DNA RESEARCH 15, 173–183, (2008)

Invited Review
doi:10.1093/dnares/dsn020

Genomics of Aspergillus oryzae: Learning from the History of Koji Moldand Exploration of Its Future
Masayuki MACHIDA1, *, Osamu YAMADA2, and Katsuya GOMI3(Received 14 June 2008; accepted on 1 August 2008)

Abstract
At a time when the notion of microorganisms did not exist, our ancestors empirically establishedmethods for the production of various fermentation foods: miso (bean curd seasoning) and shoyu (soysauce), both of which have been widely used and are essential for Japanese cooking, and sake, amagical alcoholic drink consumed at a variety of ritual occasions, are typical examples. A ﬁlamentous
fungus, Aspergillus oryzae, is the key organism in the production of all these traditional foods, and its
solid-state cultivation (SSC) has been conﬁrmed to be the secret for the high productivity of secretoryhydrolases vital for the fermentation process. Indeed, our genome comparison and transcriptome analysis
uncovered mechanisms for effective degradation of raw materials in SSC: the extracellular hydrolase genesthat have been found only in the A. oryzae genome but not in A. fumigatus are highly induced during
SSC but not in liquid cultivation. Also, the temperature reduction process empirically adopted in the
traditional soy-sauce fermentation processes has been found to be important to keep strong expressionof the A. oryzae-speciﬁc extracellular hydrolases. One of the prominent potentials of A. oryzae is that it
has been successfully applied to effective degradation of biodegradable plastic. Both cutinase, responsible
for the degradation of plastic, and hydrophobin, which recruits cutinase on the hydrophobic surface toenhance degradation, have been discovered in A. oryzae. Genomic analysis in concert with traditional
knowledge and technology will continue to be powerful tools in the future exploration of A. oryzae.
Key words: Aspergillus oryzae; genome sequence; solid-state culture; secretory hydrolase; non-syntenic block;comparative genomics

1.

Historical backgrounds

Aspergillus oryzae is a fungus widely used in traditional Japanese fermentation industries, including
soy sauce, sake, bean curd seasoning and vinegar
production. Filamentous fungi generally have theability to produce various and vast amounts ofenzymes in a secretory manner. Among ﬁlamentous*

Edited by Katsumi Isono
To whom correspondence should be addressed. Tel. þ81 29861-6164. Fax. þ81 298-61-6174. E-mail: m.machida@aist.go.jp# The Author 2008. Kazusa DNA Research Institute.

fungi, A. oryzae is known to have prominent potential
for the secretory production of various enzymes. In
addition, developments in genetic engineering technology have led to the application of A. oryzae in the
production of industrial enzymes in modern biotechnology. Aspergillus oryzae was used for the ﬁrst
example of commercial production of heterogonous
enzyme, the lipase for laundry detergent in 1988.1
1.1. Solid-state cultivation
One of the distinctive features of the use of A. oryzae
in traditional Japanese fermentation is the use of

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Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST),
Central 6, 1-1, Higashi, Tsukuba, Ibaraki 305-8566, Japan1; National Research Institute of Brewing, 3-7-1
Kagamiyama, Higashi-Hiroshima, Hiroshima 739-0046, Japan2 and Graduate School of Agricultural Science,
Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, Miyagi 981-8555, Japan3

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Genomics of Aspergillus oryzae

Figure 1. A historical signboard of a producer of A. oryzae
conidiospores. Aspergillus oryzae conidiospores are industrially
produced and are distributed to fermentation companies. Two
suppliers were established 600 years ago (Muromachi period).
No other suppliers were established before A.D. 17–18th. The
ﬁgure shows a photograph of an original signboard, Kuro-ban(black stamp), prepared under the license of Koji-za, theassociation of A. oryzae conidiospores suppliers during the
Muromachi period. Currently there are ﬁve major distributors inJapan supplying A. oryzae conidiospores to 4500 sake (Japanese
alcoholic beverage, ca. 1900 brewers), miso (soybean paste, ca.
1200 brewers) and shoyu (soy sauce, ca. 1500 brewers) brewers
in Japan, excluding several of the biggest soy-sauce companies.
The three characters called Hiragana, which were originallydeveloped in Japan; on the signboard have pronunciations, “mo”,“ya” and “shi” from top to bottom. Moyashi means the A. oryzae
conidiospores used mainly by sake brewers.

