A alkali-fast sorangium cellulosum and uses of the same in producing epothilone

A technology of Cystobacter fibroblastus and alkali-resistant fibers, which is applied in the biological field and can solve the problems of low yield of secondary metabolites, contamination of bacteria, and slow growth rate of Cystobacter fibrioides

Inactive Publication Date: 2009-04-08
SHANDONG UNIV
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AI Technical Summary

Problems solved by technology

[0005] 1. The growth rate of S. cellulosus is very slow, and the generation time is more than 16 hours (it is the longest group of all myxobacteria at present), so the fermentation cycle is long, and it is easy to cause bacterial contamination in liquid culture, such as E. The cycle of bleomycin fermentation is usually about 14 days;
[0006] 2. The yield of secondary metabolites of wild S. cellulosum is relatively low, generally 0.5-20 mg/L. For example, the yield of epothilone A fermented by So ce90 strain of S. cellulosus is about 23 mg/L;
In 2004, Boddy et al. reported that the last four modules of the epothilone synthesis gene were successfully expressed in Escherichia coli to produce epothilone C, but the complex substrates (C9-C21 fragments of C9-C21 fragments) must be provided outside the host bacteria. N-acetyl-cysteine ​​thioester, SNAC)
In 2006, Mutka SC et al. reported that the entire...

Method used

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  • A alkali-fast sorangium cellulosum and uses of the same in producing epothilone
  • A alkali-fast sorangium cellulosum and uses of the same in producing epothilone
  • A alkali-fast sorangium cellulosum and uses of the same in producing epothilone

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Experimental program
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Effect test

Embodiment 1

[0089] Embodiment 1 Utilizes inducing acclimation to obtain the alkali-resistant Sonocystis cellulosus bacterial strain of high-yield epothilones

[0090] Isolation and purification of starting strains: The isolated soil samples come from the shore soil of Chenghai Lake in the middle of Yongsheng County, Lijiang City, Yunnan Province. The sampling site is 1503 meters above sea level, with an average temperature of 18.7°C, no frost throughout the year, and the water quality is alkaline (pH8.6 ), the light is sufficient, the pH of the soil sample is 9.2, and it is air-dried for later use. Spread sterile filter paper on the CNST plate, the pH of the culture medium is 7.2, and add filter-sterilized cycloheximide with a concentration of 25 μg / ml, culture it upright at 30°C, and observe the myxobacteria under a dissecting microscope every day after 2 days The expansion situation, and the myxobacteria that appeared were picked and expanded and touched to the fresh CNST medium for cul...

Embodiment 2

[0098] Example 2 Sequencing of the 16S rRNA gene of Sorangium cellulosum So0157-2CCTCC NO: M 208078 strain

[0099] The alkali-resistant Sonocystis cellulosus So0157-2 of high-yielding epothilones obtained by the induction and acclimation of Example 1, i.e. the CCTCC NO: M 208078 bacterial strain, was entrusted to Shanghai Sangon Biological Engineering Technology and Service Co., Ltd. , Ltd.) for 16S rRNA gene sequencing.

[0100] The experimental method is: mix the bacteria of CCTCC NO: M 208078 strain with a spinner bottle, centrifuge, and suspend with STE solution (approximately 0.5ml of STE solution per 0.1g of bacteria); add 0.5ml of bacteria into 5ml of centrifuged In the tube, mix well with a shaker, add 400μl 10% SDS, 30μl 20mg / ml proteinase K, mix well, keep warm at 37°C for 1 hour; add 0.4ml 5M NaCl, mix well and let stand for 10 minutes, then add 0.4 ml CTAB / NaCl, mix well, incubate at 65°C for 20 minutes; take out the centrifuge tube and extract with an equal volu...

