Tools to make Stachybotrys chartarum genetically amendable: Key to unlocking cryptic biosynthetic gene clusters.

The soil and indoor fungus Stachybotrys chartarum can induce respiratory disorders, collectively referred to as stachybotryotoxicosis, owing to its prolific production of diverse bioactive secondary metabolites (SMs) or mycotoxins. Although many of the toxins responsible for the harmful effects on animals and humans have been identified in the genus Stachybotrys, a number of secondary metabolites remain elusive. Through in silico analyses, we have identified 37 polyketide synthase (PKS) genes, highlighting that the chemical profile potential of Stachybotrys is far from being fully explored. Additionally, by leveraging phylogenetic analysis of known SMs produced by non-reducing polyketide synthases (NR-PKS) in other filamentous fungi, we showed that Stachybotrys possesses a rich reservoir of untapped SMs. To unravel natural product biosynthesis in S. chartarum, genetic engineering methods are crucial. For this purpose, we have developed a reliable protocol for the genetic transformation of S. chartarum and applied it to the ScPKS14 gene cluster. This cluster is homologous to the already known Claviceps purpurea CpPKS8 gene cluster, responsible for the production of ergochromes. While no novel SMs were detected, we successfully applied genetic tools, such as generating deletions and overexpressing single cluster genes. This toolbox can now be readily employed to unravel not only this particular biosynthetic gene cluster but also other candidate biosynthetic gene clusters present in S. chartarum, making this fungus accessible for genetic engineering.