86NISR RESEARCH GRANT
2002 Young Investigator Research Grant

Transcriptional Analysis of Genes for Energy Catabolism andHydrolytic Enzymes in the Filamentous Fungus Aspergillus
oryzae Using cDNA Microarrays
Hiroshi MAEDA
Department of Molecular and Cell Biology, Graduate School of Agricultural Science, Tohoku University
Aspergillus oryzae is a fungus used extensively in thefermentation industry. We constructed cDNA microarrays
comprising 2070 highly expressed cDNAs selected fromthe 6000 nonredundant expressed sequence tags (ESTs)in the A. oryzae EST database. Using the cDNA microarrays, we analyzed the gene expression profiles of A. oryzae
cells grown under the glucose-rich (AC) and glucose-depleted (AN) liquid culture conditions. In particular, transcription levels of most catabolic genes of the glycolytic
pathway (EMP) and tricarboxylic acid cycle (TCA) werehigher under AC than AN conditions, suggesting that A.
oryzae uses both EMP and TCA for glucose metabolism
under AC conditions. Therefore, the gene expression profiles for glucose metabolism in A. oryzae differ from thoseobserved for Saccharomyces cerevisiae and Trichoderma
reesei. We further studied the gene expression of hydrolytic
enzymes and those of energy catabolism by using three industrial solid-phase biomass media. The wheat-bran culture
gave the richest gene expression profile of the hydrolytic
enzymes and the lowest expression levels of the catabolic
genes among the three media. The low expression levels of
catabolic genes in the wheat-bran culture may releasecatabolite repression, consequently leading to the rich expression profiles of the hydrolytic enzymes.
Methods
We used Aspergillus oryzae RIB40 (ATCC 42149),
which was used for the A. oryzae EST project and the A.
oryzae genome sequencing project. The mycelia were cultured in YPD (the glucose-rich “AC condition”). The
mycelia in the YPD culture were washed and transferred toCzapek-Dox medium without a carbon source and wereused after cultivation (the glucose-depleted “AN condition”). The solid-phase media, wheat-bran, rice bran, andsoybean-curd refuse, then were inoculated with 5106
conidia and incubated at 30°C. The set of 2070 amplified
DNAs was used for preparation of cDNA microarrays.
From separate mRNA samples, cDNA was prepared and labeled with the fluorochromes cyanine-3 (Cy3) and cyanine5 (Cy5).
1.

Glycolytic pathway (EMP)
Figure 1 shows the expression of selected glycolyticgenes as determined by the cDNA microarray. The genes inthe EMP were highly expressed in the presence of glucose(AC). In comparison, the expression of the genes for acetaldehyde dehydrogenase (aldA), acetyl-CoA synthase( facA), and alcohol dehydrogenase II (adhB) was significantly higher in the absence of glucose (AN) than in its
presence (AC).
2.

Tricarboxylic acid (TCA) cycle

We compared the expression levels of genes encoding
enzymes of the TCA cycle under AC and AN conditions(Fig. 1). Except for that of the genes in TCA were upregulated under AC conditions. Whereas we found that thegenes for succinate dehydrogenase and malate dehydrogenase were almost constitutively expressed in A. oryzae, glucose in the culture media of Saccharomyces cerevisiae
strongly represses the transcription levels of all eight TCA
cycle genes. Under AN conditions, the pckA gene wasspecifically expressed as well as those observed in S. cerevisiae and in the filamentous fungus T. reesei.
3.

Biomass-degrading enzymes

Nontoxigenic Aspergillus oryzae and Aspergillus sojae
are industrially cultured on cereals for enzyme production.
For these reasons, it is important to elucidate the gene expression profiles of Aspergilli fungi on solid-phase surfaces. In the food industry, three biomass materials, such as
wheat bran, rice bran, and soybean-curd refuse, are generated in great quantities as waste from wheat, rice, and soybeans. These three waste materials have been used for further enzyme production and have been considered assources for ethanol production after their enzymatic or
chemical degradation. To address enzyme production forbiomass use, we used our cDNA microarrays to compare
the gene expression profiles of A. oryzae cells grown on
these three materials. To optimize fermentation, industrial
applications ideally require simultaneous monitoring of theexpression levels of target genes and those for cellular energy metabolism; therefore, we measured the transcripts ofgenes in the glycolytic pathway and TCA cycle in addition
to those of hydrolytic enzymes (Fig. 2A).
Various genes encoding dietary fiber-degrading enzymes
were transcribed in A. oryzae cells grown in the wheat-bran

87Fig. 1. Expression profiles of genes encoding enzymes for the glycolytic pathway (EMP), and tricarboxylic acid (TCA) cycle in A. oryzae grown inglucose-rich (AC) and glucose-depleted (AN) media.

solid-phase culture. Soybean-curd refuse generated relatively higher expression levels of proteolytic genes. Proteolytic genes such as oryzin and aspergillopepsin O werewell transcribed in the wheat-bran and rice-bran cultures,and the soybean-curd refuse culture showed induced expression of genes for oryzin and dipeptidyl peptidase. In
light of these results, the wheat-bran solid-phase medium isbetter than the other two biomass media for industrial enzyme production. Therefore, our findings validated the useof wheat bran as the medium for Aspergilli fungi by mostof the enzyme industry using solid-phase cultures.Further,in the cells grown on the wheat-bran solid-phase culture,
the transcription levels of most genes in the EMP and TCA
cycle were rather repressed compared with those in thecells grown on the two other biomass media (Fig. 2B). Thisdecreased gene expression might alleviate catabolite repression, which is known to downregulate the transcription levels of many of the hydrolytic enzymes we described.