Construction of a Recyclable Genetic Marker and Serial Gene Deletions in the Human Pathogenic Mucorales<i>Mucor circinelloides</i>

Abstract

<i>Mucor circinelloides</i> is a human pathogen, biofuel producer, and model system that belongs to a basal fungal lineage; however, the genetics of this fungus are limited. In contrast to ascomycetes and basidiomycetes, basal fungal lineages have been understudied. This may be caused by a lack of attention given to these fungi, as well as limited tools for genetic analysis. Nonetheless, the importance of these fungi as pathogens and model systems has increased. <i>M. circinelloides</i> is one of a few genetically tractable organisms in the basal fungi, but it is far from a robust genetic system when compared to model fungi in the subkingdom Dikarya. One problem is the organism is resistant to drugs utilized to select for dominant markers in other fungal transformation systems. Thus, we developed a blaster recyclable marker system by using the <i>pyrG</i> gene (encoding an orotidine-5'-phosphate decarboxylase, ortholog of <i>URA3</i> in <i>Saccharomyces cerevisiae</i>). A 237-bp fragment downstream of the <i>pyrG</i> gene was tandemly incorporated into the upstream region of the gene, resulting in construction of a <i>pyrG-dpl237</i> marker. To test the functionality of the <i>pyrG-dpl237</i> marker, we disrupted the <i>carRP</i> gene that is involved in carotenoid synthesis in <i>pyrG</i>^- mutant background. The resulting <i>carRP</i>::<i>pyrG-dpl237</i> mutants exhibit a white colony phenotype due to lack of carotene, whereas wild type displays yellowish colonies. The <i>pyrG</i> marker was then successfully excised, generating <i>carRP-dpl237</i> on 5-FOA medium. The mutants became auxotrophic and required uridine for growth. We then disrupted the calcineurin B regulatory subunit <i>cnbR</i> gene in the <i>carRP</i>::<i>dpl237</i> strain, generating mutants with the alleles <i>carRP</i>::<i>dpl237</i> and <i>cnbR</i>::<i>pyrG</i> These results demonstrate that the recyclable marker system is fully functional, and therefore the <i>pyrG-dpl237</i> marker can be used for sequential gene deletions in <i>M. circinelloides</i>.