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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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Author: Gouka, R.J. · Hessing, J.G.M. · Stam, H. · Musters, W. · Hondel, C.A.M.J.J. van den
Institution: Centraal Instituut voor Voedingsonderzoek TNO
Source:Current Genetics, 6, 27, 536-540
Identifier: 67195
doi: DOI:10.1007/BF00314444
Keywords: Biology · Amino Acid Sequence · Aspergillus · Base Sequence · DNA, Fungal · Gene Transfer Techniques · Genes, Fungal · Genetic Vectors · Molecular Sequence Data · Mutagenesis, Site-Directed · Restriction Mapping · Transformation, Genetic · Aspergillus awamori · Aspergillus niger · Fungi


A homologous gene transfer system for Aspergillus awamori for site-specific integration is described, based on two components. First, a defined A. awarnori 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. awarnori 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 pyrC locus.