Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Effects of manR disruption and overexpression on Aspergillus oryzae gene expression

ABSTRACT: Fungal endo-M-NM-2-mannanases (M-NM-2-mannanases) are widely used as industrial enzymes; however, no transcriptional regulator of M-NM-2-mannanases has been identified in fungi or other eukaryotic cells to date. To identify a transcriptional regulator of M-NM-2-mannanases in Aspergillus oryzae, a gene-disruptant library of transcriptional regulators was screened for mutants exhibiting reduced M-NM-2-mannanase activity by using konjac glucomannan as the substrate, and ManR, a Zn(II)2Cys6 type DNA binding protein was identified. Moreover, a manR-overexpressing strain showed significantly increased M-NM-2-mannanase activity. DNA microarray analysis of the manR-disruptant strain and the manR-overexpressing strain further indicated that when konjac glucomannan is used as the carbon source, ManR positively regulates the gene expression of not only M-NM-2-mannanase, but also the enzymes involved in the degradation of galactomannans and glucomannans such as M-NM-1-galactosidase, M-NM-2-mannosidase, acetylmannan esterase, and M-NM-2-glucosidase. Therefore, we conclude that ManR is a positive regulator of the M-NM-2-mannan utilization system in A. oryzae. manR disruptant, manR-overexpressing strain and A. oryzae RkuptrP2-1M-bM-^HM-^FAF/P (derivative of A. oryzae RIB40) were cultivated in minimal medium containing 1% konjac glucomannan as the sole carbon source. After 6 h cultivation, total RNAs from the mycelia were extracted, and DNA microarray analysis was carried out. The analysis of manR disruptant was conducted with 4 biological replications, the analysis of manR overexpressing strain was conducted with 3 biological replications.

ORGANISM(S): Aspergillus oryzae RIB40

SUBMITTER: Masahiro Ogawa

PROVIDER: E-GEOD-40590 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

ManR, a novel Zn(II)2Cys6 transcriptional activator, controls the β-mannan utilization system in Aspergillus oryzae.

Ogawa Masahiro M Kobayashi Tetsuo T Koyama Yasuji Y

Fungal genetics and biology : FG & B 20121011 12

Fungal endo-β-mannanases (β-mannanases) are widely used as industrial enzymes; however, no transcriptional regulator of β-mannanases has been identified in fungi or other eukaryotic cells to date. To identify a transcriptional regulator of β-mannanases in Aspergillus oryzae, a gene-disruptant library of transcriptional regulators was screened for mutants exhibiting reduced β-mannanase activity by using konjac glucomannan as the substrate, and ManR, a Zn(II)(2)Cys(6) type DNA binding protein was ...[more]

PMID: 23063954

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Project description:The potential for sexual reproduction in Aspergillus oryzae was assessed by investigating the presence and functionality of MAT genes. Previous genome studies had identified a MAT1-1 gene in the RIB40 reference strain. We now report the existence of a complimentary MAT1-2 gene and sequencing of an idiomorph region from A. oryzae strain AO6. This allowed the development of a PCR diagnostic assay, which detected isolates of MAT1-1 and MAT1-2 genotype among 180 strains assayed including industrial tane-koji isolates. Strains used for sake and miso production showed a near 1:1 ratio of MAT1-1 and MAT1-2 mating-types, whereas strains used for soy sauce production showed a significant bias towards the MAT1-2 mating type. MAT1-1 and MAT1-2 isogenic strains were then created by genetic manipulation of the resident idiomorph, and comparisons were made of gene expression by DNA microarray and RT-PCR methodologies under conditions when MAT genes were expressed. 33 genes were found to be up-regulated greater than 10-fold in either the MAT1-1 host or MAT1-2 gene replacement strains relative to each other, showing that both MAT1-1 and MAT1-2 genes functionally regulate gene expression in A. oryzae in a mating-type dependent manner, the first such report from a supposedly asexual fungus. MAT1-1 expression specifically up-regulated an a-pheromone precursor gene, but most genes affected were of unknown function. Results are consistent with a heterothallic breeding system in A. oryzae, and prospects for the discovery of a sexual cycle are discussed. On a condition that induces mating-type genes descrived in growth protocol section, each cogenic MAT1-1 and MAT1-2 mating-type strains were processed into RNA extraction and hybridization on Affymetrix microarrays. In a speculation that genes involved in putative mating process were reguated in mating-type dependent manner, we designed the cogenic strains that differs only in mating-type gene locus to analyze differential gene expression of MAT1-1 vs MAT1-2 strains.

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Dataset Information

Effects of manR disruption and overexpression on Aspergillus oryzae gene expression

Publications

ManR, a novel Zn(II)2Cys6 transcriptional activator, controls the β-mannan utilization system in Aspergillus oryzae.

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