Full text of DOI:10.1007/BF02942182

Biotechnol. Bioprocess Eng. 2000, 5: 253-262
ꢀ
Current Progress in the Analysis of Transcriptional Regulation in
the Industrially Valuable Microorganism Aspergillus oryzae
Keiichi Nakajima, Motoaki Sano, and Masayuki Machida*
Molecular Biology Department, National Institute of Bioscience and Human-Technology, Higashi 1-1, Tsukuba,
Ibaraki 305-8566, Japan
Abstract Aspergillus oryzae is considered to be an attractive host for heterologous protein
production because of its safety and ability to secrete large amounts of proteins. In order to
obtain high productivity, thus far promoters of amylases have been most widely used in A.
oryzae. Recent progress in cloning and expression analysis, including EST sequencing, re-
vealed that glycolytic genes represent some of those most strongly expressed in A. oryzae.
Therefore, promoters of glycolytic genes could be important alternatives to promoters of
amylases because lower amounts of proteases are produced in the presence of glucose. Sev-
eral A. oryzae transcription factors responsible for the induction and/or maximum expression
of many industrially important genes encoding amylases and proteases have been cloned
and characterized. In addition to the transcriptional regulatory factors, the gene encoding
the largest subunit of RNA polymerase II, constituting the basic transcription machinery,
has also been cloned from A. oryzae. This recently acquired understanding of the details of
transcriptional regulatory mechanisms and factors will facilitate engineering flexible con-
trols for the expression of proteins important for the fermentation industries.
Keywords: Aspergillus oryzae, transcriptional regulation, transcriptional regulatory factor,
promoter
INTRODUCTION
promoters from the genes encoding industrially impor-
tant enzymes with high levels of expression have been
extensively analyzed to date. Alternatively, the im-
provement of the expression level of the target gene
may be achieved by increasing the number of expression
cassettes, each of which is composed of a promoter and
the gene to be expressed. Productivity from the trans-
formant having multiple copies of the cassette rises de-
pending on the increasing number of cassettes in the
genome. Increasing the number of regulatory elements
(cis-elements) in the promoter instead of increasing the
number of cassettes may also be effective provided that
detailed knowledge of these regulatory elements is
available [8]. However, in either case, production levels
from strains possessing excess copies of the transcrip-
tional regulatory element (on average, more than 5-6
copies) did not increase further in response to increasing
the number of cassettes or regulatory elements [1,8].
Furthermore, the expression of other genes (perhaps
regulated in a similar way by the promoter used for the
over-expression), may be significantly reduced, probably
because of exhaustion of the regulatory factors within
the cell.
A filamentous fungus, Aspergillus oryzae, has played
an important role in Japan for many years in the fer-
mentation industries producing soy sauce and miso
(soybean paste), as well as sake (alcoholic beverage). In
addition to the traditional uses, A. oryzae is considered
an attractive host for heterologous protein production
for the following reasons: First, A. oryzae is considered
to be safe. Because it has been used in food industries
for a long time, this organism is listed GRAS (generally
recognized as safe) by the Food and Drug Administra-
tion (FDA) of the USA. Second, A. oryzae has the ability
to secrete large amounts of enzymes such as amylases,
proteases and nucleases. The development of molecular
genetic techniques including DNA transformation sys-
tems has made it possible to overproduce many extra-
cellular proteins [1] including aspartic protease (Rhizo-
mucor miehei) [2], lipases (R. miehei) [3], thaumatin
(Thaumatococcus danielli) [4], lactoferrin (human) [5],
chymosin (calf) [6] and lysozyme (human) [7] by this
organism.
In order to increase protein productivity, the use of a
promoter with strong transcriptional activity is gener
ally most promising and effective. Therefore, strong
Many species of Aspergillus produce extracellular pro-
teases, which are utilized for protein degradation in the
fermentation industries. Strains with high protease
production have been sought for many years to enhance
productivity in soy source manufacture. On the other
hand, protease degrades the protein to be produced
*Corresponding author
Tel: +81-298-61-6214 Fax: +81-298-61-6240
e-mail: machida@nibh.go.jp

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Biotechnol. Bioprocess Eng. 2000, Vol. 5, No. 4
from the engineered strain. To date, therefore, many
attempts have been made to reduce protease activities
by classical mutagenesis. The disruption of transcription
factors necessary for the expression of protease is effi-
cient in this respect, as well as disruption of the prote-
ase structural gene itself. Therefore, understanding the
transcriptional regulatory mechanisms in detail, in-
cluding the factors regulating transcription, will facili-
tate the engineering of flexible control of the expression
of enzymes important in the fermentation industries.
