Transformation of Aspergillus awamori by Agrobacterium tumefaciens-mediated homologous recombination

Agrobacterium tumefaciens is known to transfer part of its tumor-inducing ( Ti) plasmid to the filamentous fungus Aspergillus awamori by illegitimate r ecombination with the fungal genome. Here, we show that when this Ti DNA sh ares homology with the A. awamori genome, integration can also occur by hom ologous recombination, On the basis of this finding, we have developed an e fficient method for constructing recombinant mold strains free from bacteri al DNA by A. tumefaciens-mediated transformation. Multiple copies of a gene can be integrated rapidly at a predetermined locus in the genome, yielding transformants free of bacterial antibiotic resistance genes or other forei gn DNA. Recombinant A. awamori strains were constructed containing up to ni ne copies of a Fusarium solani pisi cutinase expression cassette integrated in tandem at the pyrG locus. This allowed us to study how mRNA and protein levels are affected by gene copy number, without the influence of chromoso mal environmental effects. Cutinase mRNA and protein were maximal with four gene copies, indicating a limitation at the transcriptional level. This tr ansformation system will potentially stimulate market acceptance of derived products by avoiding introduction of bacterial and other foreign DNA into the fungi.