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Method for increasing output of erythrocin by SACE_5717 gene of saccharopolyspora erythraea

A technology of saccharopolyspora and erythromycin, which is applied in other directions of inserting foreign genetic material, genetic engineering, plant genetic improvement, etc.

Active Publication Date: 2019-02-12
ANHUI AGRICULTURAL UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there are still few studies on the regulatory genes of Saccharopolyspora red mold, only bldD (SACE_2077), SACE_7040, SACE_0012, SACE_5599, SACE_3986, SACE_3986 and SACE_5388 and other regulatory genes are reported.

Method used

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  • Method for increasing output of erythrocin by SACE_5717 gene of saccharopolyspora erythraea
  • Method for increasing output of erythrocin by SACE_5717 gene of saccharopolyspora erythraea
  • Method for increasing output of erythrocin by SACE_5717 gene of saccharopolyspora erythraea

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Embodiment 1

[0033] Construction of embodiment 1 SACE_5717 gene inactivation mutant:

[0034] The pUCTSR plasmid is a 1360bp thiostrepton resistance gene (tsr) inserted between the BamH I and Sma I restriction sites of pUC18. In order to knock out the SACE_5717 gene in Saccharopolyspora, use PUp1 / PUp2 and PD1 / PD2 as primers and the Saccharopolyspora genome as a template to amplify the homology arms of about 1.5 kb upstream and downstream of the SACE_5717 gene by PCR SU and SD DNA fragments.

[0035] The primers for PCR amplification of SU DNA fragments are PUp1, PUp2, the nucleotide sequence of PUp1 is shown in SEQ ID NO.3, and the nucleotide sequence of Pup2 is shown in SEQ ID NO.4; wherein, the " "AAGCTT" is the restriction site of HindIII, and "TCTAGA" in the sequence of Pup2 is the restriction site of Xbal.

[0036] The primers for PCR amplifying SD DNA fragments are PD1, PD2, the nucleotide sequence of PD1 is shown in SEQ ID NO.5, and the nucleotide sequence of PD2 is shown in SEQ ID ...

Embodiment 2

[0039] The construction of embodiment 2 SACE_5717 gene reversion bacterial strain:

[0040] In order to introduce the SACE_5717 gene in ΔSACE_5717, PCR amplification SACE_5717 gene primers were designed, namely P3 and P4; the nucleotide sequence of P3 is shown in SEQ ID NO.9, and the nucleotide sequence of P4 is shown in SEQ ID NO.10 where, "CATATG" in the P3 sequence is the NdeI restriction site, and "TCTAGA" in the P4 sequence is the XbaI restriction site.

[0041] The SACE_5717 gene was amplified from the Saccharopolyspora Rhododerma A226 genome, inserted between the Nde I and Xba I restriction sites of pIB139, and the expression plasmid pIB139-5717 was constructed, and then pIB139 was transformed into pIB139 by PEG-mediated protoplast transformation -5717 was imported into ΔSACE_5717. After preliminary screening with apramycin, the apramycin resistance gene (apr) was identified by PCR, and the obtained revertant strain was named ΔSACE_5717 / pIB139-5717.

Embodiment 3

[0042] Overexpression of SACE_5717 gene in embodiment 3 Saccharopolyspora erythromycetes A226:

[0043] pIB139-5717 was introduced into Saccharopolyspora Erythromycetes A226 by PEG-mediated protoplast transformation technology, and the apramycin resistance gene (apr) was used for PCR identification, and the obtained strain was named A226 / pIB139-5717.

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Abstract

The invention provides a method for increasing output of erythrocin by an SACE_5717 gene of saccharopolyspora erythraea. The method comprises the following steps of deactivating the SACE_5717 gene inthe saccharopolyspora erythraea by a gene engineering path, so as to obtain a saccharopolyspora erythraea strain with high output of erythrocin; applying the saccharopolyspora erythraea engineering strain with high output of erythrocin, and fermenting, so as to produce the erythrocin, wherein a nucleotide sequence of the SACE_5717 gene is shown in SEQ ID NO.1; an amino acid sequence coded by the nucleotide sequence is shown in SEQ ID NO.2. The method has the advantages that the SACE_5717 gene in the saccharopolyspora erythraea is deactivated by the engine engineering path, so as to obtain thestrain with high output of erythrocin; the obtained strain with high output of erythrocin is used for fermenting to produce the erythrocin, so that the output of the erythrocin is increased, and the new technical support is provided for the increasing of output of the erythrocin in industrial production.

Description

technical field [0001] The invention relates to a method for increasing the yield of erythromycin by fermentation, in particular to a method for increasing the yield of erythromycin through the gene of Saccharopolyspora erythromycetes SACE_5717. Background technique [0002] Actinomycetes are an important microbial resource that can produce secondary metabolites, and are closely related to humans. About 75% of the widely used antibiotics are produced by actinomycetes. Common vancomycin, erythromycin, lincomycin, tetracycline and other different types of secondary metabolites are produced by actinomycetes. Antibiotic synthesis in actinomycetes is controlled by synthetic gene clusters, and the transcription of synthetic gene clusters generally depends on the regulation of specific regulatory proteins or global regulatory proteins. Through the study of this regulatory protein, and the modification of the regulatory protein gene , to regulate secondary metabolism and change the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/62C12N15/74C12N15/90C12N15/31
CPCC07K14/195C12N15/74C12N15/902C12P19/62
Inventor 刘静张部昌吴杭汪焰胜陈云富
Owner ANHUI AGRICULTURAL UNIVERSITY
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