enzymes in SSC, physical barrier and low water
activity.7 Recently, practical approaches to liquid
cultivation containing solid materials succeeded in
the effective production of enzymes in a secretory
manner allowing the degradation of raw materials
with a similar efﬁciency to SSC.8 Analysis of themechanism for the secretory production of enzymes
in SSC may be important to the understanding ofhighly efﬁcient secretion.
1.2. Isolation and identiﬁcation of A. oryzae
Sequencing the genome of A. oryzae RIB40 (ATCC42149) was completed in 2005.9 The sequenced
strain is a wild-type strain, most similar to thoseused for sake brewing but still has the ability of strong
production of proteases, which is one of the mostimportant characteristics for soy-sauce fermentation.

Downloaded from https://academic.oup.com/dnaresearch/article-abstract/15/4/173/376904 by guest on 17 June 2019solid-state cultivation (SSC) (rice grain, soybean andwheat bran). This style of fermentation is thought tohave originated 3000 – 2000 years ago in China.2,3
The technology was imported into Japan during the
Yayoi period (B.C. 10th –A.D. 3rd).2 Inocula from ﬁlamentous fungi for fermentation have been commercially available as koji since the 13– 15th century(Heian and Muromachi period).4 This indicates thatkoji was cultivated without the knowledge that it iscomposed of a microorganism. Thus, the word, koji,indicates both the material fermented by A. oryzae
in the form of SSC and the A. oryzae microorganism
itself (koji mold). A key technology enabling theindustrial production and distribution of A. oryzae
was the production of conidiospores whilst keeping
them alive and uncontaminated. Traditionally, thistechnology involved the use of hardwood leaves
burned to white ashes in poor aeration. The conidiospores packed in paper bags were layered with theashes between them in a box and stored. The technology was indispensable to the avoidance of contamination by other microorganisms in a period when
desiccant or air conditioning was unavailable.4 This
technology led to the discovery that leaf ashes
added to steamed rice also allowed reliable production of conidiospores. Conidiospores preparedindustrially for sake brewing are called Moyashi.
Signboards with three Japanese characters, ‘moyashi’(or fermentation starter), indicate the supplier of
A. oryzae conidiospores for sake brewers (Fig. 1). It is currently known that alkaline pH, produced by the additionof the ash, prevents contamination by other microorganisms and that minerals contained with the ashenhance the formation of conidiospores.2,4,5 This technology has been applied to the production of
Trichoderma spores as a biological pesticide by Akita
Konno Co., Ltd, a biotech company whose origins stemfrom the traditional fermentation industry.
The ability of secretory production of proteins isfurther enhanced in solid-state culture comparedwith submerged culture. For example, A. oryzae canproduce 50 g of a-amylase from 1 kg of wheat
bran, which is roughly equivalent to 1 L of liquid
culture medium. The ﬁrst example of the industrial
application of SSC to enzyme production by
A. oryzae was Taka-diastase, developed as a stomach
medicine by Jokichi Takamine in 1894. Recently,
solid-state fermentation systems using the industrial
fungi A. oryzae and A. sojae have been introduced forthe production of various industrial enzymes (cf. amylases, proteases, lipases) and speciality chemicals.6 In
spite of the extensive use of SSC, much less is known
about SSC than for submerged cultivation, which is
widely used in modern biotechnology owing to its
easier automatic handling. Gene expression analysis
revealed important factors affecting secretion of[Vol. 15,

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M. Machida et al.

