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A novel strategy for the isolation of defined pyrG mutants and the development of a site-specific integration system for Aspergillus awamori

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Abstract

A homologous gene transfer system for Aspergillus awamori for site-specific integration is described, based on two components. First, a defined A. awamori pyrG mutant strain constructed by a selection strategy for gene-replacement in fungi. Second, a vector with a homologous pyrG selection marker containing a defined mutation at a site different from that of the mutations in the pyrG gene of the defined mutant strain. Defined mutation in the A. awamori pyrG gene, isolated from a genomic library by heterologous hybridisation with the A. niger pyrG gene as a probe, were introduced by specifically altering sequences at restriction sites in the coding region of the gene. After transformation of the A. awamori wild-type strain with vectors containing these mutated pyrG genes, and selection for 5-fluoro-orotic acid resistance (5-FOAR), on the average 60% of the 5-FOAR colonies originated from replacement of the wild-type pyrG gene by the mutated pyrG allele. After transformation of a mutant strain, carrying a mutation near the 5′ end of the pyrG gene with vectors containing a mutation near the 3′ end of the pyrG gene, 35% of the resulting transformants contained one copy of the vector at the pyrG locus.

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References

  • Alic M, Akileswaran L, Gold MH (1993) Gene replacement in the lignin-degrading basidiomycete Phanerochaete chrysosporium. Gene 136: 307–311

    Google Scholar 

  • Bennett JW, Lasure LL (1991) Growth media. In: Bennett JW, Lasure LL (eds) More gene manipulations in fungi. Academic Press, San Diego, pp 441–457

    Google Scholar 

  • Boeke JD, LaCroute F, Fink GR (1984) A positive selection for mutants lacking orotidine-5′-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet 197: 345–346

    Google Scholar 

  • Gouka RJ, van Hartingsveldt W, Bovenberg RAL, van den Hondel CAMJJ, van Gorcom RFM (1991) Cloning of the nitrate-nitrite reductase gene cluster of Penicillium chrysogenum and use of the niaD gene as a homologous selection marker. J Biotechnol 20: 189–200

    Google Scholar 

  • Gouka RJ, Van Hartingsveldt W, Bovenberg RAL, Van Zeijl CMJ, Van den Hondel CAMJJ, Van Gorcom RFM (1993) Development of a new transformant selection system for Penicillium chrysogenum: isolation and characterization of the P. chrysogenum acetyl-coenzyme A synthetase gene (facA) and its use as a homologous selection marker. Appl Microbiol Biotechnol 38: 514–519

    Google Scholar 

  • Hamer JE, Timberlake WE (1987) Functional organization of the Aspergillus nidulans trpC promoter. Mol Cell Biol 7: 2352–2359

    Google Scholar 

  • Hessing JGM, van Rotterdam C, Verbakel JMA, Roza M, van Gorcom RFM, van den Hondel CAMJJ (1994) Isolation and characterization of a 1,4-β-endoxylanase gene of A. awamori. Curr Genet 26: 228–232

    Google Scholar 

  • Kinsey JA (1989) A simple colony blot procedure for Neurospora. Fungal Genet Newslett 36: 45–46

    Google Scholar 

  • Kolar M, Punt PJ, van den Hondel CAMJJ, Schwab H (1988) Transformation of Penicilium chysogenum using dominant selection markers and expression of an Escherichia coli lacZ fusion gene. Gene 62: 127–134

    Google Scholar 

  • Miller BL, Miller KY, Roberti KA, Timberlake WE (1987) Positiondependent and-independent mechanisms regulate cell-specific expression of the SpoC1 gene cluster of Aspergillus nidulans. Mol Cell Biol 7: 427–434

    Google Scholar 

  • Punt PJ, van den Hondel CAMJJ (1993) Transformation of filamentous fungi based on hygromycin B and phleomycin resistance markers. Methods Enzymol 216: 447–457

    Google Scholar 

  • Punt PJ, Dingemanse MA, Kuyvenhoven J, Soede RDM, Pouwels PH, Van den Hondel CAMJJ (1990) Functional elements in the promoter region of the Aspergillus nidulans gpdA gene, encoding glyceraldehyde-3-phosphate dehydrogenase. Gene 93: 101–109

    Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

    Google Scholar 

  • Van Gorcom RFM, Van den Hondel CAMJJ (1988) Expression analysis vectors for Aspergillus niger. Nucleic Acids Res 16: 9052

    Google Scholar 

  • Van Gorcom RFM, Pouwels PH, Goosen T, Visser J, Van den Broek HWJ, Hamer JE, Timberlake WE, Van den Hondel CAMJJ (1985) Expression of an Escherichia coli β-galactosidase fusion gene in Aspergillus nidulans. Gene 40: 99–106

    Google Scholar 

  • Van Hartingsveldt W, Mattern IE, van Zeijl CMJ, Pouwels PH, van den Hondel CAMJJ (1987) Development of a homologous transformation system for Aspergillus niger based on the pyrG gene. Mol Gen Genet 206: 71–75

    Google Scholar 

  • Van den Hondel CAMJJ, Punt PJ (1991) Gene transfer systems and vector development for filamentous fungi. In: Peberdy JF, Caten CE, Ogden JE, Bennett JW (eds) Applied Molecular Genetics of Fungi. Cambridge University Press, Cambridge, pp 1–28

    Google Scholar 

  • Van den Hondel CAMJJ, Punt PJ, Van Gorcom RFM (1991) Heterologous gene expression in filamentous fungi. In: Bennet JW, Lasure LL (eds) More gene manipulations in fungi. Academic Press, San Diego, pp 396–428

    Google Scholar 

  • Verdoes JC, Punt PJ, Van den Hondel CAMJJ (1995) Molecular genetic strain improvement for the overproduction of fungal proteins by filamentous fungi. Appl Microbiol Biotechnol (in press)

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Gouka, R.J., Hessing, J.G.M., Stam, H. et al. A novel strategy for the isolation of defined pyrG mutants and the development of a site-specific integration system for Aspergillus awamori . Curr Genet 27, 536–540 (1995). https://doi.org/10.1007/BF00314444

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  • DOI: https://doi.org/10.1007/BF00314444

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