In this paper, we focus on the transcriptional regula-
tion of A. oryzae from the viewpoint of the fermenta-
tion industries and biotechnological applications. In the
first part of the manuscript, the promoters and highly
expressed genes, the promoters of which are thought to
have strong promoter activity, are discussed. While
amylase promoters from amyB, glaA and agdA have
been almost exclusively used for gene expression in A.
oryzae, the promoters of the glycolytic genes (gpdA and
pki) have been widely used in Aspergillus nidulans and
Aspergillus niger [9,10]. Thus, the promoters of glyco-
lytic genes recently cloned from A. oryzae could be an-
other choice for the strong expression of foreign genes.
In the second part of the manuscript, transcription fac-
tors important for the expression of the strong promot-
ers and of industrially important genes are discussed.
the control of an improved promoter showed more than
70-fold the activity of the recipient strain in the pres-
ence of glucose, and the transformant possessing muti-
ple copies of the cassette showed a 140-fold increase.
Interestingly, Taka-amylase and glucoamylase were
strongly repressed in the α-glucosidase-overproducing
transformant, which harbored multiple copies of the
region III regulatory element [8]. The promoters of
amyB and glaA, which encode Taka-amylase and glu-
coamylase, respectively, also contained an element ho-
mologous to region III. Recently, reduction of produc-
tivity of the amylases was shown to be due to exhaus-
tion of the common trans-acting factor, amyR, which
specifically binds to the consensus sequence found in the
region III [15]. The binding sequence of CreA, which
mediates catabolite repression in Aspergillus, is also pre-
sent in the amyB promoter [16]. A CCAAT sequence
found in region IIIb may also be a cis-acting element, to
which an A. oryzae AnCP-like protein binds [17,18].
Production of Industrially Important Enzymes by
using Amylase Gene Promoters of A. oryzae
Several attempts to overexpress valuable proteins to
nearly industrial level have been reported using amylase
promoters. R. miehei, Humicola lanuginosa and Candida
arctica lipases were overproduced using A. oryzae as a
heterologous host. The main use of industrial fungal
lipases is as additives in washing detergents and in the
food industries. Lipases for washing powder are partly
produced by recombinant strain of A. oryzae [19], in
which R. miehei lipase cDNA was inserted between the
A. oryzae α-amylase gene promoter and the A. niger glu-
coamylase gene terminator [3]. R. miehei aspartic prote-
ase was also successfully produced by using the same
expression system, yielding greater than 3 g/L of the
protease secreted in culture medium [2].
Other attempts to overexpress valuable proteins to
nearly industrial level have been made using the glu-
coamylase promoters. A. oryzae nuclease S1, which has
been widely used for removal of single-stranded DNA in
genetic manipulation, was overexpressed by using the
glaA promoter. The transformant secreted approxi-
mately 100-fold nuclease S1 originally produced by the
recipient strain without the expression cassette of the
enzyme [20]. Calf chymosin was successfully expressed
from its cDNA and secreted into culture medium under
control of the glaA promoter [6]. Higher level secretion
of calf chymosin (150 mg/kg wheat bran) was achieved
by the fusion of the chymosin cDNA to the A. oryzae
glucoamylase gene and with the wheat bran substrate
culture [21].
STRONG AND INDUCIBLE PROMOTERS
FROM AMYLASE GENES
The amylase promoters are among the strongest and
best characterized promoters from A. oryzae. These
promoters are known to be inducible by oligosaccha-
rides such as maltose, but are repressed by glucose in
the medium [11]. In order to overproduce proteins, the
target gene is introduced downstream of the promoter
which has desirable expression characteristics. Inducible
promoters such as the amylase promoters are advanta-
geous for the production of proteins that are either
toxic or instable. In A. oryzae, promoters from amylases
(amyB, glaA) have been well-studied and are most
widely used for strong and inducible expression (19-fold
and 15-fold induced in the presence of maltose, respec-
tively) [11].