1.3. Safety
The long history of extensive use of A. oryzae in foodfermentation industries prompted industrial applications of A. oryzae to be listed as Generally
Recognized as Safe (GRAS) by the Food and Drug

Administration (FDA) in the USA.11,12 The safety ofthis organism is also supported by World HealthOrganization (WHO).13 Although A. oryzae is genetically very close to A. ﬂavus, which is known
to produce the most potent natural carcinogen,aﬂatoxin, A. oryzae has no record of producing aﬂatoxin or any other carcinogenic metabolites.14
Fermented foods produced by A. oryzae including
A. sojae, the close relative to A. oryzae in section
Flavi, have been shown to be aﬂatoxin free.15,16 The
two species, A. oryzae and A. ﬂavus were traditionallydistinguished based on morphological, physiologicaland culture-based characteristics.17 Recent DNAbased techniques have enhanced the potential for
distinction.18 – 20 Zhang et al. reported that homologs
of aﬂatoxin biosynthesis gene cluster of A. oryzae were
not expressed even under the conditions that are
favorable to aﬂatoxin expression in A. ﬂavus andA. parasiticus.21
We have received huge beneﬁts from A. oryzae forthousands of years and may continue to receive
them in the future. At the same time, we are facing
various mysteries regarding A. oryzae: uniquecultivation method, species origin, isolation and soforth, as described above. In spite of economical
importance and scientiﬁc interest, the fact that
A. oryzae forms multinucleate conidia and lacks asexual life cycle have prevented extensive basic
studies by conventional approaches. It is very interesting to see whether and to what extent genomics
elucidates the subjects concerning ancestor, molecularmechanism for high secretion and safety, and thepotential of A. oryzae in new ﬁelds where A. oryzae
has never yet been applied.

2.

Lessons from genomics

The A. oryzae genome consists of eight chromosomes with an entire genome size of 37.6 Mb(Fig. 2). Comparison of the A. oryzae genome with
two other Aspergilli sp., A. nidulans and A. fumigatus,
sequenced in the similar timelines revealed that
A. oryzae had a 25– 30% bigger genome size thanthe other two species.9 Accordingly, A. Oryzae had
2000 – 3000 more genes than the other two
species, indicating similar gene densities of the three
species. The increase of gene number, in accordancewith the increase of genome size, mainly derived
from the gene expansion of metabolic genes. This
includes secretory hydrolases, transporters andprimary and secondary metabolism genes, among
which the expansion of secondary metabolism genes
is most prominent9 (see another review22 fordetail). Aspergillus oryzae is known to have a wide
variety of hydrolytic enzymes and a strong capacity

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Aspergillus oryzae was ﬁrst isolated from koji by
H. Ahlburg in 1876 when he was invited to theJapanese Medical College. Its original name,Eurotium oryzae, was later renamed A. oryzae by
F. Cohn because he found that it lacked the ability ofsexual reproduction. It is said that A. oryzae is a domesticated species and, therefore, that A. oryzae may befound only in a domesticated form but not in nature.
On the other hand, it has been stated, in a historicalliterature, that koji should be isolated from the moldgrowing on an ear of rice. Some reports about the isolation of A. oryzae from soil, plants and food10 support
the natural isolation of A. oryzae for fermentation.
However, these species might now be distributed innature following domestication many years ago.
There are two scenarios to be considered for theorigination of koji fermentation. In the ﬁrst scenario,
A. oryzae may have been isolated from nature independently in Japan. A literature describing naturally
fermented alcoholic beverage was found in a historical document, Harima no Kuni Fudoki, edited in AD
715.2 Another historical document described thatkoji must be isolated from the mold growing on an
ear of rice. This means that A. oryzae may haveexisted in nature before domestication and might beisolated from other dangerous species such as
A. ﬂavus through the method indicated in the historical literatures above. In the second scenario, A. oryzae
may have been imported from China during the Yayoi
period. The material used for the fermentation in East
Asia was a block of hardened raw wheat or rice
powder. The ﬁlamentous fungi that grew on theblock is thought to have been Mucor or Rhizopus.2
On the other hand, the material used in the production of Japanese alcoholic beverage was steamed
rice grain, thus, proteinous components in rice areconsiderably denatured. Mucor and Rhizopus, whichhave a low productivity of proteases allowing theassimilation of denatured proteins as nitrogensources, grow poorly on steamed rice. On the contrary,A. oryzae, producing a considerable amount of proteases, could predominantly grow in an environment
where a mixture of spores of these heterologous
species exists. The names of the traditional fermented
materials, which indicate yellow color, but not darkgray as Mucor and Rhizopus, also suggests that
A. oryzae was ﬁrst used in Japanese fermentationindustries. Therefore, the second scenario may not
be well supported.

