Using DNA-Tagged mutagenesis to improve heterologous protein production in Aspergillus oryzae.

Debbie S. Yaver, Howard Brody, Michael Lamsa, Rebecca Munds, Stephen H. Brown, Suzie M. Otani, and James A. Johnstone.


In order to identify Aspergillus oryzae genes that are important for heterologous protein production a large library of DNA-tagged mutants was generated and screened for production of a model heterologous protein. Using restriction enzyme-mediated integration (REMI) two libraries of 27,000 and 28,000 transformants were made in an A.oryzae strain producing a Thermomyces lanuginosus lipase using pyrG as the tagging DNA and either BamHI or EcoRI enzyme, respectively. The libraries were screened robotically for lipase production, and mutants with either decreases or increases in lipase production were isolated. The pyrG tagging vector along with the genomic DNA flanking the integration event has been rescued from one of the yield improved mutants (DEBY10.3), and the nucleotide sequence of the flaking DNA shares identity to the Aspergillus nidulans palB gene. As expected for a palB minus strain, the DEBY10.3 mutant is unable to grow on pH 8.0 minimal medium plates. Three lines of experimental evidence demonstrate that the increase in lipase yield in DEBY 10.3 is linked to the palB minus phenotype generated by the integration of the tag into the palBgene. These results will be presented.

abstract No: 

Fungal Genet. Newsl. 46 (Supl):

Full conference title: 

Fungal Genetics Conference 20th
    • Fungal Genetics Conference 20th (1999)