Identification of novel proteins regulating lipid droplet biogenesis by reverse genetics

Lipid droplets (LDs) are storage organelles for neutral lipids. Our knowledge about fungal LD biogenesis is limited to budding yeast. Moreover, the regulation of LD in multinucleated filamentous fungi with considerable metabolic activity is unknown. Here, 19 LD-associated proteins were identified in Aspergillus oryzae using colocalization screening of a previously established Enhanced green fluorescent protein (EGFP) fusion proteins library. Following a functional screening, 12 candidates have been identified as lipid droplet regulating (LDR) proteins, the loss of which resulted in aberrant LD biogenesis. Four LDR proteins localize to LD via the putative amphipathic helices (AHs), as demonstrated with bioinformatics, targeted mutagenesis, and imaging. Further analysis revealed that LdrA with Opi1 domain is essential for cytoplasmic and nuclear LD biogenesis via this novel AH. Phylogenetic analysis demonstrated the pattern of their evolution, which was predominantly based-on gene duplication. Our study provides substantial molecular and evolutionary insights into LD biogenesis and creates a breakthrough in using reverse genetics for identifying LD-regulating proteins.