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Genetic engineering strain for producing thymidine as well as construction method and application of genetic engineering strain

A technology for genetically engineering strains and genes, applied in the field of genetic engineering, can solve problems such as poor production capacity, and achieve the effects of clear genetic background, good industrial application prospects, and stable and efficient production.

Active Publication Date: 2022-04-29
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there are few reports on the de novo construction of thymidine-producing strains from wild-type strains, and the production capacity is generally poor

Method used

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  • Genetic engineering strain for producing thymidine as well as construction method and application of genetic engineering strain
  • Genetic engineering strain for producing thymidine as well as construction method and application of genetic engineering strain
  • Genetic engineering strain for producing thymidine as well as construction method and application of genetic engineering strain

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Experimental program
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Embodiment approach

[0042] According to a preferred embodiment of the present invention, the pyrimidine nucleoside operon pyrBCAKDFE is connected with a promoter P trc and / or the uridine kinase mutant gene pyrH D90A linked with promoter P trc and / or ribonucleoside diphosphate reductase operon gene nrdABC linked with promoter P trc ; and / or the promoter thymidylate synthase gene thyX is connected with a promoter P trc and / or the 5'-nucleotidase gene TMPase is connected with a promoter P trc .

[0043] According to the present invention, the starting strain for constructing the E. coli genetic engineering strain may be any E. coli, and according to a preferred embodiment of the present invention, the starting strain is E. coli MG1655.

[0044] In a second aspect, the present invention provides a method for constructing the Escherichia coli genetic engineering strain as described above, comprising: introducing the pyrimidine nucleoside operon gene pyrBCAKDFE of Bacillus subtilis into the startin...

Embodiment 1

[0058] Embodiment 1: Construction of Escherichia coli genetic engineering strain E.coli THY3-5

[0059] 1. Enhance uridine acid synthesis pathway

[0060] The pyrimidine nucleoside operon pyrBCAKDFE (comprising eight genes of pyrB, pyrC, pyrAA, pyrAB, pyrK, pyrD, pyrF, pyrE) of Bacillus subtilis (B.subtilis A260) has a total of 9495bp, and is divided into pyr1 and pyr2 in this embodiment. The three segments of pyr3 and pyr3 were integrated into the E. coli yghX gene locus in turn, and the promoter P trc The transcriptional expression of the exogenous operon was initiated, and the strain E. coli THY1-3 was constructed. Specifically include:

[0061] 1.1P trc -Integration of pyr1

[0062] Using the E.coli MG1655 genome as a template, the upstream homology arm primers (UP-yghX-S, UP-yghX-A) and downstream homology arm primers (DN-yghX-S1, DN -yghX-A), PCR amplification of its upstream and downstream homology arm fragments; using the B. subtilis A260 genome as a template, pri...

Embodiment 2

[0096] Embodiment 2: Thymidine strain shake flask fermentation experiment

[0097] The shake flask culture method is as follows:

[0098] Slant activation culture: Streak inoculation of -80°C preserved strains on the activation slant, culture at 37°C for 12 hours, and then passage again;

[0099] Seed culture: Scrape a ring of slanted 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;

[0100] Fermentation culture: Inoculate 10-15% of the volume of seed culture solution into a 500mL Erlenmeyer flask with fermentation medium (final volume is 30mL), seal with nine layers of gauze, 37°C, 200r / min shaking culture, fermentation process The pH is maintained at 7.0-7.2 by adding ammonia water; the fermentation is maintained by adding 60% (m / v) glucose solution; the fermentation period is 24 hours.

[0101] Incline medium: glucose 1-5g / L, peptone 5-10g / L, ...

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Abstract

The invention belongs to the technical field of genetic engineering, and relates to breeding of industrial microorganisms, in particular to a genetic engineering strain for producing thymidine as well as a construction method and application of the genetic engineering strain. According to the genetic engineering strain, a pyrimidine nucleoside operon gene pyrBCAKDFE of bacillus subtilis, a uridine monophosphate kinase mutant gene pyrHD90A, a thymidylate synthase gene thyX of streptomyces coelicolor and a 5 '-nucleotidase gene TMPase of bacteriophage PBS1 are subjected to heterologous overexpression; the ribonucleoside diphosphate reductase operon gene nrdABC of the escherichia coli is over-expressed; in addition, a pyrimidine nucleoside phosphorylase gene deoA, a thymidine kinase gene tdk, a uridine phosphorylase gene udp and nucleoside hydrolase genes rihA, rihB and rihC are not expressed. From the escherichia coli genome level, modules of a uridylic acid synthesis pathway, a thymidine decomposition pathway and a thymidine branch synthesis pathway are systematically and comprehensively combined and optimized mainly through a metabolic engineering technical means, and the thymidine fermentation performance of the strain is improved.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and relates to the breeding of industrial microorganisms, in particular to a gene engineering bacterial strain for producing thymidine and its construction method and application. Background technique [0002] Pyrimidine nucleosides and their derivatives are widely used in anti-tumor and anti-virus. Thymidine is a pyrimidine nucleoside, which is used in the treatment of pyrimidine metabolism and related diseases. It is mainly used as the precursor of anti-AIDS drugs stavudine and zidovudine, and has important medicinal value. With the increase of AIDS patients in the world, the market demand for thymidine continues to expand, and there is an urgent need to develop a low-cost thymidine production process that can be applied on a large scale. [0003] The current production method of thymidine is mainly the combination of enzymatic method and chemical method, the cost of raw materials i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/31C12N15/54C12N15/53C12N15/55C12N15/56C12P17/12C12R1/19
CPCC07K14/32C12N9/1229C12N9/1007C12N9/16C12N9/0093C12N9/1077C12N9/1211C12N9/2497C12P17/12C12Y207/04022C12Y201/01148C12Y301/03005C12Y117/04001C12Y204/02004C12Y207/01021C12Y204/02003C12Y302/02003Y02A50/30
Inventor 谢希贤秦臻吴鹤云姚卓越
Owner TIANJIN UNIV OF SCI & TECH
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