The Breeding Of Productive Strain For Epothilone And Research Of Antitumor Activity

Myxobacteria are Gram-negative bacteria, with complicated multicellular social interactions, that includes one kind strain of degrading cellulose which is called Sorangium cellulosum. Sorangium strains could produce large numbers of bioactive compounds, which show anticancer, antibacterial, fungicidal, and immune-modulating effects, meanwhile, a kind of macrolide antibiotics called the epothilones, stabilizing microtubules in the same manner as Taxol, absords more attentions. So far, the epothilone medicine has been joined into the market successfully, which plays an important role in resisting tumor. Otherwise, the production of epothilone depends on chemical synthesis greater camparing the microorganism fermentation, the main restriction is due to the poor epothilone-producing ability of Sorangium strains, therefore, there is a prevalent meaning of guidance by transforming Sorangium to enhance the epothilone production.In this paper, the effective mutation and selection breeding for So 0157-2 was carried out, we selected a mutants LG3111776 that yielded high levels of epothilone compared with So 0157-2, and the mutants yielded about 5 or 3 times more epothilone A ,B than the starting strain. Additionally, the paper not only analyze the antitumor activity mechanism for the epothilone, but also elaborate mechanism of action about synergistic effect of epothilone mixture and the relation between apoptosis and autophagy induced by epothilone.In this work, in order to obtain superior strains, So 0157-2 was used to original strain, and the breeding method of pH extreme environment was applied to acclimatize the strain. After analyzing by HPLC, the yield of the epothilone A, B for tolerance LG31 strain was improved compared original strain. Then, the UV and DES was applied to the mutate LG31, uniting screening method of high-throughput screening and HPLC, we got the mutant strain LG31-11-7 which yielded more output of epothilone A, B, about 15.0 mg / L, 8.8 mg / L. In this paper, we also utilized mutation breeding of UV connecting rational screening, which made use of high concentration of precursor sodium acetate and end-product as the pressure. Differing from the following screening method, the indicator organism and TLC was carried out to select positive mutation promptly. Finally, after fermentation condition optimization, the epothilone A, B output of mutant strain LG3111776 was about 43.1 mg/L and 22.9 mg/L, which provides not only culture variousness for protoplast fusion but also excellent strain source for industrial production.The satisfied antitumor activity can be found after the epothilone acted on many varieties of tumor lines. Especially for the sensitive tumor lines of PC3, HELA, KB, K562, MCF-7and U87, we achieve not only the high inhibition rate, but also the unique synergistic effect was discovered. In order to make sure the synergistic effect, we employed three methods of the cell cycle detection, cell apoptosis detection and monitoring the cell growth for Xcelligence to prove it. Meanwhile, we took a possible conclusion that the main result causing the synergistic effect was attractive force between active protein on the cell surface and binding group of epothilone, besides, the competition between epothilone compounds played a significant role. Additionally, when the receptor cell was the U87 lines, the cytoplasmic puncta around LC3 protein (the microtubule-associated light chain 3) came out, which shows the epothilone could induce autophagy, that was one kind of pathway of programmed cell death. Autophagy is most conveniently monitored by measuring the redistribution of the microtubule-associated light chain-3 (LC3) into cytoplasmic dots (which constitute bona fide autophago(lyso)somes), by transfecting cells with a GFP-LC3 (green fluorescent LC3 protein) fusion protein. After DAPI dyeing for the cell nucleus, the observation of changing process demonstrated that there was the interdependence and intercommunication between the cell apoptosis and autophagy caused by epothilone. All these work will help us analyze the mechanism of inhibiting the tumor cell growth by epothilone drug.