A comparison of the promoter sequences of the A.
oryzae amylase genes, amyB, glaA and agdA, revealed
four highly conserved sequences in each promoter,
which were designated regions I, II, IIIa, and IIIb [12].
Deletion analysis using Escherichia coli uidA as a reporter
showed that region IIIa was involved in both high ex-
pression and maltose induction and that region I and
region IIIb were involved in enhancing expression in
conjunction with region IIIa [12-14].
Introduction of 12 tandem repeats of region III into
the agdA promoter enhanced the promoter activities 5-
and 18-fold as compared to the intrinsic promoter in
the presence of maltose and glucose as a carbon source,
respectively [8]. In practice, α-glucosidase activity from
the transformant carrying a single copy of agdA under
GLYCOLYTIC GENES AS DONORS OF
STRONG PROMOTERS
Expression of Glycolytic Genes
Some of the glycolytic genes such as phosphoglycer-

Biotechnol. Bioprocess Eng. 2000, Vol. 5, No. 4
255
recently cloned and sequenced [25]. The expression of
pdcA was also strongly induced in the presence of glu-
cose (unpublished data).
Promoters of Glycolytic Genes
Promoters repressed by glucose, such as the amylase
promoters, may sometimes be unsuitable for protein
production in glucose-containing media. However, ex-
pression in glucose would be preferable for industrial
applications because lower amounts of proteases are
produced by fungi in the presence of glucose compared
to media containing complex plant material [26]. Thus,
the promoters of glycolytic genes with strong and glu-
cose-inducible expression could be an important avenue
to pursue.
ꢀ
Most of the yeast glycolytic genes are co-regulated by
the common transcriptional regulatory genes, gcr1 and
gcr2 [27-31]. Further, many house-keeping genes include-
ing glycolytic genes are regulated by the general tran-
scriptional regulatory factor, Rap1p [32-34]. Gcr1p and
Rap1p directly bind to their recognition sequences, CT-
blocks and RPG-boxes, respectively, which exist in the
5′-flanking region of most of the glycolytic genes, re-
sulting in glucose-dependent expression [35-42]. How-
ever, an RPG-box was not found in the 5′-flanking re-
gion of the A. oryzae enoA [43] and pgkA (Ogawa et al.,
GenBank D28484) genes. Further, the sequence-specific
DNA-binding activity that recognized the yeast RPG-
box (Rap1p-binding consensus) could not be detected in
A. oryzae whole cell extracts; instead, a factor that spe-
cifically bound to a short element free of Rap1p- binding
consensus in the enoA upstream region was detected in
the same extract (unpublished data). These results sug-
gest that the mechanism of transcriptional regulation in
A. oryzae glycolytic genes is different from that in S.
cerevisiae, although the expression profiles of the glyco-
lytic genes resemble one another.
The 5′-flanking region of A. oryzae enoA and pgkA
contained a CT-rich region, which is also found in the
promoter of various highly expressed genes of filamen-
tous fungi [44]. There are two highly conserved ele-
ments designated pgk and gpd boxes, which were identi-
fied by sequence comparison among pgkA and gpdA
promoters from A. nidulans and A. niger. The A. oryzae
pgkA promoter has a sequence homologous to pgk box,
which is involved in carbon source regulation [45]. Both
pgk and gpd boxes were found in the 5′-flanking region
of A. oryzae gpdA (unpublished data) as in the A. nidu-
lans gpdA promoter [46,47]. Deletion of gpd box over an
approximately 50 bp length resulted in a two- to three-
fold reduction in expression as assessed using promoter-
reporter gene fusion in A. nidulans [46,47]. Detailed
analysis revealed that 18 bp at the 3′ end of the gpd box
was essential for the transcriptional enhancement activ-
ity in the box [48]. The A. nidulans gpdA promoter has
been widely used in vector constructions for heterolo-
gous gene expression in fungi. The intracel-lular heter-
ologous protein expressed by this promoter reached 10-
25% of total soluble proteins [10]. Despite
Fig. 1. Regulation of A. oryzae and S. cerevisiae glycolytic
genes by glucose. (a) The mRNA levels from mycelia grown in
medium containing glucose or pyruvate are compared. The
values greater than 1 on the vertical axis indicate induction
rates by glucose while the values smaller than –1 indicate re-
pression rates [24]. (b) The mRNA levels from S. cerevisiae
cells grown in medium containing glucose or lactate are com-
pared, quoted from Moore et al. [95].
