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High-yield tacrolimus streptomycete mutant strain and application thereof

A technology of mutagenic strains, tacrolimus, applied in the field of microorganisms, can solve the problems of high production cost, long fermentation cycle, low fermentation capacity, etc., and achieve the effect of increasing yield and good genetic stability

Active Publication Date: 2019-05-10
FUJIAN INST OF MICROBIOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In summary, although there are many Streptomyces that produce tacrolimus, and some strains have relatively stable genetic traits and fermentation units, some strains have low fermentation capacity, some have long fermentation cycles, and high production costs. There is a large gap between foreign countries, resulting in the current domestic tacrolimus market being dominated by countries such as the United States and Japan

Method used

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  • High-yield tacrolimus streptomycete mutant strain and application thereof
  • High-yield tacrolimus streptomycete mutant strain and application thereof
  • High-yield tacrolimus streptomycete mutant strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1, the isolation of bacterial strain FIM-07

[0020] (1) Collect soil under the forest of Mingfeng Mountain in Kunming City. Specifically, use a sampling shovel to remove the top 10cm floating soil, collect 10g-25g of soil samples at 10cm; weigh 2g of soil samples and add 10mL of sterile saline, After oscillating, let it stand for 30 minutes, take the supernatant, that is, the stock solution, and carry out gradient dilution with sterile saline, and the selected dilution is 10 -2 、10 -3 、10 -4 and 10 -5 suspension;

[0021] (2) Take 0.1mL of the stock solution and the selected suspension and apply it on the ISP4 medium plate prepared by adding 50mg / L potassium dichromate water, repeat 3 parallel plates for each sample, and then spread the coated plate Culture at 28°C, observe the colony appearance, size, color, edge shape, surface dry and wet state and other morphological characteristics;

[0022] (3) Under aseptic operation conditions, after culturing fo...

Embodiment 2

[0023] Embodiment 2, identification of bacterial strain FIM-07

[0024] (1) Physiological and biochemical characteristics identification: the obtained strain FIM-07 was streaked on the ISP4 medium plate and inserted into a cover glass, cultured at 28°C for 8-12 days, and a single colony was observed with an optical microscope, transmission and scanning electron microscope Morphological characteristics and hyphae. The main morphology and physiological and biochemical characteristics of the bacterial strain FIM-07 were obtained as follows:

[0025] There are 5-10 spores in the mature spore chain, and the shape of the spores is oval, 0.4-0.6×0.6-0.7 μm. On the plate, the colony is round, the diameter of the colony is about 5-6mm after 10 days of culture, the surface is smooth, white or light gray to gray, it grows luxuriantly in the ISP4 medium, the aerial hyphae are off-white, the hyphae in the base are poor, and do not produce pigment or Produces light pink pigment. With the...

Embodiment 3

[0030] Embodiment 3, the acquisition of bacterial strain FIM-07-23

[0031] (1), take the Streptomyces tsukubaensis FIM-07 strain and transfer it to the slant of the ISP4 medium, and cultivate it in a constant temperature incubator for 8-12 days, and the culture temperature is 28 ° C; after that, use normal saline Wash the spores on the slant of the medium, break up the glass beads, and filter through lens paper to make 10 6 Individual / mL spore suspension;

[0032] (2), the spore suspension that step (1) is made is packed in 5 sterile test tubes, carry out 60 Co radiation treatment, the doses were 300Gy, 450Gy, 600Gy, 800Gy, 1000Gy, and the dose rate was 900rad / min. The bacterial suspension before and after the mutagenesis was serially diluted and spread on the separation plate, cultured at 28°C for 10 days, observed the growth of the colonies, counted the colonies, and made the lethality curve as follows: figure 1 shown; from figure 1 It can be seen that there is an obvio...

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Abstract

The invention provides a high-yield tacrolimus streptomycete mutant strain. The mutant strain is (Streptomyces tsukubaensis)FIM-07-23, and preserved at the common microorganism center of China Committee for Culture Collection of Microorganisms on January 7th, 2019, the address is No.3, courtyard 1, the western Beichen road, Chaoyang District of Beijing, and the preservation number is CGMCC No.17137; the (Streptomyces tsukubaensis)FIM-07-23 is applied into fermentation production of tacrolimus. The (Streptomyces tsukubaensis)FIM-07-23 can ferment high-yield tacrolimus, the yield of tacrolimus is greatly increased, and the genetic stability is good.

Description

【Technical field】 [0001] The invention belongs to the field of microorganisms, and in particular relates to a streptomyces mutagenic strain with high yield of tacrolimus and application thereof. 【Background technique】 [0002] Macrolide immunosuppressants are a class of immunosuppressants with a macrolide structure that has been proved to have significant immunosuppressive effects by in vivo and in vitro studies, and its representative substances are Tacrolimus (FK506), Ascomyces Ascomycin (FK-520) and its derivatives, sirolimus (Sirolimus, Rapamycin), etc. Macrolide immunosuppressants can be used to treat organ transplant rejection and autoimmune related diseases. [0003] Tacrolimus (Tacrolimus, FK506) is a 23-membered macrolide compound containing pipecoyl group extracted from the fermentation broth of Streptomyces tsukubaensis. It has the advantages of low incidence of reaction, and has remarkable curative effect in organ transplantation and autoimmune system diseases....

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N13/00C12P17/18C12R1/465
Inventor 连云阳张祝兰严凌斌邱观荣张引王德森杨煌建黄洪祥
Owner FUJIAN INST OF MICROBIOLOGY
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