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A marine micromonospora strain fermenting high-yield rakicidin H and its application

A kind of strain and marine technology, which is applied in the field of marine Micromonospora fermenting high-yield RakicidinH strains, can solve the problem of low fermentation yield of RakicidinH, and achieve the effect of facilitating extraction and purification and meeting the needs of industrialization

Active Publication Date: 2021-03-30
FUJIAN INST OF MICROBIOLOGY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] For this reason, the technical problem to be solved by the present invention is to provide a kind of marine micromonospora strain of fermentation high-yield Rakicidin H, to solve the lower problem of Rakicidin H fermentation yield in the prior art;

Method used

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  • A marine micromonospora strain fermenting high-yield rakicidin H and its application
  • A marine micromonospora strain fermenting high-yield rakicidin H and its application
  • A marine micromonospora strain fermenting high-yield rakicidin H and its application

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Embodiment 1

[0033] The mutation breeding of embodiment 1 bacterial strain

[0034] The mutagenesis breeding method of marine Micromonospora FIM-R181103 described in the present embodiment comprises the following steps:

[0035] (1) Preparation of spore suspension: add an appropriate amount of sterile saline to the fresh slant of the mature cultured starting strain FIM02523 (preservation number CGMCC NO.12132), gently scrape it off with an inoculation spatula, and pour it into a sterile shaker with glass beads. Shake the bottle to disperse, filter the mycelia, and leave the spore suspension for later use;

[0036] (2) Nitrosoguanidine (NTG) mutagenesis: Weigh 200mg NGT into a 100ml Erlenmeyer flask, add 2ml acetone, then add 18ml Tris-aminomethane maleic acid buffer, dissolve it completely and mix evenly to obtain the concentration 20ml of 10mg / ml NTG solution; mix the NTG mother solution with the prepared bacterial suspension so that the final NTG concentrations are 1mg / ml, 2mg / ml, and 3...

Embodiment 2

[0044] Embodiment 2 mutant strain stability investigation

[0045] The slant of the high-yielding strain Micromonospora FIM-R181103 obtained after mutagenesis was continuously passaged for 6 generations, and no significant change was found in the fermentation titer of Rakicidin H. The bacteria were stored in a 20°C seed preservation cabinet, and the original strain was subcultured every two months for fermentation verification. As a result, no significant change was found in the fermentation titer of Rakicidin H in the bacteria within 12 months. The above conclusions indicate that the mutant strain FIM-R181103 has high yield and genetic stability, and is suitable for industrial production. The obtained strain Micromonospora FIM-R181103 was preserved in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures, with a preservation number of CGMCC NO. 17014 and a preservation date of December 19, 2018.

Embodiment 3

[0046] Embodiment 3 starting strain FIM02523 and mutant strain FIM-R181103 shaking flask fermentation comparison

[0047] Scrape the slant spores of freshly cultivated Micromonospora marina FIM02523 and the mutant strain FIM-R181103 and inoculate the suspension into the shake flask seed culture, cultivate at 32°C and 250rpm for 48 hours, and inoculate at 10% inoculum Put it into the shake flask fermentation medium, cultivate it at 30°C and 250rpm for 120 hours, put it into the bottle, and measure the fermentation product by HPLC (for HPLC detection, refer to Chinese patent CN108586380A).

[0048] Preparation of seed medium formula (mass fraction): 2.0% soluble starch, 1.0% glucose, 2.0% yeast powder, MgSO4 7H 2 O 0.05%, KH 2 PO 4 0.05%, CaCO 3 0.2%, prepared with tap water, adjust the pH value to 7.0-7.5, sterilize at 121°C for 30 minutes, add a certain amount of spore suspension after cooling, and carry out seed liquid cultivation at 32°C.

[0049] Preparation of fermen...

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Abstract

The invention belongs to the technical field of microbial fermentation, and in particular relates to a marine Micromonospora strain producing Rakicidin H by fermentation and application thereof. The present invention obtains a high-yield Rakicidin H mutant strain FIM‑R181103 by performing mutation breeding on the existing Rakicidins compound-producing Micromonospora marine strain FIM‑R181103, which can effectively increase the content of Rakicidin H in the fermentation broth. Potency, in the fermentation experiment of 20‑1000L fermenter, the titer of Rakicidin H produced by Micromonospora marine FIM‑R181103 was as high as 430mg / L, which greatly facilitated the extraction and purification of Rakicidin H and could meet the needs of industrialization .

Description

technical field [0001] The invention belongs to the technical field of microbial fermentation, and in particular relates to a micromonospora marine strain fermenting high-yield Rakicidin H and an application thereof. Background technique [0002] Rakicidins compounds are the fermentation products of Micromonospora marine, and are currently known antitumor active substances with clinical application value isolated from the metabolites of marine microorganisms, and are an important class of compounds. [0003] It has been reported that a series of Rakicidins compounds with anti-tumor activity or antibacterial activity have been found in Micromonospora and Streptomyces, including: For example, in 1995, Kimberly D.Mcbrien et al. discovered Rakicidin A and B; In 2000, Hu Jin-Feng et al. discovered Rakicidin C from Streptomycessp.GT61042; in 2010, Yasuhiro Igarashi et al. discovered Rakicidin D from Streptomycessp.MWW064; in 2014, Naoya Oku et al. found Rakicidin D in Micromonospo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12P21/02C12R1/29
CPCC12N1/20C12P21/02C12N1/205C12R2001/29
Inventor 周剑方志锴陈丽赵薇江宏磊林风江红连云阳
Owner FUJIAN INST OF MICROBIOLOGY
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