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Marine Micromonospora strain for fermentation production of Rakicidin B1, and application thereof

A strain and marine technology, which is applied in the field of marine Micromonospora strains fermented to produce RakicidinB1, can solve the problem of low fermentation yield, achieve the effect of increasing fermentation yield, meeting the needs of industrialization, and reducing yield

Active Publication Date: 2018-07-20
FUJIAN INST OF MICROBIOLOGY
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  • 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 B1 by fermentation, to solve the lower problem of the fermentation yield of Rakicidin B1 compound in the prior art

Method used

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  • Marine Micromonospora strain for fermentation production of Rakicidin B1, and application thereof
  • Marine Micromonospora strain for fermentation production of Rakicidin B1, and application thereof
  • Marine Micromonospora strain for fermentation production of Rakicidin B1, and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Example 1 Mutagenesis and breeding of strains

[0031] This example illustrates that the mutation breeding method of Marine Micromonospora FIM R160609 includes the following steps:

[0032] (1) Preparation of spore suspension: add proper amount of sterile normal saline to the fresh slant of the mature starting strain FIM02523 (preservation number CGMCC NO.12132), gently scrape it with an inoculating spatula, and pour it into a sterile shaker with glass beads Shake the bottle to disperse, filter the hyphae, and leave the spore suspension for later use;

[0033] (2) Nitrosoguanidine (NTG) mutagenesis: Weigh 200mg NGT in a 100ml Erlenmeyer flask, add 2ml acetone, and then add 18ml Tris-aminomethane maleic acid buffer to completely dissolve and mix uniformly to obtain a concentration of 20ml of 10mg / ml NTG solution; mix NTG mother liquor with the prepared bacterial suspension so that the final concentration of NTG is 1mg / ml, 2mg / ml, 3mg / ml, shake culture at 32℃ for 60min; after m...

Embodiment 2

[0037] Example 2 Comparison of starting strain FIM02325 and mutant strain FIM-R160609 in shake flask fermentation

[0038] Scrape the slant spores of FIM02325 and mutant strain FIM-R160609 from freshly cultured marine micromonospora and inoculate them into a shake flask seed culture. After incubating at 32℃ and 250rpm for 48 hours, the inoculum is 5%. It was inoculated into the fermentation medium in a shake flask, incubated at 30°C and 250 rpm for 120 hours, and then placed in the flask. The fermentation product was determined by HPLC.

[0039] Prepare seed culture medium formula (mass fraction): maltodextrin 2.0%, glucose 1.0%, soybean meal powder 1.0%, yeast powder 2.0%, MgSO 4 ·7H 2 O 0.05%, KH 2 PO 4 0.05%, CaCO 3 0.3%, prepared with tap water, adjusted to pH 7.0-7.5; cultured with seed liquid at 30°C.

[0040] Formulated fermentation medium formula (mass fraction): maltodextrin 5.0%, glucose 1.0%, soybean meal powder 2.0%, yeast powder 1.5%, ammonium sulfate 0.8%, MgSO 4 ·7H ...

Embodiment 3

[0045] Example 3 The mutant strain FIM-R160609 was fermented on a 20L fermentor

[0046] According to the formula in Example 2, the seed medium and the fermentation medium were prepared.

[0047] The seeds are shake flask seeds, each 500ml shake flask contains 100ml of liquid, cultivated at 32°C and 250rpm for 48 hours; after that, it is inoculated in a 20L fermenter (the actual amount of liquid is 13L) at a 5% inoculum for fermentation, and cultured at 30°C. The tank pressure is controlled to be 0.03-0.05Mpa, the starting speed is 200rpm, and after 48 hours, it is gradually adjusted to 350rpm according to the change of the fermentation parameter DO, and the aeration rate is 1:1vvm, and it is about 96-120 hours until the end of the fermentation.

[0048] During the fermentation process, the content of Rakkidins in the fermentation broth was detected by HPLC, and the final fermentation titer was: the content of Rakicidin B1 was 436.41 mg / L, the content of Rakicidin B was 11.17 mg / L, a...

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Abstract

The invention belongs to the technical field of microbial fermentation, and particularly relates to a marine Micromonospora strain for fermentation production of Rakicidin B1, and an application thereof. An existing marine micromonospora FIM02523 for producing Rakicidins compounds undergoes mutation breeding to obtain a mutant strain marine Micromonospora FIM-R160609 for highly yielding RakicidinB1. The mutant strain can effectively increase the tilter of the Rakicidin B1 in a fermentation broth. In a fermentation experiment using a 20-1000 L fermenter, the tilter of the Rakicidin B1 producedby the marine Micromonospora FIM-R160609 reaches up to about 600 mg / L, and a ratio of the component B1 to the component B in the fermentation broth is equal to or more than 97:3 so the fermentation yield of the target product Rakicidin B1 is greatly increased, the yield of other Rakicidins byproducts is effectively reduced, subsequent extraction and purification of the Rakicidin B1 are facilitated, and industrialization demands can be met.

Description

Technical field [0001] The invention belongs to the technical field of microbial fermentation, and specifically relates to a marine micromonospora strain that ferments to produce Rakicidin B1 and its application. Background technique [0002] Rakicidins are the fermentation products of marine micromonospora. The currently isolated Rakicidins mainly contain Rakicidin A, Rakicidin A1, Rakicidin B1 and Rakicidin B. The chemical structure and components are as follows: [0003] [0004] Rakicidins have attracted attention because of their 15-membered cyclic lipopeptide structure containing a rare 4-amino-2,4-pentadienoate octamide and anti-tumor cell activity. Yamazaki (2007) found that Rakicidin A has excellent hypoxia-selective anti-tumor cell activity, and the anti-colon cancer HCT-8 cell activity under hypoxia is 17.5 times that under normoxia; Takeuchi (2011) also reported for the first time Rakicidin A can induce apoptosis of myeloid chronic leukemia stem cells under hypoxic con...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P21/02C12R1/29
CPCC12N1/20C12P21/02C12N1/205C12R2001/29
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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