Targeted Activation Of The Oxytetracycline Cryptic Biosynthesis Gene Cluster In Streptomyces Rimosus M527

In previous research,the whole genome of Streptomyces rimosus M527 was sequenced,and comparison of genes（clusters）were analyzed and predicted.The result showed that the genome of S.rimosus M527 contained more than 40 biosynthetic gene clusters encoding secondary metabolites which were important sources for novel bioactive compounds and drug discovery.However,these gene clusters are not expressed or poorly expressed under normal laboratory conditions,which are called cryptic gene cluster.The key issue for development of novel bioactive compounds is effective activation of these cryptic gene clusters.Therefore,in this study a doublereporter-guided system was constructed and employed to targeted activation of the oxytetracycline（OTC）biosynthesis gene cluster in S.rimosus M527.This work will provide the basis for subsequent effective and widely applicable for targeted activation of other cryptic gene clusters encoding novel compounds.The main contents were performed as follows:Firstly,a biosynthesis gene cluster located in S.rimosus M527 genome showed a high homology identity（about 95%）with the known OTC gene cluster was found based on the bioinformation analysis.OTC,an aromatic polyketide,synthesized by bacterial type II polyketide synthases（PKS）,the minimal PKS is consist of genes oxyA,oxyB and oxyC that accepts a malonamyl-co A as starter unit and affords a poly-β-ketone backbone.The genes related to OTC biosynthesis were reported.Among them,four genes oxyA_sr,oxyC_sr,oxyDsr,and oxyPsr encoding ketosynthase,acyl carrier protein,amidotransferase,transamidase,respectively.Low transcription levels of these four genes were also confirmed by using semi-quantitative PCR,especially oxyA_sr gene.In addition,oxytetracycline in the fermentation broth was almost undetectable,and S.rimosus M527 showed no antagonistic effect activity against Escherichia.coli JM109 or Bacillus subtilis WB600,suggesting that the expression level of OTC biosynthesis gene cluster in S.rimosus M527 was very poor.The above results indicated that the OTC biosynthetic gene cluster is in silent.OTC,an aromatic polyketide,synthesized by bacterial type II polyketide synthases（PKS）,the minimal PKS is consist of genes oxyA,oxyB and oxyC that accepts a malonamyl-co A as starter unit and affords a poly-β-ketone backbone.The genes related to OTC biosynthesis were reported.Among them,four genes oxyA_sr,oxyC_sr,oxyDsr,and oxyPsr encoding ketosynthase,acyl carrier protein,amidotransferase,transamidase,respectively.Low transcription levels of these four genes were also confirmed by using semi-quantitative PCR,especially oxyA_sr gene.The above results indicated that the OTC biosynthetic gene cluster is in silent.Secondly,in order to activate the expression of OTC biosynthetic gene cluster,the native promoter of gene oxyA_sr named P_oxyA was cloned,and the reporter genes gus A（encoding β-glucuronidase）and tsr（encoding thiostrepton resistance gene）were placed under the control of promoter P_oxyA to generate double-reporter plasmid pAGT.Integration of the plasmid pAGT into genome of S.rimosus M527 by conjugation was performed to yield recombinant strain S.rimosus M527-pAGT.Then,rifampicin（Rif）was utilized for screening Rif resistant mutants of S.rimosus M527-pAGT by ribosome engineering technology.Based on the observation of dual phenotypes of Tsr resistance and GUS,the results indicated that seven strains have increased Tsr resistance levels of concentration from forty Rifr mutant strains,among them,five strains also showed GUS enzymatic activity（blue color）and antagonistic activity against E.coli and B.subtilis which were used as indicator strain.Finally,a Rifr mutant strain S.rimosus M527-pAGT-R7 showed the best performance on GUS enzymatic activity and antimicrobial activity.The cultured filtrate from the mutant S.rimosus M527-pAGT-R7 was subjected to HPLC analysis,and a distinct peak appeared at a position corresponding to that of the standard sample of OTC.Then this distinct peak was determined by MS,HPLC spectroscopic analyses,and was identified as OTC.Furthermore,sample and reaction stability,accuracy of the chromatographic detection method were evaluated after HPLC condition was optimized.Analysis and comparison of OTC production,cell growth and the relative transcriptional levels of oxygenes(oxyA_sr,oxyC_sr,oxyDsr,oxyPsr)between the Rifr mutant strain S.rimosus M527-pAGT-R7,control strain S.rimosus M527-pAGT and wild-type strain S.rimosus M527 were investigated.The fermentation results showed that the Rifr mutant strain M527-pAGT-R7 produced 10.2 times increase in OTC production（235.2 mg/L）comparing to that in control strain M527-pAGT.The q RTPCR results indicated that the relative transcription levels of oxygenes in the mutants M527-pAGT-R7 were significantly higher than those of S.rimosus M527-pAGT and S.rimosus M527.It is worth mentioning that transcription levels of oxyA_sr,oxyC_sr,oxyDsr genes were increased with the increased OTC production.In addition,there are no significant differences in cell growth of these three strains.In conclusion,in this study the double-reporter genes system was constructed and successfully applied to activate the OTC biosynthetic gene cluster,which will provide a reference for further activating other cryptic gene clusters in S.rimosus M527 in future.