Recombinant bacterium for improving lincomycin component A and reducing lincomycin component B and application thereof

A technology of recombinant engineering bacteria and lincomycin, applied in the biological field, can solve the problems of high toxicity, achieve high potency, improve economic benefits, and reduce production costs

Active Publication Date: 2015-10-21
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Component A of lincomycin is the active component of medicine, component B of lincomycin is a by-product, its activity is only 25% of that of component A, and its toxicity is relatively high

Method used

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  • Recombinant bacterium for improving lincomycin component A and reducing lincomycin component B and application thereof
  • Recombinant bacterium for improving lincomycin component A and reducing lincomycin component B and application thereof
  • Recombinant bacterium for improving lincomycin component A and reducing lincomycin component B and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] A recombinant engineering bacterium that increases component A of lincomycin and reduces component B is constructed by the following method:

[0046] (1) Using the Streptomyces lincoeum genome as a template, design the following primers: use the sequence shown in SEQ ID No.1 as the upstream primer, and use the sequence shown in SEQ ID No.2 as the downstream primer to clone the lmbW gene (GenBank: EU124663.1) ;

[0047] 5'-GCCC AAGCTT CTTCAGACGGACCGAGGTGCC-3' (the underline is the HindIII restriction site); SEQ ID No.1

[0048] 5'-GCCG GGATCC TCCGCGTGGTTCAGGGCACA-3' (the underline is the BamHI restriction site); SEQ ID No.2

[0049] The 50 μL PCR reaction system is: 10 μL of 5× buffer, 5 μL dNTPs, 5 μL DMSO, 1 μL upstream primer, 1 μL downstream primer, 1 μL genome template, 1 μL polymerase, and make up the rest with purified water.

[0050] The PCR parameters are as follows: 95°C for 5 min; 30 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 1 min; 72°C for 10 ...

Embodiment 2

[0059] The second recombinant engineering bacterium that improves the lincomycin A component and reduces the lincomycin B component is constructed by the following method:

[0060] (1) Using the Streptomyces lincoeum genome as a template, design the following primers: use the sequence shown in SEQ ID No.3 as the upstream primer, and use the sequence shown in SEQ ID No.4 as the downstream primer to clone the metK gene (GenBank: KP225159);

[0061] 5'-ACGC AAGCTT GTGTCCCGTCGTCTGTTCAC-3' (the underline is the HindIII restriction site); SEQ ID No.3

[0062] 5'-GCCG GAATTC GGGGCACCGCGTAGTCGAAGTA-3' (the underline is the EcoRI restriction site); SEQ ID No.4

[0063] The PCR reaction system and parameter settings are the same as in Example 1 (wherein the primers are different).

[0064] The metK gene fragment was amplified. Using a DNA purification kit, recover the PCR fragment.

[0065] (2) The metK gene was ligated to the pUWL201apr vector by enzyme-cut ligation to construct ...

Embodiment 3

[0070] The third kind of recombinant engineering bacteria that improves the lincomycin A component and reduces the lincomycin B component is constructed by the following method:

[0071] (1) Using the pAP04 vector obtained in Example 1 as a template, using the sequence shown in SEQ ID No.5 as an upstream primer, and using the sequence shown in SEQ ID No.2 as a downstream primer, clone the lmbW gene;

[0072] 5'-GCCG GAATTC GCCCGATGCTAGTCGCGGTTG-3' (underlined is the EcoRI restriction site); SEQ ID No.5 PCR reaction system and parameter settings are the same as in Example 1 (wherein the primers and templates are different).

[0073] The lmbW gene fragment was amplified, and the PCR fragment was recovered with a DNA purification kit.

[0074] (2) The obtained lmbW gene was connected to the pAP05 vector obtained in Example 2 by restriction enzyme digestion, and the pAP06 expression vector in which metK and lmbW were connected in series was constructed.

[0075] The operations ...

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Abstract

The invention discloses a recombinant bacterium for improving the lincomycin component A and reducing the lincomycin component B and the application thereof. The building method of the recombinant bacterium comprises the steps that 1, streptomyces lincolnensis genome is used as a template, the sequence shown in the SEQ ID No.1 is used as an upstream primer, the sequence shown in the SEQ ID No.2 is used as a downstream primer, and a lmbW gene is cloned; 2, the lmbW gene is connected to a pUML201apr carrier in an enzyme-cut and link up mode, and a pAP04 carrier is built; 3, the pAP04 carrier is converted to streptomyces lincolnensis, screening is carried out, and therefore the recombinant bacterium for improving the lincomycin component A and reducing the lincomycin component B is obtained. According to the recombinant bacterium, the fermentation potency of the lincomycin component A is high, the lincomycin component B is reduced, the downstream separation and purification process can be simplified, the production cost is reduced, energy conservation and emission reduction are achieved, and economic benefits are improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and particularly relates to a recombinant engineering bacterium for increasing lincomycin A component and reducing lincomycin B component and its application. Background technique [0002] Lincomycin is a lincosamide antibiotic produced by Streptomyces lincoi, which is mainly used to treat infectious diseases caused by Gram-positive bacteria. During the fermentation process of Streptomyces lincoeum, two components of lincomycin A and B are produced. Component A of lincomycin is a medicinally active component, component B of lincomycin is a by-product, its activity is only 25% of that of component A, and its toxicity is relatively high. The excess lincomycin B produced in the fermentation needs to be removed in the downstream separation and purification process in order to meet the requirements of the drug. Therefore, increasing the lincomycin A component and reducing the lincomycin B component is v...

Claims

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Application Information

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IPC IPC(8): C12N15/76C12N1/21C12P19/64C12R1/565
Inventor 赵广荣逄爱萍
Owner TIANJIN UNIV
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