| Myxobacteria are Gram-negative, unicellular gliding bacteria, which exhibit complicated multicellular social behavior. The microbes are capable of producing large numbers of bioactive compounds that show anticancer, antibacterial, fungicidal or immune-modulating activities. Thus far, about 100 basic structures and 500 structural analogs have been discovered in myxobacteria and have been fully characterized chemically, which account for about 3.5% of the presently known secondary metabolites of microbial origin. The diversity of the chemical structures and action mechanisms of the metabolites from myxobacteria can even be compared with those from Streptomyces and Bacillus.Sorangium cellulosum is a species of myxobacteria belonging to the Myxococcales order, Sorangineae suborder, Polyangiaceae class, Sorangium genus and it can grow by degradation of cellulose. As we know, Sorangium has the largest prokaryotic genome hitherto, for instance, Sorangium cellulosum So ce56 harbors the genome with a size of approximately 13 Mb. As reported, over half of these genes are involved in the production of secondary metabolites. In attation, almost half of the bioactive secondary metabolites of myxobacteria were identified from Sorangium, and nearly all of the Sorangium strains were found to produce these bioactive compounds. Therefore, myxobacteira are not only important in fundamental studies, but also alluring by industrial application.Epothilones, which are naturally produced by the myxobacterium Sorangium cellulosum, are anticancer agents that mimic the anticancer mechanisms of paclitaxel (i.e. microtubule stabilization), it also has activity to the tumor cells which are resistant to paclitaxel. Up to now, there are at least five epothilones or chemically modified derivatives of epothilone undergoing evaluation in clinical trials and one has already been authorized for clinical use by the U.S. Food and Drug Administration. However, the research and development of epothilone drugs is seriously limited by difficulties in their production.The Sorangium GSUV3-205 strain was obtained by mutation and high-throughput screening based on the technology of genome shuffling. Our previous bioactivity screening analysis confirmed that GSUV3-205 is a promising strain for the production of bioactive secondary metabolites. However, the epothilone production became low by GSUV3-205 because of strain degradation and the epothilone production of other epothilone producer strains was very low in contrast to the biomass and substrate in the medium. Thus, it has become the most interesting questions for researchers that how to explore the potential of epothilone production by natural producers Sorangium in fermentation system.In this thesis, we optimized and improved the culture conditions, nutrition and growth characteristic to improve the production of epothilones by S. cellulosum GSUV3-205 that had been confirmed by the previous study in our laboratory to be an epothilone producer, the results were excellent.Process improvement is an important method to improve the production of metabolites of industrial microbiology for every metabolites producer. Therefor, this article firstly optimized the nutrition and culture conditions in the process of biosynthesis of epothilone by S. cellulosum GSUV3-205 though two phases.PhaseⅠ, based on the physiological characteristics of GSUV3-205, we investigated some culture condition factors such as inoculum size, fermentation time, seed condition when inoculating, adding time of resin, shaker speed in the process of epothilone production. We determined the appropriate inoculum size and fermentation time (108/ml,9d) by formulating synthetic curve of epothilone at different inoculum size. Afterwards, we determined the appropriate seed condition (200rpm,5d) by analyzing the titer of epothilone on the different seed conditions at different inoculum size. Finally, we established the suitable adding time of resin (before sterilization) and shaker speed (200rpm) by single-factor design.PhaseⅡ, We statistical optimized the medium components in order to maximize epothilone B production by S. cellulosum GSUV3-205 using multiple steps of the response surface methodology. The optimization experiments include two steps. Firstly, based on GFM medium used in initial experiments, we extended the nutrition components of fermentation medium by single-factor design, Plackett-Burman design and Box-Behnken RSM design, and obtained a primary optimized medium which components includes dextrin 3.0g; sucrose 0.5g; glucose 0.8g; soy powder 1.7g; Slim milk powder 1.0g; MgSO4.7H2O 1.0g; CaCl2 1.0g; EDTA-Fe3+ 2ml and trace element solution 0.5ml; distilled water 1000ml; Amberlite XAD-16 resin (Rohm and Haas) 2%(v/v); pH 7.2. In this medium, compared the GFM medium, complex carbon resource is 0.3% dextrin instead of 0.2% potato power and a new component sucrose was added, and the content of most other components were adjusted. The yield of epothilone B by GSUV3-205 in this medium reached 46.5mg/L, which was about 4.1 folds than the yield in the GFM medium. Then, we optimized