58NISR Research GRANT
2007 Young Investigator Research Grant

Regulation mechanisms of cellulase gene expression inAspergillus aculeatus
Shuji TANI
Graduate School of Life and Environmental Sciences, Osaka Prefecture University

Research aims

Results

The filamentous fungus, Aspergillus aculeatus No. F-50,
secretes a broad spectrum of polysaccharide-degrading enzymes that cooperatively hydrolyze pulp with those fromHypocrea jecorina (anamorph Trichoderma reesei), which
is known to be one of the strongest cellulase producers.1–3)
Biochemical analyses of those enzymes revealed that hemicellulases and b -glucosidase secreted by A. aculeatusmainly contributed to effective saccharification of cellooligosaccharides to glucose. However, the regulation
mechanisms of those genes are still insufficiently understood in A. aculeatus.
It has been revealed that Xyr1, which is a homolog oftranscriptional activator XlnR of Aspergillus species, governed the inductions of all cellulase and hemicellulase gene
expressions in T. reesei.4) However, I revealed that an XlnRindependent signaling pathway existed and regulated cellobiohydrolase I (cbhI) and carboxymethyl cellulase 2(cmc2) gene expressions in A. aculeatus. This XlnR-independent signaling pathway is supposed to be regulated by anovel transcription factor. To better understand regulation
mechanisms of the XlnR-independent pathway in A. aculeatus, I attempted to identify physiological inducers andcis-elements involved in the XlnR-independent signaling
pathway.

1) Effects of b -linked disaccharides on the cbhI gene expression
It is predicted that b -linked disaccharides derived fromhydrolysis of cellulose could function as the physiologicalinducer for cellulase gene expression in filamentous fungi.
Therefore, I investigated which b -linked disaccharides effectively induced the cbhI expression regulated via XlnRindependent pathway by quantitative RT-PCR. RNA wasisolated from mycelia of A. aculeatus wild type grown for 3
hours in minimal medium supplemented with 3 mM glucose, 3 mM sophorose, 3 mM cellobiose, or 3 mM gentiobiose as a sole carbon source. To directly assess the effectof disaccharides on the cbhI expression, glucosidase inhibitor, nojirimycin, was added to culture during induction.
Since addition of nojirimycin might cause carbon starvation, transcripts of the cbhI were also analyzed under carbon free condition. Among tested disaccharides, cellobiose
induced the cbhI gene expression the most effectively.
However, lactose did not induce the cbhI expression. These
results suggest that the hydroxyl group on carbon 4 is crucial for the cbhI induction.
Although cellobiose effectively induced the cbhI expression, nojirimycin was necessary for the induction. Therefore, I assessed how much concentration of cellobiose wasnecessary for the sufficient cbhI expression with nojirimycin added. A significant induction of the cbhI was observed when more than 30 nM of cellobiose and nojirimycin were added. Km value of b -glucosidase for cellobiose is larger than hundred m M. These results suggestthat the cbhI gene can be induced in response to cellobiose
if a low concentration of cellobiose is supplied continuously.

Methods
Gene expression profiles were investigated by semiquantitative or quantitative RT-PCR. RNA was isolated
from mycelia grown under the following conditions: A. aculeatus wild type was grown in minimal medium (MM)supplemented with 1% glucose for 24 hours at 30°C. Then,mycelia were harvested, washed with carbon free MM, andgrown in fresh MM supplemented with various carbon
sources for 3 hours. In the case of GUS assay, mycelia weregrown in MM supplemented with 1% avicel or 1% arabinose for another 24 hours after 24 hours preincubation.
Aspergillus transformation was done by protoplast-PEG
method. Reporter plasmids possessing modified cbhI promoters were integrated at sC locus of A. aculeatus. Activities of the GUS in transformants were assayed using pNPb -glucuronide at 37°C. Proteins were extracted frommycelia using GUS assay buffer, after grinding with Micro
Smash (Tomy, Japan). Protein levels were standardizedusing Protein assay reagent (Biorad, Japan).

2) Identification of cis-elements required for the cbhI induction
Next, I performed functional analysis of truncated andmodified cbhI promoters using GUS as a reporter to identify cis-element(s) sufficient for the cbhI induction. Reporter plasmids possessing those modified promoters wereintroduced at A. aculeatus sC locus as a single copy. The attached 657 promoter region induced the GUS gene in response to an inducing carbon source, avicel. This expression level was 45-fold higher than that with a noninducing
carbon source, arabinose. This result proved that cis-element(s) sufficient for the cbhI induction lay in the attached59

promoter region. Although truncation of the promoter sequence to 469 had no effect on GUS expression, furthertruncation from 469 to 192 resulted in the reduction ofgene expression level and the loss of inducibility. Conserved sequences were searched in this region and threeconserved regions, namely A, B, and C, were found. Mutations of B and C region resulted in the reduction of the gene
expression and induction level. However, the inducibility
was lost only when all regions were mutated. These results
indicated that three regions were required for complete geneinduction by cbhI promoter. Common sequence among threeconserved regions was identified as CCGN2CCN7G which
was quite similar to CeRE found as an element required for
endoglucanase A gene induction in A. nidulans.5) Now, weare planning to identify a factor recognizing the conserved
sequence.

that cellobiose could be a physiological inducer since a low
concentration of cellobiose effectively induces the cbhI expression. This signal is transmitted to the conserved sequence, namely, CCGN2CCN7G, in the cbhI promoter region and triggered the gene induction. Now, I try to identify
the factors involved in this signal transduction pathway.
References
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Conclusion
5)

I revealed that cellobiose effectively induced the cbhI expression regulated via an XlnR-independent pathway inA. aculeatus. I assumed that cellobiose could not induce thecbhI expression since b -glucosidase would hydrolyze cellobiose immediately at first. However, my data suggested

Montenecourt, B. S., Trichoderma reesei cellulases. Trends Biotechnol. 1, 156–161 (1983)
Béguin, P., Molecular biology of cellulose degradation. Annu. Rev.
Microbiol. 44, 219–248 (1990)
Murao, S., Kanamoto, J., and Arai, M., Isolation and identification ofa cellulolytic enzyme producing microorganism. J. Ferment.
Technol., 57, 151–156 (1979)
Stricher, A. R., Grosstessner-Hain, K., Würleitner, E., and Mach, R.
L., Xyr1 (xylanase regulator 1) regulates both the hydrolytic enzyme
system and D-xylose metabolism in Hypocrea jecorina. EukaryoticCell, 5, 2128–2137 (2006)
Endo, Y., Yokoyama, M., Morimoto, M., Shirai, K., Chikamatsu, G.,
Kato, N., Tsukagoshi, N., Kato, M., and Kobayashi, T. Novel promoter sequence required for inductive expression of the Aspergillus
nidulans endoglucanase gene eglA. Biosci. Biotechnol. Biochem., 72,
312–320 (2008)