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A kind of adenine production strain and its construction method and application

A technology for producing strains and constructing methods, which is applied in the field of genetic engineering, can solve problems such as high production costs, harsh reaction conditions, and numerous synthesis steps, and achieve good fermentation stability and pollution-free effects

Active Publication Date: 2022-04-08
SUZHOU LEAD BIOTECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] The third technical problem to be solved by the present invention is that the method for producing adenine in the prior art has many synthetic steps, harsh reaction conditions, high production cost and unfriendly to the environment. Method for producing adenine with high yield, mild conditions and less environmental pollution

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  • A kind of adenine production strain and its construction method and application
  • A kind of adenine production strain and its construction method and application

Examples

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Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Construction of Bacillus subtilis expression plasmid pMA5-RihC

[0053] The target expression plasmid constructed in this example is the expression plasmid pMA5-RihC of Bacillus subtilis. In this example, the rihC gene was cloned into the downstream of the constitutive promoter HpaII carried by the Bacillus subtilis-Escherichia coli shuttle plasmid pMA5, and the target expression plasmid was constructed in Escherichia coli.

[0054] The target expression plasmid constructed in this example has the gene encoding the biological enzyme, which is introduced into the adenosine-accumulating strain through plasmid integration, thereby obtaining an adenine-producing strain.

[0055] That is, the adenosine-accumulating bacterial strain used in this example is Bacillus subtilis; as a preferred embodiment of this example, the adenosine-accumulating bacterial strain has inosinate dehydrogenase gene function loss or weakening Sexually mutated Bacillus subtilis;

[0056] ...

Embodiment 2

[0063] Example 2 Construction of Bacillus subtilis expression plasmid pMA5-Amn

[0064] The target expression plasmid constructed in this example is the expression plasmid pMA5-Amn of Bacillus subtilis. In this example, the Amn gene was cloned into the downstream of the constitutive promoter HpaII carried by the Bacillus subtilis-Escherichia coli shuttle plasmid pMA5, and the target expression plasmid was constructed in Escherichia coli.

[0065] The target expression plasmid constructed in this example has the gene encoding the biological enzyme, which is introduced into the adenosine-accumulating strain through plasmid integration, thereby obtaining an adenine-producing strain.

[0066] That is, the adenosine-accumulating bacterial strain used in this example is Bacillus subtilis; as a preferred embodiment of this example, the adenosine-accumulating bacterial strain has inosinate dehydrogenase gene function loss or weakening Sexually mutated Bacillus subtilis;

[0067] The...

Embodiment 3

[0075] Embodiment 3 Contains the construction of the Bacillus subtilis engineering bacterium of target expression plasmid

[0076] In this example, the expression plasmid constructed in Example 1 was transformed into an adenine-accumulating strain by electroporation to obtain the adenine-producing strain. The strain accumulating adenosine is Bacillus subtilis CCTCC NO: M2018227.

[0077] The construction method of described adenine producing strain comprises the steps:

[0078] (c) taking the strain accumulating adenosine, inserting it into the culture medium, culturing it until the OD value is 0.4-0.6, and then collecting the bacteria;

[0079] (d) Resuspend the bacterium collected in step (c) in the electroporation culture medium, add the target expression plasmid obtained in step (1) to it, at a voltage of 2000-3000V, a capacitance of 2-5uF, and a resistance of 100 Electric shock at -500Ω;

[0080] (e) culturing the electroporated bacteria, and screening to obtain the ad...

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Abstract

The adenine-producing bacterial strain of the present invention is obtained by introducing a gene encoding a biological enzyme into a strain that accumulates adenosine; the biological enzyme is a biological enzyme that catalyzes the hydrolysis of the β-N9-glycosidic bond of adenosine or adenosine acid. The invention also provides the construction method and application of the production strain. The adenine producing strain of the invention has many advantages such as low cost, high yield, mild conditions, less environmental pollution, etc., and has broad application prospects.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to an adenine-producing bacterial strain and its construction method and application. Background technique [0002] Adenine, namely 6-aminopurine, is an essential compound in organisms, a nucleic acid base, and one of the constituents of DNA and RNA. Adenine can be used as a pharmaceutical intermediate for the production of plant hormones 6-benzyl adenine, adefovir dipivoxil, etc. Adenine phosphate can also be used as medicine, which can promote the proliferation of leukocytes, and can be used to prevent and treat leukopenia caused by various reasons, especially leukopenia or other acute neutropenia caused by tumor chemotherapy. Due to the wide application prospects mentioned above, the global demand for adenine is also expanding in recent years. Therefore, the development of environmentally friendly and economical adenine industrialization methods has also attracted...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12P19/40C12R1/125
CPCC12N9/1077C12N9/2497C12P19/40C12Y204/02001C12Y302/02001C12Y302/02007
Inventor 谢新开徐伟王斌
Owner SUZHOU LEAD BIOTECH CO LTD
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