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Transcriptional profile asm gene cluster of high Ansamitocin P3 producing mutants derived from A. pretiosum

ABSTRACT: 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

ORGANISM(S): Actinosynnema pretiosum

PROVIDER: GSE12739 | GEO | 2008/12/01

SECONDARY ACCESSION(S): PRJNA111045

REPOSITORIES: GEO

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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:In filamentous fungi, secondary metabolism is often linked with developmental processes such as conidiation. In this study we analyzed the link between secondary metabolism and conidiation in the main industrial producer of the β-lactam antibiotic penicillin, the ascomycete Penicillium chrysogenum. Therefore, we generated mutants defective in two central regulators of conidiation, the transcription factors BrlA and StuA, respectively. Inactivation of both BrlA and StuA blocked conidiation and altered hyphal morphology during growth on solid media, as shown by light and scanning electron microscopy, but did not affect biomass production during liquid submerged growth. Genome-wide transcriptional profiling identified a complex StuA- and BrlA-dependent regulatory network, including genes previously shown to be involved in development and secondary metabolism. Remarkably, inactivation of StuA, but not BrlA, drastically down-regulated expression of the penicillin biosynthetic gene cluster during solid and liquid submerged growth. In agreement, penicillin V production was wild type-like in BrlA-deficient strains but 99 % decreased in StuA-deficient strains during liquid submerged growth as shown by HPLC analysis. Thus, among identified regulators of penicillin V production StuA has the most severe influence. Over-expression of StuA increased the transcript levels of BrlA and AbaA (another developmental regulator), de-repressed conidiation during liquid submerged growth, but did not affect penicillin V productivity. Taken together, these data demonstrate an intimate but not exclusive link between regulation of development and secondary metabolism in P. chrysogenum. Transcriptomes of PcbrlA- and PcstuA- deletion mutants were compared with expression data from recipient strain deltaPcku70 as a control Mycelia from the transformants and the reference strain were harvested at successive stages of development for RNA extraction and hybridization on Affymetrix microarrays.

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Transcriptional profile asm gene cluster of high Ansamitocin P3 producing mutants derived from A. pretiosum

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