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Applied and Environmental Microbiology, December 2008, p. 7684-7693, Vol. 74, No. 24
0099-2240/08/$08.00+0     doi:10.1128/AEM.00692-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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

Tadashi Takahashi,1* Feng Jie Jin,1 Misao Sunagawa,2 Masayuki Machida,2 and Yasuji Koyama1

Noda Institute for Scientific Research, 399 Noda, Noda City, Chiba 278-0037, Japan,1 Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology, Central 6, Higashi 1-1-1, Tsukuba, Ibaraki 305-8566, Japan2

Received 25 March 2008/ Accepted 29 September 2008

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.

* Corresponding author. Mailing address: Noda Institute for Scientific Research, 399 Noda, Noda City, Chiba 278-0037, Japan. Phone: 81-4-7123-5573. Fax: 81-4-7123-5953. E-mail: ttakahashi{at}mail.kikkoman.co.jp

{triangledown} Published ahead of print on 24 October 2008.

Applied and Environmental Microbiology, December 2008, p. 7684-7693, Vol. 74, No. 24
0099-2240/08/$08.00+0     doi:10.1128/AEM.00692-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Jin, F. J., Takahashi, T., Machida, M., Koyama, Y. (2009). Identification of a Basic Helix-Loop-Helix-Type Transcription Regulator Gene in Aspergillus oryzae by Systematically Deleting Large Chromosomal Segments. Appl. Environ. Microbiol. 75: 5943-5951 [Abstract] [Full Text]  


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