Generation of Large Chromosomal Deletions in Koji Molds Aspergillus oryzae and Aspergillus sojae via a Loop-Out Recombination

Abstract

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 Δ ku70 strains in this study. The technique described here may be broadly applicable for the genomic engineering and molecular breeding of filamentous fungi.