Preliminary Study On The Mechanism Of A Class Of Novel Regulators Involved In The Biosynthesis Of Benzoisochramquino Antibiotics

BIQ antibiotics are a class of aromatic polyketides produced by Streptomyces,possess a variety of biological activities and include medermycin(anti-tumor activity),well-studied actinorhodin(anti-bacterial activity)and so on.They are featured with a core skeleton structure composed of two aromatic rings and a chiral pyran ring.They are proposed to share a common biosynthetic pathway for the formation of their core skeletons.Medermycin and actinorhodin share a chiral pyran ring with same configurations(3S,15R).According to the similarity search,med-ORF29 in the medermycin gene cluster was proposed to encode an enoyl-reductase,involved in the formation of C15 chiral center in medermycin.Another gene,med-ORF10,is a high homologue to actVI-ORFA,a positive regulator gene for the biosynthesis of actonorhodin.The present study aimed to investigate the roles of med-ORF 10,actVI-ORFA and med-ORF29 in BIQ pathways.1)Comparative proteomics analysis between the wild type strain and actVI-ORFA-deficient strainTotal proteins isolated from the liquid cultures of the wild-type S.coelicolor and actVI-ORFA deficient mutant,respectively,were subject for two-dimensional electrophoresis(2D-PAGE).Quantitative analysis of 2D-PAGE showed that there were nearly 300 proteins showing remarkable difference at the translational level between these two strains,167 of which were up-regulated and 130 were down-regulated.These data implied that actVI-ORFA might not be a pathway-specific regulator,but a pleiotropic or global regulator.Subsequent Peptide Mass Fingerprinting(PMF)analysis revealed two proteins(actVI-ORFA and actVI-ORF4)involved in actinorhodin biosynthesis and others identified by PMF are not encoded by the actinorhodin gene cluster.At the translational level,actVI-ORFA was not expressed in the actVI-ORFA deficient strain as expectation,while actVI-ORF4 were down-regulated by 2.6 fold in this mutant strain,compared to the wild type strain.2)RT-PCR analysis of target genes in the actinorhodin gene cluster regulated by ActVI-ORFA.Since actVI-ORF4 and predicted target gene actVI-ORF1 belong to the same transcription unit,we designed primers for RT-PCR analysis of actVI-ORF4 and actVI-ORF1,aiming to compare their expression at transcriptional levels between wild type and mutant strains.After total RNAs were extracted from liquid-cultured wild-type S.coelicolor and actVI-ORFA deficient mutant strains,RT-PCR analysis was performed.Our data showed the expression of actVI-ORF4 and actVI-ORFl in actVI-ORFA deficient mutant strain was downregulated by about 75 and 65 folds respectively,compared to that in the wild-type S.coelicolor strain.Both proteomics and RT-PCR data showed that actVI-ORP1 and actVI-ORF4 are most likely the target genes of actVI-ORFA.3)Function study on med-ORF29 in the gene cluster of medermycin.Firstly,we constructed a plasmid pHSL143 for knockout of gene med-ORF29.Then,we introduced it into the wild-type medermycin-producer via conjugation.After mangy trials for screening knockout mutant strains,however,we have not obtained expected strain,while a mutant strain(LL-2)always was obtained.We suspected the presence of other homologous gene or another copy of med-ORF29)in the genome(,are preferentially knocked.HPLC analysis of the mutant LL-2 culture revealed that the yield of medermycin remained unchanged while other compounds were accumulated remarkably.Then,we conducted overexpression experiments of the med-ORF29:The gene med-ORF29 was amplified and cloned into the integration plasmid pIJ8600.The resultant plasmid pHSL144 was introduced into the wild-type strain.The fermentation and detection of target compounds of this strain are under the way.4)Study on the relationship between Med-ORF29 and med-ORF10med-ORF10 acted as a regulator involved in the medermycin biosynthesis.Here,we intended to determine whether the expression of med-ORRF29 is controlled by Med-ORF10.The prokaryotic expression plasmid for Med-ORF29 expression was constructed.After introducing it into E.coli BL21(DE3),we conducted induction expression and protein purification.Subsequently,we used the purified protein Med-ORF29 to inject rabbits for polyclonal antibody preparation.After harvesting polyclonal antibody against Med-ORF29,we detected the expression of Med-ORF29 in the Streptomyces strains by western blot.Our data showed that in the med-ORF10 deficient strain,the expression of Med-ORF29 was down-regulated,while in the med-ORF10-overexpressing strain,the expression level of Med-ORF29 increased,suggesting that the expression of Med-ORF29 is controlled by the med-ORF10.