Regulation Of The Biosynthesis Of Secondary Metabolite In Streptomyces

Streptomyces spp.are a large group of Gram-positive,well known to produce variety of secondary metabolites,many of which are antibiotics,antiturmors and immunosurpressors.The genes responsible for biosynthesis of secondary metabolites are clustered in genome of Streptomyces spp.Streptomyces hygroscopicus 10-22 was characterized to produce cyclothiazomycin,a kind of thiopeptide antibiotic,and the gene cluster was identified.The genome of S.hygroscopicus 5008 which is a close relative of 10-22,was sequenced recently.A cluster was found identical to that responsible for the biosynthesis of cyclothiazomycin in S.hygroscopicus 10-22.Streptomyces sp.FR008 was found to produce a polyketide-FR-008.The gene cluster of FR-008 was also identified.There are several regulatory genes in gene clusters of cyclothiazomycin or FR-008.In order to determine the regulation of cyclothiazomycin and FR008 biosynthesis,our study was focus on the functions of regulators in gene clusters responsible for cyclothiazomycin and FR-008.I Regulation of cyclothiazomycin biosynthesisThe genome of S.hygroscopicus 5008 was sequenced recently.A cluster was found identical to that responsible for the biosynthesis of cyclothiazomycin in S.hygroscopicus 10-22,suggesting that S.hygroscopicus 5008 has the potential to produce cyclothiazomycin.Bioassay,HPLC and MS were performed to analyze the production of cyclothiazomycin.The results showed that S.hygroscopicus 5008 can produce cyclothiazomycin and the gene cluster of S.hygroscopicus 5008 is functional.Three genes predicted to encode regulators of different families were identified in cyclothiazomycin cluster of S.hygroscopicus 5008.SHJG8833 has its counterpart(cltH)in 10-22 and it was deduced to encode a large transcriptional regulatory proteins containing a N-terminal AAA ATPase domain and a C-terminal LuxR-like HTH DNA-binding domain;SHJG8837 also has its counterpart(cltP)in 10-22 and it is predicted to encode a protein which has a N-terminal region homologous with members of the HTH-XRE family of transcriptional regulators;SHJG8838 which only exists in 5008 has a C-terminal motif characteristic of the HTH-AraC family of transcriptional regulators.To determine if the three putative proteins regulate the production of cyclothiazomycin in 5008,we generated in-frame deletions for SHJG8833,SHJG8837,and SHJG8838.The mutant strains were designated ?8833,?8837 and A8838.Production of cyclothiazomycin by three mutant strains were tested.The results indicated that A8833 was fail to synthesize cyclothiazomycin,suggesting that SHJG8833 is essential for the biosynthesis of cyclothiazomycin;in contrast,SHJG8837 or SHJG8838 is not required for cyclothiazomycin biosynthesis.Analyses of the transcription level of cyclothiazomycin cluster showed that SHJG8833 regulates the expression of seven structural genes SHJG8826-32.RT-PCR was also performed to determine if genes are cotranscribed and the results indicated that SHJG8826-27,SHJG8828-32 may form two operons.The DNA-binding domain of SHJG8833 was found to bind the promoters of SHJG8826,SHJG8827,SHJG8828 and SHJG8830 and the putative binding sites were CCCGNNNCCNGNN.Proposed pathway for regulation of cyclothiazomycin by SHJG8833 was deduced:SHJG8833 binds to the promoters of SHJG8827 and SHJG8828 to activate the transcription of SHJG8826-27,SHJG8828-32 opreon and regulate the production of cyclothiazomycin.? Regulation of FR-008 biosynthesisFR-008 is a heptaene macrolide antibiotic produced by Streptomyces sp.FR-008.The complete gene cluster responsible for FR-008 in Streptomyces sp.FR-008 was characterized.Four genes(fscR?,fscR?,fscR? and fscR?),encoding putativeregulators might be involved in the biosynthesis of FR-008.fscR? was deduced to encode a regulatory protein containing a N-terminal PAS domain and a C-terminal LuxR family HTH DNA binding domain.The proteins encoded by fscR?,fscR?,fscRIV belong to LAL family as SHJG8833 which containing a N-terminal AAA ATPase domain and a C-terminal LuxR family HTH DNA binding domain.fscRI was in-frame deleted to generate AfscR1.Bioassay and HPLC analyses were performed to analyze the production of FR-008 by mutant strain.The results showed that AfscR1 abrogated the production of FR-008,suggesting that fscR1 is essential for the biosynthesis of FR-008.RT-PCR and Real-time PCR were performed to analyze the transcription level of genes in FR-008 cluster.All the structural genes were down-regulated except fscO and pabC in AfscR?;the regulatory gene fscRIV was also down-regulated,but no transcriptional changes of fscR?,fscR? and fscR?were observed.EMSAs were performed to test the target genes of FscRI.The results showed that FscRI can bind to the promoter of fscR?/MI,fscPabAB,fscA,fscB,fscD.We found the same conserved binding sites on the promoter of fscR?/NI,fscA,fscB,fscD but not pabAB,and the EMSAs confirmed that FscRI binds the sites(CTVGGGAWWTCCCBAG)specifically.FscRI was found to regulate the expression of fscRIV.fscR? was deleted to generate AfscRIV.The production of FR-008 in AfscRIV was decreased,compared to that in wild type strain and the complemented strain,suggesting that fscRIV is a positive regulator.RT-PCR and Real-time PCR were performed to analyze the transcriptional levels of genes in FR-008 cluster.The results indicated that all the structural genes but fscO were regulated by FscRIV;fscR? was also regulated by FscRIV,but there were no influence on the expression of fscR? or fscR?.The other two regulatory genes-fscP?,fscR?-were deleted to generate ?fscR?,?fscR?.The production of FR-008 by two mutant stains were lost.We deduced that fscR? or fscR? is crucial for the biosynthesis of FR-008.Real-time PCR was carried out to analyze the transcriptional levels of genes in FR-008 cluster.The results indicated that all the structural genes but fscO were regulated by FscRII.All the structural genes were regulated by FscRIII,and fscE,fscD,fscM? were regulated dramatically,implying these genes are target of FscRIII.The regulatory patterns of other genes by FscR? were close to that of FscRII and FscRIV,which is less than FscRI.FscR?,FscR? and FscRIV regulate the transcription of fscR?,suggesting that they may regulate the structural genes by FscRI.In addition,FscRII regulates the expression of fscR? negatively and FscR? also regulate the expression of fscR?negatively.Above all,it is supposed that FscR?,FscR?,FscR? and FscR? involve in a complex regulatory network to control the production of FR-008.