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Method for improving yield of erythromycin by modifying saccharopolyspora erythraea SACE_4682 gene

A technology of saccharopolyspora and erythromycin, applied in the field of genetic engineering, can solve problems such as being unsuitable for wide application, time-consuming, uneconomical and the like

Active Publication Date: 2020-05-12
ANHUI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The traditional method improves the yield of erythromycin by optimizing the fermentation conditions, but this method is time-consuming and uneconomical, and is not suitable for wide application

Method used

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  • Method for improving yield of erythromycin by modifying saccharopolyspora erythraea SACE_4682 gene
  • Method for improving yield of erythromycin by modifying saccharopolyspora erythraea SACE_4682 gene
  • Method for improving yield of erythromycin by modifying saccharopolyspora erythraea SACE_4682 gene

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Experimental program
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Effect test

Embodiment 1

[0035] Construction of SACE_4682 gene deletion mutants:

[0036] Such as figure 2 As shown, in order to knock out the SACE_4682 gene in Saccharopolyspora red mold, use 4682-P1, 4682-P2, 4682-P3 and 4682-P4 as primers, Saccharopolyspora red mold A226 genome as template, PCR amplify SACE_4682 Homologous fragments of about 1.5 kb upstream and downstream of the gene.

[0037]The upstream and downstream fragments of the above SACE_4682 were respectively connected to the pUCTSR vector to complete the construction of the plasmid pUCTSRΔ4682. Using PEG-mediated protoplast transformation technology, the large fragment of tsr-Δ4682 was transformed into protoplasts of Rhodomycetes saccharopolyspora. Based on homologous recombination technology of chromosome fragments, positive mutants were screened by thiostrepton resistance, and SACE_4682 was obtained. Genetically engineered strains whose genes are replaced by tsr. 4682-P5 and 4682-P6 were used as identification primers, the plasmid...

Embodiment 2

[0039] Construction of SACE_4682 gene reversion and overexpression strains:

[0040] The SACE_4682 gene was amplified using the designed primers 4682-P5 and 4682-P6, and recovered by electrophoresis. The recovered SACE_4682 gene fragment and pIB139 were double-enzymatically digested and recovered using NdeI and XbaI endonucleases, and then extracted by T4DNA ligase. The SACE_4682 gene fragment was connected to pIB139, and the plasmid pIB139-4682 was successfully obtained. Then pIB139 and pIB139-4682 plasmids were introduced into A226 and ΔSACE_4682 protoplasts by PEG-mediated protoplast transformation technology. Through preliminary screening with apramycin, the apramycin resistance gene (apr) was identified by PCR. The obtained positive reverting strain was named ΔSACE_4682 / pIB139-4682, the positive reverting empty control strain was named ΔSACE_4682 / pIB139; the obtained positive overexpression strain was named A226 / pIB139-4682, and the positive overexpressing empty control ...

Embodiment 3

[0042] HPLC detection of fermentation products of Saccharopolyspora red mold:

[0043] Inoculate Saccharopolyspora red mold series strains into TSB medium, and after shaking culture at 30°C for 48 hours, transfer to R5 liquid medium, shake culture at 30°C for 6 days, then use organic solvent to extract the fermentation broth, use a water bath After the pot is evaporated to dryness, add 1mL of methanol to dissolve and use a 0.22μm organic filter membrane to process, and then use the machine to detect the content of erythromycin A in the sample, such as image 3 Shown in B.

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Abstract

The invention discloses a method for improving the yield of erythromycin by modifying a saccharopolyspora erythraea SACE_4682 gene. In the saccharopolyspora erythraea, a TetR family transcription regulation gene SACE_4682 is deleted by a genetic engineering method to obtain an erythromycin high-yield engineering strain, and the obtained strain is fermented to produce the erythromycin, so that a new technical support is provided for improving the yield of the erythromycin in industrial production.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for improving the yield of erythromycin by modifying the gene of Saccharopolyspora erythromycetes SACE_4682. Background technique [0002] Erythromycin is a class of broad-spectrum macrolide antibiotics produced by Saccharopolyspora, which has good activity against Gram-positive bacteria and low gastrointestinal side effects. Since it was first isolated from Saccharopolysporaerythraea in 1952, the utilization rate of erythromycin has been at the forefront of antibiotics in the world. Among the components of erythromycin fermented by Saccharopolyspora, erythromycin A has the highest antibacterial activity, and azithromycin, clarithromycin, roxithromycin and telithromycin derived from erythromycin are also It is the mainstream medical antibiotic today. It can be seen that improving the output of erythromycin is an urgent problem to be solved at present. [0...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/15C12P19/62C12R1/645
CPCC07K14/37C12P19/62
Inventor 张部昌陈昱宏吴杭王静刘英
Owner ANHUI UNIVERSITY
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