175

176

Genomics of Aspergillus oryzae[Vol. 15,for degrading various materials. The gene expansionof secretory hydrolases explains these abilities well.
The multiple transporters may be used for efﬁcientdegradation or further conversion of degradedmaterials, but their main role may be to excrete
toxic compounds for A. oryzae to grow. In fermentation, A. oryzae is exposed to vast amounts and highconcentrations of compounds derived from a single
material, rice, soybean, wheat, etc., which may
include phytoalexin or other compounds that inhibit
growth. The unusual environment that was artiﬁciallyintroduced might allow A. oryzae to acquire additionaltransporters.
2.1. Insights into the molecular mechanism of safety
Interestingly, the expanded genes indicated above
were highly enriched on the non-syntenic blocks(NSBs) that were determined by the comparison ofgene orders on the genomes between A. oryzae andA. nidulans or A. fumigatus. The NSBs were distributed
through the entire A. oryzae genome in a mosaic
manner. The expressed sequence tag (EST) analysis23
showed that the transcriptional expression of the
NSB genes was considerably weaker than the genes

on syntenic blocks (SBs) (P ¼ 4.1  102134).9 The
difference of absolute levels of the gene expression
between the two blocks was conﬁrmed by DNA
microarray.24 Therefore, the secondary metabolism
genes, which are highly enriched on NSBs, are notexpressed in the culture conditions examined
including SSC. Consequently, the EST analysis showeda striking contrast in expression of the aﬂatoxin
biosynthesis gene homologs; whereas ESTs for all the
25 gene homologs25 were found in A. ﬂavus.26 No
ESTs except for aﬂJ and norA were found in A. oryzae.23
These results suggest that the long history of industrial
use of A. oryzae has removed genes unfavorable
for human consumption. Alternatively, A. oryzae, bysome means, might have been selected as a safe
mutant from the beginning. Another industrial
species, A. sojae, which is genetically very close to
A. parasiticus, the aﬂatoxin producer, lacks the ability
to produce aﬂatoxin, and so is similar to A. oryzae,although it harbors aﬂatoxin gene cluster homologs.27 One of the possible reasons explaining theinability of the expression of the aﬂatoxin biosynthesis
gene homologs in the two species is mutation of theaﬂR gene. However, Takahashi et al. showed thatcomplementation of the mutated A. sojae aﬂR geneDownloaded from https://academic.oup.com/dnaresearch/article-abstract/15/4/173/376904 by guest on 17 June 2019

Figure 2. Contigs and NSBs/SBs maps Contigs (SCnnn; n, decadecimal number) and NSBs (black bars) are mapped on the A. oryzae
chromosomes.9 Arrows and values in parentheses indicate direction and length of the contigs in mega base, respectively. Adaptedfrom Machida et al.9

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M. Machida et al.