ate kinase and enolase genes from yeast and filamen-
tous fungi are known to have promoters with strong
expression. Generally, the glycolytic enzymes constitute
30-60% of cellular protein. A. oryzae glycolytic genes are
also highly expressed in the presence of glucose, as has
also been found in yeast. The level of expression of A.
oryzae enoA encoding one of the glycolytic enzymes,
enolase [22], reached approximately 3% of total mRNA
when induced by glucose. This level is comparable to
that of Taka-amylase, which is known to be one of the
most highly expressed inducible genes [23]. As expected,
glycolytic genes were found frequently in the A. oryzae
ESTs (expressed sequence tags) (Hagiwara et al. unpub-
lished data, URL http://www.aist.go.jp/RIODB/ffdb/
index.html). These results suggest that the promoters
of A. oryzae glycolytic genes are important for industrial
applications.
Recently, all the glycolytic genes were cloned and se-
quenced. With the exception of pfkB and fbpA, most of
the A. oryzae glycolytic genes were induced to different
extents by glucose [24](Sano et al. in press). The genes
most highly induced by glucose were phosphofructoki-
nase (pfkA) and pyruvate kinase (pkiA), which were
induced 16- and 4-fold, respectively. Interestingly, the
expression profile of A. oryzae glycolytic genes was very
similar to that of Saccharomyces cerevisiae (Fig. 1). A py-
ruvate decarboxylase gene, pdcA, which is probably one
of the most highly expressed genes in A. oryzae, was

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Ta b l e 1 . Experimentally characterized transcription regulatory factors from A. oryzae
Tr a n s c r i pt i o n
regulatory
factors
Amino acid identity
to the A. nidulans
Genes under
controla
Homologous DNA-Binding
Property
References
genesb
domain
homologue
AmyR
Positive regulator of
amylase genes
agdA, amyB, glaA
62.3%
70.0%
68.9%
70.9%
MALR
C6 type zinc [15,49]
finger
Area
Positive regulator of
nitrogen metabolism genesꢀ
Nitrogen
metabolism genes
GAT1
C4 type zinc [52]
finger
FacB
Positive regulator of
acetate utilization genes
(acuD), (acuE),
(amdS), (facA)
C6 type zinc [54]
finger
CAT8
AmdR
Positive regulator of omega
amino acid and lactam
utilization genes
(amdS), (gabA),
(gatA), (lamA/B)
-
C6 type zinc
Finger
[58]
PacC
Regulator of pH inducible
Genes
(gabA), (ipnA),
(pacA), (palD),
(prtA)
72.3%
RIM1
C2H2 type
zinc finger
unpublished
data
a The genes in parentheses are deduced from the results by A. nidulans.
b Homologus genes from S. cerevisiae having an E value smaller than 10-20 in the BLASTX search.
wide use for heterologous gene expression and extensive
analyses, no data on transcriptional regulatory factors
have been reported for the A. nidulans gpdA promoter.
Because A. oryzae pgkA and gpdA were frequently ob-
served in ESTs, promoters from those genes are ex-
pected to be very strong and useful for heterologous
gene expression.
lated gene was designated AmyR. The amyR gene en-
codes a 604-amino acid protein containing a GAL4-type
zinc finger motif at the N-terminus.
Searches in the databases showed that the central
part of AmyR (residues 242-492) can be aligned with
regulatory proteins involved in maltose utilization in-
cluding MALR [50] from S. cerevisiae and SUC1 [51]
from Candida albicans, which are involved in both mal-
tose and sucrose utilization. These two proteins also
contain a GAL4-type zinc finger cluster highly homolo-
gous to the one found in AmyR. The DNA-binding do-
main of AmyR binds to two types of sequences found
in a number of promoters. One type of binding site is
characterized by two CGG triplets separated by eight
nucleotides, and the other type has only one CGG trip-
let which is followed by the sequence AAATTTAA.
