Aspergillus oryzae

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Aspergillus oryzae is a filamentous fungus, or mold, that is used in East Asian (particularly Japanese and Chinese) food production, such as in soybean fermentation. A. oryzae is utilized in solid-state cultivation (SSC), which is a form of fermentation in a solid rather than a liquid state. This fungi is essential to the fermentation processses because of its ability to secrete large amounts of various degrading enzymes, which allows it to decompose the proteins of various starches into sugars and amino acids. [4]

A. oryzae morphology. [1]

Classification

Higher Order Taxa

Eukarya; Fungi; Eurotiomycetes; Eurotiales; Tricocomaceae

Species

NCBI: A. oryzae Taxonomy

Aspergillus oryzae

Genome

The full genome of A. oryzae RIB40 contains eight chromosomes and the mitochondrion (which is circular, rather than linear) and is estimated to be 37.6μb, or 37,878,829 bp, in size.[5] It contains 12,074 genes, and is 7-9μb longer (or 25-30% larger) than other members of the Aspergillus genus, namely the species A. nidulans and A. fumigates. A. oryzae's linear genome is made up of 48.25% GC-content, or guanine-cytosine content, which is an indicator of a higher melting point. There are 12,084 ORFs (open reading frames) within the genome, which may potentially code for essential proteins or peptides. Coding regions account for 44.02% of the genome, whereas there are only 7.48% intronic regions. Additionally, the A. oryzae genome contains 270 tRNA genes, and only 3 rRNA genes.[6]

When comparing the three Aspergillus species, it was found that in A. oryzaea combination of syntenic blocks derived from a singular ancestral region and blocks specific to A. oryzae arranged mosaically comprised the full genome. The A. oryzae-specific sequence codes for metabolite synthesis and specific gene expansion for secreting hydrolytic enzymes when used in SSF, or solid-state fermentation, which makes it such an effective microbe in fermentation processes.[7]

Close relatives of Aspergillus oryzae, Aspergillus flavus and Aspergillus niger contain syntenic genes from a singular ancestor, such as a set of twenty-five proteins which code for a pathway for the poisonous aflatoxin. Yet unlike in relatives Aspergillus flavus and Aspergillus niger, these genes fail to be expressed in Aspergillus oryzae, indicating that they were inactivated during its specific evolution.[8] Because A. oryzae has been domesticated, it is possible that gene expansion is due to horizontal gene transfer, as is seen in A. oryzae-specific genes, which use clonal lines to transfer chromosomes.[9]

The fermentation of A. oryzae to make sake, a Japanese alcohol. Photo credit to Kazuo Kikuchi.[2]

Cell Structure, Metabolism and Life Cycle

It was previously thought that A.oryzae could only reproduce asexually through mitosis by dispersing spores using conidiophores. Yet, it was recently found to contain an alpha mating-type gene within its genome which implicates a heterosexual mating process. Despite this, asexual reproduction is favored in all conditions, and rarely will sexual reproduction be utilized. A.oryzae grows under warm temperatures and moist environments, as most fungi and mold do. As it matures, the filaments grow longer into a white, fluffy texture. [10]

A.oryzae, along with most other members of the Aspergillus family, have a hyphae that is hyaline and septate, and conidiophore, which ends at a round-shaped vesicle. From the vesicle extend long filaments called a conodial chain. The spore-bearing cells, or asci, are produced within the ascocarp, or the fruiting body.

A.oryzae receives its energy through contact with energy sources. Once it makes contact with an energy source (eg. a grain or starch, such as soybeans or rice), it secretes enzymes that degrade the proteins and peptide bonds within the starch and convert them into amino acids and sugars for consumption. The primary enzyme secreted by the filamentous fungi is called amylase, which lends a sweet taste to the food it is fermented into. This enzyme is most efficient at a temperature of 35-40 degrees Celcius. Most other enzymes found in A.oryzae grow at a temperature of around 30-35 degrees Celcius.[11]

Ecology and Pathogenesis

As a component of SSF,

The habitat that the A. oryzae live in are oxygen rich environments. It is highly aerobic and grow as molds on the surface of a substrate. They are contaminants of starchy foods such as bread and potatoes and may grow on plants and or trees as well. Members of its genus can also be used for medications that treat diseases. A. oryzae seems to have evolved by domestication from Aspergillus flavus, a wild-type species commonly found in soil and litter, several thousand years ago. [6]

