycoside, etc., to treat cancer or diseases caused by viruses. Uridine has very important practical value, and the market has a large demand for uridine, so there is an urgent need for a low-cost uridine production process that can be produced on a large scale....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/54C12N15/70C12P19/38
CPCC12N9/1077C12N15/70C12P19/385
Inventor 谢希贤陈宁吴鹤云李国梁李强范晓光徐庆阳张成林李燕军马倩
Owner TIANJIN UNIV OF SCI & TECH
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