TRANSCRIPTIONAL REGULATORY
FACTORS FROM A. ORYZAE
Whereas there has been relatively little work on the
general transcriptional machinery in Aspergillus in com-
parison with yeast, increasing numbers of transcrip-
tional regulatory proteins have been identified in A.
oryzae. Most A. oryzae genes encoding transcription fac-
tors except amyR [49] were cloned by hybridization us-
ing the corresponding A. nidulans gene as a probe. Be-
cause the A. nidulans genes were initially isolated by
complementation using mutants, much genetic data
and information on the phenotypes of the mutants has
been accumulated in this species. Transcriptional regu-
latory factors from A. oryzae, which have been experi-
mentally examined to date are summarized in Table 1.
2) AreA [52,53]
The areA gene encodes the major regulatory protein
which activates transcription of many structural genes
encoding enzymes for nitrogen source catabolism under
conditions of limited nitrogen availability. The areA
gene of A. oryzae was cloned by cross-hybridization
with the A. nidulans areA gene and was found to encode
an 866-amino-acid protein that is very similar to other
fungal nitrogen regulatory proteins. The areA protein
contains a single CX2CX17CX2C DNA binding motif
which recognizes and binds to DNA sequences contain-
ing a core 5′-GATA-3′ sequence. Functional analyses
indicated that the N-terminal region of the A. oryzae
AreA protein was dispensable for function and revealed
a probable acidic activation domain in the protein. C-
terminal truncation of the protein resulted in derepres-
sion of several nitrogen-controlled activities in A. nidu-
lans, while deletions extending into the conserved
Transcriptional Regulatory Factors from A. oryzae
1) AmyR [15,49]
Recently, the transcriptional activation factor respon-
sible for maltose induction of the amylase genes was
isolated from A. oryzae by the complementation of a
regulatory mutant of amylases [49] and by the rever-
sion of the reduced expression of amyB in the transfor-
mant having mutiple copies of region III [15]. The iso-

Biotechnol. Bioprocess Eng. 2000, Vol. 5, No. 4
257
GATA-type zinc finger region abolished the activator
function. The DNA binding domain and extreme C-
terminal residues of these proteins are highly conserved.
In A. oryzae areA mutants, the NADP-glutamate de-
hydrogenase levels were reduced, whereas the gluta-
mine synthetase levels were not affected. The AreA
protein may play an important role in the regulation of
nitrogen assimilation in addition to its previously estab-
lished regulatory role in nitrogen catabolism.
Consensus sequences for the binding of A. nidulans
CreA (5′-SYGGRG-3′) were found in the 5′-flanking
region of the A. oryzae amyB and fbpA genes, expression
of which was repressed in the presence of glucose.
6) PacC
PacC is a wide-domain regulatory factor implicated in
modulating gene expression in response to ambient pH
[64-66]. pacC itself is an alkaline-expressed gene, sub-
jected to autogenous transcriptional activation, ampli-
fying the alkaline ambient pH signal. PacC protein pos-
sesses three elements which resemble zinc fingers of the
C2H2 class and bind to GCCARG promoter sites. Re-
cently, the pacC gene was cloned from A. oryzae (Sano et
al., unpublished data).
3) FacB [53,54]
The facB gene of A. oryzae encodes the major regula-
tor of genes involved in acetate utilization. Sequencing
of the facB gene revealed that it encodes a protein that
contains an N-terminal GAL4-like Zn(II)2Cys6 binu-
clear cluster for DNA binding, leucine zipper-like heptad
repeat motifs and central and C-terminal acidic α-
helical regions, consistent with a function as a DNA-
binding transcriptional activator. The Zn(II)2Cys6 clus-
ter shows strong similarity with those of the S. cere-
visiae carbon metabolism regulatory proteins CAT8 [55]
and SIP4 [56]. A significant level of similarity with
CAT8 is found throughout the length of the protein,
suggesting at least partial functional homology. The
FacB-binding sequences derived from the footprints are
TCC/GN8-10C/GGA and GCA/C N8-10T/GGC. Both bin-
ding sites show imperfect rotational symmetry. In A.
nidulans, facB mutants isolated by resistance to fluoro-
acetate, grew poorly on acetate as a sole carbon source
and showed decreased levels of the gluconeogenic en-
zymes phosphoenolpyruvate carboxykinase (PEPCK)
and fructose-1,6-bisphosphatase (FBP) [57].
