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Fermentation process for preparing tiancimycin-A and derivatives thereof by streptomyces sp

A fermentation process, the technology of Tiancimycin, which is applied in the field of biomedicine, can solve the problems of many steps and large consumption of organic solvents, and achieve the effect of high-end drug prospect

Active Publication Date: 2019-06-04
CHANGSHA CHARISM BIOSCIENCES CO LTD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the separation and purification of TNM-A from the fermentation broth still uses traditional methods such as extraction of the fermentation supernatant with ethyl acetate, breaking the bacteria with acetone, and then extraction with ethyl acetate. There are many steps and a large amount of organic solvents are consumed in the process.

Method used

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  • Fermentation process for preparing tiancimycin-A and derivatives thereof by streptomyces sp
  • Fermentation process for preparing tiancimycin-A and derivatives thereof by streptomyces sp
  • Fermentation process for preparing tiancimycin-A and derivatives thereof by streptomyces sp

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1: Cultivation and fermentation of Streptomyces CB03234 and detection of biological activity of TNM-A

[0057] Streptomyces CB03234 was inoculated to Gaoshi No. 1 (G1) solid medium (the G1 solid medium was: 10g / L soluble starch, 0.5g / LMgSO 4 ·7H 2 O, 0.5g / LK 2 HPO 4 , 1g / L NaCl, 1g / L KNO 3 , 0.01g / L FeSO 4 ·7H 2 (0, 20g / L agar, pH=7.0) slant, cultivate about 8-15 days under 30 ℃ of constant temperature conditions, collect spores with aseptic 20% glycerin solution and obtain spore suspension and refrigerate in-80 ℃ for subsequent use. In order to obtain the target product TNM-A, 50 μ LCB03234 spore suspension was inoculated into 50 mL tryptone soybean broth (TSB) seed medium (the TSB seed medium was: 17 g / L tryptone, 3 g / L plant peptone, 2.5 g / L K 2 HPO 4 , 5g / L NaCl, 2.5g / L glucose, pH=7.3), after culturing for 48 hours at 30°C and 200rpm, 5mL seeds were transferred to 50mL production medium (the production medium was: 10g / L soluble starch, 5g / L cott...

Embodiment 2

[0058] Embodiment 2: Separation and purification and HPLC detection of TNM-A

[0059] The 50mL fermented liquid obtained in Example 1 is centrifuged and the supernatant and thalline are collected respectively, and the supernatant is extracted with ethyl acetate (EA) (50mL*3 times); the thalline is extracted with 50mL acetone, and the extract is passed through Concentrate on a rotary evaporator and then extract with EA water mixed solvent (1:1, volume ratio) (50mL*3 times), combine the above extraction phases, concentrate and dry again, and finally redissolve in methanol for HPLC detection. HPLC analysis conditions and procedures are as follows: mobile phase A is 99.9% deionized water and 0.1% formic acid; mobile phase B is 99.9% methanol and 0.1% formic acid, flow rate is 1.0mL / min, UV detector wavelength is 540nm, linear gradient analysis The program was: 0-5 minutes, 90% A to 5% A; 5-9 minutes, 5% A; 9-13 minutes, 5% A to 90% A; 13-15 minutes, 90% A.

Embodiment 3

[0060] Embodiment 3: the addition of different types of resins in the fermentation process of Streptomyces CB03234

[0061] TNM-A is extremely toxic, and as its concentration increases in the later stage of fermentation, it will rapidly cause the death and autolysis of the bacteria that produce it, causing the pH value of the fermentation broth to rise sharply; while TNM-A will increase its concentration when the pH exceeds 7.5 Under certain conditions, it begins to decompose, and with the further increase of pH value, it accelerates the decomposition until it disappears. Therefore, adding macroporous resin to adsorb TNM-A during the fermentation process can effectively eliminate the toxic and side effects of TNM-A on its own strains to maintain stable biomass for TNM-A synthesis, and by reducing TNM-A in the fermentation broth concentration to further induce its synthesis.

[0062] Therefore, the selection includes styrene-based macroporous strong acid cation exchange resin ...

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Abstract

The invention relates to a fermentation process for preparing tiancimycin-A and derivatives thereof by streptomyces sp., wherein the streptomyces sp. is streptomyces sp. CB03234-S, is preserved in China Center for Type Culture Collection on September 25, 2017, and has the preservation number of CCTCC M 2017538. By systematically optimizing the fermentation culture medium of the TNM-A high-yieldingstrain CB03234-S, the optimized culture medium suitable for high-efficiency synthesis of the target product TNM-A is determined; on the basis, pilot scale amplification of the microbial fermentationpreparation method is carried out in a fermentation tank, and the microbial fermentation preparation process of the TNM-A is established and optimized by control of the temperature, the rotating speedand the pH value, control of raw material supplementation and other control means; finally, through the gradient control of dissolved oxygen in fermentation broth in the fermentation process (the dissolved oxygen in the fermentation broth is controlled at 60-80% in the early stage of fermentation, the dissolved oxygen in the fermentation broth is controlled at 40-60% in the middle and later stageof fermentation, and the pH is 8.0-9.0) and feeding control, the fermentation yield of the TNM-A is further improved, and the yield of the TNM-A breaks through 20 mg / L.

Description

technical field [0001] The invention relates to a fermentation process of Streptomyces, in particular to a fermentation process of high-yield Streptomyces for preparing Tiancimycin-A and derivatives thereof. It belongs to the technical field of biomedicine. Background technique [0002] Enediyne natural products have a unique conjugated alkyne-ene-yne ​​molecular structure and strong biological activity, and are the most promising class of anti-tumor antibiotics. According to their core structure, they can be divided into neocarzinotatins and neocarzinotatins. , NCS), lidamycin (C-1027), kedarcidin, and maduropepti, etc., and nine-membered cycloenediynes including calicheamicin (CAL), esperamicin, dynemicin (DYN) and uncialamycin (UCM ) and other ten-membered cycloenediynes. The biological activity of enediynes antitumor antibiotics mainly depends on the DNA damage mechanism induced by them, that is, the formation of temporary benzene ring difree radicals through electroni...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P17/18C12R1/465
Inventor 段燕文沈奔朱湘成黄勇庄周康
Owner CHANGSHA CHARISM BIOSCIENCES CO LTD
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