Project description:This series of microarry is the study of the transcriptional profile asm gene clusster of high Ansamitocin P3 producing mutants derived from isolating rifampicin resistant mutants. Background The Actinomycete Actinosynnema pretiosum ssp. auranticum has commercial importance due to its production of ansamitocin P-3 (AP-3), a potent antitumor agent. One method to improve the productivity of A. pretiosum is to genetically alter the regulation of ansamitocin biosynthesis by manipulating selected genes. To identify potential targets for genetic engineering, the transcriptional profile of an A. pretiosum mutant with enhanced AP-3 yield was compared to the parental strain. High-producing mutants were isolated from rifampicin-resistance screens using a plate-based bioassay. Transcriptional profiling of genes in the ansamitocin biosynthetic cluster was carried out using a custom-designed A. pretiosum microarray and verified with quantitative RT-PCR. Results Genes involved in the synthesis of the AP-3 precursor, 3-amino-5-hydroxybenzoic acid (AHBA), including asm43, asm45 and asm47, were significantly up-regulated (P-value<0.05) by more than two-fold in the high-producing mutant relative to the parental strain. In addition, genes involved in polyketide synthesis (asmB and asmD), bicyclomycin resistance (asm35) and transcriptional regulation (asm29) were up-regulated, while the transcriptional repressor, asm2, was down-regulated. Interestingly, the two AHBA synthase gene homologues, asm24 and asm43, had divergent expression profiles despite their strong sequence similarity and functional complementarities. The rifampicin-resistance mutation associated with increased AP-3 production was mapped to the gene encoding RNA polymerase beta subunit, rpoB. While distinct multiple mutations in RpoB were noted for each of the mutants sequenced, a common H436R mutation was identified in cluster I of rpoB in all four mutants sequenced. Conclusions The transcriptional analysis has provided useful insights into the mechanism underlying the increased AP-3 production in the high-producer mutant and expanded our understanding of the function and regulation of genes in the ansamitocin biosynthetic cluster. In addition, the differentially expressed genes identified are potential targets for genetic manipulation; alternatively, they can be used in reporter-based selections to isolate mutants with greater AP-3 productivity. Keywords: genetic modification, time course, boutique microarray, quantile normalization Microarray chip was hybdridized with cDNA obtained from samples vs sheared A. pretiosum gDNA. cDNA obtained from cultures of A. pretiosum (ATCC:31565) and r50D1 mutant were used. Cell pellets were harvested on days 2,4,6 and 8 of each culture.Three biological replicates were performed for each cell line with two technical replicates for each biological replicate:

Project description:This series of microarry is the study of the transcriptional profile asm gene clusster of high Ansamitocin P3 producing mutants derived from isolating rifampicin resistant mutants. Background The Actinomycete Actinosynnema pretiosum ssp. auranticum has commercial importance due to its production of ansamitocin P-3 (AP-3), a potent antitumor agent. One method to improve the productivity of A. pretiosum is to genetically alter the regulation of ansamitocin biosynthesis by manipulating selected genes. To identify potential targets for genetic engineering, the transcriptional profile of an A. pretiosum mutant with enhanced AP-3 yield was compared to the parental strain. High-producing mutants were isolated from rifampicin-resistance screens using a plate-based bioassay. Transcriptional profiling of genes in the ansamitocin biosynthetic cluster was carried out using a custom-designed A. pretiosum microarray and verified with quantitative RT-PCR. Results Genes involved in the synthesis of the AP-3 precursor, 3-amino-5-hydroxybenzoic acid (AHBA), including asm43, asm45 and asm47, were significantly up-regulated (P-value<0.05) by more than two-fold in the high-producing mutant relative to the parental strain. In addition, genes involved in polyketide synthesis (asmB and asmD), bicyclomycin resistance (asm35) and transcriptional regulation (asm29) were up-regulated, while the transcriptional repressor, asm2, was down-regulated. Interestingly, the two AHBA synthase gene homologues, asm24 and asm43, had divergent expression profiles despite their strong sequence similarity and functional complementarities. The rifampicin-resistance mutation associated with increased AP-3 production was mapped to the gene encoding RNA polymerase beta subunit, rpoB. While distinct multiple mutations in RpoB were noted for each of the mutants sequenced, a common H436R mutation was identified in cluster I of rpoB in all four mutants sequenced. Conclusions The transcriptional analysis has provided useful insights into the mechanism underlying the increased AP-3 production in the high-producer mutant and expanded our understanding of the function and regulation of genes in the ansamitocin biosynthetic cluster. In addition, the differentially expressed genes identified are potential targets for genetic manipulation; alternatively, they can be used in reporter-based selections to isolate mutants with greater AP-3 productivity. Keywords: genetic modification, time course, boutique microarray, quantile normalization

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Actinosynnema pretiosum

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