Prediction,Identification And Transcriptional Regulatory Mechanism Of The Internal Promoters From The Epothilone Operon In Myxococcus Xanthus

Epothilones are a class of macrolide compound produced by Sorangium cellulosum,the epothilone gene cluster is 56 kb in length and only has a terminator at the end.Theoretically,it will be transcribed into a huge mRNA,which also points out the fragility of the gene cluster transcription process and the difficulty in regulating the balance of transcription of multiple genes.Therefore,internal promoters may be present to coordinate the transcription of different genes.There also be mutual regulation between multiple internal promoters.In order to answer this question,this paper discussed the transcriptional regulation of internal promoters from the epothilone operon,and revealed the rules and partial mechanisms of mutual regulation between internal promoters.Our results provide a reference for the research on transcriptional regulation of large metabolite gene clusters and the design of gene circuits with promoters as core elements.In this paper,we predicted the presence of at least six internal promoters from the epothilone gene cluster in Myxococcus xanthus.Then we constructed a promoter activity detection vector in E.coli.We detected the transcriptional activity of these internal promoters by CAT ELISA,which further verified the existence of these internal promoters.Moreover,we activated these internal promoters separately by CRISPRa in E.coli.and successfully increased the expression level of the downstream reporter gene.Subsequently,we applied this CRISPRa system to Myxococcus xanthus.We activated 6 internal promoters separately,and then selected the sites with better activation effect to conduct combined activation experiments.At the same time,we selected multiple epothilone-producing strains for experiments.The date showed that the strain ZE5-DEF exhibited the best activation effect:the transcription level of the epothilone gene cluster was increased by 3-50 times,and the yield of epothilones was increased 15-fold,from the original 0.31mg/L to 4.42mg/L.More importantly,we analyzed the transcriptional map of the entire gene cluster and found that in the non-activation site,the transcription of the gene has also changed.The above results all showed that the transcriptional regulation of internal promoters is crucial for biosynthetic pathways of secondary metabolites encoded by a big operon,and even the transcriptional activity of a single internal promoter can significantly change the entire transcriptional map of gene cluster.This result has important guiding significance for the research of transcriptional regulation of large secondary metabolite gene clusters and the optimization of product yield.In above experiments,we also found that these internal promoters have differences in activity between the operon form and the independent working form,and also show different trends after being activated by CRISPRa.Combined with our previous literature survey,we speculated that the promoter is more complexly regulated in the operon,and there is mutual regulation between multiple tandem promoters.Therefore,we simulated the structure of the internal promoters in the epothilone gene cluster and established a tandem double-promoter gene switch model.We selected two inducible promoters and two fluorescent protein reporter genes to form a dual-promoter and dual-gene operon.The switch and activity of the promoters are controlled by the amount of the inducer,and the expression levels of the report genes were analyzed.Finally,we found the regulation rules in the operon with two internal promoters:when the upstream promoter is low in activity,it will block the downstream gene,and when the upstream promoter is in high activation,the inhibitory effect can be relieved,and even the downstream gene can be enhanced;At the same time,under the premise that the upstream promoter works,a high active downstream promoter also enhances the expression of upstream genes.Subsequently,we also further explored the mechanism of this regulatory mode.We screened multiple influencing factors,and finally found one of the key elements.The results showed that the topology of the DNA double helix is one of the key factors causing the mutual influence of tandem promoters.In the de novo design of complex circuits for synthetic biology,the transcriptional regulation of promoter is of key significance,given that it is one of the essential elements for the construction of operon.Our experimental results have important reference value for scientific and rational design of synthetic biology.