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-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.
Gouka
R J
RJ
Department of Molecular Genetics and Gene Technology, TNO Nutrition and Food Research Institute, Rijswijk, The Netherlands.
Hessing
J G
JG
Stam
H
H
Musters
W
W
van den Hondel
C A
CA
D000595:Amino Acid Sequence; D001230:Aspergillus / Q000235:genetics*; D001483:Base Sequence; D004271:DNA, Fungal / Q000235:genetics; D018014:Gene Transfer Techniques; D005800:Genes, Fungal; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015183:Restriction Mapping; D014170:Transformation, Genetic
D004271:DNA, Fungal