177

2.2. Secrets of solid-state fermentation
Transcriptome analysis using cDNA microarrayrevealed that the carbohydrate metabolism showed
typical characteristics of gluconeogenesis in SSC in
spite of the presence of rich nutrients in koji
culture.31 The lower expression levels of catabolic
genes of the glycolytic pathway and the tricarboxylic
acid cycle in solid-state culture led to a release fromcatabolite repression, and consequently to high-level
expression of hydrolytic enzymes. Low water activity
may cause inefﬁcient degradation and uptake ofnutrients resulting in starvation.
Recently, the expression regulation of the NSB genes
were analyzed by DNA microarray comprised morethan 11 000 oligonucleotide probes of the A. oryzae
genes. The signiﬁcantly lower expression level of the
NSB genes when compared with the SB genes in an
average was conﬁrmed by the DNA microarray experiments (Fig. 3A). Besides the difference in absolute
expression levels between NSBs and SBs, the expression
regulation showed striking difference between the two
blocks.24 The genes on NSBs were signiﬁcantly
repressed at heat shock (428C), whereas those on SBs
were induced in an average. Interestingly, the genes
on NSBs appeared globally up-regulated in SSC, but
those on SBs did not display global characteristics of
up- or down-regulation under the same conditions(Fig. 3B).24 High induction ratios of the genes encoding
extracellular enzymes, which belong to the metabolism of the COG meta-category, contributed mostto the global induction of the NSB genes. In contrast,

Figure 3. Characteristic transcriptional expression of the genes on
NSBs and SBs. (A) Approximately 1500 genes on chromosome
6 were mapped with even spacing with their absolute
expression levels at 428C in YPD medium, measured by DNA
microarray. The gray boxes under the horizontal zero axis
indicate NSBs. (B) Averages of the logarithmic (log2) expression
ratios of the genes on the SBs (dotted line) and NSBs (boldline). Wheat Meal/Defatted Soybean, Wheat Meal/Defatted
Soybean against CD; Wheat Bran, Wheat Bran against CD;
Dried Yeast, CD containing 2% dried yeast against CD; Carbon
Starvation, CD without glucose against YPD; Heat Shock, YPD at
428C against YPD at 308C; High Osmotic Pressure, CD
containing 0.8 M NaCl against CD. See further details in theprevious reports. Reproduced from Tamano et al., 2008.24

the global up-regulation of the SB genes at heatshock was mainly due to the induction of the genes
belonging to the meta-category of Information
Storage and Processing. The two meta-categories
played an important role for the characteristic global
up-regulation of the genes on each block showed
obviously bigger occupation in comparison between
the two blocks. Furthermore, the NSB genes that did
not have genes with sequence similarity to the genes
on SBs showed higher induction ratios on averagecompared with those from the NSB genes that had
homologs on SBs. Therefore, the genes that had similarity between the two blocks after the removal of
the genes speciﬁc in each block showed global
expression proﬁles which are more similar than using
entire genes on each block.24
Hydrolases occupied a major part of the genes highly
induced in two SSC (9 out of the top 29 induced NSB
genes and 10 out of the top 47 induced SB genes).
The genes include those encoding hydrolases

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(truncation by nonsense mutation) was insufﬁcient for
the expression of aﬂatoxin cluster homologs.27 Further,base substitutions in the two elemental sequences(AreA and FacB binding sites) in the A. oryzae aﬂR
promoter alone do not seem to be key for theabsence of aﬂR mRNA.28 These results suggest asilencing mechanism similar to that observed in theregulation of aﬂatoxin biosynthesis gene homologs
in A. sojae.15,27,28 Recently, a global regulator of
secondary metabolism genes, laeA,29 was found.
The fact that the predicted laeA gene seems intact in
A. oryzae may suggest the existence of a global regulation mechanism of secondary metabolism byother transcriptional regulatory factor(s). Greaterthan 40% of the A. oryzae strains deposited in the
National Research Institute of Brewing lack morethan half of the entire cluster of aﬂatoxin biosynthesis
gene homologs (Group 2 and other strains), but
the remaining strains have the near intact cluster(Group 1 strains).30 It is thought that the latterstrains have at least double or more safety locks
preventing induction of aﬂatoxin biosynthesis gene
homologs outside the cluster.

