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Identification of a basic helix-loop-helix-type transcription regulator gene in Aspergillus oryzae by systematically deleting large chromosomal segments.

ABSTRACT: 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 Delta5 and Delta7 displayed clear phenotypic changes involving growth and conidiation. In particular, the Delta5 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 Delta5 mutant phenotype. The AO090011000215 gene was predicted to encode a helix-loop-helix binding protein belonging to the bHLH family of transcription factors. These results illustrate the potential of the approach for identifying novel functional genes.

SUBMITTER: Jin FJ

PROVIDER: S-EPMC2747867 | biostudies-other | 2009 Sep

REPOSITORIES: biostudies-other

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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 Feng Jie FJ Takahashi Tadashi T Machida Masayuki M Koyama Yasuji Y

Applied and environmental microbiology 20090724 18

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 constr ...[more]

PMID: 19633118

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Project description:Basic-region helix-loop-helix (bHLH) proteins are a superfamily of transcription factors that are often involved in the control of growth and differentiation. Recently, it was reported that the bHLH transcription factor DevR is involved in both asexual and sexual development in Aspergillus nidulans and regulates the conidial melanin production in Aspergillus fumigatus In this study, we identified and characterized an Aspergillus oryzae gene that showed high similarity with devR of A. nidulans and A. fumigatus (AodevR). In the AodevR-disrupted strain, growth was delayed and the number of conidia was decreased on Czapek-Dox (CD) minimal agar plates, but the conidiation was partially recovered by adding 0.6 M KCl. Simultaneously, the overexpression of AodevR was induced and resulted in extremely poor growth when the carbon source changed from glucose to polysaccharide (dextrin) in the CD agar plate. Scanning electron microscopy (SEM) indicated that the overexpression of AodevR resulted in extremely thin aberrant hyphal morphology. Conversely, the deletion of AodevR resulted in thicker hyphae and in more resistance to Congo red relative to the control strain. Quantitative reverse transcriptase PCR (RT-PCR) further indicated that AoDevR significantly affects chitin and starch metabolism, and importantly, the overexpression of AodevR inhibited the expression of genes related to starch degradation. A yeast one-hybrid assay suggested that the DevR protein possibly interacted with the promoter of amyR, which encodes a transcription factor involved in amylase production. Importantly, AoDevR is involved in polysaccharide metabolism and affects the growth of the A. oryzae strain.IMPORTANCEAspergillus oryzae is an industrially important filamentous fungus; therefore, a clear understanding of its polysaccharide metabolism and utilization is very important for its industrial utilization. In this study, we revealed that the basic-region helix-loop-helix (bHLH) transcription factor AoDevR is importantly involved in chitin and starch metabolism in A. oryzae The overexpression of AodevR strongly suppressed the expression of amylase-related genes. The results of a yeast one-hybrid assay suggested that the DevR protein potentially interacts with the promoter of amyR, which encodes a transcription factor involved in amylase production and starch utilization. This study provides new insight for further revealing the regulation mechanism of amylase production in A. oryzae.

Dataset Information

Identification of a basic helix-loop-helix-type transcription regulator gene in Aspergillus oryzae by systematically deleting large chromosomal segments.

Publications

Identification of a basic helix-loop-helix-type transcription regulator gene in Aspergillus oryzae by systematically deleting large chromosomal segments.

Similar Datasets

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