Transformation of Aspergillus awamori
by Agrobacterium tumefaciens−mediated homologous recombination
Robin J. Gouka1, Casper Gerk1, Paul J.J. Hooykaas2, Paul Bundock2, Wouter Musters1, C. Theo Verrips1
& Marcel J.A. de Groot1, 3
1
Unilever Research Vlaardingen, Olivier van Noortlaan 120,
3133 AT Vlaardingen, The Netherlands.
2
Institute of Molecular Plant Sciences, Clusius Laboratory, Leiden University,
Wassenaarseweg 64, 2333 AL Leiden, The Netherlands.
3
Present address: Senter, Grote Marktstraat 4, 2500 GS Den Haag, The Netherlands.
Correspondence should be addressed to Robin J. Gouka robin.gouka@unilever.comtargetingintegrationcutinasetitration
Agrobacterium tumefaciens is known to transfer part of its tumor-inducing (Ti) plasmid to the filamentous fungus Aspergillus awamori by illegitimate recombination with the fungal genome. Here, we show that when this Ti DNA shares homology with the A. awamori genome, integration can also occur by homologous recombination. On the basis of this finding, we have developed an efficient method for constructing recombinant mold strains free from bacterial 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 foreign DNA. Recombinant A. awamori strains were constructed containing up to nine 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 chromosomal environmental effects. Cutinase mRNA and protein were maximal with four gene copies, indicating a limitation at the transcriptional level. This transformation system will potentially stimulate market acceptance of derived products by avoiding introduction of bacterial and other foreign DNA into the fungi.
