Analysis Of Extracellular Induced Mechanism Of Epothilone Biosynthesis

Firstly,we have successfully established a liquid co-culture method refer to the method from literature for research the interaction between microorganisms.We have screened 36 kinds of microorganisms by this co-culture method,only assisted microbial LDF#-actinomycetes 02 can promote the synthesis of epothilones by 6% to 9%,based on molecular biology identification of the microorganism identified as Streptomycs microflavus 5406.The use of DMSO,methanol and ethyl acetate were prepared by the actinomycetes extracts,and promoted by adding three kinds of experiments to explore the crude extract of epothilone synthesis.The results show that the methanol extracts a significant promotion effect.Further more,we find that in a range of different adding amount,about 0.02% to 0.3%,on the equivalent to promote epothilone synthesis.And the promotion of methanol and methanol crude extract are equivalent from the experiment of adding methanol and crude extract separately,that is the methanol as the induction of small molecules in the promotion of epothilone synthesis plays an important role.Through the screening of small molecules to the influence of epothilone synthesis,we find that the role of methanol on Sorangium cellulosum producing epothilone is relatively compared with other small molecule.We can exclude the role of methanol as carbon and energy sources of Sorangium cellulosum in growth and metabolism,because the amount of methanol is so little about 0.05%.It is more specific methanol as signaling molecules or some enzyme inducer changed sorangial one or a few metabolic pathways leading to the increase of epothilone A and B.Through the further optimization of the methanol addition time and concentration showed that add volume ratio 0.05% methanol before inoculation are the largest and most stable for epothilones synthesis,at the same time we found that the promotion of methanol on epothilones metabolism is related to the overall metabolic intensity and production capacity,enhancement of methanol is greater when the yield is higher up to about 40%,that is to say methanol has amplification effect to improve the yield of epothilones,this is more conducive to the epothilones yield greatly improved based on stable high yield production process has been identified.Adding of methanol has no significantly affect on the expression level of several key enzyme which are common in growth and metabolism of Sorangium cellulosum by RT-q PCR.Then,the analysis of transcriptome gene expression difference shows that adding of methanol affect the expression level of gene which are related to the pathway of epothilones synthesis,for the further analysis significance screening according to the conditions which |log2(fold change)| and FDR<0.05 indicates that only two gene,ATP-dependent chaperone Clp B and sodium-translocating pyrophosphatase,express significant up-regulation.The final result caused by methanol affect the two up-regulated in Sorangium cellulosum should be raise the leader peptide modification process of the signaling molecules of QS system,in order to strengthen the metabolic flux related to synthesis of epothilones,the specific data and the final conclusion needs to be verified by RT-q PCR.The data and conclusions obtained in this study will provide material and research direction for clarify the small molecules inducers on over-expression of epothilones synthesis in vitro.