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Mutant strain for producing sisomicin

A technology of sisomicin and mutant strains, applied in bacteria, microorganisms, fermentation, etc., can solve the problems of slow progress in the application of Micromonospora

Inactive Publication Date: 2012-08-01
FUZHOU UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Streptomyces cell engineering and Streptomyces genetic engineering, which emerged in the 1980s, have made some progress in the cloning of antibiotic biosynthesis genes, yield improvement, component improvement, and research on hybrid antibiotics. Applications in sporozoites are progressing slowly

Method used

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  • Mutant strain for producing sisomicin
  • Mutant strain for producing sisomicin
  • Mutant strain for producing sisomicin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] The acclimation screening of bacterial strain HP388 of the present invention needs at least more than 10 cycles, and each cycle mainly includes the following processes:

[0024] Take out the cultivated slant surface of T125 strain T125 (M. inuuuumosa var. Chengjiang varietal T125), add 3ml of sterile water, gently scrape off the spores with an inoculation needle, take out the spore suspension, and shake it fully on the shaker, so that Disperse into single spores, followed by serial dilution (10 0 、10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 ), spread the flat plate, and after ultraviolet irradiation (10, 20, 30, 40, 50 seconds), culture at 37°C to isolate a single colony. When a single colony grows enough to distinguish its characteristics (25 days), pick colonies with different morphological characteristics and carry out submerged fermentation culture (6 days).

[0025] Take the above-mentioned high-quality strains, culture mycelia, and prepare protoplasts....

Embodiment 2

[0028] Example 2 Obtaining and DNA Sequencing of Sisomicin Biosynthetic Gene Cluster in Mutant Strain HP388

[0029] 1 material

[0030] 1.1 Strains and plasmids E. coli DH5α and cosmid carrier Supercos-1 (purchased from Shanghai Sangong); E. coli LE392 was purchased from Stratagene Company.

[0031] 1.2 Reagents Restriction endonuclease, proteinase K, calf intestinal alkaline phosphatase (CIAP enzyme), T4 DNA ligase and Taq enzyme were purchased from TAKARA company; lysozyme was purchased from Shanghai Sangong; phage packaging protein extract (Gigapack Ⅲ XL packaging extract) was purchased from Stratagene.

[0032] 1.3 Culture medium Bacterial culture medium LB, add ampicillin (final concentration: 100 μg / ml) and kanamycin (final concentration: 25 μg / ml) as needed; Micromonospora einuatus culture medium.

[0033] library construction method

[0034] (a) Construction of Micromonas eugenus gene library: cosmid vector Supercos-1 with restriction endonuclease Nhe ...

Embodiment 3

[0043] Example 3: Preparation of sisomicin, a metabolite of Micromonas eugenus HP388

[0044] The high-yield and stable-yield engineering bacterium Micromonospora eneurica HP388 provided by the invention is directly used to produce sisomicin as an antibacterial drug and to synthesize netilmicin.

[0045] 1. Fermentation and Culture of Micromonospora eneurica HP388 Strain

[0046] Slant medium (%): by weight percentage, soluble starch 1.5%, bran 1.5%, MgSO 4 ·6H 2 O 0.03%, KNO 3 0.3%, K 2 HPO 4 3H 2 O 0.03%, NaCl 0.05%, CaCO 3 0.4%, agar 2%, pH7.5.

[0047] Seed medium: glucose 0.1%, sweet potato starch 1.0%, corn flour 1.5%, peptone 0.2%, soybean cake powder 1.0%, KNO 3 0.05%, CaCO 3 0.5%, pH7.0.

[0048] Fermentation medium: sweet potato starch 6.0%, corn flour 1.0%, peptone 0.4%, soybean cake powder 2.0%, KNO 3 0.01%, (NH 4 ) 2 SO 4 0.1%, CaCO 3 0.5%, amylase 0.025%, pH7.5.

[0049] Fermentation of Micromonas eneurica HP388. Before fermentation, h...

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Abstract

The invention relates to a mutant strain for producing sisomicin, which is applied to antibiotic manufacture and belongs to the field of antibiotic pharmacy. The mutant strain is micromonosporainyoensis HP388 registered and preserved in a general microorganism center of the Chinese microscobial preservation management committee on March 19, 2012, and the preservation number is CGMCCNo.5921. The mutant strain HP388 has the most remarkable advantages of high fermentation unit, stable production process, convenience in control, low oxygen consumption, low cost, low pollution and fine product quality.

Description

technical field [0001] The invention belongs to the field of antibiotic pharmacy, and relates to a sisomicin-producing mutant strain, which is used in the manufacture of antibiotics. Background technique [0002] Sisomicin is primarily indicated for the treatment of serious infections caused by sensitive Gram-negative bacilli such as Pseudomonas aeruginosa, Proteus (indole positive and negative), Escherichia coli, Klebsiella, Neonatal sepsis, sepsis, central nervous system infection (including meningitis), urinary tract infection, respiratory tract infection, gastrointestinal tract infection, etc. caused by Enterobacter, Serratia and Citrobacter Peritonitis, biliary tract infection, skin or bone infection, otitis media, sinusitis, soft tissue infection, listeriosis, etc. [0003] Micromonospora can produce abundant secondary metabolites, especially aminoglycoside antibiotics, such as gentamicin, sisomicin and fortimicin. The distribution of these micromonospora is peculiar...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P19/48C12R1/29
Inventor 洪文荣张国华宋逸婷沈斌彬黄俊龙
Owner FUZHOU UNIVERSITY
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