Streptomycete strain mutagenesis preparation method for high-yield production of tacrolimus

A technology of tacrolimus and streptomyces, applied in the biological field, can solve the problems of low chemical synthesis efficiency and high cost, and achieve the effects of low production cost, improved lipase activity, and simple operation

Inactive Publication Date: 2020-06-26
浙江弘盛药业有限公司
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  • Summary
  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

The chemical synthesis of tacrolimus has low efficiency and high cost, so it is considered to be prepared by microbial fermentation

Method used

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  • Streptomycete strain mutagenesis preparation method for high-yield production of tacrolimus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The spores of Streptomyces tsukuba No. 9993 (S. tsukubaensis No. 9993) on the slant medium were washed with physiological saline and made into a spore suspension, treated with 0.9 mg / mL NTG for 25 min, and coated with a serially diluted dilution of physiological saline. Spread on the agar plate, cultivate at 30 °C for 8 days, select the high-yielding strains that survive after fermentation and screening; wash the slant spore culture of the high-yielding strains to make 10 6 / mL of spore suspension, take the dilution and spread it on a separation agar plate containing 3 mg / mL phlegmatin solution, and culture on the agar plate at 30 °C for 8 days; the slanted spores are washed with 8 mL of 10% sterile glycerol. And make a suspension, inoculate 0.2mL suspension in 100mL seed medium (500mL conical flask), 30 ℃, 260r / min shaking culture for 3 days. Filled with 100mL fermentation medium (500mL conical flask) and inserted with 20% of the seed amount, 30°C, 260r / min shaking cul...

Embodiment 2

[0030] 8 mg / mL of tenascin was selected as the selection pressure, and the operation steps were the same as those in the above-mentioned Example 1.

Embodiment 3

[0032] The concentration of craterin was selected to be 12 mg / mL as the selective pressure, and the operation steps were the same as those of the above-mentioned Example 1.

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Abstract

The invention provides a streptomycete strain mutagenesis preparation method for high-yield production of tacrolimus. According to the invention, mutagenesis breeding is performed with nitrosoguanidine (NTG), and then active substances of vibralactone and ethyl malonyl coenzyme A are used as selection pressures to achieve the aim of increasing the yield of tacrolimus. According to the invention, the yield of tacrolimus prepared by taking the active substances of vibralactone and ethyl malonyl coenzyme A synergistically as selection pressures is obviously increased compared with the yield of mutagenesis breeding by using a single selection pressure. Streptomyces tsukubaensis prepared by taking the active substances of vibralactone and ethyl malonyl coenzyme A as selection pressures has thecharacteristics of high activity of ethyl malonyl coenzyme A and high activity of lipase, and the yield of tacrolimus is improved due to high activity of ethyl malonyl coenzyme A and high activity oflipase, and generation of by-products is reduced. The yield of combined use of vibralactone and ethyl malonyl coenzyme A is obviously higher than the yield of mutagenesis breeding by using a single selection pressure.

Description

【Technical field】 [0001] The invention relates to the field of biotechnology, in particular to a method for preparing a high-yielding tacrolimus-producing Streptomyces strain by mutagenesis. 【Background technique】 [0002] Tacrolimus, also known as FK-506, is a 23-ring macrolide antibiotic (erythromycin family) isolated from the fermentation product of a Streptomyces strain in the soil of Tsukuba, Japan in 1979. Immune rejection after liver and kidney transplantation can be produced by a variety of Streptomyces species. Its immunosuppressive efficacy is 10 to 100 times that of cyclosporine A, but the adverse reactions are smaller. Its mechanism of action is similar to that of cyclosporine, by inhibiting the activity of calcineurin to reduce the T-cell-mediated immune response produced by interleukin-2, but its effect is 10 to 100 times stronger than that of cyclosporine. In recent years, the sales of FK506 in the immunosuppressant market have accounted for more than 20% of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/01C12N1/36C12P17/18C12R1/465
CPCC12N1/36C12N15/01C12P17/188
Inventor 汪炳英金志敏葛晶晶赵如慧李孙平顾勤城
Owner 浙江弘盛药业有限公司
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