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Transformation of Aspergillus awamori by Agrobacterium tumefaciens–mediated homologous recombination

Nature Biotechnology volume 17, pages 598–601 (1999)Cite this article

Abstract

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.

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Figure 1: Experimental design for targeted integration of multiple copies of a gene into Aspergillus awamori.
Figure 2: Plasmid pUR5755.
Figure 3: Southern blot of 11 transformants of Aspergillus awamori AWCSCE.
Figure 4: (A) Northern blot of RNA isolated from Aspergillus awamori AWCSCE transformants, containing different cutinase gene copy numbers (indicated above the lanes), after 24 h of induction in minimal medium with 5% D-xylose.
Figure 5: Cutinase production levels from cultures of Aspergillus awamori AWCSCE transformants with a different number of cutinase gene copies (varying from one to nine, corresponding to the transformants shown in Fig. 3) integrated at the pyrG locus.

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Acknowledgements

J.W. Chapman is acknowledged for critical reading of this manuscript.

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Author notes

  1. Marcel J.A. de Groot

    Present address: Senter, Grote Marktstraat 4, 2500 GS, Den Haag, The Netherlands

Authors and Affiliations

  1. Unilever Research Vlaardingen, Olivier van Noortlaan 120, Vlaardingen, 3133 AT, The Netherlands

    Robin J. Gouka, Casper Gerk, Wouter Musters, C. Theo Verrips & Marcel J.A. de Groot

  2. Institute of Molecular Plant Sciences, Clusius Laboratory, Leiden University, Wassenaarseweg 64, Leiden, 2333 AL, The Netherlands

    Paul J.J. Hooykaas & Paul Bundock

Authors
  1. Robin J. Gouka
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  2. Casper Gerk
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Correspondence to Robin J. Gouka.

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Gouka, R., Gerk, C., Hooykaas, P. et al. Transformation of Aspergillus awamori by Agrobacterium tumefaciens–mediated homologous recombination. Nat Biotechnol 17, 598–601 (1999). https://doi.org/10.1038/9915

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