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 l

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
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Example Embodiment

[0019] Example 1. Isolation of strain FIM-07

[0020] (1) Collect soil under the forests of Mingfeng Mountain in Kunming. Specifically, use a sampling shovel to remove about 10 cm of floating soil on the surface, and collect 10 g to 25 g of soil samples at 10 cm; weigh 2 g of soil samples and add 10 mL of sterile physiological saline. After shaking, let it stand for 30 minutes, take the supernatant, the stock solution, and dilute it gradually with sterile saline, and choose a dilution of 10 -2 , 10 -3 , 10 -4 And 10 -5 Suspension

[0021] (2) Separately take 0.1 mL of the stock solution and the selected suspension and spread them on an ISP4 medium plate prepared with 50 mg / L potassium dichromate water, repeat 3 parallel plates for each sample, and then apply the coated plate Cultivate at 28℃ and observe the appearance, size, color, edge shape, dry and wet state of the colony and other morphological characteristics;

[0022] (3) Under aseptic operating conditions, after culturing for...

Example Embodiment

[0023] Example 2. Identification of strain FIM-07

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

[0025] The mature spore chain has 5-10 spores, and the spore shape is oval, 0.4-0.6×0.6-0.7μm. On the plate, the colony is round, the colony is cultured for 10 days, the diameter is about 5-6mm, the surface is smooth, white or light gray to gray, it grows luxuriantly on the ISP4 medium, the aerial hyphae are gray-white, the intrabasal hyphae are poor, and they do not produce pigment or Produce light pink pigment. With the extension of culture time, the colony changes fro...

Example Embodiment

[0030] Example 3. Obtaining strain FIM-07-23

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

[0032] (2). Divide the spore suspension prepared in step (1) into 5 sterile test tubes, and proceed 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 applied to the separation plate by gradient dilution, cultured at 28°C for 10 days, the growth of the colony was observed, the colony count was performed, and the lethality curve was prepared as figure 1 Shown; from figure 1 It can be seen that there is a significant dose-effect...

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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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