Compartmentalized co-cultivation and temporal transcriptomics reveal SexM-mediated crosstalk between pheromone signaling and metabolic reprogramming in the industrial carotenoid producer Blakeslea trispora.

Sexual reproduction in industrial β-carotene producer Blakeslea trispora requires (+)/(-) mating-type interaction through diffusible trisporoid pheromones, triggering carotenogenesis as a secondary metabolic response. However, the molecular mechanisms linking pheromone perception to mating-type-specific metabolic reprogramming remain unclear due to technical constraints. Here, we established a compartmentalized co-cultivation system to physically separate (-)-type hyphae from (+) partners while retaining diffusible metabolite exchange. The system allows transcriptomic analysis for each mating type separately while maintaining the natural pheromone signaling dynamics that occur during mating. Time-resolved RNA-seq of the isolated (-) strain revealed widespread transcriptional reprogramming, with a total of 887 core genes responding consistently throughout mating process. Through temporal expression clustering indicated that mating response unfolds in three distinct phases: early-phase activation of transporters and signal transduction, mid-phase metabolic rewiring that links primary metabolism to terpenoid biosynthesis, and late-phase initiation of DNA repair mechanisms. The transcription factor landscape showed remarkable changes during mating. Out of 293 differentially expressed transcription factors representing 103 families, we found a core regulatory module of 42 up- and 12 downregulated TFs, with SexM most strongly induced. Network analysis placed SexM as a central hub linking pheromone signaling to carotenogenesis, connecting early genes such as hmgr and ggpps to late biosynthetic genes carRA and carB. G-protein signaling and oxidoreductases were highly responsive, supporting roles for GPCR-MAPK pathways and redox homeostasis during metabolic reorganization. These results reveal how gene regulatory network is organized to control sexual carotenogenesis and suggest new strategies for enhancing β-carotene production in the sexual development pathway.