Effect Of Promoter Tandem And Overexpression Of Key Genes On The Production Of Epothilone In Heterologous Expression Strains

Myxobacteria is a gram-negative bacterium with capability of superior secondary metabolite synthesis,considered as an important source for the development of new synthetic drugs.Sorangium cellulosum belongs to the suborder of Myxococcus which utilize cellulose from nature as a carbon source to meet its own growth needs.Due to production of several biologically active secondary metabolites,S.cellulosum become point of interest in research field.S.cellulosum produces epothilone that has antitumor activity and cytotoxicity.However,the long-term generation and unclear genetic background of Sorangium cellulosum,it is difficult to do the genetic modification.Therefore,heterologous expression of secondary metabolic gene clusters is a better method to solve these problems.M.xanthus DZ2 has a similar genetic background with S.cellulosum.In our laboratory,the epothilone gene cluster and its upstream and downstream sequences were randomly inserted into the M.xanthus DZ2 strain,resulting in ZE strains.But the highest yield of ZE strains was only 1 mg/L.Therefore,we are trying to improve the epothilone production by genetic engineering methods.Up to now,there are few reports about the modification of the epothilone promoter.But still there is lack of research to improve epothilone production by selecting and designing effective promoters.Firstly,based on the prediction and analysis results of the epothilone gene cluster promoter in the early stage of the laboratory,we constructed 1-3 fragments of the core region(EP and SP),and detected by using the GFP reporter gene.The results showed that the activity of both the EP promoter clusters and SP promoter clusters showed an upward trend with the increase of the number of tandem promoters.Afterwards,we inserted the promoter clusters ahead of epothilone native promoter in the heterologous expression strains ZE9,ZE10 and KE17,to detect the effect of epothilone production in the expression strain.Finally,we obtained 10 mutants,and the production of epothilones in the 9-1SP,9-1EP,and 10-3SP strains increased by 13%,72%,and 113%,respectively.The highest yield of 10-3SP is as high as about 20 mg/L,especially.In addition,wo also proved that substrate supply is an important for the biosynthesis of epothilones.Acyl-CoA synthetase(ACS)and the S-adenosylmethionine synthetase(SAMs)are the substrates for the synthesis of epothilones.We overexpressed the ACS and the SAMs to increase the two substrates in Myxococcus.Results showed that the production of epothilone increased synchronously with the corresponding over-expressed enzyme in most strains,except 10-3SP-19-ACS strain which is abnormal in growth.This result also further proved that ACS and SAMs may play an important role in promoting the biosynthesis of epothilone.Overexpression of this two genes can increase the production of epothilone.