| Epothilones are kind of polyketone secondary metabolites with antitumor activity,and they are originally produced by some strains of Sorangium cellulosum.Epothilones mimicks the taxol in the polymerization of tubulin and disturbing the formation of spindle during mitosis to inhibit the growth of tumor cells.Compared with taxol,epothilones have smaller molecular weight,better water solubility,excellent cytotoxicity on multidrug-resistant tumor cells and can be fermentated for mass production,which make them the replacement products for taxol.As potential anti-cancer drugs,epothilones have attracted great attention of researchers from different fields,and become a hot cake in biological,chemical and clinical researches.Because of the low yield and low production rate,the cost of producing epothilones is pretty high,which makes epothilones one of the most expensive drugs in clinical anticancer drugs.In recent years,epothilones have been reported to be expressed successfully in different hosts,such as Streptomyces,Escherichia coli,Myxococcus xanthus and Burkholderiales.However,the production in heterologous hosts was far from that of S.cellulosum.As a host more proximal from S.cellulosum,the shorter doubling time,rich precursor supply and mature genetic operating system make M.xanthua friendly fermentation host for epothilones.In M.xanthus,it may be the most direct way to solve the low yield problem by improving the expression level of epothilone gene cluster.However,the international studies on the transcription of the gene cluster and its effect on the yield of epothilone are still in blank.In previous work,we transformed the epothilone gene cluster with flanking sequence into M.xanthus DZ2 to produce ZE strain and realize the efficient production of epothilones.With such an efficient host platform,we preliminary optimized the fermentation conditions of ZE strains and carried out a series of genetic modification study on the epothilone gene cluster and host to further improve epothilone yield.We firstly optimized the fermentation conditions of ZE strains and determined the optimum inoculation quantity and optimum fermentation period.Addition of methyl oleate in to the medium can not only promote the growth of strains,but also improve the transcription of the gene cluster and epothilone yield in ZE strain by about 10 times.To regulate the synthesis of epothilone more directly,we tried to seek the related regulatory genes.We studied an unknown gene adjacent to the epothilone gene cluster,whose transcription is found to be negatively correlated with the transcription of epothilone gene cluster and the final yield and thus named it as esi(epothilone synthesis inhibitor)gene.Combining bioinformatics analysis and EMSA experimental results,we concluded that esi can bind to the epothilone promoter to regulate the transcription of the whole gene cluster negatively,and the best binding site was a special 17-bp sequence downstream of the transcription start site.After knockout of the esi gene in ZE strain,the transcriptional levels of all epothilone genes were up-regulated at different levels,and the yield of epothilones was increased by about 38%.This is the first and only reported transcriptional regulatory factor for epothilone synthesis.Promoters are central to regulating genes' transcription,so we tried to replace the original epothilone promoter(Pepo)with a stronger promoter.We chose the promoters of two endogenous genes with high expressional level as the candidates.Using the green fluorescent protein gene(gfp)as a reporter,the PpilA and PgroEL1 showed much higher transcriptional activity than Pepo in E.coli.However,when replaced the Pepo with PpilA and PgroELi respectively,we were surprised to find that the epothilone production was decreased by 80%.In order to resolve the detailed mechanism,we analyzed the time patterns of three promoters expressing gfp gene and epothilone gene cluster in M.xanthus.The result showed that the Pepo is stable but less active during the early stage of incubation and functioned majorly after the exponential growth stage.While PpilA and PgroELi are highly efficient in the growth stage,but markedly decreased in the late growth stage.The low-high transcriptional mode of Pepo is consistent with the epothilone production,i.e.Pepo guides the epothilone gene expression majorly after the growth of cells,which is suggested to accumulate more epothilone products.Our research reveals a mechanism some strong promoters are inapposite to express secondary metabolites,and this suggests that in addition to the strength of the promoter,the time-pattern should also be considered as an important index in promoter modification to overexpress desired genes.During studying the transcription of epothilone gene cluster in ZE strain,we found that there are significant differences among the transcriptions of the seven constitutive genes.The genes with relatively low transcriptional level may be the rate-determining step in epothilone synthesis.Therefore,we also tried to insert extra promoters inside the epothilone gene cluster to improve the transcription level of different epothilone genes.Insertion of PpilA in front of epoB gene increased the trscriptional level of epoB,epoC and epoD by 8.5-fold,4.1-fold and 2.6-fold,respectively.The total yield of epothilones was improved by 34%.Insertion of Paph in the same place resulted in similar but leak influence.Besides,we noticed that insertion of extra promoter have an adverse affect on the transcription of upstream genes.The transcription of epoP-epoD were dramatically decreased by insertion of Paph in front of epoE gene.Accordingly,the production of epothilones declined by more than 90%.Anyway,this experiment demonstrated the feasibility of regulating the production of epothilone by coordinating the transcriptional levels of different genes,thereby seeking the rate-determining step in the epothilone synthesis.Except for promoter modification to improve epothilone production by increasing transcriptional level of epothilone gene cluster,we also modified the host by deactivating the synthesis of endogenous secondary metabolites which will compete for precursor and energy in M.xanthus to further excavate its potential to synthesize epothilones.According to the the analysis of genome sequence,there may be 24 secondary metabolic gene clusters in M.xanthus DZ2,among which 6 gene clusters have been studied and identified for the corresponding products.We made 5 gene cluster synthesizing products with relative higher yields a priority to deactivate by deleting the reported key genes and finally obtained 4 mutant strains.The similar growth curves of different mutants mean that inactivation of the selected secondary metabolic gene clusters had no significant effect on the growth of host.However,the yield of epothilones in two mutant strains increased significantly(increased by 34%and 19%),while decreased in the other two mutant strains(decreased by 61%and 80%).The above results indicated that inactivation of endogenous secondary metabolic gene clusters may promote or impede epothilone synthesis in M.xanthus,which reflects the complex secondary metabolic regulatory networks.There is still a large number of endogenous secondary metabolic genes remaining to be studied in the genome of host strain.Single inactivation of each gene cluster can help us to avoid adversely factors and screen out favourable factor that can be further accumulated by multi-inactivation of different gene clusters,which could also establish the preliminary experimental basis for construction of chassis host for epothilone production.In conclusion,after fermentation condition optimization of the ZE strain,we effectively improved the epothilone yield in M.xanthus by identification and deletion the negative transcriptional regulator,promoter modification of epothilone gene cluster and inactivation of endogenous secondary metabolic gene clusters.All three strategies could promote epothilone synthesis in varying degrees,and we expect that,by integrating different strategies,the promoting effect may be accumulated to realize the greatly improved of epothilone production in heterologous host,which may rival or even better than the original producers.The high-yielding heterologous expression strains can not only realize large-scale production of epothilones and creating huge economic value and social benefits,but also make the directional transformation of epothilone to develop novel anti-tumor drug feasible by genetically engineering. |
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