CCAAT-binding Protein
The CCAAT motif, which is found in many fungal
promoters as well as in higher eukaryotes, is also pres-
ent in many A. oryzae promoters including the Taka-
amylase promoter. A complex designated HAP was first
shown to bind to the CCAAT sequence in S. cerevisiae
[67]. In recent years, HAP-like complexes have been
identified in filamentous fungi. CCAAT-binding factors
of A. nidulans have been independently found to bind to
the acetamidase (amdS), Taka-amylase (amyB) and
isopenicilline N-acyltransferase (aat) genes, and were
designated A. nidulans CCAAT factor (AnCF) [68], A.
nidulans CCAAT-binding protein (AnCP) [17] and peni-
cillin-regulatory protein (PENR1) [69], respectively.
Three CCAAT-binding complexes appeared to be the
same, and AnCF/AnCP were counterparts of the S. cere-
visiae HAP complexes [70]. Recently, HAP-like com-
plexes were also found in A. oryzae [71]. In vivo determi-
nation of the A. oryzae α-amylase activity in A. nidulans
was used as a reporter system to measure the effects of
directed mutagenesis of the CCAAT box. When the
CCAAT sequence was mutated to CGTAA, amyB ex-
pression was reduced to 30% of that observed with the
wild-type construct. Further mutagenesis of this motif
made binding of AnCP impossible and abolished its ex-
pression when using A. nidulans as an intermediate host
[18]. These results illustrate the positive regulatory
function of the CCAAT motif confers on amyB expres-
sion [18]. Region IIIb, com-prising a CCAAT sequence,
is also likely to be a general cis-acting element.
4) AmdR [58]
The amdR gene encodes a positively acting regulatory
protein which mediates omega amino acid induction of
the amdS, gatA (GABA transaminase), and gabA (GABA
permease) genes. The amdR gene contains a Zn(II)2Cys6
DNA-binding domain at the N-terminus, and four acti-
vation domains in the middle and at the C-terminus
which are required for full activation. The amdR inacti-
vated mutant resulted in inability to grow on GABA as
a carbon and/or nitrogen source(s) indicating that
GABA utilization is amdR-dependent in A. oryzae as it is
in A. nidulans.
5) CreA [59]
The creA gene encodes a wide-domain regulatory pro-
tein mediating carbon catabolite repression of many
genes such as polysaccharide hydrolysis, ethanol me-
tabolism and proline utilization [60-62]. CreA has two
zinc-finger regions of the Cys2His2 class, which bind to
a 5′-SYGGRG-3′ target sequence, and bear strong simi-
larity to the S. cerevisiae Mig1p [63] glucose repressor
protein. CreA inhibits transcription of many target
genes by binding to the specific sequences in the pro-
moter. The creA gene has not been cloned from A. oryzae
to date. The DNA-binding domain of creA expressed as
a fusion protein with maltose-binding protein could
bind to the A. oryzae amylase promoter[16].
Basal Apparatus for Transcription from
Aspergillus
Studying the elements and factors important for
transcriptional regulation of each gene provides useful
information to improve promoter activity and charac-
terize the organism. However, understanding the basic
apparatus for transcription as a common regulatory
machinery affected by all the transcription factors is
also important. While transcriptional regulatory factors
have been studied regarding expression, little is known
of the basal transcriptional apparatus, RNA polymerase
II (polII) and general transcription factors (GTFs) in

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Biotechnol. Bioprocess Eng. 2000, Vol. 5, No. 4
any Aspergilli. There are reports only on RNA polymer-
ase II (polII) and TATA-box binding protein (TBP)
[72,73]. TBP isolated from A. nidulans was functional in
S. cerevisiae. The polypeptide components of polII and
motifs of GTFs are well-conserved among eukaryotes
from yeast to human. Therefore, the other components
of Aspergillus basal transcriptional apparatus are
thought to be functionally compatible with yeast.
