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Nocardiopsis for producing ansamycin P-3 and 15-hydroxyl derivatives as well as preparation method and application of nocardiopsis

A technology of Nocardioides, hydroxy derivatives, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc.

Inactive Publication Date: 2015-09-02
XINJIANG INST OF ECOLOGY & GEOGRAPHY CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003]The only reported ansamycin P-3 producing strain is Actinosynnema pretiosum subsp. pretiosum No. C-15003 (Higashide et al., 1977) and Actinosynnema pretiosum subsp. aurantium ATCC 31565 and its mutants, while other genera have not been reported

Method used

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  • Nocardiopsis for producing ansamycin P-3 and 15-hydroxyl derivatives as well as preparation method and application of nocardiopsis
  • Nocardiopsis for producing ansamycin P-3 and 15-hydroxyl derivatives as well as preparation method and application of nocardiopsis
  • Nocardiopsis for producing ansamycin P-3 and 15-hydroxyl derivatives as well as preparation method and application of nocardiopsis

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment one: Pseudo-Nocardia ( Nocardiopsis sp. ) Isolation, cultivation and identification of EGI 80425 CGMCC No. 9969

[0040] 1. Strain isolation:

[0041] The soil samples were collected from the natural high-saline land in the Junggar Basin. After the soil samples were collected, they were put into sterilized sealed polyethylene bags and brought back to the laboratory for storage at 4°C. 2g of the soil samples were diluted in 18 mL of sterile water, and 200 Spread the μL 10-6 and 10-7 dilutions on the ISP2 plate (medium composition (g / L): yeast extract 4.0, malt extract 4.0, glucose 10.0, NaCl 30; trace salts 1 mL, pure water 1000 mL; After the sterilization, the pH was adjusted to 10.0 with 10 N NaOH), and after culturing at 37 °C for 3 weeks, a single colony was picked and purified by streaking to obtain a strain of white air hyphae, the number of which was EGI 80425.

[0042] 2. Morphological characteristics of the strain:

[0043] The colonies are dry,...

Embodiment 2

[0048] Embodiment two: Nocardiosis ( Nocardiopsis sp. ) Sequence determination and phylogenetic analysis of EGI 80425 CGMCC No.9969

[0049] 1. DNA extraction: A small amount of CGMCC 9969 genomic DNA was extracted by lysozyme method; the strain was inoculated in ISP2 medium (medium components: yeast extract 4.0 g, malt extract 4.0 g, glucose 10.0 g, NaCl 30 g; trace salts 1 mL, pure water 1000 mL; pH 7.5; sterilize at 121°C for 30 minutes.), shake at 37°C for 36 h. Take 1.5ml of the culture and centrifuge at 12000rpm for 2min; add 480 μL of TE buffer and 20 μL of lysozyme (20 mg / mL) to the precipitate, mix well and incubate overnight on a shaker at 37°C and 200 rpm; then add 50 μL of 10% SDS and 15 μL of proteinase K, mix well, and incubate at 55° C. for 1 h.

[0050] Add 550 μL of phenol / chloroform / isoamyl alcohol (25:24:1, v / v), shake and mix well, centrifuge at 12,000 rpm for 15 min, take the supernatant aqueous phase into a 1.5 mL centrifuge tube, and repeat the extr...

Embodiment 3

[0058] Embodiment three: pseudo-nocardia ( Nocardiopsis sp. ) EGI 80425 CGMCC No.9969 for the production of Ansamycin P-3 and its 15-hydroxyl derivatives

[0059] 1. Seed culture:

[0060] (1) Prepare seed medium: yeast extract 4.8g, glucose 4.8g, malt extract (paste) 12.0g, trace salt 1.2mL (formula: FeSO 4 .7H 2 O 0.1g; MnCl 2 .4H 2 O 0.1g; ZnSO 4 .7H 2 O 0.1g; distilled water 1L), pure water 1200mL, pH adjusted to 7.2-7.4. Divide into ten 500mL Erlenmeyer flasks evenly, and extinguish at 121°C for 30 minutes.

[0061] (2) Cultivation of seeds: Inoculate the strain EGI 80425 into the seed medium, and cultivate at 28° C. and 200 rpm for 36 hours.

[0062] 2. Fermentation culture:

[0063] (1) Preparation of fermentation medium: 30.4 g of yeast extract, 30.4 g of glucose, 76.0 g of malt extract (paste), 7.6 mL of trace salt (formula: FeSO 4 .7H 2 O 0.1g; MnCl 2 .4H 2 O 0.1g; ZnSO 4 .7H 2 O 0.1g; distilled water 1L), pure water 7594mL, pH adjusted to 7.2-7.4...

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Abstract

The invention discloses an alkali-resisting nocardiopsis for producing ansamycin P-3 and 15-hydroxyl derivatives as well as a preparation method and the application of the nocardiopsis. The provided strain is nocardiopsis EGI 80425, and the collection number of the nocardiopsis EGI 80425 is CGMCC No. 9969; a good technical effect can be obtained in the application of the strain in preparing the ansamycin P-3 and the 15-hydroxyl derivatives thereof. Tests prove that a remarkable and obvious technical effect can be obtained in the application of the strain nocardiopsis EGI 80425 CGMCC No. 9969 in producing the ansamycin P-3 and the 15-hydroxyl derivatives thereof, and therefore, the strain has excellent application prospect in producing the ansamycin P-3 and the 15-hydroxyl derivatives thereof.

Description

technical field [0001] The invention relates to the technical field of industrial microorganisms, in particular to the technical field of Nocardioides and its production of ansamycin P-3 and its 15-hydroxyl derivatives. Background technique [0002] ANSA-type antibiotics have various biological activities such as anti-cancer, anti-bacteria, anti-fungal, anti-virus, anti-tuberculosis, and hypolipidemic, and have become a research hotspot in many scientific research institutions and pharmaceutical companies in recent years. Ansamycin (also known as Ansamectin, Ansamitocin ) is a class of maytansinoids derived from microorganisms, and its components are P-0, P-1, P-2, P-3, P-3', P-4 and P-4'. Among them, Ansamycin P-3 ( Ansamitocin P-3) is the main fermentation product, which prevents cell mitosis and causes cell death by hindering the formation of microtubules, and has a significant anti-tumor effect in vitro and in tumor-bearing animals. [0003] The only reported ansamy...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P17/18C12R1/01
CPCC12N1/20C12P17/188C12N1/205C12R2001/01
Inventor 张永光李文均刘晴宋永相鞠建华王宏飞李丽张元明郭建伟
Owner XINJIANG INST OF ECOLOGY & GEOGRAPHY CHINESE ACAD OF SCI
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