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Genomics of Aspergillus oryzae

regulators are basically the same. Aspergilli haveHK8 histidine kinase family, which is absent inother sequenced ﬁlamentous fungi belonging to
Sordariomycetes and Dothideomycetes.9 Further,A. oryzae generally has greater numbers of HK6,
MAPK, MAPKK and MAPKKK than other ﬁlamentous
fungi.9 The complex signal transduction cascade may
be necessary in order to regulate genes expanded in
the A. oryzae genome and may acquire the ability toadapt various growth conditions.
The genes speciﬁcally expressed in SSC have beencloned and sequenced by preparing the subtracted
cDNA library in which the genes speciﬁc to SSC wereenriched. Approximately half of the genes expressed
speciﬁcally in SSC encoded secretory or intracellularenzymes and transporter proteins. Most of theremaining genes were functionally unknown.23,37
Recent proteomic analysis showed that A. oryzae produced a much larger amount of total proteins in SSC(wheat bran) than in submerged cultivation (wheatbran suspension supplemented with ammonium
sulfate and phosphate) although more variety ofsecreted proteins over 50 kDa was produced in submerged culture than in solid-state culture.38
Secretion of a large amount of enzymes was thought
to be necessary in order to obtain nutrients under
the low water activity. More than half of the entire
amount of the visible proteins in the 2D gel electrophoresis is a-amylases and their proteolytic products.
Interestingly, A. oryzae mycelia grown on a nylon
membrane placed over a Czapek – Dox medium plateshowed signiﬁcant induction of the alternative glucoamylase gene, glaB, which is known to be one of thetypical genes speciﬁc to SSC. Small pore size
(0.2 mm) of the membrane and high concentrationof maltose (50%) in the medium were important forstrong induction.39 Environmental factors responsible
for transcriptional induction speciﬁc to SSC arethought to have low water activity, low diffusion ofnutrients and gases, continuity of medium distribution and physical barriers for hyphal extension.39 – 41
This method is very useful because of its SSC-like
conditions and mycelia can be much more easily
prepared than native SSC.
2.3. Genome evolution of A. oryzae
The comparison of three Aspergillus spp. genomes
from spp. A. oryzae, A. nidulans and A. fumigatus
revealed expansion of genes and genome size. The
expansion reached 20% of the A. oryzae genome.
At least two scenarios must be taken into consideration accounting for the mechanism of the expansion;(i) the ancestor might have a small genome size similar
to A. nidulans and A. fumigatus, and might haveacquired genetic materials to make the A. oryzae

Downloaded from https://academic.oup.com/dnaresearch/article-abstract/15/4/173/376904 by guest on 17 June 2019[cf. leucine aminopeptidase (Chien et al., 2002)32 andneutral protease II (Tatsumi et al., 1991)]33 on NSBs
and alkaline protease (Murakami et al., 1991)34, glucoamylase (Hata et al., 1998)35 on SBs, which play
an important role in the degradation of proteins andstarch in the raw materials for fermentation. Anothernotable feature of the highly induced genes in SSC is
the high abundance of transporters in the major facilitator superfamily (MFS). These transporters might benecessary for the growth in vast amounts of the limited
variety of raw materials, where trace amounts of toxic
materials in the raw materials might be condensed.
As mentioned above, the transcriptional level ofthe NSB genes are signiﬁcantly lower than the SB
genes. However, some NSB genes, neutral protease(AO090103000310)
and
MFS
transporter(AO090010000493) genes, for example, showed an
expression strength comparable to alkaline protease(AO090003001036) and translation elongation
factor (AO090120000080), which showed thehighest expression levels among the SB genes (4sigma bigger than the average of entire genes). Thus,
the genes above may make a large contribution to
the degradation of raw materials. The bigger induction
ratios of the genes speciﬁc to NSBs suggest that the
products of these genes might be important to theeffective degradation of the raw materials throughbreaking minor chemical linkage, which loosen theconformation of polymers for example. During solidstate fermentation, the materials with A. oryzae
growing on their surface are mixed upside down for awhile. This process, which is called Teire, is necessary
to keep the temperature below 408C and to
improve aeration during fermentation while A. oryzae
is growing vigorously. The results of the expression
analysis above suggest that this process is importantin preventing the A. oryzae-speciﬁc homologs ofvarious hydrolases on NSBs from being repressed bytemperature increase.
Interestingly, no genes encoding polyketide synthase(PKS) or non-ribosomal peptide synthetase (NRPS)(Cramer et al., 2006)36 on NSBs were induced in SSC,
although on average the NSB genes were highly
induced. Similarly, of the SB genes, no genes encoding
PKS and NRPS were induced except one PKS gene
(AO090701000530). In contrast, the genes encodinghydrolases and transporters, which were highly
enriched on NSBs as well, were induced as describedabove.24 This indicates that the expression proﬁles of
the NSB genes are classiﬁed into at least two groups
and might suggest the mechanism of gene regulationof A. oryzae in acquiring a high potential for degrading
raw materials with a high level of safety. Aspergilli
possess more sensor histidine kinases (13 – 15) thanyeast (1 – 3), whereas the numbers of histidinecontaining phosphotransfer factors and response[Vol. 15,

