Research On The Mechanism Of MarR Regulated By SACE?Lrp In Saccharopolyspora Erythraea

Lrp family and Mar R family are two group of transcriptional regulators widely distributed among prokaryotes.However,the hierarchical-regulatory relationship between the Lrp family and the Mar R family remains unknown.Our previous study found that the Lrp family regulatory factor SACE?Lrp in Saccharopolyspora erythraea indirectly repressed the biosynthesis of erythromycin,but the specific metabolic regulatory mechanism is not very clear,so it is necessary to find the downstream target genes directly regulated by SACE?Lrp.In this paper,a downstream target gene SACE?6745 that is directly regulated by SACE?Lrp was mined by whole-genome alignment of homologous target sequences.The gene belongs to the Mar R family.In order to further analyze the regulation mechanism and function of this gene,firstly,we found that SACE?Lrp directly regulates the expression of mar R through the specific binding precise site OM(5'-CTCCGGGAACATT-3')through the in vitro EMSA experiment of site-directed mutagenesis probe.Meanwhile,the direct interaction between SACE?Lrp protein and P_{mar R} was verified by green fluorescence reporting system in vivo.Then,mar R gene was knocked out by large fragment homologous recombination technique in S.erythraea A226,and a complementary strain of mar R was constructed.In the analysis of erythromycin yield of?mar R,we found that mar R knockout increased erythromycin yield by 45%,but mar R deletion did not affect the biomass and morphological differentiation of S.erythraea.The q PCR results showed that the transcriptional level of erythromycin biosynthesis gene in?mar R,ery gene group was significantly increased by 1.7-4.6 times compared with A226 respectively.These results suggested that mar R could affect the yield of erythromycin by inhibiting the expression of erythromycin biosynthesis gene.Finally,by detecting the transcription level of SACE?2701-2702 in?mar R,constructing the mutant strain with deletion and overexpression of the gene SACE?2701-2702 and detecting the production of erythromycin in and out of the cell,it was found that mar R negatively regulates the expression of the antibiotic exogenous gene SACE?2701-2702.It was confirmed that ABC transporter SACE?2701-2702 was responsible for the cellular exocrine of erythromycin,and mar R regulated the production of erythromycin by coordinating the biosynthesis and excretion of erythromycin in cells.Furthermore,by expressing in vitro expressed Mar R protein and conducting EMSA experiments,we found that Mar R can directly bind to erythromycin biosynthesis gene and the promoter region of SACE?2701-2702.These results suggest that Mar R directly controls genes of erythromycin biosynthesis,export and resistance.In summary,this study characterized a novel Mar R family protein(SACE?6745)from S.erythraea,which is controlled by SACE?Lrp,and found that this regulatory factor plays a direct regulatory role in erythromycin biosynthesis,export and resistance.This study provides a new basis for revealing the cascade regulatory relationship between Lrp and Mar R proteins,and provides a new approach and method for coordinating antibiotic biosynthesis and transport in actinomycetes through the combined application of regulatory elements.