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

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...

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 epothilone NRPS / PKS gene cluster was expressed in Escherichia coli to produce epothilone C and D, but the yield was only at a level that could just be detected by LC-MS

A huge problem faced by these works is that epothilones have strong cytotoxic activity against non-Solexis cellulosus hosts, but the resistance mechanism of epothilones is not clear, and related genes have not been found, making heterologous expression low yield;

[0008] 4. Another problem encountered during the separation and purification of epothilones is that there are too many homologues. For example, the So ce90 strain can produce nearly 50 structurally similar epothilones during fermentation, of which only a few It has good activity and can be developed into a drug

However, after searching the literature and patents, there are no literature or patent reports on related technologies and methods using this idea at home and abroad.

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