Abstract
Diverse tools are available for performing genetic modifications of microorganisms. However, new methods still need to be developed for performing precise genomic engineering without introducing any undesirable side-alteration. Indeed for functional analyses of genomic elements, as well as for some industrial applications, only the desired mutation should be introduced at the locus considered. This article describes a new approach fulfilling these requirements, based on the use of selection systems consisting in truncated genes encoding dominant-negative transcription factors. We have demonstrated dominant-negative effects mediated by truncated Gal4p and Arg81p proteins in Saccharomyces cerevisiae, interfering with galactose and arginine metabolic pathways, respectively. These genes can be used as positive and negative markers, since they provoke both growth inhibition on substrates and resistance to specific drugs. These selection markers have been successfully used for precisely deleting HO and URA3 in wild yeasts. This genetic engineering approach could be extended to other microorganisms.