FLP/FRT-Mediated Recombination for Resistance Marker Recycling.

The availability of selection markers for metabolic engineering of Ustilago species is limited. The inducible flippase (FLP) recombinase system allows for the recycling of a selection marker after a successful recombination event. Following a traditional marker-based homologous recombination of DNA for deletion or insertion of a gene using special constructs with two flanking Flippase-Recognition Targets (FRT) at both sides of the selection marker, FLP-mediated recombination allows for marker removal after transformation. This enables its recycling for a further transformation approach. In Ustilago maydis, the FLP protein has been codon optimized and configured under the control of an arabinose-inducible promoter in a self-replicating plasmid for episomal expression of the recombinase. This FLP/FRT system facilitates advanced genetic modifications in Ustilago spp. by enabling repeated genetic manipulation with one marker, thus streamlining molecular genetic methodologies.