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Applied and Environmental Microbiology, November 2007, p. 7240-7245, Vol. 73, No. 22
0099-2240/07/$08.00+0     doi:10.1128/AEM.01839-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Transient Marker System for Iterative Gene Targeting of a Prototrophic Fungus{triangledown}

Michael L. Nielsen,{dagger} Willem A. de Jongh,{dagger},{ddagger} Susan L. Meijer, Jens Nielsen, and Uffe H. Mortensen*

Center for Microbial Biotechnology, BioCentrum-DTU, Technical University of Denmark, Building 223, DK-2800 Kongens Lyngby, Denmark

Received 8 August 2007/ Accepted 24 September 2007

Auxotrophic microorganisms are often used for genetic engineering, because their biosynthetic deficiency can be complemented by the transforming DNA and allows selection for transformants that have become prototrophic. However, when complementation is obtained by ectopic expression this may lead to unpredictable side effects on the phenotype and, consequently, misinterpretation of experimental data. There are various ways to overcome the problem of auxotrophy, but the most reliable is to restore the function of the defective biosynthetic gene at the native genomic locus. This can be done by either sexual crossing or further genetic engineering. For fungal species lacking a perfect state or situations in which gene targeting is generally cumbersome we have developed a concept that allows transient disruption of pyrG. When the gene is in the disrupted state, multiple rounds of gene targeting can be performed with the strain. Once the desired genome engineering is completed, pyrG function can be rapidly returned to wild type by a simple selection scheme.

* Corresponding author. Mailing address: Center for Microbial Biotechnology, BioCentrum-DTU, Technical University of Denmark, Building 223, DK-2800 Kongens Lyngby, Denmark. Phone: 45 4525 2701. Fax: 45 4588 4148. E-mail: um{at}biocentrum.dtu.dk

{triangledown} Published ahead of print on 5 October 2007.

{dagger} M.L.N. and W.A.D. contributed equally to the work.

{ddagger} Present address: Pharmexa A/S, Kogle Allé 6, DK-2970 Hørsholm, Denmark.

Applied and Environmental Microbiology, November 2007, p. 7240-7245, Vol. 73, No. 22
0099-2240/07/$08.00+0     doi:10.1128/AEM.01839-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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