Generation of large chromosomal deletions in koji molds Aspergillus oryzae and Aspergillus sojae via a loop-out recombination.

We established a technique for efficiently generating large chromosomal deletions in the koji molds Aspergillus oryzae and A. sojae by using a ku70-deficient strain and a bidirectional marker. The approach allowed deletion of 200-kb and 100-kb sections of A. oryzae and A. sojae, respectively. The deleted regions contained putative aflatoxin biosynthetic gene clusters. The large genomic deletions generated by a loop-out deletion method (resolution-type recombination) enabled us to construct multiple deletions in the koji molds by marker recycling. No additional sequence remained in the resultant deletion strains, a feature of considerable value for breeding of food-grade microorganisms. Frequencies of chromosomal deletions tended to decrease in proportion to the length of the deletion range. Deletion efficiency was also affected by the location of the deleted region. Further, comparative genome hybridization analysis showed that no unintended deletion or chromosomal rearrangement occurred in the deletion strain. Strains with large deletions that were previously extremely laborious to construct in the wild-type ku70(+) strain due to the low frequency of homologous recombination were efficiently obtained from Delta ku70 strains in this study. The technique described here may be broadly applicable for the genomic engineering and molecular breeding of filamentous fungi.

Transcriptional regulation of genes on the non-syntenic blocks of Aspergillus oryzae and its functional relationship to solid-state cultivation.

Transcriptome analysis revealed close relationship between solid-state cultivation and the transcriptional regulation of the genes on the non-syntenic blocks (NSBs), which were characterized by the comparison of Aspergillus oryzae genome with those of Aspergillus fumigatus and Aspergillus nidulans. Average expression ratio of the genes on NSBs in solid-state cultivation was significantly higher than that on the syntenic blocks (SBs). Of the induced genes, the genes relating to metabolism, which are highly enriched on NSBs, most contributed to the NSB-specific induction. The analysis using the SB- and NSB-genes that had sequence similarity between the two blocks significantly decreased the difference of average expression ratios between the two blocks. In spite of remarkably high averaged expression ratio of the NSB genes encoding extracellular enzymes, no induction of PKS and NRPS genes on NSBs were observed in solid-state cultivations. These results strongly suggest that the genes on NSBs play an important role on solid-state fermentation.