in detail the components of the primary optimized medium. Based on this new incubation system, we elucidated the medium components that significantly affect epothilone B production with a fractional factorial design (FFD), and found three factor (Dextrin, Slim milk powder, MgSO4) were important for epothilone B production. These significant ingredients were optimized by central composite design (CCD). A statistical model: (Y=51.69+17.03×X2+1.17×X6+0.70×Xg-4.55×X2×X2+7.01×X2×X6-2.48×X6×X6-0.69×X2×X8-2.01×X6×X8+0.52×X8×X8) was established for the description of the relationships between the medium components and epothilone B production using statistical analysis system. The statistical signification of the model indicated that the experimental model was in good agreement with the experimental results. The optimal medium for producing epothilone B was obtained by RSM and canonical analysis of mathematical model using SAS. The components of the optimal medium includes dextrin 6.3g; sucrose 0.5g; glucose 0.8g; soy powder 1.7g; Slim milk powder 2.6g; MgSO4.7H2O 3.2g; CaCl2 1.0g; EDTA-Fe3+ 2ml and trace element solution 0.5ml; distilled water 1000ml; Amberlite XAD-16 resin (Rohm and Haas) 2%(v/v); pH 7.2. Epothilone B production in the optimal medium reached 82.0mg/L about 76.3% more than that in the primary optimized medium by GSUV3-205. For the first time, we optimized the process of biosynthesis of epothilone in the natural producer Sorangium. Meantime, we greatly improved the yield of epothilone and provided a way to the industrial production of epothilone.Based on the literatures reported, we investigated some small molecules to examine the influences on biosynthesis of epothilone. The results showed that epothilone biosynthesis was inhibit when the small molecules, such as iron ions, betaine, acetate, propionate, methylmalonic acid, succinic acid and cysteine, was excess in the medium. However, the existence of some small molecules could stimulate the biosynthesis of epothilone by GSUV3-205 while they were added with appropriate amount at appropriate time. For instance, the epothilone production increased 16% when 0.05% of betaine hydrochloride was added to the medium in the third day of fermentation, and the yield of epothilone A and B increased 68% and 270% respectively while10 mM solution including acetate, methylmalonic acid and cysteine was added to the medium. In addition, we, for the first time, added methylmalonic acid which had a same carbon chain skeleton to the precursor of epothilone B methylpropionyl-CoA to the fermentation medium and achieved good results that the yield of epothilone B increased 86% in contrast to the control while 5 mM methylmalonic acid was added to the medium. These results provided a basis for further use of continuous fermentation technology with adding precursors, but also provide a guideline for selecting the metabolites from GSUV3-205 product.S. cellulosum GSUV3-205 is agglomerate in the liquid culture; this character is a formidable inconvenience for the industrial production and is a difficult problem for genetic research on the strain. Accordingly, we hope to use biological methods to make GSUV3-205 dispersed in liquid culture while maintaining the ability of epothilone synthesis. This can increase the biomass to improve epothilone production, thus speeding up the process of its industrial application, on the other hand, it can bring great convenience when we further explore GSUV3-205 though molecular biology methods.We tried to get a mutant that was dispersed in liquid culture through biological domestication and genetic manipulation on GSUV3-205. First, we designed a special domestication process of GSUV3-205, after 8 cycles of domestication; we finally got one mutant GSUV3-205XH which was dispersed in M26 liquid medium. Although GSUV3-205XH grow very slow and lost the ability to synthesize epothilone, we can use this strain to proceed protoplast fusion with the strain which has good ability to synthesize epothilone to get a mutant which both has the ability to synthesize epothilone and grow dispersed in liquid culture on basis of theory of genome shuffling in future.Afterwards, we chose epsD and pilA gene, which might be closely related to the aggregation of GSUV3-205 and had not reported in Sorangium yet, to built two insertion inactivation plasmid pCCPA and pCCED by cloning the homologous fragments. We obtained 70 resistant strains by conjugation, then verified the status of these strains in liquid culture and found that all of these resistant strains had no ability to grow dispersed. In addition, we tested the insertion site of plasmids by the method plasmid rescue and the results showed that the plasmid did not insert to the right site of the genome of GSUV3-205. Thus, we proposed a corollary that Sorangium could absorb and random integrate exogenous DNA into the genome of chromosome at ambient pressure which caused the large genome of Sorangium. However, we were lack of more conclusive evidence to clarify the specific mechanism of this theory. |
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