Combinatorial biosynthesis of cyclic peptide natural products in Amanita mushrooms by a versatile macrocyclase

Amatoxins and phallotoxins are cyclic peptide natural products isolated from Amanita mushrooms that include α-amanitin, a potent inhibitor of RNA polymerase II, and phalloidin, which binds F-actin. Biosynthesis of the amatoxins and phallotoxins occurs ribosomally, in which the peptides are encoded by the ‘MSDIN’ family of genes and expressed as 34 or 35-amino acid proproteins. Post-translational modifications to these precursors give rise to the mature bioactive forms. Beyond the amatoxins and phallotoxins, Amanita bisporigera and A. phalloides encode at least 30 additional MSDIN proproteins, which are characterized by having conserved sequences at the N- and C-termini flanking an internal variable region that contains the amino acids found in the final products. The conserved structure among the MSDIN sequences enables their use as scaffolds for the combinatorial biosynthesis of cyclic peptides using the same core biochemical machinery. This talk will focus on the discovery and characterization of the MSDIN pathway and prolyl oligopeptidase B (POPB), the enzyme responsible for macrocyclization of the precursors. We show that POPB is a versatile macrocyclase capable of synthesizing a variety of cyclic peptides in vitro from both natural and synthetic substrates, and demonstrate the enzyme’s applications in biotechnology through the production of combinatorial cyclic peptide libraries and naturally occurring cyclic peptides with potential medicinal value.