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M. Machida et al.

genome bigger, (ii) the ancestor might have a genome
size similar to A. oryzae, and the other two species
might have lost their genes (Fig. 4A and B).
Phylogenomic analysis of the three species revealed
that A. nidulans branched off earlier than speciationof A. oryzae and A. fumigatus.42 Therefore, if A. nidulans
and A. fumigatus lost their genes as in the second
scenario, they have lost the genes independently after
speciation. However, considering that the gene organization between the two species seems very similar as
shown by the synteny map of the two species andthat the number of genes lost from the ancestor
reaches nearly 20%,9 the second scenario is unlikely
to be accepted. Mosaic distribution of phylogeneticallyanomalous genes on the chromosomes and apparently
similar phylogenetic distances of the expanded genes
to their homologs on SBs suggest a horizontal genetransfer (HGT) mechanism for the gene acquisition.9
Recently, Fedorova et al. proposed a gene duplicationand divergence (GDD) mechanism43,44 for the acquisition of the A. fumigatus speciﬁc genes.45 Nearly halfof the A. fumigatus speciﬁc genes with paralogs
localized in the subtelomeric regions that occupied,20% of the entire genome. The result together withthe signiﬁcant decrease of gene size and intron
number of the A. fumigatus speciﬁc genes strongly
suggests the GDD scenario.45 This suggests that mostof the A. oryzae expanded genes might have beenacquired by this mechanism (Fig. 4C). Apparent localization of NSBs to the chromosomal ends (see Fig. 2)
could support the idea. Nevertheless, there is aclear exception involving two A. oryzae speciﬁc
genes clustered into the bacterial clades with thehighest similarity to Agrobacterium tumefaciens genes(AGR_L_1864, AGR_L_1866).9 Large number of theextra genes in the A. oryzae genome might have been
expanded by the combination of the two mechanisms,
HGT and GDD.
The most recent effort for sequencing the A. ﬂavus
genome has made it possible to analyze the historyof its genome evolution and to evaluate the potentialof cellular function of the two species by comparing
the two genomes. Aspergillus ﬂavus had genome size
(36.8 Mb) and gene number (12 197) very similar
to those of A. oryzae (36.7 Mb, 12 079, respectively).
Surprisingly, the number of genes found uniquely ineach genome was only 350 indicating that thegenes of the two species are highly similar in termsof amino acid sequence and organizations.46,47 This
clearly indicates that the expanded genes in
A. oryzae existed from the beginning, i.e. before domestication. Detailed comparative analysis will shedlight on the important and interesting subjects mentioned above. Aspergillus ﬂavus, which utilizes nutrients from plants, may require redundant and various
secretory hydrolases. During the long history of domestication, the ancestor of the two species lacked theability to produce poisonous compounds, aﬂatoxin
for example, and these enzymes might have been
applied to effective degradation of raw materials
during fermentation.
2.4. Applied genomics of A. oryzae
Genomics has facilitated the application of A. oryzae
to recycling of biodegradable plastic. Maeda et al.48
found that A. oryzae could grow with poly-(buthylene
succinate) (PBS) as a sole carbon source by degrading
it with cutinase, CutL1. They applied DNA microarray31 to investigate the additional factor(s) involvedin efﬁcient PBS degradation, and found that the rolA
(rodA-like) gene was highly transcribed speciﬁcallyin the presence of PBS.49 This mechanism appears to
involve cutinase recruitment to a solid surface, dependent on the conformation of the RolA bound to thehydrophobic surface. Interestingly, they found that
RolA bound to the solid PBS surface and speciﬁcallyrecruits CutL1 to the surface, which stimulatedhydrolysis of PBS by CutL1.49 Speciﬁc interactions
between RolA adsorbed on the hydrophobic surface,and CutL1 was conﬁrmed by quartz crystal microbalance50 and immunostaining methods. The interaction dramatically facilitated the PBS degradation
catalyzed by CutL1. Furthermore, they found thathigh levels of ﬂuorescence recovery was observed
after photobleaching51 of ﬂuorescein isothiocyanatelabeled RolA, suggesting lateral mobility of RolA on

