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Engineering strain for producing beta-thymidine, and application thereof

A technology of engineering strains and strains, applied in the biological field, can solve problems such as high cost and long fermentation cycle, and achieve the effects of reducing production costs, reducing environmental pollution, and low separation costs

Active Publication Date: 2018-07-20
SHANGHAI ACEBRIGHT PHARMA GRP +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the fermentation production process using glycerol as a carbon source has problems such as high cost and long fermentation cycle.

Method used

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  • Engineering strain for producing beta-thymidine, and application thereof
  • Engineering strain for producing beta-thymidine, and application thereof
  • Engineering strain for producing beta-thymidine, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Using E.coli BL21 as the original strain, knock out three genes encoding thymidine phosphorylase (deoA), thymidine kinase (tdk) and uridine phosphorylase (udp) in the pyrimidine replenishment pathway of BL21, and truncate thymidine The breakdown pathway allows thymidine to accumulate.

Embodiment 2

[0086] Using E.coliBL21 as the original strain, the mutant strain BL21-U2 obtained by knocking out a single gene of udp, the mutant strain BL21-UD2 obtained by knocking out the udp and deoA genes, and the mutant strain BL21-UD2 obtained by knocking out the udp, deoA and tdk genes Mutant strain BL21-UDT2 (see Table 1), was cultured for thymidine production as follows.

[0087] Table 1 Genotypes of E.coli BL21 mutants

[0088]

[0089] Inoculate the E.coliBL21 strain and each mutant strain in 5ml of M9 enriched medium (supplemented with 0.1M K 2 HPO 4 ,0.01M Fe 2 (SO 4 ) 3 and 2g / L amino acid) at 37°C overnight. Inoculate 40ul of overnight cultured cells into 4ml of M9 enriched medium and culture at 37°C for 5h (220rpm), while using blank M9 enriched medium as a control. After the completion of the culture, the cultured cells were centrifuged (3500rpm, 10min, 4°C) to collect the supernatant, and the content of thymidine, thymine, 2-deoxyuridine, uridine and uracil in the ...

Embodiment 3

[0094] The pgi gene of the BL21-UDT2 bacterial strain in Example 2 is knocked out by the method of gene recombination to obtain BL21-UDT2P, which is cultured in a 50ml centrifuge tube for 72 hours and then the thymidine content is measured, and the effect on the bacterial strain thymidine before and after the pgi gene knockout is compared. The effect of glycoside content, the results are shown in Table 3.

[0095] Table 3 Thymidine production ability of strains before and after pgi gene knockout

[0096] strain

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Abstract

The invention provides an engineering strain for producing beta-thymidine, and an application thereof. The engineering strain is obtained through genetic modification, such as gene knockout or gene integration, has a high stable beta-thymidine production capability, and can be used for producing the beta-thymidine.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically relates to an engineering strain for producing β-thymidine and application thereof. Background technique [0002] β-thymidine (hereinafter referred to as thymidine) is the key intermediate of anti-AIDS zidovudine. Zidovudine is the world's first anti-AIDS drug approved by the US FDA. Because of its definite curative effect, it has become the most basic component of the "cocktail" therapy. [0003] Due to the dramatic increase in the number of AIDS patients, cheap, mass-produced thymidine processes have received widespread attention. The production of thymidine by biological fermentation mainly uses the biosynthetic pathway of microbial strains to produce thymidine. The production of thymidine by biological methods has the advantages of simple process and low cost of subsequent separation, especially the current genetic engineering breeding technology and high-yield optimization...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N1/21C12P19/38C12R1/19
CPCC12N9/1077C12N9/1211C12P19/385C12Y204/02003C12Y204/02004C12Y207/01021C12N15/70C12P19/38C12N1/205C12R2001/19
Inventor 施明安王涛K·基拉奇克M·布拉奇克
Owner SHANGHAI ACEBRIGHT PHARMA GRP
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