Novel Anthraquinone Chlorination Contributes to Pigmentation and ATP Formation in Thermomyces dupontii

Fungal Pigments, particularly Anthraquinones, play critical roles in food industrial applications due to their vivid hues and bioactive properties. Despite their significance, the functional mechanisms underlying pigment variation and their natural functions remain poorly understood. Here, we investigate the genetic and biochemical basis of anthraquinone-mediated pigmentation in the thermophilic fungus Thermomyces dupontii. Through a combination of transcriptomic, biochemical and chemical analyses, we identified the flavin-dependent halogenase gene (hal) as a key controller of anthraquinone chlorination, a process essential for Fungal pigmentation and metabolic responses to low temperature stress. Disruption of hal abolished chlorinated anthraquinone production, reduced colony pigmentation and impaired ATP production. We characterised novel chlorinated and dichlorinated Anthraquinones, highlighting chlorination's role in enhancing the structural diversity of Anthraquinones and Fungal pigmentation. Detailed bioassays indicated that hal-mediated anthraquinone chlorination contributes to Fungal survival under cold stress by enhancing two distinct energy modes. This study provides new insights into the ecological significance of Fungal pigmentation and opens avenues for the biotechnological exploitation of fungal Pigments in food industrial and medical research.