Screening of Promoters for the Heterologous Gene Expression in Ganoderma lucidum
Rutuja Nandre and Hyeon-Su Ro*
Department of Bio&Medical Bigdata, Gyeongsang National university, Jinju, Korea. .
Heterologous gene expression in mushroom has been of interest for its ability to grow on various substrates with rapid growth rate. However, it has been challenging because of scarcity in molecular tools such as vector plasmid, transformation, and selection method. Moreover, the fact that mushroom uses non-homologues end joining (NHEJ) to repair damaged DNA makes it difficult to incorporate a gene-of-interest to the target site. Fortunately, our recent studies have shown that a Cas9-gRNA mediated gene editing technology enables the insertion of a foreign DNA into the target site through homologous recombination (HR). Based on these results, we have applied the gene editing method to screen promoters which facilitate gene expression at various conditions. For this, we constructed various promoter-EGFP containing HR fragments, flanked by 300 bp upstream and downstream sequences of pyrG of Ganoderma lucidum. Disruption of pyrG enables survival-and-death screening on 5-fluoroorotic acid (5-FOA) -containing agar medium. Transformation with the HR fragments and Cas9-gRNA targeting pyrG yielded multiple transformants which carry HR fragment at the pyrG site. Characterization of the transformants has been underwent and will be reported at the conference.