Using DNA-tagged mutagenesis to improve heterologous protein production inAspergillus oryzae

Restriction enzyme-mediated integration (REMI) has been employed as a mutag en to generate two insertion libraries in an Aspergillus oryzae strain expr essing a Thermomyces lanuginosus lipase, The REMI libraries were created us ing linearized plasmid containing the A. oryzae pyrG and either BamHI or Ec oRI enzyme. The libraries were screened for lipase production, and mutants with increased production were isolated, The genomic DNA flanking the integ ration event was cloned from one of the mutants with increased lipase titer s (DEBY10.3). Nucleotide sequence of the flanking DNA revealed similarity t o the Aspergillus nidulans palB gene. Disruption of the palB gene in a stra in producing lipase resulted in increased lipase expression. Additionally, complementation of the palB phenotype of DEBY10.3 led to a decrease in lipa se production. These lines of evidence demonstrate that the increase in lip ase yield in DEBY10.3 is linked to the palB phenotype generated by the inte gration of the pyrG gene into the palB gene. The results also demonstrated that tagged mutagenesis with REMI can be used to identify genes that influe nce expression of heterologous proteins. (C) 2000 academic Press.