The RNAi machinery regulates lovastatin biosynthesis via microRNA-like RNAs in industrial Aspergillus terreus

The RNA interference (RNAi) machinery regulates diverse cellular processes in fungi, but its functions in industrial Aspergillus terreus remain unknown. Here, we systematically investigated two industrial strains: the itaconic acid-producing A. terreus LYT10 and the lovastatin-producing A. terreus HZ01. Both strains possess RNAi machinery core components, yet the phenotypic consequences of RNAi disruption differed between the two strains. In strain LYT10, RNAi deficiency had negligible effects on growth, conidiation and itaconic acid production. Conversely, in strain HZ01, it caused severe growth impairment, dysfunctional conidiation, reduced lovastatin production, and activated by-product geodin biosynthesis. Notably, the dicer-like 2 (dcl2) deletion mutant exhibited the most severe phenotype among all mutants. Transcriptomic analysis linked these phenotypic changes to strong repression of the lovastatin biosynthetic gene cluster, downregulation of acetyl-CoA flux, and partial induction of the geodin biosynthetic gene cluster in strain HZ01. Small RNA profiling identified 14 DCL2-dependent microRNA-like RNAs (milRNAs) in the Δdcl2 mutant. The milRNA-mRNA network analysis revealed that these DCL2-dependent milRNAs target genes involved in the regulation of glucose uptake, maintenance of fatty acid metabolic homeostasis, and suppression of geodin biosynthesis, thereby indirectly influencing lovastatin biosynthesis. Collectively, the RNAi machinery via DCL2 and milRNAs to exert precise post-transcriptional regulation on target genes expression, ensuring normal development and efficient production of lovastatin in industrial A. terreus HZ01. This study reveals that the regulatory roles of RNAi machinery differ between the two industrial A. terreus strains and elucidates a milRNA-mediated regulatory mechanism in strain HZ01, offering new insights into fungal RNAi biology and its biotechnological applications.