The Study Of Fed-Batch Fermentation Of Epothilone

The epothilones, are16-membered macrolides produced by the gram-negativemyxobacterium, sorangium cellulosum. The compounds were firstly studied as plantprotection product due to their properties of antimycotic. They were not noticed untiltheir cytotoxic activity come to light. And epothilone then was classified as tubulinpolymerization agent. Now the epothilone is known as a novel class of non-taxanemicrotubule-stabilizing agents and the epothilones are more potent than the taxanesand active in some taxane-resistant models.The epothilones are more potent than the taxanes; active against tumorsrefractory to taxanes; and more soluble with enhanced oral bioavailability. Epothiloneobtained from the fermentation of the cellulose degrading myxobacteria, Sorangiumcellulosum. As a result, the epothilones are becoming promising antineoplastic drugs.However, the poor epothilone-producing ability of Sorangium strains became thebiggest difficulty in epothilone research. Because Sorangium strains are difficult tomanipulate, researchers have attempted to transfer entire epothilone gene clusters toheterologous hosts. However, due to the toxicity of epothilones to heterologous hosts,the yield of epothilones is low. Research of focusing on improving the synthesis ofepothilones in Sorangium cells turns out to be an effective method.In this experiment, the basic kinetic parameters of the epothilone-producingstrains in both shake-flask scale fermentation and50L fermenter scale fermentationwere studied. Based on that, optimization of fed-batch fermentation using responsesurface method was carried out. Finally, a set of efficient craft of epothilonefermentation was established by progressively larger test of bioreactors.In this study, the basic kinetic parameters of the epothilone-producing strainswere studied. It turned out that the rapid amplification of the strains coupled with theconsuming of reducing sugar. As a secondary metabolite, the epothilones wereproduced mainly in the stabilization period, lagging behind the burst of the cells. Theconsuming of amino nitrogen in the fermentation process was not regular. Thereforethe reducing sugars, the biomass and the yield of epothilones remained as the basicmeasuring kinetic parameters.There might be a big change of the product obtained and its production when thefermentation was carried out in the fermentation tank using Shake flask experiment conditions, especially for the myxobacteria, which is extremely sensitive to theenvironment. Fermentation basic dynamic parameters may also be enlarged becauseof the change of experimental conditions. A preliminary study and research onfed-batch fermentation focusing on the influence of single factor, such as glycerin andsodium propionate, on the growth of Sorangium and the yield of the epothilones wascarried out in order to approach large-scale production. The feeding time andconcentration of glycerol and sodium propionate were optimized using responsesurface methodology. Confirmation experiments were carried out. And underconditions of feeding10mL/L glycerin,0.4mg/mL sodium propionate at the4th dayof fermentation, about13.7%increase of epothilone B yield was obtained.Feeding conditions are established. However, whether the parameters of thefermentation process conditions being affected has become the first problem. Changesin temperature and pH have a great influence on the enzymatic reaction in the processof growth of microorganisms. And the inoculated amount will affect the proliferationof bacterial cells in the fermentation process and the yield of the objective product.Experiments studied the temperature, initial pH and amount of inoculation under theconditions of fed-batch fermentation, trying to find out the optimum conditions. Itproved that the initial pH within the range of7.2~7.5, the inoculation amountranging between10%to20%, and fermentation temperature of20°C are the mostsuitable feeding conditions for bacterium proliferation and epothilone synthesis.The amplified experiments were successfully in500-L stirring type bioreactorsand500-L gas-lift fermenters. Under the given fed-batch conditions, high-densityculture is able to achieve. And the yield of epothilone B reached a point of22.09mg/L on the10th day of fermentation, leading an increase of more than29.9%compared with the original batch fermentation conditions.