Tools and Techniques Cre-loxP-based system for removal and reuse of selection markers in Ashbya gossypii targeted engineering Tatiana Q. Aguiar 1 , Cláudia Dinis 1 , Lucília Domingues ⇑ CEB – Centre of Biological Engineering, University of Minho, 4710-057 Braga, Portugal article info Article history: Received 8 August 2013 Accepted 21 April 2014 Available online 2 May 2014 Keywords: Ashbya gossypii Cre-loxP system Gene targeting Marker recycling Recombinase expression abstract The filamentous ascomycete Ashbya gossypii is amenable to genetic manipulation and is an excellent model system for studying eukaryotic cell biology. However, the number of selection markers in current use for both targeted gene integration and disruption in this fungus are very limited. Therefore, the Cre-loxP recombination system was adapted for use in A. gossypii and its effectiveness in recycling marker genes was demonstrated by constructing both single and double deleted Agura3 and Agade1 auxotrophic strains free of exogenous markers. In spite of its wide use, this is the first report in which the Cre-loxP system was applied to A. gossypii, opening new perspectives for targeted engineering of this fungus with several promising biotechnological applications. Ó 2014 Elsevier Inc. All rights reserved. 1. Introduction The Cre-loxP recombination system of the bacteriophage P1 has been shown to mediate efficient recombination between directly repeated loxP sites flanking selectable markers in several organ- isms, resulting in the excision of marker genes (Sternberg and Hamilton, 1981; Sauer and Henderson, 1988; Orban et al., 1992; Zhang et al., 2009). Cre-loxP-based systems have been widely used to study gene function and to remove selection markers in fungi, among which Saccharomyces cerevisiae (Sauer, 1987; Güldener et al., 1996), Kluyveromyces lactis (Steensma and Ter Linde, 2001), Kluyveromyces marxianus (Ribeiro et al., 2007), Schizosaccharomyces pombe (Iwaki and Takegawa, 2004), Yarrowia lipolytica (Fickers et al., 2003), Hansenula polymorpha (Qian et al., 2009), Cryptococcus neoformans (Patel et al., 2010), Aspergillus nidulans (Forment et al., 2006), Trichoderma reesei (Steiger et al., 2011), Neotyphodium coenophialum, N. uncinatum and Epichloë festucae (Florea et al., 2009). Ashbya gossypii (syn. Eremothecium gossypii) is a flavinogenic fil- amentous ascomycete (Ashby and Nowell, 1926; Kurtzman, 1995) used for the industrial production of riboflavin (Stahmann et al., 2000), which shares remarkable genomic similarities with the budding yeast S. cerevisiae (Brachat et al., 2003; Dietrich et al., 2004), being therefore considered an excellent model to study filamentous growth (Wendland and Walther, 2005; Schmitz and Philippsen, 2011). The high degree of gene homology and gene order conservation (synteny) between the A. gossypii genome and the S. cerevisiae genome have facilitated the assignment of poten- tial functions to A. gossypii genes (Brachat et al., 2003; Dietrich et al., 2004). Moreover, other A. gossypii features have made the genetic manipulation of this fungus to be quite straightforward when comparing to other filamentous fungi, among which: highly efficient homologous recombination, which allows the use of PCR-based gene targeting tools (Steiner et al., 1995; Wendland et al., 2000); haploid nuclei and unicucleated spores (Steiner et al., 1995), which facilitate the isolation of stable homokaryotic strains; and ability to freely replicate plasmids containing S. cerevisiae autonomously replicating sequences (ARS) (Wright and Philippsen, 1991) and to regulate gene expression using S. cerevisiae promoters (Wendland et al., 2000; Kaufmann, 2009; Ribeiro et al., 2010; Magalhães et al., 2014), making possible the use of several tools already available to manipulate S. cerevisiae. Although a wide range of molecular tools exist to genetically engineer A. gossypii, the number of selection markers in current use is limited. The absence of a known sexual cycle in A. gossypii has hindered the use of classical genetics approaches in this fungus (Wendland et al., 2011). Thus, the molecular characterization of A. gossypii genes has relied on reverse genetics approaches. However, only four marker genes are available for PCR-based gene targeting in A. gossypii, which has hampered the disruption and/or integra- tion of multiple genes in the same strains. The existent markers are: (1) the S. cerevisiae LEU2 gene fused with its own promoter and terminator (Alberti-Segui et al., 2001), which can only be used as auxotrophic marker for gene manipulations in stable Agleu2 http://dx.doi.org/10.1016/j.fgb.2014.04.009 1087-1845/Ó 2014 Elsevier Inc. All rights reserved. ⇑ Corresponding author. Fax: +351 253 604429. E-mail address: luciliad@deb.uminho.pt (L. Domingues). 1 These authors contributed equally to this work. Fungal Genetics and Biology 68 (2014) 1–8 Contents lists available at ScienceDirect Fungal Genetics and Biology journal homepage: www.elsevier.com/locate/yfgbi