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Method for producing high-activity and high-purity rifamycin SV

A technology of rifamycin and cytochrome, applied in the field of using the bacteria, can solve the problems such as not very clear

Active Publication Date: 2011-12-21
CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] However, the last step (maybe several steps) in the rifamycin synthesis pathway, that is, the genes used for the conversion from Rf_SV to Rf_B and the mechanism of the enzyme-catalyzed reaction, are not very clear so far

Method used

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  • Method for producing high-activity and high-purity rifamycin SV
  • Method for producing high-activity and high-purity rifamycin SV
  • Method for producing high-activity and high-purity rifamycin SV

Examples

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Effect test

Embodiment 1

[0118] Example 1: Whole-genome sequencing of the rifamycin Rf_SV strain U32 and its sequence comparison with the rifamycin B strain

[0119] The rif16 gene was sequenced by the inventors after sequencing the entire genome of a strain of A. mediterranei strain U32 producing rifamycin Rf_SV and comparing it with three wild-type strains producing rifamycin B. discovered during the process. in particular:

[0120] Using the second-generation 454 sequencer, combined with SOLID, Sanger sequencing methods, and 6-8k plasmid library and fosmid library construction, the complete gene sequence of 10,236,715bp Amycobacterium mediterranei U32 with an error value of less than 0.5 / 100,000 was finally obtained . After the rifamycin synthesis gene cluster rif was obtained by BLASTP annotation, it was compared with the rif synthesis gene cluster of the Rf_B-producing strain S699 studied by Floss. The inventors discovered 41 SNPs and 8 INDEL sites, which altogether affected the promoter regi...

Embodiment 2

[0130] Embodiment 2: transfer rif16 of ATCC 21789 to U32

[0131] Using conventional molecular biology methods, the PCR product of the rif16 gene containing its own promoter region in ATCC 21789 (the primers used are shown in SEQ ID Nos: 19 and 20) was cloned into the episomal multi-copy plasmid of A. mediterranei On the EcoR V site of pDXM4 (refer to Ding Xiaoming 2001), a plasmid pDXM4-P450 containing wild-type ATCC 21789 rif16 gene was obtained. Using the empty plasmid as a control, pDXM4-P450 containing rif16 was transferred to U32 by electroporation (refer to Ding Xiaoming 2001). The result is as Figure 5 shown.

Embodiment 3

[0132] Embodiment 3: The stability of U32 complementation plasmid pDXM4-P450 is transformed into the influence of Rf_B by Rf_SV

[0133] The plasmid pDXM4-P450 can be stabilized in the U32 strain if the antibiotic apramycin is added to the medium, otherwise the plasmid will be lost during the proliferation of the bacteria. Taking the addition of apramycin antibiotics in the medium as a control, we took samples at the time points of 2 days, 3 days, and 5 days in the U32 fermentation broth without adding apramycin antibiotics, and did it together with the fermentation broth with antibiotics added. HPLC-MS detection. The results found that with the loss of the complementing plasmid pDXM4-P450, Rf_SV and Rf_S in the fermentation broth accumulated rapidly. It is verified from the side that rif16 plays a role in the conversion of Rf_SV to Rf_B ( Figure 6 ).

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Abstract

The invention relates to a method for producing high-activity and high-purity rifamyicn SV(Rf_SV). The invention particularly relates to the use of a cytochrome P450 gene or a transketolase gene or proteins coded by the cytochrome P450 gene and the transketolase gene in regulation of the conversion of rifamyicn SV into rifamyicn B. The method produces the high-activity and high-purity rifamyicn SV by deactivating two or one of key enzymes (Rif15 and Rif16) required for participating in conversion from Rf_SV into Rf_B in a rifamyicn synthesis process in a rifamyicn B (Rf_B) producing strain.

Description

technical field [0001] The invention relates to the fields of bioengineering and pharmacy. More specifically, the present invention relates to the use of cytochrome P450 gene or transketolase gene or the protein encoded by them in regulating the transformation of rifamycin SV into rifamycin B, and the production of high activity obtained by bioengineering method 1. A bacterial strain of high-purity rifamycin SV, a method for preparing the bacterial strain, and a method for producing high-activity, high-purity rifamycin SV using the bacterial strain. Background technique [0002] Amycolatopsis mediterranei was isolated from soil samples near St.Raphael, France in 1957. It is a kind of bacteria that can produce the important antibiotic rifamycin of actinomycetes. [0003] Rifamycin belongs to ansamycins and is mainly used to treat tuberculosis and leprosy caused by Mycobacterium tuberculosis and Mycobacterium leprae. It is also very effective in various infections caused by...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N15/09C12N15/53C12N15/54C12P17/18C12R1/01
CPCC12Y202/01001C12P17/189C12R1/01C12N9/0077C12N9/1022C12N1/205C12R2001/01
Inventor 赵国屏赵维袁华钟怡覃重军
Owner CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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