Development of a promoter shutoff system in Aspergillus oryzae using a sorbitol-sensitive promoter.
Promoter shutoff is a general method for analyzing essential genes, but in the fungus Aspergillus oryzae, no tightly repressed promoters have been reported. To overcome the current limitations of conditional promoters, we examined sorbitol- and galactose-responsive genes using microarrays to identify regulatable genes with only minor physiological and genetic effects. We identified two sorbitol-induced genes (designated as sorA and sorB), cloned their promoters, and built a regulated egfp and brlA expression system. Growth medium-dependent enhanced green fluorescence protein (EGFP) fluorescence and conidiation were confirmed for egfp and brlA under the control of their respective promoters. We also used this shutoff system to regulate the essential rhoA, which demonstrated the expected growth inhibition under repressed growth conditions. Our new sorbitol promoter shutoff system developed can serve as a valuable new tool for essential gene analyses of filamentous fungi.
a Division of Fundamental Research , National Research Institute of Brewing (NRIB) , Hiroshima , Japan.
Ken
K
Oda
a Division of Fundamental Research , National Research Institute of Brewing (NRIB) , Hiroshima , Japan.
Shiho
S
Terado
a Division of Fundamental Research , National Research Institute of Brewing (NRIB) , Hiroshima , Japan.
Rieko
R
Toyoura
a Division of Fundamental Research , National Research Institute of Brewing (NRIB) , Hiroshima , Japan.
Hisashi
H
Fukuda
a Division of Fundamental Research , National Research Institute of Brewing (NRIB) , Hiroshima , Japan.
Moriyuki
M
Kawauchi
a Division of Fundamental Research , National Research Institute of Brewing (NRIB) , Hiroshima , Japan.
Kazuhiro
K
Iwashita
D001236:Aspergillus oryzae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics*; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics*; D005690:Galactose / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D049452:Green Fluorescent Proteins / Q000235:genetics; D010088:Oxidoreductases / Q000235:genetics*; D011401:Promoter Regions, Genetic; D013012:Sorbitol / Q000378:metabolism*; D013172:Spores, Fungal / Q000235:genetics / Q000254:growth & development; D020742:rhoA GTP-Binding Protein / Q000235:genetics
D002352:Carrier Proteins; D005656:Fungal Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D013012:Sorbitol; D010088:Oxidoreductases; D020742:rhoA GTP-Binding Protein; D005690:Galactose
Aspergillus oryzae; essential gene analysis; promoter shutoff; sorbitol metabolism