Recombinant strain for synthesizing hypoxanthine and construction method and application of recombinant strain

A technology of hypoxanthine and recombinant bacteria, applied in the field of genetic engineering, can solve the problems of difficult to achieve purine accumulation, long synthetic route, and difficult to meet the needs of large-scale biosynthesis of purine compounds

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

AI Technical Summary

Problems solved by technology

Although the biosynthetic route of purine in cells has been found out for a long time, its synthetic route is long and closely regulated at different levels in the cell, so it is difficult to realize the accumulation of purine in the natural state.
At present, researchers have only achieved the synthesis of hypoxanthine at the micromolar level in Corynebacterium glutamicum, which is difficult to meet the demand for a large amount of biosynthesis of purine compounds
Escherichia coli is one of the preferred hosts in the field of microbial synthesis because of its low culture cost, fast growth rate, and easy gene manipulation. However, there is no report on the production of hypoxanthine by engineered Escherichia coli

Method used

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  • Recombinant strain for synthesizing hypoxanthine and construction method and application of recombinant strain
  • Recombinant strain for synthesizing hypoxanthine and construction method and application of recombinant strain
  • Recombinant strain for synthesizing hypoxanthine and construction method and application of recombinant strain

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Experimental program
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Effect test

Embodiment 1

[0064] Embodiment 1: Construction of recombinant bacteria

[0065] 1. Gene knockout (P1 phage transduction method, the principle is as follows Figure 4 shown)

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

[0067] The transcriptional repressor gene purR (Gene ID: 945226) of E. coli W3110 genome was knocked out by using P1 phage transduction method, using P1 phage to infect the donor strain JW1650 of the Keio Collection library, and preparing the cleavage library of the donor strain, using JW1650 The cleavage library was used to transduce the recipient strain Escherichia coli W3110, and the mutant strain W3110△purR knocking out the transcriptional repressor gene purR was obtained;

[0068] 1) Phage activation

[0069] 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 is derived from the Keio Collection library and can be purcha...

Embodiment 2

[0087] The shake flask fermentation test of embodiment 2 recombinant strains

[0088] The present embodiment carries out two groups of experiments altogether, to illustrate the effect that the present invention can obtain, concrete experimental method is as follows:

[0089] Control group: wild strain E.coli W3110,

[0090] Experimental group: recombinant strain Q2955,

[0091] 1) Inoculate the activated wild strain E.coli W3110 and the recombinant strain Q2955 into a 250mL shake flask containing 50mL fermentation medium (containing 50mg / L chloramphenicol) at a ratio of 1:100, 37°C, 180rpm Shaking culture under conditions. OD 600 When it reached about 0.6, 100 μM IPTG was added to induce expression, and after induction, culture was continued at 30° C. and 180 rpm for 96 hours until the end of fermentation.

[0092] 2) Take 1 mL of fermentation broth, centrifuge at 12000 rpm for 10 min at 4°C, take the supernatant, filter it with a 0.22 μm filter membrane, and detect the fe...

Embodiment 3

[0094] The fermenter experiment of embodiment 3 recombinant strains

[0095] 1) The recombinant strain Q2955 was inoculated into 3 mL of liquid LB medium, shaken overnight at 37°C and 180 rpm, and cultured as the primary seed solution.

[0096] 2) Inoculate the primary seed solution of the activated culture into a 250mL shake flask containing 50mL of fermentation medium (containing 50mg / L of chloramphenicol) according to the ratio of 1:100, shake and cultivate at 37°C and 180rpm for 4h, as a secondary seed solution.

[0097] 3) The secondary seed liquid is inoculated into the fermenter according to the inoculation amount of 2% of the medium volume, the culture temperature is 37°C, the stirring speed is 400-800rpm, 20% dissolved oxygen is related to the speed, and it is automatically adjusted by using ammonia water and 10% sulfuric acid The pH was about 7.0, and the recombinant cells were cultured to an OD600 of 10, and then the inducer IPTG was added to a final concentration ...

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Abstract

The invention discloses a recombinant strain for synthesizing hypoxanthine and a construction method and application of the recombinant strain, and belongs to the technical field of genetic engineering. According to the recombinant strain, escherichia coli is used as an original strain, a transcription repressor protein gene purR, a phosphogluconate anhydrase gene edd, an adenylosuccinic acid synthetase gene purA and an inosinic acid dehydrogenase gene guaB on an escherichia coli genome are respectively knocked out, and besides, D128A mutated PRPP synthetase gene prs and K326Q as well as a P410W double-mutated PRPP transamidase gene purF are subjected to overexpression. Besides, the invention further provides a preparation method of the recombinant strain and a method for producing the hypoxanthine through the recombinant strain. The efficient biosynthesis of the hypoxanthine in engineering escherichia coli is realized for the first time. The recombinant strain is suitable for production of the hypoxanthine through fermentation.

Description

technical field [0001] The invention relates to a recombinant bacterium for synthesizing hypoxanthine and its construction method and application, belonging to the technical field of genetic engineering. Background technique [0002] Purine is a class of important life-active substances, which participate in the synthesis of genetic information substances in cells. The intermediate metabolites in the process of purine biosynthesis play an important role in the transfer of genetic material, signal transport and energy metabolism in the process of cell life. In addition, purine and its derivatives are also widely used in the field of medicine, such as the diagnosis and treatment of diseases and research fields such as life sciences, which has aroused people's strong interest in the synthesis of purine and its derivatives. At present, derivatives with purine as the backbone have been widely used in drug research, for example, purine compounds can induce the release of interfer...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P19/40C12R1/19
CPCC12P19/40C12N15/70
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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