The Journey to the Treasure: Secondary metabolite gene clusters and histone modifications in the genus Aspergillus

Secondary metabolites (SMs) are small, bioactive molecules produced by a diverse range of organisms, especially fungi. While not essential for normal growth and reproduction, they are crucial for interactions with other organisms and environmental adaptation. Fungal SMs, in particular, have been invaluable across various applications, including medicine, agriculture, food preservation, and cosmetics, due to their diverse bioactivities. Despite the extensive diversity of SMs and their biosynthetic pathways encoded in fungal genomes, only a limited number of SMs have been discovered and widely utilized. This gap is primarily due to the challenges in accurately expressing the enzymes responsible for SM biosynthesis in laboratory settings, as these enzymes are tightly regulated and only produced under specific natural conditions that are difficult to replicate. Chapter 1 of this book introduces the concept of SMs and the biosynthesis gene clusters (BGCs) responsible for their production. I detail the three-tier regulatory mechanisms governing SM-BGC expression, emphasizing histone post-translational modifications (PTMs). These chemical modifications of histone proteins, which are part of the DNA-protein complex called chromatin, play a significant role in organizing genetic information within the eukaryotic nucleus and influencing gene expression. Histone modifiers—enzymes that write, erase, and read PTMs—regulate chromatin organization into 'open' and 'closed' states, affecting gene expression. Histone PTMs are key to unlocking the vast, untapped potential of fungal SMs. In Chapter 2, I explore the abundance and diversity of SM BGCs across the genus Aspergillus. Utilizing a phylogenetic approach, I reveal the diversity of SM backbone genes coding for key biosynthetic enzymes. These genes are abundant in Aspergillus and exhibit significant differences across taxonomic sections. Furthermore, I find that SM backbone genes are frequently localized in low-synteny regions, including sub-telomeric regions, which correlates with higher gene expression variability likely linked to specific histone PTMs. Chapter 3 focuses on the evolutionary history of genes responsible for PTM formation and regulation across Aspergillus. I summarize known histone modifiers from model species and conduct phylogenetic analyses of these complexes in diverse Aspergilli. These complexes, comprising both catalytic and accessory subunits, are highly conserved, indicating a complex repertoire of PTMs. Notably, the PRC2 complex, responsible for methylations on the histone protein H3 at lysine 27 (H3K27me), is absent in Aspergilli, suggesting alternative mechanisms for chromatin conformation and gene expression regulation. To confirm the presence of PTMs and uncover additional modifications in Aspergilli, I perform in Chapter 4 an unbiased quantitative proteomics study using mass spectrometry. This study, focused on three different Aspergillus species, reveals 23 single and 23 co-occurrent PTMs, including lysine methylations and acetylation. My findings support computational predictions and reveal novel PTMs, highlighting species-specific differences in PTM abundance and patterns. Chapter 5 introduces the concept of the 'histone code in Aspergilli,' proposing that specific PTMs compensate for the absence of H3K27me and may regulate SM biosynthesis. Overall, my research provides insights into the epigenetic regulation of SM biosynthesis in fungi, paving the way for future studies to harness the full potential of fungal SMs.