Synthesis and biological evaluation of coprinoferrin, an acylated tripeptide hydroxamate siderophore.

Coprinoferrin (CPF), originally isolated from a genetically engineered strain (ΔlaeA) of the mushroom fungus Coprinopsis cinerea, is an acylated tripeptide hydroxamate consisting of tandem aligned N5-hexanoyl-N5-hydroxy-L-ornithine with modifications of N-acetyl and C-carboxamide. These unique chemical properties make CPF an iron(III) binder (siderophore), which helps in iron acquisition from the environment and promotes hyphal growth as well as fruiting body formation in C. cinerea. However, CPF's detailed mode of action remains enigmatic. In this study, we have accomplished the synthesis of CPF from N-Boc-L-glutamic acid 5-benzyl ester. The physicochemical characteristics, spectroscopic features, and biological activity observed in the synthetic CPF closely match those of natural CPF. This alignment provides unequivocal confirmation of the proposed chemical structure, facilitating a deeper understanding of its physiological role in nature, particularly in fruiting body formation.

Uncovering hidden sesquiterpene biosynthetic pathway through expression boost area-mediated productivity enhancement in basidiomycete.

Basidiomycetes are known to biosynthesize many biologically interesting compounds, including terpenoids. However, they are notoriously difficult to manipulate. Previously, we identified the gene cluster encoding enzymes responsible for the biosynthesis of lagopodins, cuparene-type sesquiterpenoid quinone natural products in Coprinopsis cinerea. In this study, we attempted to increase the productivity of lagopodin B (1) and related pathway products by overexpressing the terpene cyclase gene cop6 in C. cinerea to determine the details of the complex lagopodin and hitoyol biosynthetic pathway. Random integration of the cop6 into the genome of the ku70-deficient C. cinerea strain resulted in an ~2.4-fold increase in the production of 1. However, integration of cop6 into a highly transcribed position within the chromosome we designated as an expression boost area (EBA) resulted in an ~14-fold greater production of 1. Furthermore, the EBA-integration strain allowed us to isolate a previously undetected product 2, which we determined to be the known compound, hydroxylagopodin B. This finding expanded our understanding of the lagopodin-hitoyol biosynthetic pathway and allowed us to hypothesize a possible mechanism for the biosynthesis of a related homodimeric compound, lagopodin C. Our results demonstrate the potential of targeting EBA to integrate key biosynthetic genes into the genome for enhancing the production of difficult-to-obtain compounds for studying the biosynthesis of complex secondary metabolites in basidiomycetes and other complex eukaryotic organisms.

Genomic Mushroom Hunting Decrypts Coprinoferrin, A Siderophore Secondary Metabolite Vital to Fungal Cell Development.

LaeA is a positive global regulator of secondary metabolism in Ascomycetes, but its role in Basidiomycetes, including medicinal mushrooms, remains uncharacterized. Here, knockout of laeA in the model mushroom Coprinopsis cinerea unexpectedly upregulated the biosynthesis of a novel siderophore, coprinoferrin. Furthermore, knockout of the nonribosomal peptide synthetase-encoding cpf1 responsible for coprinoferrin biosynthesis resulted in growth defect and loss of fruiting body formation, indicating the complex role that this natural product plays in fungal cell development.