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Recombinant bacteria producing xanthine and construction method and application thereof

A technology of recombinant bacteria and xanthine, applied in the field of genetic engineering, can solve problems such as low efficiency, long synthetic route, and unstable strains

Active Publication Date: 2020-01-07
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Summary
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  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

At present, the production strains of purine compounds are mainly obtained by mutagenesis screening technology, but it is often time-consuming, low in efficiency, and the obtained strains are unstable.
Although the biosynthetic route of purine in cells has long been identified, its synthetic route is relatively long and is regulated by intracellular transcriptional repression, transcriptional attenuation, and substrate feedback inhibition of enzymes. Purines are mainly used to synthesize intracellular genetic material. , it is difficult to accumulate in the cell
Simultaneously, existing technology still does not have the report of recombinant bacterium preparation xanthine

Method used

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  • Recombinant bacteria producing xanthine and construction method and application thereof
  • Recombinant bacteria producing xanthine and construction method and application thereof
  • Recombinant bacteria producing xanthine and construction method and application thereof

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Effect test

Embodiment 1

[0063] Embodiment 1: Construction of recombinant bacteria

[0064] 1. Gene knockout (P1 phage transduction method, the principle is as follows figure 2 )

[0065] The transcriptional repressor protein gene purR (Gene ID: 945226), the phosphoglucose isomerase gene pgi (Gene ID 948535), and the phosphogluconate dehydratase gene edd (Gene ID: 946362), adenosuccinate synthase gene purA (Gene ID: 948695) and GMP synthase gene guaA (Gene ID 947334).

[0066] The knockout of the transcriptional repressor protein gene purR was carried out as follows:

[0067] 1) Phage activation

[0068] Add 4ml of heated and melted 0.4% agar medium to a 10ml sterile EP tube, add 400 μL of overnight cultured donor strain JW1650 (the donor strain JW1650 is derived from the Keio Collection library, and can be purchased through commercial channels, Keio collection library construction method Such as "Baba T, et al. Construction of Escherichiacoli K-12 in-frame, single-gene knockout mutants: the Keio...

Embodiment 2

[0088] The fermentation test of embodiment 2 recombinant strains

[0089] 1) Inoculate the recombinant strain ZG-2960 into LB liquid medium, and shake overnight at 37°C and 180rpm.

[0090] 2) The overnight culture of step 1) was inoculated into a 250mL shake flask containing 50mL fermentation medium (containing 50mg / L chloramphenicol) according to the ratio of 1:100, and shaken at 37°C and 180rpm for 4h, as a seed solution.

[0091] 3) Inoculate the seed liquid in step 2) into the fermenter according to the inoculation amount of 2% of the culture medium volume, the culture temperature is 37°C, the stirring speed is 300-700rpm, 20% dissolved oxygen is related to the rotation speed, and ammonia water and 10% Sulfuric acid automatically adjusted the pH to about 7.0, and the recombinant cells were cultured to an OD600 of 8, then the inducer IPTG was added to a final concentration of 100 μM, and the fed-feed fermentation was continued for 96 hours using a glucose stock solution w...

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Abstract

The invention discloses recombinant bacteria producing xanthine and a construction method and application thereof, and belongs to the technical field of genetic engineering. The recombinant bacteria use escherichia coli as original strains, the transcriptional repressor protein purR, phosphoglucose isomerase gene pgi, glucogluconate dehydratase gene edd, adenylosuccinate synthetase gene purA and GMP synthase gene guaA on escherichia coli genome are knocked out, and D128A mutated PRPP synthase gene prs and K326Q and P410W double mutated PRPP amidotrasferase purF are subjected to overexpression.Meanwhile, the invention further provides a method for preparing the recombinant bacteria and a method for producing xanthine by using the recombinant bacteria. For the first time, the high-efficiency biosynthesis of xanthine in recombinant bacteria is realized. The recombinant bacteria are suitable for producing xanthine by fermentation.

Description

technical field [0001] The invention relates to a recombinant bacterium for producing xanthine and its construction method and application, belonging to the technical field of genetic engineering. Background technique [0002] Purine bases and their nucleoside products are widely used in the fields of medicine and food. Such products can affect the activity of the nervous system, produce cardiovascular effects, and can play a role in sedation, dilating blood vessels, and lowering blood pressure. The development of new drugs such as nucleosides and their derivatives with anti-tumor and anti-viral activities has become one of the current research hotspots. In addition, it can also be used to produce various functional foods, including cold-resistant foods, diet foods, etc. Xanthine is a purine base widely distributed in the organs and body fluids of the human body and other organisms. It is often used as a mild stimulant and bronchodilator, especially for the treatment of as...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P17/18C12R1/19
CPCC07K14/245C12N9/90C12N9/88C12N9/93C12N9/1235C12N9/104C12P17/182C12Y504/02002C12Y402/01012C12Y603/04004C12Y603/05002C12Y207/06001
Inventor 赵广刘敏咸漠高文杰
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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