Studies On The Biosyntheses Of Epothilones And Cephalosporins In Their Producers

Antibiotics have close connection with human beings while saving countless lives in clinical treatments. The research in antibiotics is improving due to the extensive attentions, and the antibiotics are upgrading to achieve better treatment towards patients. Due to the importance of antibiotics production, optimizing the production methods of antibiotics becomes a critical area in scientific research. In antibiotics production area, one focal point is on developing new drugs, the other one is on increasing production and reducing costs. The improving of production methods are mainly in the strain transformation and fermentation condition optimization. In this research, two antibiotics, the Epothilones and the Cephalosporins, were researched, and the production methods of these antibiotics were optimized.Firstly, the heterologous expression of Epothilone modifying enzymes was detected. The expression performance and the fermentation products of glycosylase, halogen and esterase enzyme were detected respectively. The expression amounts of glycosylase and halogen were higher than esterase enzyme. In the fermentation of strains with glycosylase, efforts were carried out in the addition of different carbohydrate, but no glycosylation product was generated. However, no modified Epothilones product was detected in fermentation broth for the reason of low yield and additional impurities. Therefore, efforts were made to enhance the yield of Epothilones and ruduce the producing of impurities. Due to its complex compositions, the effect of yeast extract was detected. In fact, yeast extract couldn’t affect the yield of Epothilones and the producing of impurities. Based on the results in our previous research of Sorangium cellulosum SoO 157-2, ZE series Myxococcus xanthus were tested in fermentation of Epothilones. Different compounds were added into fermentation medium to improve the yield of Epothilones. Indole acetic acid, methacrylic acid, methyl oleate and triolein were studied respectively. Among that, the addition of methacrylic acid could enhance the yield of Epothilones in both Myxococcus xanthus and Sorangium cellulosum, so methacrylic acid was presumably involved in the process of synthesis of epothilone. Indole acetic acid showed enhancement in Sorangium cellulosum but inhibition in Myxococcus xanthus during Epothilones production, showing that it might be involved in the specific metabolic pathways in Sorangium cellulosum. Methyl oleate led to great improvement in Epothilones yield. However, methyl oleate and triolein could inhibite cell growth in early time of cultivation. Thus, the time point of methyl oleate and triolein adding was further researched, and 36 h was confirmed to be the best time point to add methyl oleate. The amount of Epothilones produced was 13.45 mg/L and 29.45% better than adding methyl oleate at 0 h. The adding amount of methyl oleate was researched at 24 h, and 20 uL/mL was confirmed to be the best adding amount of methyl oleate. The amount of Epothilones produced was 15.97 mg/L and 53.71% better than adding 10 μL/mL methyl oleate at 0 h. These results present basis for the selection of the best producing strain and the research in synthesis mechanism of Epothilones.For the Cephalosporins-producing strain Acremonium chrysogenum, the strain transformation methods were optimized in this research. Experiments were carried out in cell culture and the preparation, regeneration and transformation of protoplast. Optimal condition of spore culture was confirmed that the strain was cultured using pre-dried slant for 13 days. The best method in mycelium culture was to inoculate dispersed spore suspension filtrate in SGY liquid medium and cultured for 48 h. Protoplast preparation was catalyzed by 0.02 g/mL Snailase, and the hydrolysis time was 1.5-2 h. SGY solid medium was used in protoplast regeneration. Considerable efforts were attempted in transformation, but no transformant was gained.However, the optimizations of culture conditions and transformation provide the basis and reference for the future realization of strain genetic engineering.In this work, problems in Epothilones and Cephalosporins productions were researched. Due to the limited experimental conditions, the fermentation in this research was achieved with shake flasks, thus the controllable parameter was finite and distinct from those in real fermenter. In further research, fermenter could be tried in fermentation experiments to control other producing conditions such as dissolved oxygen rate, agitation rate and feed medium as well as to increase the volume of fermentation. In this way, the enhanced yield and the richer products would be achieved. Through series of experiments, basis and references in Epothilones production and new Cephalosporins drugs development were provided.