Genomics of Aspergillus oryzae: learning from the history of Koji mold and exploration of its future

Abstract

At a time when the notion of microorganisms did not exist, our ancestors empirically established methods for the production of various fermentation foods: miso (bean curd seasoning) and shoyu (soy sauce), both of which have been widely used and are essential for Japanese cooking, and sake, a magical 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 secretory hydrolases 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 genes that 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 expression of 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 to enhance 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.

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, the association of A. oryzae conidiospores suppliers during the Muromachi period. Currently there are five major distributors in Japan 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 originally developed 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.

Figure 2

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

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 42°C 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 (bold line). 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 42°C against YPD at 30°C; High Osmotic Pressure, CD containing 0.8 M NaCl against CD. See further details in the previous reports. Reproduced from Tamano et al., 2008.

Figure 4

Possible mechanism of gene and genome size expansion of A. oryzae. (A) The common ancestor of the three Aspergillus species (A. oryzae, A. fumigatus and A. nidulans) is assumed to have smaller genome size of A. oryzae and similar size to A. fumigatus and A. nidulans. Aspergillus oryzae might have acquired genetic materials (represented by hatched boxes) during evolution. (B) The common ancestor is assumed to have 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 the three species is indicated. (C) The A. oryzae genome size might have been expanded by the gene duplication followed by divergence of one of the duplicated genes (represented by hatched boxes).