Genetically engineered bacterium for high-yield production of hydroxytetrahydropyrimidine as well as construction method and application of genetically engineered bacterium

A technology of hydroxy ectoine and genetically engineered bacteria, which is applied in the field of genetic engineering, can solve the problems of failing to meet the industrial production requirements of hydroxy ectoine, high requirements for fermentation equipment, and large loss, and achieve separation and purification, and operability Strong performance and high production efficiency

Inactive Publication Date: 2018-08-24
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The production methods of hydroxy ectoine reported at present all have low yields, which cannot meet the industrial production requirements of hydroxy ectoine. In addi...

Method used

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  • Genetically engineered bacterium for high-yield production of hydroxytetrahydropyrimidine as well as construction method and application of genetically engineered bacterium
  • Genetically engineered bacterium for high-yield production of hydroxytetrahydropyrimidine as well as construction method and application of genetically engineered bacterium
  • Genetically engineered bacterium for high-yield production of hydroxytetrahydropyrimidine as well as construction method and application of genetically engineered bacterium

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Construction of strain E.coli HECT06

[0060] 1 Methods of gene editing

[0061] 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.

[0062] The concrete steps of this method are as follows:

[0063] 1.1. pGRB plasmid construction

[0064] The purpose of constructing the plasmid p...

Embodiment 2

[0122] Utilize the method for the fermentative production of hydroxy ectoine by genetically engineered bacteria E.coli HECT06 as follows:

[0123] (1) Shake flask fermentation:

[0124] Slant culture: Streak inoculation of -80°C preserved strains on the activated slant, culture at 37°C for 12 hours, and passage once;

[0125] 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;

[0126] 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 (use phenol red as indicator, when the color of the fermentatio...

Embodiment 3

[0140] Fermentation experiment of strain E.coli HECT06 on 5L fermenter:

[0141] Slope activation culture: Scrape a ring of strains from the -80°C refrigerator bacteria preservation tube, 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;

[0142] Seed cultivation: 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;

[0143]Fermentation culture: add fresh fermentation medium according to 15% inoculation amount, start fermentation, control pH to be stable at around 7.0 during fermentation, maintain temperature at 37°C, and dissolve oxygen between 30-40%; 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 relates to a genetically engineered bacterium for high-yield production of hydroxytetrahydropyrimidine as well as a construction method and application of the genetically engineered bacterium. The strain integrates RNA (Ribonucleic Acid) polymerase which is controlled by a xylose promoter and is derived from T7 bacteriophage; a thrA gene for encoding homoserine dehydrogenase I is knocked out; a lysC gene derived from corynebacterium glutamicum, an ectABC gene cluster derived from halomonas elongate and an ectD gene for encoding tetrahydropyrimidine hydroxylase are integrated andare promoted by a strong promoter PT7; a hydroxytetrahydropyrimidine synthesis way is reconstructed; the ectD gene is integrated at a sucCD site of succinyl coenzyme A synthetase and is started by thestrong promoter PT7; the genetically engineered bacterium has the highest level of producing the hydroxytetrahydropyrimidine by a fermentation method which is reported at present.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a genetically engineered bacterium with high yield of hydroxyectoine and its construction method and application. Background technique [0002] Hydroxytetrahydropyrimidine (1,4,5,6-tetrahydro-2-methyl-5-hydroxy-4-pyrimidinecarboxylic acid) is a polar, readily soluble, uncharged small molecule in the physiological pH range Organic matter, originally discovered in Streptomyces parvulus. In recent years, ectoine has been extensively studied as a compatible solute in microbial cells. Studies have found that hydroxytetrahydropyrimidine not only has the function of osmotic adjustment, but also can protect and stabilize the structure of nucleic acid, cell membrane, protein and other substances in adverse environments such as high temperature, dryness, hypertonicity, freezing, etc., and the protection of hydroxyectoine on protein The effect is better than that of ectoine an...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N15/90C12P17/12C12R1/19
CPCC12N9/0006C12N9/0071C12N9/1247C12N9/22C12N9/2462C12N9/93C12N15/70C12N15/902C12P17/12C12Y101/01003C12Y114/11C12Y207/07006C12Y302/01017C12Y602/01005
Inventor 谢希贤王宁马倩陈宁范晓光刘旭峰徐庆阳张成林李燕军
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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