An Efficient Marker Recycling System For Sequential Gene Deletion In A Deep Sea-Derived Fungus Acremonium Sp. Hdn16-126

Acremonium species are prolific producers of therapeutic molecules which include the widely used beta-lactam antibiotic, cephalosporin. In light of their significant medical value, an efficient gene disruption method is required for the physiological and biochemical studies on this genus of fungi. However, the number of selection markers that can be used for gene targeting is limited, which constrain the genetic analysis of multiple functional genes. In this study, we established a uridine auxotrophy based marker recycling system which achieves scarless gene deletion, and allows the use of the same selection marker in successive transformations in a deep seaderived fungus Acremonium sp. HDN16-126. We identified one homologue of Acremonium chrysogenum pyrG (also as a homologous gene of the yeast URA3) from HDN16-126, designated as pyrG-A1, which can be used as a selection marker on uridine free medium. We then removed pyrG-A1 from HDN16-126 genome via homologous recombination (HR) on MM medium with 5-fluoroortic acid (5-FOA), a chemical that can be converted into a toxin of 5-flurouracil by pyrG-A1 activity, thus generating the HDN16-126-.pyrG mutant strain which showed auxotrophy for uridine but insensitivity to 5-FOA and enabled the use of exogenous pyrG gene as both positive and negative selection marker to achieve the scarless deletion of target DNA fragments. We further applied this marker recycling system to successfully disrupt two target genes pepL (encodes a putative 2OG-Fe (II) dioxygenase) and pepM (encodes a putative aldolase) identified from HDN16-126 genome, which are proposed to be functional genes related to 2-aminoisobutyric acid metabolism in fungi. This work is the first application of uridine auxotrophy based scarless gene deletion method in Acremonium species and shows promising potential in assisting sequential genetic analysis of filamentous fungi.