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A kind of marine Micromonospora strain producing rakicidin A by fermentation and its application

A kind of strain and marine technology, applied in the field of marine Micromonospora strains fermented to produce RakicidinA, can solve the problem of low fermentation yield, achieve the effect of increasing potency and meeting the needs of industrialization

Active Publication Date: 2019-03-05
FUJIAN INST OF MICROBIOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] For this reason, the technical problem to be solved by the present invention is to provide a kind of marine micromonospora strain that produces Rakicidin A by fermentation, to solve the lower problem of Rakicidin A fermentation yield in the prior art

Method used

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  • A kind of marine Micromonospora strain producing rakicidin A by fermentation and its application
  • A kind of marine Micromonospora strain producing rakicidin A by fermentation and its application
  • A kind of marine Micromonospora strain producing rakicidin A by fermentation and its application

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Embodiment 1

[0029] The mutation breeding of embodiment 1 bacterial strain

[0030] The mutagenesis breeding method of marine Micromonospora FIM R150103 described in the present embodiment comprises the steps:

[0031] (1) Preparation of spore suspension: add an appropriate amount of sterile saline to the fresh slant of the mature 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;

[0032] (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 3mg / ml respectively,...

Embodiment 2

[0044] Embodiment 2 mutant strain stability investigation

[0045] The slant of the high-yielding strain Micromonospora FIM-R150103 obtained after mutagenesis was continuously passaged for 6 generations, and no significant change was found in the fermentation titer of Rakicidin A. 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 Rakicidin A fermentation titer of the bacteria within 12 months. The above conclusions indicate that the mutant strain FIM-R150103 has high yield and genetically stable traits, and is suitable for industrial production. Gained bacterial strain Micromonospora FIM-R150103 is preserved in China Microbiology Culture Collection Management Committee General Microorganism Center, and its preservation number is CGMCCNO.14822, and preservation date is on October 16, 2017.

Embodiment 3

[0046] Embodiment 3 starting strain FIM02523 and mutant strain FIM-R150103 shaking flask fermentation comparison

[0047] Scrape the slant spores of freshly cultured Micromonospora marina FIM02523 and the mutant strain FIM-R150103 and inoculate the suspension into the shake flask seed culture, culture at 32°C and 250rpm for 48 hours, and inoculate at 5% inoculum Put it into the shake flask fermentation medium, cultivate it at 30° C. and 250 rpm for 120 hours, put it into the bottle, and measure the fermentation product by HPLC.

[0048] Preparation of seed medium formula (mass fraction): 2.0% soluble starch, 1.0% glucose, 2.0% yeast powder, 1.0% peptone, MgSO 4 ·7H 2 O 0.05%, FeSO 4 ·7H 2 O 0.005%, CuSO 4 ·5H 2 O 0.005%, CoCl 2 ·6H 2 O 0.0005%, CaCO 3 0.2%, prepared with tap water, adjust the pH value to 7.0-7.5, and carry out seed liquid cultivation at 28-32°C.

[0049] Preparation of fermentation medium formula (mass fraction): 4.0% soluble starch, 1.0% sucrose, 3....

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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 A by fermentation and application thereof. The present invention obtains a high-yield Rakicidin A mutant strain FIM-R150103 by performing mutation breeding on the existing Rakicidins compound-producing Micromonospora marine strain FIM-R150103, which can effectively increase the content of Rakicidin A in the fermentation broth. Potency, in the fermentation experiment of 20‑1000L fermenter, the titer of Rakicidin A produced by Micromonospora marina FIM‑R150103 was as high as 300 mg / L, which greatly facilitated the extraction and purification of Rakicidin A and could meet the needs of industrialization .

Description

technical field [0001] The invention belongs to the technical field of microbial fermentation, in particular to a marine Micromonospora strain producing Rakicidin A by fermentation and application thereof. Background technique [0002] Rakicidins are the fermentation products of Micromonospora marine. The currently isolated Rakicidins mainly contain Rakicidin A, Rakicidin A1, Rakicidin B1 and Rakicidin B. Their chemical structures and components are as follows: [0003] [0004] Rakicidins have attracted attention because their 15-membered cyclic lipopeptide structure contains a rare 4-amino-2,4-pentadienoic acid octanamide and has anti-tumor cell activity. Yamazaki (2007) found that Rakicidin A has excellent hypoxic selective anti-tumor cell activity, and the anti-colon cancer HCT-8 cell activity under hypoxic conditions is 17.5 times that under normal oxygen conditions; Takeuchi (2011) also reported for the first time Rakicidin A can induce the apoptosis of myeloid chr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12P17/14C12R1/29
CPCC12N1/20C12P17/14C12N1/205C12R2001/29
Inventor 周剑林风江红连云阳江宏磊陈丽赵薇方志锴
Owner FUJIAN INST OF MICROBIOLOGY
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