By using the combination of a ku70-deficient strain and bidirectional markers of the koji molds Aspergillus oryzae and Aspergillus sojae, we established a fundamental technology for efficiently generating large chromosomal deletions. Previously, we have reported that the genomic deletion of a 60-kb region including the aflatoxin gene cluster was efficiently carried out in an A. sojae strain (1). In this study, we investigated the limitations of the possible deletion range in one deletion cycle and the efficiency of 2 deletion methods, the loop-out (resolution-type recombination) and direct (replacement-type recombination) deletions. The large genomic deletions generated by a loop-out deletion mechanism enable us to make multiple deletions by using marker recycling. As a result, over 300-kb regions of chromosome 3 including the aflatoxin biosynthetic gene cluster and those of chromosome 4 have been completely deleted from the koji molds. Moreover, no additional sequence remained in the resultant deletion strains, which makes this technique suitable for the breeding of food-grade microorganisms. In addition, we recently discovered that the limitation of deletion range in the replacement-type recombination method was highly expanded in ku70 mutant strains. Thus far, replacement-type recombination between 2 homologous regions separated by a large nonhomologous sequence has been practically impossible due to the low frequency of recombination in wild-type koji mold strains. In this study, we have reported the successful deletion of regions larger than 200 kb in chromosome 3, including the aflatoxin gene cluster, and 470 kb in chromosome 8 with high efficiency by using ku-deficient strains of the koji molds A. sojae and A. oryzae. The results obtained in this study indicate that the ku70 mutation elevated not only the conventional gene-targeting frequency but also the frequency of recombination between 2 distant sites. The technology described here is applicable for genomic engineering and molecular breeding of industrially used filamentous fungi by artificially generating large-scale genomic deletions and marker-free strains. 1 Takahashi et al. (2006) In: Abstracts of ECFG8, PP272.
Full conference title:
9th EUROPEAN CONFERENCE ON FUNGAL GENETICS
- ECFG 9th (2008)