Internal promoters of the epothilone biosynthetic gene cluster and their activation in Myxococcus xanthus by CRISPR-dCas9

Abstract Background Multiple genes involving in a complex pathway are often clustered into a giant operon with no transcription terminator before the end, and this leads to frangibility of the transcriptional process and arduous engineering work to control the transcription of operon genes. Internal promoters might occur in operon to coordinate the transcription of individual genes, but their effects on the transcription of operon genes have been less investigated. Results Epothilones are a kind of polyketides synthesized by seven multifunctional enzymes, which are encoded by a 56-kb operon of the myxobacterium Sorangium cellulosum. In this study, we determined that the epothilone operon contained multiple internal promoters. These promoters were activatable by the CRISPRa technique, and the yields of epothilones were accordingly increased. However, the activation efficiencies of promoters in operon and separate forms were greatly different. Further, we found that the transcriptional levels of the epothilone genes were always increased at a greater extent than the epothilone yields, which suggested that the transcriptional activation of single genes probably had a weak effect on the final epothilone yield, and higher yield required an overall transcriptional increase of the multiple operonic genes. Finally, we combined the activation of the starting promoter PepoA together with internal promoters in different epothilone-producing strains, and obtained the highest 15-fold increase of epothilone yield in Myxococcus xanthus ZE5. Conclusions This is the first time to report the internal promoters in epothilone gene clusters in Myxococcus xanthus and the first time to assay the activation effects of these internal promoters by CRISPR-dCas9. Our results highlight that tuning internal promoter activities is critical to control the transcription of operon genes and the production efficiency of microbial secondary metabolites.