Yoshiki Hanya

Identification of species in Aspergillus section Flavi based on sequencing of the mitochondrial cytochrome b gene.

The partial sequences of the mitochondrial (mt) cytochrome b gene (402 bp) were determined for species of Aspergillus section Flavi. On the basis of identities of DNA sequences, 77 strains were divided into seven DNA types, from D-1 to D-7. The type strains of A. sojae, A. parasiticus, A. flavus and A. oryzae together, A. tamarii, and A. nomius were placed in DNA types D-1. D-2, D-4, D-5 and D-7, respectively. These species could be differentiated from each other. Furthermore, two other DNA types, D-3 and D-6 were found. DNA type D-3 was closely related to A. parasiticus (D-2) and included one strain that deposited as A. flatus var. flavus and produced aflatoxins B and G. DNA types D-6 included one strain named A. flavus and closely related to A. tamarii. The observations of conidial wall texture by SEM (Scanning Electron Microscopy) supported the relationships derived from the cytochrome b gene. The production of aflatoxins was also examined. Using the DNA sequence of cytochrome b gene, several strains were reidentified. The derived amino acids sequences were all the same in the studied strains. The mt cytochrome b gene is useful and reliable in distinguishing and identifying the species in Aspergillus section Flavi.

Absence of aflatoxin biosynthesis in koji mold (Aspergillus sojae)

Abstract. 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.

Draft Genome Sequencing and Comparative Analysis of Aspergillus sojae NBRC4239

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.

Cryptococcus nodaensis sp nov, a yeast isolated from soil in Japan that produces a salt-tolerant and thermostable glutaminase

An anamorphic basidiomycetous yeast, which produced a salt-tolerant and thermostable glutaminase, was isolated from soil in Japan and classified in the genus Cryptococcus. Its substrate specificity suggests that this enzyme is an L-glutaminase asparaginase (EC 3.5.1.38). The strain, G60, resembles Cryptococcus laurentii in the taxonomic criteria traditionally employed for yeasts, however it can be distinguished as a separate species based on DNA–DNA reassociation experiments and sequence analysis of the large sub-unit rDNA. Phenotypically, the isolate can be differentiated from C. laurentii by the inability to utilize arbutin as a sole source of carbon. Based on sequence analysis, the strain is related to a group of hymenomycetous yeasts including Bulleromyces albus, Bullera unica, C. laurentii and C. skinneri. The strain, which is formally described as Cryptococcus nodaensis, is industrially important for the formation of the umami taste during production of proteolytic seasonings.

Identification of the Glutaminase Genes of Aspergillus sojae Involved in Glutamate Production during Soy Sauce Fermentation

Glutaminase, an enzyme that catalyzes the conversion of L-glutamine to L-glutamate, enhances the umami taste in soy sauce. The Aspergillus sojae genome contains 10 glutaminase genes. In this study, we estimated that approximately 60% of the glutamate in soy sauce is produced through the glutaminase reaction. To determine which glutaminase is involved in soy sauce glutamate production, we prepared soy sauces using single and multiple glutaminase gene disruptants of A. sojae. The glutamate concentration in soy sauce prepared using the ΔgahA-ΔgahB-ΔggtA-Δgls disruptant was approximately 60% lower than that in the control strain, whereas it was decreased by approximately 20–30% in the ΔgahA-ΔgahB disruptant. However, the glutamate concentration was unchanged in the soy sauces prepared using the ΔgahA-ΔggtA-Δgls and ΔgahB-ΔggtA-Δgls disruptants. These results indicate that four glutaminases are involved in glutamate production in soy sauce, and that the peptidoglutaminase activities of GahA and GahB increase the glutamate concentration in soy sauce.

Targeted Tandem Duplication of a Large Chromosomal Segment in Aspergillus oryzae

ABSTRACT We describe here the first successful construction of a targeted tandem duplication of a large chromosomal segment in Aspergillus oryzae. The targeted tandem chromosomal duplication was achieved by using strains that had a 5′-deleted pyrG upstream of the region targeted for tandem chromosomal duplication and a 3′-deleted pyrG downstream of the target region. Consequently, strains bearing a 210-kb targeted tandem chromosomal duplication near the centromeric region of chromosome 8 and strains bearing a targeted tandem chromosomal duplication of a 700-kb region of chromosome 2 were successfully constructed. The strains bearing the tandem chromosomal duplication were efficiently obtained from the regenerated protoplast of the parental strains. However, the generation of the chromosomal duplication did not depend on the introduction of double-stranded breaks (DSBs) by I-SceI. The chromosomal duplications of these strains were stably maintained after five generations of culture under nonselective conditions. The strains bearing the tandem chromosomal duplication in the 700-kb region of chromosome 2 showed highly increased protease activity in solid-state culture, indicating that the duplication of large chromosomal segments could be a useful new breeding technology and gene analysis method.