Genome Mining for Unknown-Unknown Natural Products

Genome mining of biosynthetic pathways with no identifiable core enzymes can lead to discovery of the so-called unknown (biosynthetic route)-unknown (molecular structure) natural products. In this work, we focused on a conserved fungal biosynthetic pathway (ank) that lacks a canonical core enzyme, and used heterologous expression to identify the associate natural product to be a highly modified cyclo-arginine-tyrosine dipeptide (cRY). Biochemical characterization of the ank pathway led to identification of a new arginine-containing cyclodipeptide synthase (RCDPS), which was previously annotated as a hypothetical protein (HP) and has no sequence homology to nonribosomal peptide synthetase (NPRS) or bacterial cyclodipeptide synthase (CDPS). RCDPS homologs are widely encoded in fungal genomes and we showed other members of this family can synthesize diverse cyclo-arginine-Xaa dipeptides. Characterization of a cyclo-Arg-Trp (cRW) RCDPS showed the enzyme is aminoacyl-tRNA dependent, and represents the first report of such CDPS-like enzyme from fungi. Further characterization of the biosynthetic pathway anchored by the cRW synthase led to discovery of new compounds of which the structures would not have been predicted without knowledge of RCDPS function.