Insertional Mutagenesis in the Vitamin B 2 Producer Fungus Ashbya gossypii

The ascomycete Ashbya gossypii, a filamentous fungus, is a natural overproducer of vitamin B2 and is currently exploited for the industrial production of this vitamin. Classical mutagenesis and selection of mutants showing improved production capacities have been routinely applied with success to the development of industrial strains of A. gossypii that overproduce vitamin B2. However, this approach does not allow the subsequent isolation and identification of affected genes in organisms such as A. gossypii that lack a sexual life cycle, thus hampering further improvements of the strains on the basis of rational strategies derived from knowledge of the function of the genes involved in the process. Here, we describe an efficient in vitro Himar1based transposition reaction of minitransposon elements on chromosomal A. gossypii DNA, which is subsequently reintroduced into A. gossypii by electrotransformation. Sequencing of the minitransposon insertion point in numerous transposition mutants and a limited screening of the insertional mutants demonstrate that this method can lead to the exhaustive mutagenesis of A. gossypii, allowing—for the first time—a genomic-scale mutational analysis of this biotechnologically important fungus.