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Figure 4. Possible mechanism of gene and genome size expansionof A. oryzae. (A) The common ancestor of the three Aspergillus
species (A. oryzae, A. fumigatus and A. nidulans) is assumed tohave smaller genome size of A. oryzae and similar size to A.
fumigatus and A. nidulans. Aspergillus oryzae might haveacquired genetic materials (represented by hatched boxes)
during evolution. (B) The common ancestor is assumed tohave the genome size similar to A. oryzae. The other two
species might have lost genetic materials (represented by open
boxes) during evolution. The phylogenetic relationship of thethree species42 is indicated. (C) The A. oryzae genome size
might have been expanded by the gene duplication followedby divergence of one of the duplicated genes (represented byhatched boxes).

179

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Genomics of Aspergillus oryzae

Table 1. Internet resources for fungal genomics
Organization

URL

Broad Institutehttp://www.broad.mit.edu/
annotation/fgi/

Genoscope

http://www.genoscope.cns.fr/spip/
Fungi-sequenced-at-Genoscope.html

Joint Genome Institute(JGI)

http://genome.jgi-psf.org/euk_home.
html

J. Craig Venter Institute(JCVI)

http://pfgrc.jcvi.org/

National Center for
Biotechnology
Information (NCBI)

http://www.ncbi.nlm.nih.gov/
genomes/FUNGI/funtab.html

National Institute ofTechnology and
Evaluation (NITE)

http://www.bio.nite.g.o.jp/dogan/Top

Sanger Center

http://www.sanger.ac.uk/Projects/
Fungi/

Washington University
Genome Sequencing
Centerhttp://genome.wustl.edu/
sub_genome_group_index.
cgi?GROUP=5

Comparative genomics and functional genomics incombination with knowledge gained from traditional
fermentation technologies should be extremely valuable as a means for both the basic and industrial
research of A. oryzae.
Acknowledgements: The authors are grateful to
Dr Yuichiro Murai of Kojiya Sanzaemon, Co., Ltd
and Harunatsuakifuyu Sousyo for providing thephotograph of the historical signboard. We wish to
thank Dr Hiroshi Konno of Akita Konno Co., Ltd forthe introduction on traditional technologies for theindustrial production of Moyashi. We also thankDr Katsumi Isono for critical reading and extensive
review.
Supplementary Data: Supplementary data are
available online at www.dnaresearch.oxfordjournals.org.

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the PBS surface.49 The mechanism could imply anovel tactic for fungal invasion of plants, insects andother animals. Without the genomic approach, RolA,
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various metabolites and will elucidate mutationsindispensable for industrial use. To date sequencingsof 50 fungal genomes have been completed or
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