We conducted genome sequencing of the filamentous fungus Aspergillus sojae NBRC4239 isolated from the koji used to prepare Japanese soy sauce. We used the 454 pyrosequencing technology and investigated the genome with respect to enzymes and secondary metabolites in comparison with other Aspergilli sequenced. Assembly of 454 reads generated a non-redundant sequence of 39.5-Mb possessing 13 033 putative genes and 65 scaffolds composed of 557 contigs. Of the 2847 open reading frames with Pfam domain scores of >150 found in A. sojae NBRC4239, 81.7% had a high degree of similarity with the genes of A. oryzae. Comparative analysis identified serine carboxypeptidase and aspartic protease genes unique to A. sojae NBRC4239. While A. oryzae possessed three copies of α-amyalse gene, A. sojae NBRC4239 possessed only a single copy. Comparison of 56 gene clusters for secondary metabolites between A. sojae NBRC4239 and A. oryzae revealed that 24 clusters were conserved, whereas 32 clusters differed between them that included a deletion of 18 508 bp containing mfs1, mao1, dmaT, and pks-nrps for the cyclopiazonic acid (CPA) biosynthesis, explaining the no productivity of CPA in A. sojae. The A. sojae NBRC4239 genome data will be useful to characterize functional features of the koji moulds used in Japanese industries.

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Ten strains isolated from industrial soy sauce producing koji mold were identified as Aspergillus sojae and distinguished from Aspergillus parasiticus morphologically and physiologically. There was no detectable aflatoxin in any culture extracts of A. sojae strains. Strain 477 was chosen as a representative strain of industrial A. sojae for further molecular analysis. All enzymatic activities associated with the aflatoxin biosynthesis were not detected or negligible in strain 477 compared with that of the A. parasiticus strain. Southern analysis suggested that the genomic DNA of strain 477 contained aflatoxin biosynthetic pathway genes. In contrast, all industrial strains lacked detectable transcripts of aflR, the main regulatory gene for aflatoxin biosynthesis, under the aflatoxin-inducing condition. Our data suggest that defects in aflR expression cause the lack of expression of aflatoxin-related genes which results in the absence of aflatoxin biosynthesis in A. sojae strains.

Glutaminase, an enzyme that hydrolyzes L-glutamine to L-glutamate, plays an important role in the production of fermented foods by enhancing the umami taste. In this study, we found ten glutaminase genes in the Aspergillus sojae genome by conducting a BLAST search of the characterized glutaminase sequence. We subsequently constructed glutaminase gene disruptants. The glutaminase activity of the gahB disruptant was decreased by approximately 90 % in A. sojae and Aspergillus oryzae, indicating that this enzyme (GahB) accounted for the majority of the glutaminase activity in Aspergillus species. Subsequently, GahB protein was purified from the AsgahB-overexpressing transformant and characterized. The molecular mass was estimated to be approximately 110 and 259 kDa by SDS-PAGE and gel filtration chromatography, respectively, indicating that the native form of AsGahB was a dimer. The optimal pH was 9.0, and the optimal temperature was 50 °C. Analysis of substrate specificity revealed that AsGahB had peptidoglutaminase-asparaginase activity, similar to AsGahA, but preferred free L-glutamine to free L-asparagine, C-terminal glutaminyl, and asparaginyl residues in peptides.

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