Identification of novel proteins regulating lipid droplet biogenesis in filamentous fungi

Lipid droplets (LDs) are storage organelles for neutral lipids which are critical for lipid homeostasis. Current knowledge of fungal LD biogenesis is largely limited to budding yeast, while LD regulation in multinucleated filamentous fungi which exhibit considerable metabolic activity remains unexplored. In this study, 19 LD-associated proteins were identified in the multinucleated species Aspergillus oryzae using a colocalization screening of a previously established enhanced green fluorescent protein (EGFP) fusion library. Functional screening identified 12 lipid droplet-regulating (LDR) proteins whose loss of function resulted in irregular LD biogenesis, particularly in terms of LD number and size. Bioinformatics analysis, targeted mutagenesis, and microscopy revealed four LDR proteins that localize to LD via the putative amphipathic helices (AHs). Further analysis revealed that LdrA with an Opi1 domain is essential for cytoplasmic and nuclear LD biogenesis involving a novel AH. Phylogenetic analysis demonstrated that the patterns of gene evolution were predominantly based on gene duplication. Our study identified a set of novel proteins involved in the regulation of LD biogenesis, providing unique molecular and evolutionary insights into fungal lipid storage.