Gene engineering bacteria containing high isovaleryl spiramycin principal component

A technology of isovalerylspiramycin and genetically engineered bacteria, applied in the field of genetically engineered bacteria

Active Publication Date: 2010-12-15
SHENYANG TONGLIAN GRP CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0016] According to the present invention, the cyB2 regulatory gene is linked with the isovaleryltransferase gene ist and co-expressed in the spiramycin-producing bacteria to construct a high-content isovalerylspiramycin main component strain. So far, no relevant reports have been seen.

Method used

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  • Gene engineering bacteria containing high isovaleryl spiramycin principal component
  • Gene engineering bacteria containing high isovaleryl spiramycin principal component
  • Gene engineering bacteria containing high isovaleryl spiramycin principal component

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] "Example 1": _Construction of ist-acyB2 double gene recombinant plasmid pSET52-ia

[0029] From the recombinant plasmid pSW4 originally constructed in our laboratory [Shang Guangdong et al. Bioengineering Journal 1999, 15(2): 171], a 1820bp fragment containing the complete ist gene and a part of the acyB2 gene was obtained by digesting with SmaI-BamHI. The total DNA of Streptomyces thermotolarences CGMCC4.1501 provided by the General Microbiology Center of the Culture Collection Management Committee was used as a template, and the primers p1: 5'-CGCTCAGGGACGCAAGACC-3' and P2: 5'- were designed according to the acyB2 sequence published by NCBI CC GGAATTC GCCCCGTGACCTCACCGTC-3'(EcoRI), a 1013bp fragment of the cyB2 partial gene was obtained by PCR reaction, the PCR product was digested with BamHI-EcoRI to obtain a 934bp fragment, and the above SmaI-BamHI (1820bp), BamHI-EcoRI (934bp) fragments were ligated into The SmaI-EcoRI restriction site of pBluesript II KS (+) vec...

Embodiment 2

[0030] "Example 2": Recombinant plasmid pSET52-ia transforms spiramycin-producing bacteria

[0031] Spiramycin-producing bacteria (S.spiramyceticus) strains are derived from the Culture Collection Center (CGMCC4.1118) of the Institute of Medical Biotechnology, Chinese Academy of Medical Sciences, and the description of the strains has been published [Acta Microbiology 1982, 22 (1): 13-16]. Bacteria in slant medium [2.0% soybean cake powder, 1.0% glucose, 3.0% starch, CaCO 3 0.5%, NaCl 0.4%, agar 2.0%], cultured at 28°C for 7-10 days, and prepared protoplasts according to the method described in the literature [D.A.Hopwood et al. Genetic manipulation of Streptomyces, ALaboratory Manual, Norwich; John Innes Foundation UK, 1985]. Specifically, inoculate strains into sucrose-containing R2YE medium [10.3% sucrose, 1.0% glucose, 0.4% yeast extract, 0.2% tryptone, 0.4% peptone, 0.4% casamino acid, K 2 SO 4 0.025%, CaCl 20.216%, KH 2 PO 4 0.005%, MgCl 2 6H2O 1.012%], in 100m...

Embodiment 3

[0032] "Example 3": WSJ-195-IA bacterial strain fermentation extraction product identification

[0033] The WSJ-195-IA strain was cultured in the above-mentioned slant medium at 28°C for 7-10 days, and the excavated pieces were inoculated in the seed medium: soybean cake powder 1.5%, starch 3.0%, NaCl 0.4%, CaCO 3 0.5%, fish peptone 0.3%, KH 2 PO 4 0.05%, cultured at 28°C for 48 hours, transferred to fermentation medium: glucose 0.5%, starch 6.0%, yeast powder 0.5%, fish meal 2.0%, NH 4 NO 3 0.6%, NaCl 1.0%, CaCO 3 0.5%, KH 2 PO 4 0.05%, MgSO 4 0.1%, 28 ℃ fermentation culture for 96 hours. Filter the fermentation broth, adjust the pH to 8.5, extract with an equal amount of ethyl acetate, concentrate the ethyl acetate extract to dryness, and dissolve in methanol. WSJ-195-IA strain fermentation extract product was analyzed by HPLC, using Kromasil C 18 Column (4.5mm×150mm, 5μm); mobile phase: methanol: sodium dihydrogen phosphate solution (volume ratio 53:47); dete...

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Abstract

The invention relates to gene engineering bacteria containing a high-antibiotic content principal component constructed by utilizing a regulator gene. The gene engineering bacteria are products that an acyB2 regulator gene and an isovaleryl transferase gene ist are linked and co-expressed in a spiramycin producing strain. Experimental study shows that the strain can obviously improve the ratio ofthe principal component isovaleryl spiramycin by 213 to 232 percent and provide important guarantee for industrial production and clinical application.

Description

Technical field: [0001] The invention relates to a genetically engineered bacterium for constructing a high-content antibiotic main component by using a regulating gene. Background technique: [0002] Bitespiramycin (formerly known as Shengjimycin) [patent number: ZL97104440.6] is a spiramycin derivative developed by genetic engineering technology. Spiramycin is a macrolide antibiotic, its main core is a lactone ring composed of 16-membered ring, which is homogeneous with Pratt lactone, and contains three sugar molecules phylosamine, mycaminose Amines and mycaminose. And the main component of bitspiramycin is a derivative of spiramycin carmycose 4' hydroxyl group esterified with isovaleryl. Since the 3-position R hydroxyl group of the spiramycin lactone ring can be esterified by acetyl or propionyl to form the corresponding components of spiramycin I, II, and III, so bitspiramycin also contains at least isovalerylspiramycin I, II, III three components. The chemical struc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/63C12R1/465
Inventor 王以光姜洋戴剑漉郝玉有杨生武林灵赫卫清周红霞倪四阳武临专
Owner SHENYANG TONGLIAN GRP CO LTD
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