The Regulation Of TetR Family Transcription Factors On The Biosynthesis Of Oligomycin In Streptomyces Pactum Act12

Stremptomyces pactum Act12 is is a pleiotropic biocontrol actinomycete,which has been prepared as a commercial bacterial agent.Whole-genome sequencing showed that this strain has abundant secondary metabolite biosynthetic gene clusters.Studies have shown that the secondary metabolism of Streptomyces is regulated by multiple levels,such as single-component signal regulation system and two-component signal regulation system.An important family of single-component signaling systems?Tet R family transcriptional regulators(TFRs),widely regulate various physiological processes,such as antibiotic biosynthesis,drug efflux,quorum sensing,and primary metabolism.spa7074 and spa0520 are two genes encoding Tet R family transcription factors in Stremptomyces pactum Act12.The production of oligomycin in the fermentation broth of the deletion mutants MU7074 and MU0520 is greatly increased,and the inhibitory effect on some common plant pathogenic fungi is significantly enhanced.To clarify the molecular mechanism of Spa7074 and Spa0520 regulating the biosynthesis of oligomycin is helpful for further metabolic engineering of Act12 bacterial agents,which has good theoretical significance and application value.Existing research on the regulation mechanism of TFRs shows that the ligands of TFRs are diverse,and the most direct way to regulate the synthesis of antibiotics is to combine with the promoter region in the antibiotic biosynthesis gene cluster.So in this experiment,the sequence characteristics of Spa7074 and Spa0520 and the structural characteristics of oligomycin biosynthesis gene clusters were analyzed,and the prokaryotic expression system of spa7074 and spa0520 was constructed.The in vitro binding experiment?Electrophoretic mobility shift assay(EMSA)was used to explore the specific binding promoter sequences of Spa7074 and Spa0520 in the oligomycin biosynthesis gene cluster.In addition,RNA-seq data analysis was performed on strain Act12,deletion mutant strains MU7074 and MU0520,and overexpression strain OE7074 of spa7074 to further explore the regulatory mechanism of oligomycin biosynthesis.Mainly made the following research progress:1.Constructed the E.coli expression system of spa7074 and spa0520,induced expression and purification at low temperature,and obtained high purity and high activity transcription factors;2.Using anti SMASH to predict the entire genome of strain Act12,a total of 30 secondary metabolite gene clusters were obtained.Analyzed the oligomycin biosynthesis gene clusters,and predicted the promoter sequences that Spa7074 and Spa0520 may bind.EMSA experiments showed that Spa7074 and Spa0520 cannot specifically bind to possible promoter sequences on the oligomycin biosynthesis gene cluster,but Spa7074 can specifically bind to its own promoter region sequence to inhibit the production of oligomycin;3.The spa235 gene encodes a cyclic nucleotide binding domain protein,and the expression level in MU7074 and MU0520 is lower than Act12,and Spa7074 and Spa0520 can specifically bind to the promoter sequence of spa235.It was speculated that Spa7074 and Spa0520 indirectly regulate the synthesis of oligomycin by regulating the synthesis of Spa235;4.Based on RNA-seq sequencing technology,it is found that there are Tet R family transcription factors and ABC transporters upstream of the oligomycin synthesis gene cluster,which provided new ideas and research directions for the subsequent further study of the regulation mechanism of oligomycin biosynthesis.