Gene series technology for increasing main component content of gene engineering isovaleryl selectomycin

A technology for isovalerylspiramycin and spiramycin is applied in the field of gene tandem technology for improving the content of main components of genetic engineering isovalerylspiramycin, and achieves the effects of improving control, simplifying extraction process and reducing production cost

Active Publication Date: 2010-02-17
SHENYANG TONGLIAN GRP CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The gene concatenation technology for improving the content of the main component of genetic engineering isovalerylspiramycin mentioned in the present invention, so far, has not yet seen relevant reports at home and abroad

Method used

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  • Gene series technology for increasing main component content of gene engineering isovaleryl selectomycin
  • Gene series technology for increasing main component content of gene engineering isovaleryl selectomycin
  • Gene series technology for increasing main component content of gene engineering isovaleryl selectomycin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] : The concatenation of two double-copy ist genes with their own promoters

[0040] For the tandem of double-copy ist genes 1 and 2, use the repeated tandem method including its own promoter, and design P1-P2 and P3-P4 primers at the ist gene start codon ATG upstream - 129kb and terminator TAG downstream - 41 (Table 1), using the pSW4 plasmid containing the ist gene as a template, two genes of about 1.3 kb with different restriction sites were amplified by PCR, namely the ist1 gene (KpnI-BamH I) and the ist2 gene (BamHI- Xba I) fragment, the same BamH I restriction site can be used to carry out the tandem connection between the ist gene with its own promoter in two copies.

Embodiment 2

[0041] : Construction of double-copy ist gene tandem transcription

[0042] Design P5-P6 primers at the ist gene start codon ATG upstream -111bp and terminator TAG downstream -24bp (excluding the transcription termination sequence), and design P7 at the ATG upstream -105bp and terminator downstream -80bp (including the transcription termination sequence) -P8 primer (Table 1), double copy ist gene tandem primer design site see figure 1 . Using the pSW4 plasmid containing the ist gene as a template, the ist 3 gene (containing BamHI-XbaI site) and ist 4 gene (containing XbaI-SalI site) fragments were respectively obtained by PCR, and the same XbaI restriction site was used to perform double A tandem of copies of the ist gene.

Embodiment 3

[0043] : construction of homologous recombination double exchange ist gene double copy plasmid (pKC1139-5)

[0044] Using P9-P10 primers to pCG4 plasmid as a template, the homologous fragment H of about 1.1 kb was amplified by PCR 1 , using P11-P12 primers to pCG4 plasmid as a template, the homologous fragment H of about 1.1kb was amplified by PCR 2 , used to construct fragments of homologous recombination double crossover on both sides of the ist gene. Digest the pWS1 plasmid with Xba I-Pst I [Shang Guandong et al The J of antibiotics 200154 (1): 66-73] to obtain a 1.3kb thiostrepton resistance gene (Tsr r ) fragment as a screening marker for homologous recombination double crossover. H 1 Fragment (EcoR I-Kpn I), ist1 gene (Kpn I-BamH I), ist 2 gene (BamHI-Xba I) described in Example 1, and Tsr r Genes (XbaI-PstI) and H 2 The (PstI-Hind III) fragment was directly connected to the thermosensitive plasmid vector pKC1139 plasmid [Bierman M, et al.Gene, 1992, 116: 43-49] on ...

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Abstract

The invention relates to the application of a gene engineering technology in increasing an antibiotic component, in particular to a gene series technology for increasing the main component content ofgene engineering isovaleryl selectomycin. The gene series technology comprises the following steps: connecting ist genes which are closely related to selectomycin isovaleryl acylation in Beta selectomycin gene engineering bacteria in series; increasing the bacterial isovaleryl acylation generating capability by increasing gene dosage; using a promoter erythrocin resistance gene erm promoter sequence with strong promotion activity to replace the original ist gene promoter sequence so as to increase the expression of the series ist genes, and improve the proportion of isovaleryl selectomycin main components of the gene engineering bacteria from a headstream.

Description

Technical field: [0001] The invention relates to the application of genetic engineering technology in improving antibiotic components. Background technique: [0002] Bitespiramycin (formerly known as Shengjimycin) [patent number: ZL97104440.6] is a spiramycin derivative developed by our laboratory using genetic engineering technology. Spiramycin is a macrolide antibiotic. Its main core is a 16-membered ring consisting of a lactone ring that is homogeneous to Pratt lactone. Mycose, and the main component of spiramycin is a derivative of spiramycin mycose 4' hydroxyl group esterified with isovaleryl. Since the R hydroxyl group at the 3-position of the spiramycin lactone ring can be esterified by acetyl or propionyl to form the corresponding spiramycin I, II, and III components, so the spiramycin contains at least isovaleryl spiramycin I , II, III three components, the chemical structure of the main component of Bitespiramycin is as follows: [0003] [0004] Folosamine P...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/76C12P19/62C12R1/465
Inventor 王以光赫卫清戴剑漉李瑞芬杨永红范迎春武临专
Owner SHENYANG TONGLIAN GRP CO LTD
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