Overexpression of rttA leads to activation of the fumicycline biosynthesis cluster including the O-acetyltransferase encoding fccF.

The opportunistic mold Aspergillus fumigatus, a WHO-priority fungal pathogen, causes life-threatening invasive infections in immunocompromised patients. A major antifungal drug target is the ergosterol biosynthesis pathway, primarily regulated by the transcription factors SrbA and AtrR. Recently, we identified RttA as an additional regulator of sterol homeostasis that mediates the activation of the sterol C24-methyltransferase-encoding gene erg6. Here, we demonstrate that overexpression of rttA leads to elevated production of the secondary metabolite fumicycline via induction of fccR, coding for the principal transcriptional activator of fumicycline biosynthesis. Lack of fccR blocked activation of fumicycline biosynthetic genes (fccA-fccE) during rttA overexpression, highlighting the indispensable role of FccR for activation of this cluster. In contrast, rttA inactivation did not abolish fumicycline production during fccR overexpression or during co-cultivation with Streptomyces rapamycinicus. In addition to fccA-fccE, our findings reveal a crucial role of FccR for the upregulation of a gene located adjacent to the fumicycline biosynthesis cluster, here termed fccF. Deletion of fccF severely diminished the conversion of fumicycline B into fumicycline C, uncovering FccF as fumicycline B O-acetyltransferase. Intriguingly, during fccR overexpression and resulting fumicycline overproduction, inactivation of fccF caused growth defects, likely due to precursor toxicity. Together, these findings broaden our understanding of fungal secondary metabolite regulation and reveal a novel role of RttA in the activation of fccR encoding the cluster-specific regulator that activates fumicycline production, including FccF, the terminal acetyltransferase in the pathway.IMPORTANCEAspergillus fumigatus is a major opportunistic fungal pathogen that causes severe infections in immunocompromised individuals and represents a significant global health threat. Understanding the regulatory networks controlling fungal metabolism is important for identifying potential therapeutic vulnerabilities. Here, we uncover a previously unrecognized role for the sterol homeostasis regulator RttA in activating fumicycline production through induction of the pathway-specific transcription factor FccR. We further show that FccR controls expression of an additional biosynthetic gene, fccF, and identify FccF as the terminal O-acetyltransferase required for conversion of fumicycline B to fumicycline C. Disruption of this final biosynthetic step impairs fungal growth during metabolite overproduction, indicating that accumulation of fumicycline B or related intermediates can negatively affect fungal growth. Together, these findings expand our understanding of fungal secondary metabolite regulation and provide new insights into the organization and function of the fumicycline biosynthetic pathway.