Streptomyces tsukubaensis mutant strain and application thereof in co-production of tacrolimus and diene tacrolimus

A technology of Streptomyces tsukuba and tacrolimus is applied in the field of microorganisms, which can solve the problems of low chemical synthesis efficiency and high cost, and achieve the effects of stable fermentation unit, improved fermentation yield and good genetic stability.

Active Publication Date: 2022-03-22
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, due to the low efficiency and high cost of chemical synthesis of tacrolimus, its raw material production still comes from microbial fermentation
At present, there is no patent report on the efficient production of diene tacrolimus by microbial fermentation

Method used

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  • Streptomyces tsukubaensis mutant strain and application thereof in co-production of tacrolimus and diene tacrolimus
  • Streptomyces tsukubaensis mutant strain and application thereof in co-production of tacrolimus and diene tacrolimus
  • Streptomyces tsukubaensis mutant strain and application thereof in co-production of tacrolimus and diene tacrolimus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Configuration of RP solid medium (g / L): cooked soybean powder 10; soluble starch 20; valine 0.5; agar 18.0; KH 2 PO 4 0.5; NaCl2; CaCO 3 3; MgSO 4 ·7H 2 O 0.5, pH adjusted to 7.2.

[0066] The configuration of the seed medium (g / L): soybean powder 20; soluble starch 10; glucose 5; yeast powder 7; peptone 7; NaCl 3; KNO 3 3; MgSO 4 ·7H 2 O 0.7; CaCO 3 1. Adjust the pH to 7.2.

[0067] Configuration of fermentation medium (g / L): soybean powder 35; glucose 30; soybean oil 15; soluble starch 10; yeast powder 2.5; peptone 3; CaCO 3 3.5, the pH was adjusted to 7.2.

[0068] All the above-mentioned media need to be sterilized at 121°C and 0.1MPa for 20min.

Embodiment 2

[0069] The acquisition of embodiment 2 Streptomyces tsukuba SL2021

[0070] A strain with high tacrolimus production was selected by ultraviolet mutagenesis.

[0071] The UV mutagenesis flow chart is as follows figure 1 shown.

[0072] Precautions for UV mutagenesis process:

[0073] 1. The bacterial strain is cultured on a solid medium for 10-14 days. After the slant spores mature, wash the spores from the slant with sterile water;

[0074] 2. Insert the spores into the seed medium, activate and cultivate them in a shaker at 35°C and 200rpm for 6-8h;

[0075] 3. Filter the seed solution with filter paper, wash twice with sterile normal saline, estimate the number of spores with a hemocytometer, and finally make 100% of the spores with sterile normal saline. 7 Individual / mL spore suspension;

[0076] 4. After the mutagenesis treatment, the mutagenized spore suspension should be refrigerated in the dark to prevent the occurrence of refolding;

[0077] 5. The entire mutag...

Embodiment 3

[0079] The fermentation verification of embodiment 3 Streptomyces tsukuba SL2021

[0080] Cultivate Streptomyces tsukuba SL2021 on solid medium at 28°C for about 14 days until the spores turn black gray. Use an inoculation loop to scrape a full ring of spores from the solid plate and inoculate it into a 250mL Erlenmeyer flask containing 40mL of seed medium. Cultivate it at 28°C and 220rpm for 48h. At this time, the seed liquid is viscous. There are fine sandy mycelium hanging on the bottom of the bottle.

[0081] The cultured seed solution was inoculated into a 250 mL Erlenmeyer flask containing 40 mL of fermentation medium at an inoculum size of 10%, and cultured at 28° C. and 220 rpm for 168 h.

[0082] The tacrolimus production of Streptomyces tsukuba SL2021 was 550mg / L at 168 hours of fermentation.

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Abstract

The invention discloses a streptomyces tsukubaensis mutant strain for high yield of tacrolimus and diene tacrolimus, the name of the strain is SL2021, the classification name is streptomyces tsukubaensis, the preservation number is CGMCC No.23925, the preservation date is CGMCC No.23925, the preservation unit is China General Microbiological Culture Collection Center, and the preservation unit is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. According to the invention, a high-yield strain of tacrolimus and dientacrolimus is obtained, the strain can be used for fermenting high-yield tacrolimus and dientacrolimus, the highest yield of tacrolimus and dientacrolimus can reach 582mg / L after shake flask fermentation, the yield of dientacrolimus can reach 181mg / L, the fermentation yield of tacrolimus is greatly improved, the genetic stability is good, the fermentation unit is relatively stable, and the strain is suitable for industrial production. The industrial application prospect is very good.

Description

technical field [0001] The invention belongs to the technical field of microbes, in particular to a Streptomyces tsukuba mutagenic strain, a method and an application of high-yielding tacrolimus and diene-tacrolimus. Background technique [0002] Tacrolimus (Tacrolimus, FK506), a new type of 23 yuan Dacrolimus extracted from the fermentation broth of Streptomyces tsukubaensis No.9993 isolated from the soil of Mt. Cyclic lactone immunosuppressants. Tacrolimus is 10 to 100 times more effective than cyclosporine at the same dose in preventing rejection of various organ transplants, and has fewer side effects, and has low mortality, high transplant survival rate, and high transplant survival rate. It has the advantages of high success rate, less dosage and less dependence. Tacrolimus has rapidly increased its market share within a short period of time after it was launched in China, and has become a clinical first-line drug for rejection after liver and kidney transplantation....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N13/00C12P17/18C12R1/465
CPCC12N1/20C12N13/00C12P17/188
Inventor 刘欢欢舒林李贞景郭庆彬许慧敏成颖李居行赵娇娇
Owner TIANJIN UNIV OF SCI & TECH
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