Draft genome sequencing and comparative analysis of Aspergillus sojae NBRC4239

Abstract

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.

Figure 1.

Phylogenic analysis of serine carboxypeptidase in five Aspergillus species. ORFs of serine carboxypeptidase with Pfam domain scores of >150 were analysed by ClustalW and were drawn by TreeView for five Aspergillus species. Filled circles, A. sojae; filled squares, A. oryzae; filled diamonds, A. flavus; triangle, A. nidulans; inverted triangles, A. fumigatus. Blue lines indicate the genes in common with all five Aspergillus species. Green lines indicate the genes in common with three species in Aspergillus section Flavi. Red shows the gene unique to A. sojae. Oranges show extra homologues in A. oryzae reported in Machida et al.

Figure 2.

Phylogenic analysis of aspartic protease in five Aspergillus species. ORFs of aspartic protease with Pfam domain scores of >150 were analysed by ClustalW and were drawn by TreeView for 5 Aspergillus species. Filled circles, A. sojae; filled squares, A. oryzae; filled diamonds, A. flavus; triangle, A. nidulans; inverted triangles, A. fumigatus. Blue lines indicate the genes in common with all five Aspergillus species. Green line indicates the genes in common with three species in Aspergillus section Flavi. Red shows the gene unique to A. sojae. Oranges show extra homologues in A. oryzae reported in Machida et al.

Figure 3.

Comparison of α-amylase genes and their flanking regions. Map of α-amylase and flanking regions. Grey box, conserved regions; shaded box, regions unique to A. sojae; white box, regions unique to A. oryzae ; black box, inversed regions; black arrow, α-amylase gene ORF; black triangle, PCR primer-binding site.

Figure 4.

Analysis of transposons surrounding α-amylase genes in A. oryzae RIB40 and A. sojae NBRC4239. (A) Expected insertion site of A. oryzae Tao1 transposon. (B) Map of transposons surrounding A. oryzae and A. sojae α-amylase genes.

Figure 5.

Comparison of A. oryzae predicted secondary metabolite clusters with A. sojae. Secondary metabolite Clusters 38 (A) and 47 (B) which were predicted from A. oryzae RIB40 by SMURF were compared with sequences in A. sojaeNBRC4239 by Harr plots.

Figure 6.

Comparison of CPA biosynthesis gene cluster regions between A. sojae NBRC4239, A. flavus NRRL3357, and A. oryzae RIB40. Nucleotide sequences of CPA biosynthesis gene cluster regions were compared between A. sojae NBRC4239, A. flavus NRRL3357, and A. oryzae RIB40 by BLASTN and are presented diagrammatically.

Figure 7.

Comparison of aflatrem cluster regions in A. sojae NBRC4239 and A. flavus. Diagram of aflatrem cluster sequence comparison between A. flavus NRRL3357 and A. sojae NBRC4239 strains. (A) ATM1 region. (B) ATM2 region.