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

## TL;DR

This study reveals how a gene in a heat-loving fungus controls pigment production and energy generation, helping the fungus survive cold stress.

## Contribution

The study identifies the hal gene's role in anthraquinone chlorination, linking it to pigmentation, ATP production, and cold stress adaptation in Thermomyces dupontii.

## Key findings

- Disruption of the hal gene reduces pigmentation and ATP production in Thermomyces dupontii.
- Chlorinated anthraquinones enhance structural diversity and fungal survival under cold stress.
- Hal-mediated chlorination supports two energy modes: intracellular ATP production and extracellular Fenton chemistry.

## Abstract

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.

This diagram illustrates the chlorination of anthraquinones and its biological functions in Thermomyces dupontii, elucidating how chlorination‐mediated fungal pigmentation promotes energy production and stress adaptation. (1) Extracellular Fenton Chemistry: Involves superoxide anion (O2
−) and ferrous ion (Fe2+), which are two fundamental elements for extracellular Fenton chemistry, which is strongly enhanced by polyketide‐derived emodin (5) and carviolin A (1) that facilitate the formation of Fe2+ and O2
− and export Fe2+. The extracellular Fenton chemistry can degrade organic matter to generate energy and elevate the ambient temperatures, collectively enabling fungal adaptation to low‐temperature conditions. (2) Anthraquinone Chlorination: Halogenase (Hal) in
T. dupontii
can introduce chlorine (Cl) to emodin and carviolin A, generating yellow monochlorinated intermediates: carviolin A‐Cl (2) and emodin‐Cl (4). These two intermediates were further fused to form a new dark purple dimeric anthraquinone. (3) Linkage to Two Energy Modes: The chlorination of anthraquinones enhances intracellular ATP production and extracellular Fenton chemistry via releasing protons and electrons from anthraquinone and Cl−, thereby contributing to fungal colour and vitality under low temperature stress. In summary, this figure illustrates how
T. dupontii
employs anthraquinone chlorination (driven by the hal gene) to enhance pigmentation, energy production and cold stress tolerance. This connection bridges chemical transformations with ecological adaptation, highlighting the biotechnological potential of fungal pigments in environmental ecology research.