Recently, the largest subunit of RNA polymerase II
(polIIL) was cloned from A. oryzae, which was the first
example of polIIL cloned from a filamentous fungus. As
a distinguishing feature, polIIL possesses a carboxyl-
terminal domain (CTD) composed of heptapeptide re-
peats (YSPTSPS). Deletion studies demonstrate that
CTD is essential for cell growth in mouse and S. cere-
visiae [74-76]. CTD is known to interact directly with
the basal transcription factors, TATA-box binding pro-
tein (TBP) [77] and with mRNA processing factors [78].
Although the numbers of repeats in CTDs from yeast
and A. oryzae polIILs were similar (25-29 repeats) (Fig.
2) [79,80], the amino acid sequence of the repeats in A.
oryzae polIIL was much less conserved than in yeast
polIIL.
The compatibility of polIILs and CTDs from several
different organisms has been examined using yeast mu-
tants lacking polIIL function. Mouse polIIL [81] and
yeast polIIL having hamster CTD instead of their origi-
nal CTD [76] successfully complemented the rpb1 phe-
notype, but yeast polIIL, the CTD of which was re-
placed by Drosophila polIIL CTD [76], and A. oryzae
rpbA (unpublished data) failed to do so. The low level of
conservation of heptapeptide repeats in A. oryzae polIIL
as in Drosophila pollIIL, especially at positions 1, 2 and 5
which were important for the viability of yeast cells
[82,83], may be functionally distinguished from polIILs
from other species that contain highly conserved hep-
tapeptide repeats.
In recent years, novel glycosylated proteins modified
by N-acetylglucoxamine nomosaccharide through O-
glycosidic linkage (O-GlcNAc) have been demonstrated
in various eukaryotic organisms [84] although most of
the O-GlcNAc-modified protein was found in higher
eukaryotes. Unlike other glycoproteins, the O-GlcNAc-
bearing proteins have been found to be localized in the
nuclear and cytoplasmic compartments of the cell
[85,86]. Many transcription factors for genes tran-
scribed by RNA polymerase II, including CTD of
mammalian polIIL, were found to be involved in this
class of modification [87-90]. It has been shown that O-
GlcNAc turns over more rapidly than the protein itself
[91,92]. It was therefore postulated that O-GlcNAc is a
regulatory modification analogous to phosphorylation.
The fact that the human transcription factor, SP1, re-
quires O-GlcNAc modification for the transcription
enhancement activity in vitro strongly suggests that O-
GlcNAc is one of the key regulatory modifications for
transcription by RNA polymerase II [93]. Interestingly,
several DNA-binding proteins that are modified by O-
Fig. 2.
Comparison of PolIILs CTD. The numbers of the hep-
tapeptide repeats in CTD (YSPTSPS) are 52, 45, 29, 26 (or 27)
and 25 times in mouse, Drosophila, Schizosaccharomyces pombe,
S. cerevisiae and A. oryzae polIILs, respectively. The repeats
perfectly matched to the consensus sequence are indicated by
black boxes[96].
GlcNAc have been detected in lower eukaryotes, such as
A. oryzae [94].
Conclusion and Outlook
A. oryzae is a very important microorganism for
the fermentation and biotechnology industries.
Lack of basic knowledge and incompatibility in
genetics have made the extensive use of this or-
ganism difficult. Recent development of tech-
nologies for genetic engineering and for genetic
analysis are reducing this disadvantage and ren-
dering this organism more attractive. Studying
transcriptional regulation is one of the most
important contributors to this situation. How-
ever, available information on transcriptional
regulation, including cis-elements and trans-
acting factors of A. oryzae, is still insufficient for
the extensive use of this organism. Ge

Biotechnol. Bioprocess Eng. 2000, Vol. 5, No. 4
259
nomic research including large scale EST analysis will
accelerate the accumulation of basic knowledge of
this organism. Most of the transcription factors
found in A. oryzae to date show high homology in
amino acid sequence to yeast factors with similar
functions. However, the factor(s) or their recognition
sequence(s) for the regulation of glycolytic genes ap-
pear different in the two organisms. EST and genome
sequencing of Aspergilli will be of great importance
not only for industrial applications but also for the
elucidation of the differences in transcriptional regu-
latory mechanisms between filamentous fungi and
yeast.
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[Received May 18, 2000; accepted August 14, 2000]