The Effects Of Adding Plant Source Oil Ester On The Production Of Epothilone In Myxobacteria

Myxobacteria is a kind of gram-negative bacteria with complex multi-cell behavior in nature,such as sliding on a solid surface and possessing a variety of signal regulation pathways.Most Myxobacteria can produce a variety of secondary metabolites with biological activity and higher values for drug development and application,which makes the Myxobacteria become the attractive microorganism only after actinomyces in the new drug development.But due to its group growth,long generation,the difficulties in large scale cultivation and genetic manipulation,it is difficult to explore its functional genes and valuable compounds,thus hindering the research progress of Myxobacteria.Sorangium cellulosum is a Myxobacteria that can degrade cellulose.Its secondary metabolite epothilones is a kind of polyketone macrolide compound with antitumor activity.The epothilones could promote the polymerization of microtubule,which is similar to anti-cancer drug paclitaxel on tumor cells.In addition,epothilones have the advantages of better water-solubility and smaller cytotoxicity.This has aroused the attention and research of the researchers on epothilones.At present,epothilones have been applied to clinic at abroad,but due to the low production of the epothilones and the immature production technology,which cause the epothilones have not been applied and developed at home.This phenomenon is caused by the long generation,difficulties in cultivation and genetic manipulation and low cell production of epothilones-producing strains.So our lab focus on the genetic operations of the epothilones gene clusters and the optimization of epothilones fermentation to solve this problem.In recent years,oils have been widely applied to the process of biological fermentation,including the biological fermentation of some antibiotics.The antibiotics have been reported which use plant oils for fermentation,the mainly antibiotics include erythromycin,tylosin,Spinosad,cephalosporin C and others.Adding the plant oils improve the production of antibiotics.This study examined the effects of oils on epothilones production and explored its impact mechanisms.The high yield strains of epothilones gene mutations are made by lab researchers through the genetic operation of epothilones gene clusters.In this thesis,we carried out three aspects of work as follows:1.To explore the effects of the addition of oils on the epothilones fermentation.According to early optimization of the basis fermentation medium of epothilones,confirmed the medium group including casein peptone 1%,yeast extraction powder 0.5%,MgSO·7H20 0.1%,10 mM MOPS,adsorption resin 2%(v/v),pH 7.5.Fermentation culture of the mutant strains was carried out in environment of 30 ?,200 rpm,after eight days,the production of epothilones is about 1.5 mg/L.On the basis of the optimized fermentation medium,the addition of methyl oleate and plant oils can increase the yield of epothilones,and the yield of epothilones increased to about 10 mg/L.The addition of oils has solved the problem of low yield and not easy to be detected.2.The effects of different ways of adding oils on the fermentation of epothilones by mutant strains.When added mix oils to the fermentation medium,the production of epothilones was increased.Added the methyl oleate and rapeseed oil into the fermentation,the epothilones production increased to 15 mg/L.After the addition of oils for a period of time in 0 h,it was found that adding a small amount of oils could further increase the production of epothilones,and the yield was higher than 15 mg/L.If the optimum amount of oils was added in batches,we found that the addition of oils in batches can't increase the production of epothilones.Adding different concentrations of plant oils in batches,when peanut oil was added with 0.2%concentration,the yield of epothilones was the highest and the maximum yield of epothilones detected was up to 18 mg/L.The effect of peanut oil is better than that of other oils.When added the different concentrations of oils in batched,meanwhile added the appropriate amount of sodium propionate and trace elements can increase the production of epothilones..3.Preliminarily exploring the mechanism of oils to improve the production of epothilones.Through detected the change of the bacteria concentrations,the production of epothilones,the level of epothilones gene clusters transcription and the content of bacteria lipase in the process of the fermentation,preliminary determined the role of oils in the fermentation process.The study found that the addition of oils increased the concentration of fermentation broth,promoted the growth of bacteria and slowed down the aging time of bacteria.The results of RT-qPCR showed that the addition of oils promoted the transcription level of the epothilones gene clusters,and the transcription of each gene module increased significantly.The change of lipase content in the fermentation process of adding oils was detected by ELISA-double antibody sandwich method.According to the test results,the content of lipase was increased by adding oils.It provides reference for the study of the mechanism of oils in fermentation.