Construction Of Gene Transformation System For Sorangium Cellulosum And The Study In Biological Fermentation And Purification Technics Of Epothilones

Epothilones, the secondary metabolites produced from the myxobacterium Sorangium cellulosum, are the potential anticancer drugs that stabilize microtubules and inhabit mitosis of cells in a manner similar to paclitaxel (Taxol). Epothilones have superior features relative to paclitaxel, including more water solubility, simpler molecular structure, non-endotoxicity and effectivity against tumors resistant to paclitaxel. These advantages make epothilones likely successors to paclitaxel and become a hotspot of clinic research. Great progresses have been made in total chemical synthesis, biological fermentation, biosynthesis and derivation, relation between activity and conformation and heterologous expression of the gene clusters. Currently five Epothilone derivatives are been initiated to phase II or III clinical trials to finally evaluate the anticancer potential. Being surrogates to replace taxol in treatment of multidrug resistant tumors, it will not be long before epothilones become marketable.However, S. cellulosum, the producer of epothilones, is difficult to engineer, due to the long doubling time, the specificity of bacterial strain, the broad drugs resistance, the aggregating growth, the low efficiency of introducing DNA into the bacterium and the limited number of molecular tools and markers that have been developed. There is no inkling as to the regulation and control of the epothilones biosynthesis gene clusters and the model of operon. Moreover, no endogenous plasmid had been found in S. cellulosum, and few attempts to transfer an autonomously replicating exogenous plasmid into this species have been reported. All these restricted the research and application of the abundant secondary metabolites with bioactivities produced by the bacterial.In our research, S. cellulosum So ce90, a positive strain for epothilone, was used. The research involved its fermentation conditions, purification technics of epothilone in the fermented solution, construction of plasmid pRP-GFP derived from pRK415 with a green fluorescent protein gene and an EpoPro fragment spanning from -890 to -1 relative to the start codon (GTG) of epoA (a part of epothilone synthase gene clusters), and the establishment of gene transformation system for the bacterial. The main conclusions are as follows:①To select the optimum medium and the conditions of culture, S. cellulosum So ce90 was cultured in nine medium, and the production of epothilone in the fermented solutions was measured by HPLC. The optimum medium included 2g/L soy grits, 10g/L potato starch, 8g/L skim milk powder, 2g/L yeast extract, 2g/L glucose, 8 mg/L Na-FeIII-EDTA, 1 g/L MgSO4 ? 7H2O, 1g/L CaCl2 ? 2H2O, 11.5g/L HEPES,2%(v/v) the resin XAD-16. The pH of the medium was adjusted to 7.4 with KOH before autoclaving. The optimum seed cultures, which were obtained from the fermented solution cultured in 180rpm, 30oC for 4 days, were added to the mediums by 5%. The maximum of epothilone could be gained after 8 days fermentation.②The separation and purification technics of epothilone are as follows. Firstly, the active materials were eluted with 100% isopropanol. And then, the active materials were extracted with ethyl acetate and petroleum ether (1:1). Lastly isolation of pure epothilone A and epothilone B were achieved by chromatography on a SOURCETM 30RPC colunm and C18 reversed-phase column. Structure of epothilone A and epothilone B were further confirmed by MS, UV and IR.③Plasmid pRP-GFP was transform from E.coli S17-1 to S. cellulosum So ce90 by conjugation, and the transformants were selected by tetracycline (50μg/ml) and kanamycin sulfate (10μg/ml). The results of the Southern blots analyses confirmed that the plasmid could be replicated autonomously without being integrated into the chromosome of S. cellulosum So ce90. To date, it is the first plasmid which has been described in myxobacteria. Therefore, the work will provide a powerful tool for isolating and analyzing the genes involved in epothilone metabolism. In this sense, this work may be a breakthrough in the genetics and molecular biology of S. cellulosum So ce90.④The conditions that might induce an increase in the transfer frequency were investigated. The frequency did not change by heating the S. cellulosum cells after transconjugation. But the mating time was more important for transfer frequency, of which the highest frequency was observed in the range of 60-65 h. The frequency of transfer to S. cellulosum So ce90 (calculated per viable Sorangium cell) was in the range 9×10-6. However, when the mating time was less than 40 h or more than 70 h, few transformants were found.⑤The stability of the S. cellulosum So ce90 transformants containing the plasmid pRP-GFP was tested. The fluorescence found in the subculture indicated that the plasmid was stably maintained in the bacteria with selective pressure. However, only 73% of the cells retained fluorescence at the 50th generation in the case of the antibiotic-free incubation. ⑥In this study, the GFP reporter system in S. cellulosum was also first established. The stable expression of GFP in S. cellulosum was a rapid and convenient marker for the conjugation screening of the transformants of the bacterium.⑦The comparison of the expression of gfp within pRP-GFP with that within pRK415-GFP (without EpoPro fragment) in S. cellulosum confirmed that the EpoPro fragment had the function of a promoter that could drive the expression of the gene in the bacterium. This would be a foundation for the analyses of the operon of epothilone synthase gene clusters and the research about the regulation and control of the gene.