The Regulatory Mechanism Of A TetR Family Regulator GdmR? In The Biosynthesis Of Geldanamycin And Elaiophylin

Geldanamycin is a benzoquinone ansamycin antibiotic with strong anti-tumor activity,and elaiophylin is a macrolide antibiotic that possesses good antibacterial activity.These two antibiotics are co-produced in several Streptomyces strains.However,the regulatory mechanism of their biosynthesis is not fully understood yet,nor the relationship between these two biosynthetic pathways.Herein,the function of a TetR family regulator GdmRIII,located in the biosynthetic gene cluster of geldanamycin,was studied to understand the regulatory mechanism of geldanamycin and elaiophylin biosynthesis in Streptomyces autolyticus CGMCC0516.The study would lay a foundation for further improving the production of geldanamycin and elaiophylin through genetic engineering.In order to understand the function of GdmRIII,a ?gdR? mutant was constructed by using an one-step PCR method,and its fermentation products were analyzed.In the AgdmR?mutant,the yield of geldanamycin decreased significantly,while the yield of other three compounds greatly increased.The purification and structural illustration of these compounds showed that they were elaiophylin and its analogues,which implied that GdmRIII not only played a positive regulatory role in the biosynthesis of geldanamycin,but also played a negative role in the elaiophylin biosynthesis.To find the target gene(s)regulated by GdmRIII,reverse transcription PCR and real-time PCR were used to analyze the regulation of GdmRIII on the transcription of genes in the geldanamycin and the elaiophylin biosynthesis pathway.It was found that GdmRIII affected the expression of gdmAI,gdmF,gdmM,gdmN,gdmH,gdmK,gdmP,gdmRI,and gdmR? in the geldanamycin biosynthesis gene cluster and elaE,elaF,elaG,elal,and elaO in the elaiophylin biosynthesis gene cluster.Thus,GdmRIII regulated the biosynthesis of geldanamycin and elaiophylin by directly or indirectly affecting the expression of these genes.The GdmRIII was expressed in Escherichia coli and purified.The probable binding sites of GdmRIII in the promoter regions of genes whose expression was affected by GdmRIII were analyzed by EMSA,the result showed that GdmRIII might directly regulate the tracscription of gdmN and elaF by binding to their promoter regions.Further analyses by using DNase ? foot-printing showed that there was a conserved sequence"5 '-ATNGAGGNC-3'" in the binding sites,however no palindromic sequence that often exists in the binding sites of such kind of regulatory proteins was found among the conserved binding sites.The complete genomic sequence of S.autolyticus CGMCC 0516 was obtained by using the Illumina HiSeq 4000 sequencing platform and the Pacbio RS? platform,which consists of a 10,029,028 bp linear chromosome and seven circular plasmids.The geldanamycin,autolytimycin,and reblastatin biosynthetic gene clusters were located on the left arm(2.06 ?2.15 Mb)of the chromosome,and the elaiophylin gene cluster was located on the right arm(9.45-9.53 Mb).In the whole genome,fifty-seven putative biosynthetic gene clusters for secondary metabolites were predicted.Among these gene clusters,tewelve putative gene clusters showed high similarity to important antibiotic biosynthetic gene clusters,nine showed moderate similarity,twenty-six showed low similarity,and ten were not similar to any known gene clusters.The presence of these cryptic biosynthetic gene clusters for secondary metabolites implies that S.autolyticus CGMCC0516 has the potential to produce new antibiotics.To further understand the global regulatory role of GdmR? in S.autolyticus CGMCC0516,transcriptome analyses of the wild-type strain and the ?gdmR? mutant were performed.The results showed that 73%of the genes whose expression substantially changed in the ?gdmR? mutant when compared to that in the wild-type strain were enriched in metabolic pathways,with the highest proportion in the biosynthetic pathways for secondary metabolites and pathways for metabolism in different environment.This is consistent with the fact that S.autolyticus CGMCC 0516 mainly produces geldanamycin and elaiophylin during fermentation.By analyzing the 100 genes whose expression most changed,we found that they were mainly located in seven secondary metabolite biosynthetic gene clusters,including echosides,hygrocin,meridamycin,galbonolides,bafilomycin,merochlorin and lankacidin.These results showed that there are other biosynthetic gene clusters for secondary metabolites expressed in S.autolyticus CGMCC 0516,and it has the potential to produce these antibiotics and their derivatives.Furthermore,GdmRIII might also affected the biosynthesis of these antibiotics.In summary,GdmRIII could simultaneously regulate the biosynthesis of geldanamycin and elaiophylin,in which it has opposite effects.This study not only helps us to further understand the complex regulatory mechanism of biosynthesis of secondary metabolites in Streptomyces,but also provides a basis for improving the production of geldanamycin and elaiophylin through subsequent genetic engineering.At the same time,the ample biosynthetic gene clusters for sencondary metabolites found in S.autolyticus CGMCC0516 provides a theoretical basis for futher mining more natural products from this microorganism.