Expression analysis of alternative oxidase gene (aox1) with enhanced green fluorescent protein as marker in citric acid-producing Aspergillus niger.
In a citric acid-producing filamentous fungus Aspergillus niger WU-2223L, a cyanide- and antimycin A-insensitive and salicylhydroxamic acid-sensitive respiratory pathway functions in the mitochondria besides the cytochrome pathway and is catalyzed by alternative oxidase (AOX). We constructed an A. niger transformant strain AOXEGFP-1 expressing a fusion gene, aox1-egfp, encoding AOX and enhanced green fluorescent protein (EGFP) to visually analyze the expression levels of aox1 without disruption of mycelia. In strain AOXEGFP-1, the localization of the fusion protein AOX-EGFP in the mitochondria was clearly confirmed because the sites of the green fluorescence by AOX-EGFP were in agreement with those of the red fluorescence of the mitochondria stained with MitoTracker Red CMXRos. When strain AOXEGFP-1 was cultivated with antimycin A, which inhibits the cytochrome pathway at the level of cytochrome bc(1) to cytochrome c and increases the expression level of aox1, EGFP fluorescence intensity increased with an increase in AOX activity measured as duroquinol oxidase activity. Moreover, EGFP fluorescence was detected in strain AOXEGFP-1 regardless of the glucose concentration in the cultivation media: for example, when cultivations were performed with 10, 30, 60 and 120 g/l glucose, EGFP fluorescence was usually detected in the mitochondria. These results indicate that aox1 was constitutively expressed regardless of the glucose concentration in A. niger.
Kirimura
Kohtaro
K
Department of Applied Chemistry, School of Science and Engineering, Waseda University, 3-4-1 Ohkubo, Tokyo, Japan. kkohtaro@waseda.jp
Ogawa
Satoshi
S
Hattori
Takasumi
T
Kino
Kuniki
K
D001234:Aspergillus niger / Q000166:cytology / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D019343:Citric Acid / Q000378:metabolism; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D015966:Gene Expression Regulation, Fungal / Q000235:genetics; D005819:Genetic Markers; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D024101:Mitochondrial Proteins; D010088:Oxidoreductases / Q000096:biosynthesis / Q000235:genetics*; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D000099188:Alternative Oxidase
D019343:Citric Acid; D005656:Fungal Proteins; D005819:Genetic Markers; D049452:Green Fluorescent Proteins; D024101:Mitochondrial Proteins; D010088:Oxidoreductases; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D000099188:Alternative Oxidase