The close relationship between A. oryzae and A. flavus and the production of highly toxic mycotoxins by the latter has resulted in careful examination of the toxigenic potential of A. oryzae. However, A. oryzae, as a koji mold, has toxigenic potential in its own right. here are two possible concerns for human health hazards associated with A. oryzae. The first, which is directly tied to A. oryzae, is the potential for mycotoxin production with extended fermentation. A variety of toxins can be produced, with the most common being the moderately toxic kojic acid. Other more potent toxins may only be produced by a few strains or in lesser quantities. These mycotoxins seem to be produced only under conditions of extended fermentation, and therefore, their production could be averted under proper fermentation conditions i.e., short fermentation times.The potential for toxin production is the main environmental hazard issue of concern for A. oryzae. If there were a method to distinguish between toxicogenic and non-toxicogenic strains, there would be no environmental concern for A. oryzae. the issues for environmental hazards are similar to those for human health hazards. The primary hazard concerns are for toxin production by A. oryzae strains. Aspergillus oryzae is considered a Class 1 Containment Agent under the National Institute of Health (NIH) Guidelines for Research Involving Recombinant DNA Molecules. The highest risk for workers when exposed to Aspefillus oryzae is when it is air born. [4]

A. oryzae shown here growing on grains of rice. Photo by Forrest O.[3]

Significance and Application

As A. oryzae is a fungus native to humid East Asian regions, it is a microorganism that is primarily used in Japanese and Chinese food production. [12] A. oryzae is utilized in solid-substrate cultivation (or SSF) which is a fermentation process used to make various different kinds of foods, from soy sauce to sake and vinegar due to its ability to secrete a multitude of useful enzymes. A. oryzae is said to have the greatest potential in efficient production of enzymes of those within the Aspergillus genus, and is therefore taken advantage of in the fields of genetic engineering and biotechnology to create industrial enzymes for even more profitable manufacturing.[13]

In solid-substrate cultivation, A. oryzae is sprinkled over rice, barley, or soybeans and fermented at a specific temperature ideal for fungus growth. The A. oryzae is sprinkled on the grain at a temperature under forty-five degrees Celcius, and the fungus (called tane koji or "seed koji" by the Japanese) grows on the steamed rice, which then raises in temperature and moisture level to allow the fungus to propagate. The enzymes it secretes break down the starches and proteins within the grains and convert it into amino acids and sugars. A grain with properly-grown fungi mycelium is characterized by fluffy, white filaments covering the outside.[14]

The production of koji, the product of the filamentous fungus A. oryzae and the chosen grain, and the techniques to cultivate it are kept a secret by each koji company.[15]

References

Machida, M., Yamada O., and Gomi K. "Genomics of Aspergillus oryzae: Learning from the History of Koji Mold and Exploration of Its Future." Oxford Journals: DNA Research. Volume 15(4). p. 173-183
Machida M., Asai K., Sano M., Tanaka T., Kumagai T., Terai G., Kusumoto K., Arima T., Akita O., Kashiwagi Y., Abe K., Gomi K., Horiuchi H., Kitamoto K., Kobayashi T., Takeuchi M., Denning D. W., Galagan J. E., Nierman W. C., Yu J., Archer D. B., Bennett J. W., Bhatnagar D., Cleveland T. E., Fedorova N. D., Gotoh O., Horikawa H., Hosoyama A., Ichinomiya M., Igarashi R., Iwashita K., Juvvadi P. R., Kato M., Kato Y., Kin T., Kokubun A., Maeda H., Maeyama N., Maruyama J., Nagasaki H., Nakajima T., Oda K., Okada K., Paulsen I., Sakamoto K., Sawano T., Takahashi M., Takase K., Terabayashi Y., Wortman J. R., Yamada O., Yamagata Y., Anazawa H., Hata Y., Koide Y., Komori T., Koyama Y., Minetoki T., Suharnan S., Tanaka A., Isono K., Kuhara S., Ogasawara N., Kikuchi H. Genome sequencing and analysis of Aspergillus oryzae. Nature 2005. Volume 438. p.1157-1161.
"Aspergillus oryzae Final Risk Assessment." Biotechnology Program under the Toxic Substances Control Act (TSCA). United States Environmental Control Agency. February 1997.
Rokas, A. "The effect of domestication on the fungal proteome." Trends Genetics 2009. Volume 25(2). p.60-63.
Goffeau, A. "Genomics: Multiple moulds". Nature 2005. Volume 438 (7071). p. 1092–1093.
Fujita, Chieko. "Koji, an Aspergillus." The Tokyo Foundation. Dec 16, 2008. Accessed April 28 2015.
"Aspergillus oryzae RIB40 (= NBRC 100959)." National Institute of Technology and Evaluation. DOGAN. January 2014. Accessed April 28 2015.

Author(s)

Page authored by Hannah Nanavaty and Matt Ogledzinski, students of Prof. Jay Lennon at Indiana University.