Molecular Genetics and Genomics

, Volume 272, Issue 3, pp 344–352 | Cite as

Efficient gene disruption in the koji -mold Aspergillus sojae using a novel variation of the positive-negative method

  • T. Takahashi
  • O. Hatamoto
  • Y. Koyama
  • K. Abe
Original Paper


When no phenotypic screen is available, gene disruption in the koji -mold Aspergillus sojae is a time-consuming process, owing to the low frequency of homologous recombination. To achieve efficient gene disruption in the koji -mold, we developed a novel positive-negative selection method to enrich for homologous recombinants. The pyrG gene from A. sojae was used as a positive selection marker for transformants, and the oliC31 gene of A. nidulans, which codes for a mutant form of subunit 9 of the F1FO-ATPase, was employed as a negative selection marker to facilitate elimination of non-homologous recombinants among the transformants. The positive-negative selection markers, in combination with a pyrG deletion strain as a host, enabled enrichment for homologous recombinants, and disruption of the genes niaD, areA and tannase was successfully demonstrated. In order to examine whether the positive-negative selection technique is effective for targeting any locus, even in the absence of information on gene function or phenotype, we attempted to disrupt the aflR gene of A. sojae, which codes for a putative transcription factor for the aflatoxin biosynthetic pathway, using the method. Despite the fact that this gene is not transcribed in A. sojae, aflR disruptants were efficiently obtained, suggesting that the method is indeed capable of targeting any locus, without additional ectopic integration, and is thus applicable for functional genomics studies in filamentous fungi, including A. sojae.


Transformation Gene targeting Screening Selection  oliC31 



We thank Genryou Umitsuki and Kenichiro Matsushima for helpful discussions, and Kuniko Shiraishi and Hideko Osawa for their technical assistance


