Genetic regulation of L-tryptophan metabolism in Psilocybe mexicana supports psilocybin biosynthesis.

Psilocybin, a psychedelic alkaloid, can account for up to 2% of the dry mass of Psilocybe mushrooms, creating a high demand for its precursor L-tryptophan during carpophore (fruiting body) formation. Using Psilocybe mexicana, researchers found that genes for L-tryptophan biosynthesis (trpE1, trpD, trpB) were upregulated in carpophores, while genes for L-tryptophan-consuming pathways (idoA, iasA) were massively downregulated. The IasA enzyme was characterized as the first microbial L-tryptophan-preferring acetaldehyde synthase. Comparison with Psilocybe cubensis revealed species-specific differences in regulation. This coordination of primary and secondary metabolism routes L-tryptophan toward psilocybin production, providing initial insight into how Basidiomycota manage metabolic flux.

Study at a glance

Abstract

Although Basidiomycota produce pharmaceutically and ecologically relevant natural products, knowledge of how they coordinate their primary and secondary metabolism is virtually non-existent. Upon transition from vegetative mycelium to carpophore formation, mushrooms of the genus Psilocybe use L-tryptophan to supply the biosynthesis of the psychedelic tryptamine alkaloid psilocybin with the scaffold, leading to a strongly increased demand for this particular amino acid as this alkaloid may account for up to 2% of the dry mass. Using Psilocybe mexicana as our model and relying on genetic, transcriptomic, and biochemical methods, this study investigated if L-tryptophan biosynthesis and degradation in P. mexicana correlate with natural product formation. A comparative transcriptomic approach of gene expression in P. mexicana psilocybin non-producing vegetative mycelium versus producing carpophores identified the upregulation of L-tryptophan biosynthesis genes. The shikimate pathway genes trpE1, trpD, and trpB (encoding anthranilate synthase, anthranilate phosphoribosyltransferase, and L-tryptophan synthase, respectively) were upregulated in carpophores. In contrast, genes idoA and iasA, encoding indole-2,3-dioxygenase and indole-3-acetaldehyde synthase, i.e., gateway enzymes for L-tryptophan-consuming pathways, were massively downregulated. Subsequently, IasA was heterologously produced in Escherichia coli and biochemically characterized in vitro. This enzyme represents the first characterized microbial L-tryptophan-preferring acetaldehyde synthase. A comparison of transcriptomic data collected in this study with prior data of Psilocybe cubensis showed species-specific differences in how L-tryptophan metabolism genes are regulated, despite the close taxonomic relationship. The upregulated L-tryptophan biosynthesis genes and, oppositely, the concomitant downregulated genes encoding L-tryptophan-consuming enzymes reflect a well-adjusted cellular system to route this amino acid toward psilocybin production. Our study has pilot character beyond the genus Psilocybe and provides, for the first time, insight in the coordination of mushroom primary and secondary metabolism.