A NOVEL STRATEGY FOR THE ISOLATION OF DEFINED PYRG MUTANTS AND THE DEVELOPMENT OF A SITE-SPECIFIC INTEGRATION SYSTEM FOR ASPERGILLUS-AWAMORI

A homologous gene transfer system for Aspergillus awamori for site-spe cific integration is described, based on two components. First, a defi ned 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 fr om that of the mutations in the pyrG gene of the defined mutant strain . Defined mutation in the A. awamori pyrG gene, isolated from a genomi c library by heterologous hybridisation with the A. niger pyrG gene as a probe, were introduced by specifically altering sequences at restri ction 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-FOA(R )), on the average 60% of the 5-FOA(R) colonies originated from replac ement of the wild-type pyrG gene by the mutated pyre allele. After tra nsformation 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 t he pyrG gene, 35% of the resulting transformants contained one copy of the vector at the pyrG locus.