Embodiment 3

[0113] Example 3 Application of Sorangium cellulosum So0157-2CCTCC NO: M 208078 in the preparation of epothilone

[0114] (1) Strain selection: select Sorangium cellulosum So0157-2CCTCCNO: M 208078 of the present invention;

[0115] (2) Activation of slant culture: inoculate the bacteria on the slant medium, and culture it statically for 96 hours at 32°C, and set it aside;

[0116] (3) Seed cultivation: With the strain cultivated in step (2), under aseptic conditions, use an inoculation loop to scrape off all the bacteria on a slope, inoculate it in 50mL (250mL triangular flask) liquid seed medium, set the rotation speed On a shaker at 180 rpm with a radius of rotation of 35 mm, culture at 32°C for 72 hours to obtain a seed solution;

[0117] (4) Fermentation culture:

[0118] Shake flask fermentation: with the inoculum amount of 10% volume ratio, inoculate the seed liquid in the shake flask that 120mL (500mL Erlenmeyer flask) fermentation medium is housed, 32 ℃, rotating sp...

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Abstract

The invention discloses an alkali proof Sorangium cellulosum strain named Sorangium cellulosum So0157-2, which is preserved in China Center for Type Culture Collection (CCTCC) with a preservation number of CCTCC No. M208078 on May 27, 2008. The invention also discloses the application of the alkali proof Sorangium cellulosum to the preparation of Epothilones. The Epothilones generated by the fermentation of the alkali proof Sorangium cellulosum has high yield stable fermenting property of strains and short fermentation period, is not easy to cause bacteria contaminants and does not generate Epothilones C, D, E, F and other homologues, thus greatly reducing the difficulty in subsequent separation and purification and being an Epothilones producing strain with high value in research and development.

Description

technical field [0001] 本发明涉及一株纤维堆囊菌及其应用,尤其涉及一株可高产埃博霉素的耐碱纤维堆囊菌菌株及其在制备埃博霉素中的应用,属于生物技术领域。 Background technique [0002] 促微管聚合类化合物紫杉醇(Paclitaxel, )及其类似物Docetaxel( )是目前临床上最为成功的抗肿瘤化疗药物,被用于卵巢癌、胸腺癌、结肠癌、肺癌和肝癌等实体癌的治疗,是美国食品与药物监督局(U.S.Food and DrugAdministration,FDA)认定的实体瘤化疗的首选药物。在与紫杉醇具有相同或相似生物学功能的多种天然化合物中,只有埃博霉素(Epothilones),[1S-[1R * ,3R * (E),7R * , 10S * , 11R * , 12R * ]]-7,11-二羟基-8,8,10,16-四甲基-3-[1-甲基-2-(2-甲基-4-噻唑基)乙烯基]-4-氮杂-17-氧杂双环[14.1.0]十七烷-5,9-二酮),来源于微生物的发酵产物,并被认为是紫杉醇的更新换代产品,是更好的抗癌药物。至今,已有五种埃博霉素类化合物进入了不同阶段的临床试验,包括Bristol-Myers Squibb公司(BMS)的埃博霉素B的两个半合成类似物BMS-247550和BMS-310705;Novartis公司的埃博霉素B(patupilone);Roche公司的埃博霉素D(KOS-862);以及最近刚刚进入临床试验的Berlex公司的ZK-EPO。它们均为粘细菌(myxobacteia)中纤维堆囊菌(Sorangiumcellulosum)的发酵产物或者发酵后的简单化学衍生物。美国食品与药物监督局于2007年10月批准了Bristol-Myers Squibb公司的 (ixabepilone,BMS-247550)作为治疗乳腺癌的药品上市。 [0003] 目前,埃博霉素的制备方法主要有化学合成法,生物合成法和化学修饰法三类。埃博霉素的化学全合成路线有十几种,如埃博霉素A的合成路线有6种,埃博霉素B有11种,埃博霉素C和D的全合成路线有2种。但是由于埃博霉素化学全合成步骤繁多,得率较低,与其它方法相比尚不具备优势,没有工业化生产的前景。通过对发酵生产的天然埃博霉素进...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P17/18C12R1/01
Inventor 李越中胡玮刘红
Owner SHANDONG UNIV
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