Transient marker system for iterative gene targeting of a prototrophic fungus.

Abstract:

: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.

journal_name

Appl Environ Microbiol

authors

Nielsen ML,de Jongh WA,Meijer SL,Nielsen J,Mortensen UH

doi

10.1128/AEM.01839-07

subject

Has Abstract

pub_date

2007-11-01 00:00:00

pages

7240-5

issue

22

eissn

0099-2240

issn

1098-5336

pii

AEM.01839-07

journal_volume

73

pub_type

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