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

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

Example Embodiment

[0022] Example 1 :

[0023] The domestication and screening of strain HP388 of the present invention requires at least 10 cycles, and each cycle mainly includes the following processes:

[0024] Take out the cultivated slope of the hair strain T125 (Micromonospora inulis Chengjiang variety T125), add 3ml of sterile water, gently scrape the spores with the inoculating needle, take out the spore suspension, and shake it on a shaker to make Disperse into single spores, and then perform a gradient dilution (10 0 , 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 ), spread the plate, irradiate it with ultraviolet rays (10, 20, 30, 40, 50 seconds), then incubate at 37°C to isolate a single colony. When a single colony grows to be able to distinguish its characteristics (25 days), pick the colonies with different morphological characteristics, carry out submerged fermentation culture (6 days), after the fermentation, detect the fermentation unit, save the higher unit for the next st...

Example Embodiment

[0028] Example 2 Obtaining and DNA sequence determination of sisomicin biosynthetic gene cluster of mutant strain HP388

[0029] 1 material

[0030] 1.1 Strains and plasmids Micromonas enu HP388, E.coli DH5α and cosmid carrier Supercos-1 (purchased from Shanghai Shenggong); E.coli LE392 was purchased from Stratagene.

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

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

[0033] Library construction

[0034] (A) Construction of the gene library of Micromonospora enulis: cosmid vector Supercos-1 with restriction endonuclease...

Example Embodiment

[0043] Example 3: Preparation of sisomicin, a metabolite of Micromonospora enulis HP388

[0044] The high-yield and stable-yield engineering bacterium Micromonospora indus HP388 provided by the present invention is directly used for the production of sisomicin as an antibacterial drug and synthetic netilmicin.

[0045] 1. Fermentation culture of Micromonospora enulis HP388 strain

[0046] Slant medium (%): Calculated 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%, pH 7.5.

[0047] Seed medium: glucose 0.1%, sweet potato starch 1.0%, corn flour 1.5%, peptone 0.2%, soybean meal 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 meal 2.0%, KNO 3 0.01%, (NH 4 ) 2 SO 4 0.1%, CaCO 3 0.5%, amylase 0.025%, pH 7.5.

[0049] Fermentation of Micromonospora enulis HP388. Before fermentation, separate high-quali...

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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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