The Molecular Regulation Mechanism Of Chlortetracycline Biosynthesis

Chlortetracycline was isolated from soil microorganismStreptomyces aureofacines by Duggar in 1948.Chlortetracycline is a broad-spectrum antibiotic andis the first generation natural antibiotic in the tetracycline family.It is function with the bacterial 30S ribosomal subunit by specifically binding to aminoacyl—tRNA,which can prevent the extension of the peptide chain to inhibit bacterial protein synthesis.Thus chlortetracycline plays an important role in animal feeds.On the basis of chlortetracycline biosynthetic studies,we found regulate gene ctcS,ctcB and ctcA.The homologousanalysis shows that they aresimilar to oxyTA1,dacT1 and dacT3,respectively.However,their function in the chlortetracycline biosynthesis is still unclear.The MarR family transcriptional regulator ctcS gene disrupted Streptomyces aureofaciens F3 was constructed by double crossover recombination.The distruption mutant LJIA03 produced less tetracycline and chlortetracycline,which was restored by in complementation of the ctcS gene.The amplicons of RT-PCR were designed to covering of the adjacent genes for verification of the operons in chlortetracycline biosynthetic cluster.We found six transcription units that can reduce the work to confirm the target gene.Quantitative realtime RT-PCR indicatedthat the transcriptionsof efflux genes were higher than thatin the wild type.EMSA experiments confirmed CtcS binds to the intergenic region between ctcR-ctcS,which meansfunction on the gene ctcR encoding efflux proteins.DNase I Footprinting experiments found twotarget binding sites,suggesting that CtcS is a dimer to keep its binding activity.The SARP family transcriptional regulator ctcB gene disrupted Streptomyces aureofaciens F3 was constructed by double crossover recombination.Tetracycline and chlortetracycline are abolished in the fermentation of LJIA02.Quantitative realtime RT-PCR indicated thatctcB was directly activate five promoters from ctcG-D,ctcH-K,ctcN-P,ctcW-T and ctcQ.According to the characteristics of the SARP binding sites,we analyzed the prediction CtcB binding regions of ctcG-D,ctcQ,ctcN-P and ctcT-W.There are conserved tandem repeats,which show the active structure needs two copy of CtcB.However,we found a sequence besides the-10region of ctcH-K.Thesequence is different from the binding regions we predicted,which may result in differenttranscription.We confirmed thatCtcBis an essential activator as aSARP family transcriptional regulatorin thechlortetracycline biosynthetic gene cluster.The LuxR family transcriptional regulator ctcA gene disrupted Streptomyces aureofaciens F3 was constructed by double crossover recombination.The disruption mutant LJIA01 lost the ability to produce tetracycline and chlortetracycline.Quantitative realtime RT-PCR indicated thatctcAcan activate the biosynthetic genesof chlortetracycline.In order to confirm whetherCtcA directlyfunction on the synthetic gene or not,we made the heterologous expression of CtcA.N-His₆-Tag CtcA was successfully purificated.In conclusion,by using combination of in vivogene deletions,in silico analysis,quantitative realtime RT-PCR analysis of genetranscripts from chlortetracycline biosynthetic pathwayand in vitrobiochemical analysisof protein-DNA binding by electrophoretic mobility shift assay and DNase I footprint assay,revealedthree genes-ctcSctcB and ctcAas positive regulator were associated with chlortetracycline biosynthesis in Streptomyces aureofaciens F3.Theseinvestigationsnot only expand the understanding of chlortetracycline biosynthesis mechanism,but also provided a foundation for improving chlortetracycline productionin Streptomyces aureofaciens F3by genetic engineering.