Expression Analysis of Alternative Oxidase Gene (aox1) with Enhanced Green Fluorescent Protein as Marker in Citric Acid-Producing Aspergillus niger(MICROBIAL PHYSIOLOGY AND BIOTECHNOLOGY)

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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, aoxl-egfp, encoding AOX and enhanced green fluorescent protein (EGFP) to visually analyze the expression levels of aoxl 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 aoxl, 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 120g/l glucose, EGFP fluorescence was usually detected in the mitochondria. These results indicate that aoxl was constitutively expressed regardless of the glucose concentration in A. niger.
社団法人日本生物工学会の論文
2006-09-25

Expression Analysis of Alternative Oxidase Gene (aox1) with Enhanced Green Fluorescent Protein as Marker in Citric Acid-Producing Aspergillus niger(MICROBIAL PHYSIOLOGY AND BIOTECHNOLOGY)

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