  1. Barratt RW (1974) Neurospora crassa. In: King RC (ed) Handbook of genetics, vol 1: bacteria, bacteriophages and fungi. Plenum Press, New York and London, pp 511–529Google Scholar
  2. Bird D, Bradshaw R (1997) Gene targeting is locus dependent in the filamentous fungus Aspergillus nidulans. Mol Gen Genet 255:219–225CrossRefPubMedGoogle Scholar
  3. Caddick MX, Arst HN Jr, Taylor LH, Johnson RI, Brownlee AG.(1986) Cloning of the regulatory gene areA mediating nitrogen metabolite repression in Aspergillus nidulans. EMBO J 5:1087–1090PubMedGoogle Scholar
  4. Chang P-K, Yu J, Bhatnagar D, Cleveland TE (1999) The carboxy-terminal portion of the aflatoxin pathway regulatory protein AFLR of Aspergillus parasiticus activates GAL1::lacZ gene expression in Saccharomyces cerevisiae. Appl Environ Microbiol 65:2508–2512PubMedGoogle Scholar
  5. Chang XB, Wilson JH (1987) Modification of DNA ends can decrease end joining relative to homologous recombination in mammalian cells. Proc Natl Acad Sci USA 84:4959–4963PubMedGoogle Scholar
  6. Christensen T, Hynes MJ, Davis MA (1998) Role of the regulatory gene areA of Aspergillus oryzae in nitrogen metabolism. Appl Environ Microbiol 64:3232–3237PubMedGoogle Scholar
  7. Clutterback AJ (1974) Aspergillus nidulans. In: King RC (editor) Handbook of genetics, vol 1: bacteria, bacteriophages and fungi. Plenum Press, NewYork and London, pp 447–510Google Scholar
  8. Crawford NM, Arst HN (1993) The molecular genetics of nitrate assimilation in fungi and plants. Annu Rev Genet 27:115–146CrossRefPubMedGoogle Scholar
  9. De Ruiter-Jacobs YM, Broekhuijsen M, Unkles SE, Campbell EI, Kinghorn JR, Contreras R, Pouwels PH, van den Hondel CA. (1989) A gene transfer system based on the homologous pyrG gene and efficient expression of bacterial genes in Aspergillus oryzae. Curr Genet 16:159–163PubMedGoogle Scholar
  10. Harris SD, Morrell JL, Hamer JE (1994) Identification and characterization of Aspergillus nidulans mutants defective in cytokinesis. Genetics 136:517–532PubMedGoogle Scholar
  11. Hatamoto O, Watarai T, Kikuchi M, Mizusawa K, Sekine H (1996) Cloning and sequencing of the gene encoding tannase and structural study of the tannase subunit from Aspergillus oryzae. Gene 175:215–221CrossRefPubMedGoogle Scholar
  12. Hatamoto O, Umitsuki G, Arai A, Kajiyama N, Masuda T (2003) Cloning and expression of tannase gene from Aspergillus oryzae. In: Abstracts of the Annual Meeting 2003, Society for Biotechnology, Japan, PP148Google Scholar
  13. Mansour SL, Thomas KR, Capecchi MR (1988) Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature 336:348–352CrossRefPubMedGoogle Scholar
  14. Matsushima K, Yashiro K, Hanya Y, Abe K, Yabe K, Hamasaki T (2001) Absence of aflatoxin biosynthesis in koji mold ( Aspergillus sojae). Appl Microbiol Biotechnol 55:771–776CrossRefPubMedGoogle Scholar
  15. Oakley BR, Rinehart JW, Mitchell BL, Oakley CE, Carmona C, Gray GL, May GS (1987) Cloning, mapping and molecular analysis of the pyrG (orotidine-5′-phosphate decarboxylase) gene of Aspergillus nidulans. Gene 61:385–399CrossRefPubMedGoogle Scholar
  16. Platt A, Ravagnani A, Arst H Jr, Kirk D, Langdon T, Caddick MX (1996a) Mutational analysis of the C-terminal region of AREA, the transcription factor mediating nitrogen metabolite repression in Aspregillus nidulans. Mol Gen Genet 250:106–114CrossRefPubMedGoogle Scholar
  17. Platt A, Langdon T, Arst HN Jr, Kirk D, Tellervey D, Sanchez JM, Caddick MX (1996b) Nitrogen metabolite signaling involves the C-terminus and the GATA domain of the Aspergillus transcription factor AREA and the 3′ untranslated region of its mRNA. EMBO J 15:2791–2801PubMedGoogle Scholar
  18. Santerre RF, Allen NE, Hobbs JN Jr, Rao RN, Schmidt RJ (1984) Expression of prokaryotic genes for hygromycin B and G418 resistance as dominant-selection markers in mouse L cells. Gene 30:147–156CrossRefPubMedGoogle Scholar
  19. Schroppel K, Soll DR (1995) The frequency of integrative transformation at phase-specific genes of Candida albicans correlates with their transcriptional state. Mol Gen Genet 246:342–352PubMedGoogle Scholar
  20. Stankovich M, Platt A, Caddick MX, Langdon T, Shaffer PM, Arst HN Jr (1993) C-terminal truncation of the transcriptional activator encoded by areA in Aspergillus nidulans results in both loss-of-function and gain-of-function phenotypes. Mol Microbiol 7:81–87PubMedGoogle Scholar
  21. Takahashi T, Chang PK, Matsushima K, Yu J, Abe K, Bhatnagar D, Cleveland TE, Koyama Y (2002) Nonfunctionality of Aspergillus sojae aflR in a strain of Aspergillus parasiticus with a disrupted aflR gene. Appl Environ Microbiol 68:3737–3743CrossRefPubMedGoogle Scholar
  22. Thomas BJ, Rothstein R (1989) Elevated recombination rates in transcriptionally active DNA. Cell 56:619–630PubMedGoogle Scholar
  23. Thyagarajan B, Johnson B, Cambell C (1995) The effect of target site transcription on gene targeting in human cells in vitro. Nucleic Acids Res 23:2784–2790PubMedGoogle Scholar
  24. Unkles SE, Campbell EI, de Ruiter-Jacobs YM, Broekhuijsen M, Macro JA, Carrez D, Contreras R, van den Hondel CA, Kinghorn JR (1989) The development of a homologous transformation system for Aspergillus oryzae based on the nitrate assimilation pathway: a convenient and general selection system for filamentous fungal transformation. Mol Gen Genet 218:99–104Google Scholar
  25. Ushijima S, Nakadai T (1987) Breeding by protoplast fusion of koji-mold, Aspergillus sojae. Agric Biol Chem 51:1051–1057Google Scholar
  26. Ushijima S, Nakadai T, Uchida K (1990) Breeding of new koji-molds through interspecific hybridization between Aspergillus oryzae and Aspergillus sojae by protoplast fusion. Agric Biol Chem 54:1667–1676Google Scholar
  27. Ward M, Turner G (1986) The ATP synthase subunit 9 gene of Aspergillus nidulans: sequence and transcription. Mol Gen Genet 205:331–338PubMedGoogle Scholar
  28. Ward M, Wilkinson B, Turner G (1986) Transformation of Aspergillus nidulans with a cloned, oligomycin-resistant ATP synthase subunit 9 gene. Mol Gen Genet 202:265–270PubMedGoogle Scholar
  29. Watson AJ, Fuller LJ, Jeenes DJ, Archer DB (1999) Homologs of aflatoxin biosynthesis genes and sequence of aflR in Aspergillus oryzae and Aspergillus sojae. Appl Environ Microbiol 65:307–310PubMedGoogle Scholar
  30. Wernars K, Goosen T, Wennekes BM, Swart K, van den Hondel CA, van den Broek HW (1987) Cotransformation of Aspergillus nidulans: a tool for replacing fungal genes. Mol Gen Genet 209:71–77PubMedGoogle Scholar
  31. Wu TS, Linz JE (1993) Recombinational inactivation of the gene encoding nitrate reductase in Aspergillus parasiticus. Appl Environ Microbiol 59:2998–3002PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • T. Takahashi
    • 1
    • 2
  • O. Hatamoto
    • 1
    • 2
  • Y. Koyama
    • 1
  • K. Abe
    • 1
    • 3
  1. 1.Research and Development DivisionKikkoman CorporationNoda CityJapan
  2. 2.Noda Institute for Scientific ResearchNoda CityJapan
  3. 3.Graduate School of Agricultural SciencesTohoku UniversitySendaiJapan

Personalised recommendations