Transcriptional regulation of genes on the non-syntenic blocks of Aspergillus oryzae and its functional relationship to solid-state cultivation

Tamano, Koichi; Sano, Motoaki; Yamane, Noriko; Terabayashi, Yasunobu; Toda, Takeshi; Sunagawa, Misao; Koike, Hideaki; Hatamoto, Osamu; Umitsuki, Genryou; Takahashi, Tadashi; Koyama, Yasuji; Asai, Ryoichi; Abe, Keietsu; Machida, Masayuki

doi:10.1016/j.fgb.2007.09.005

Cited by 43 publications

(36 citation statements)

References 24 publications

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“…This method assumes that all essential genes exist in homologous form in all genomes and all nonessential genes will be absent in one or more cases. Based on this theory, we focused mainly on NSBs, which were identified by comparison of the A. oryzae genome with those of A. fumigatus and A. nidulans (20). We predicted that the deletion of these A. oryzae-specific regions would have no significant impact on growth rate and other phenotypic changes.…”

Section: Discussionmentioning

confidence: 99%

“…We focused on chromosome 7 which contains a large number of nonsyntenic blocks (NSBs). These NSBs were detected by comparison of the A. oryzae genome with those of Aspergillus fumigatus and Aspergillus nidulans, and they contain abundant genes involved in secondary metabolism and A. oryzae-specific genes (20). Analysis of chromosome 7 deletions identified a novel gene that has been predicted to encode a basic helix-loop-helix (bHLH) protein on the basis of its conserved motif.…”

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Identification of a Basic Helix-Loop-Helix-Type Transcription Regulator Gene in Aspergillus oryzae by Systematically Deleting Large Chromosomal Segments

Jin

Takahashi

Machida

et al. 2009

Appl Environ Microbiol

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We previously developed two methods (loop-out and replacement-type recombination) for generating large-scale chromosomal deletions that can be applied to more effective chromosomal engineering in Aspergillus oryzae. In this study, the replacement-type method is used to systematically delete large chromosomal DNA segments to identify essential and nonessential regions in chromosome 7 (2.93 Mb), which is the smallest A. oryzae chromosome and contains a large number of nonsyntenic blocks. We constructed 12 mutants harboring deletions that spanned 16-to 150-kb segments of chromosome 7 and scored phenotypic changes in the resulting mutants. Among the deletion mutants, strains designated ⌬5 and ⌬7 displayed clear phenotypic changes involving growth and conidiation. In particular, the ⌬5 mutant exhibited vigorous growth and conidiation, potentially beneficial characteristics for certain industrial applications. Further deletion analysis allowed identification of the AO090011000215 gene as the gene responsible for the ⌬5 mutant phenotype. The AO090011000215 gene was predicted to encode a helix-loophelix binding protein belonging to the bHLH family of transcription factors. These results illustrate the potential of the approach for identifying novel functional genes.Aspergillus oryzae is regarded as an industrially important koji mold utilized in commercial enzyme production and the traditional fermentative manufacture of foods such as sake, soy sauce, and miso in Japan (8). In spite of its industrial significance and recently available genome sequence (10, 12), techniques for gene manipulation in A. oryzae have not yet been developed extensively. In particular, gene-targeting efficiency is very low, making gene disruption difficult. Recently, this problem was solved by disrupting the ku genes involved in the nonhomologous end-joining pathway (2,5,14). Reportedly, the ku-disrupted strains markedly enhance the gene-targeting frequency compared to the A. oryzae parent strain (19).Using a ku70-disrupted strain, we have developed two methods (loop-out and replacement-type recombination) to efficiently construct large-scale chromosomal deletion mutants of A. oryzae (16,17). Using the two methods, we have successfully constructed aflatoxin cluster-deleted A. oryzae. Generally, the replacement-type recombination (direct-deletion) method could be used for single deletions, while the loop-out-type recombination (5-fluoroorotic acid [5-FOA] selection) method enables construction of multiple deletions in a single strain by marker recycling. The potential merits of functional analysis by large-scale deletion include (i) identification of essential genes and construction of an experimental minimal gene set, (ii) detection of phenotypic change by simultaneously deleting multiple genes, (iii) greater convenience and increased efficiency than the single-gene disruption technique, and (iv) the possibility of isolating an industrially useful strain.Here, we demonstrate a novel approach for identifying functional genes in A. oryzae chromos...

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Identification of a Basic Helix-Loop-Helix-Type Transcription Regulator Gene in Aspergillus oryzae by Systematically Deleting Large Chromosomal Segments

Jin

Takahashi

Machida

et al. 2009

Appl Environ Microbiol

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“…DNA microarray experiments were performed as described previously (Tamano et al, 2008). Two cDNA samples labeled with Cy5 and Cy3 for each were mixed and applied onto 12 K A. oryzae oligonucleotide microarrays (Fermlab/NovusGene, Tokyo, Japan) for hybridization.…”

Section: Mrna Preparation and Dna Microarray Experimentsmentioning

confidence: 99%

Identification and characterization of genes responsible for biosynthesis of kojic acid, an industrially important compound from Aspergillus oryzae

Terabayashi

Sano

Yamane

et al. 2010

Fungal Genetics and Biology

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153

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“…These data seem to indicate that a large number of metabolites are present and actually await their discovery and structural elucidation. On the other hand transcriptional inventories of Ascomycetes carried out have provided evidence that many secondary metabolite genes have not been expressed under the respective cultivation conditions, and even shifts of conditions like solid-state fermentation did not lead to the expression of complex metabolite genes (Tamano et al, 2008). Molecular genetic genome mining approaches so far have utilized gene disruption approaches (Chiang et al, 2008) or the manipulation of transcriptional regulators (Bok and Keller 2004;Bok et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

A survey of nonribosomal peptide synthetase (NRPS) genes in Aspergillus nidulans

Döhren

2009

Fungal Genetics and Biology

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Transcriptional regulation of genes on the non-syntenic blocks of Aspergillus oryzae and its functional relationship to solid-state cultivation

Cited by 43 publications

References 24 publications

Identification of a Basic Helix-Loop-Helix-Type Transcription Regulator Gene in Aspergillus oryzae by Systematically Deleting Large Chromosomal Segments

Identification of a Basic Helix-Loop-Helix-Type Transcription Regulator Gene in Aspergillus oryzae by Systematically Deleting Large Chromosomal Segments

Identification and characterization of genes responsible for biosynthesis of kojic acid, an industrially important compound from Aspergillus oryzae

A survey of nonribosomal peptide synthetase (NRPS) genes in Aspergillus nidulans

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