Discovery of Uncommon Tryptophan-Containing Diketopiperazines from Aspergillus homomorphus CBS 101889 Using an Aspergillus nidulans Heterologous Expression System

Fungal secondary metabolite (SM) biosynthetic gene clusters (BGCs) containing dimethylallyltryptophan synthases (DMATSs) produce structurally diverse prenylated indole alkaloids with wide-ranging activities that have vast potential as human therapeutics. To discover new natural products produced by DMATSs, we mined the Department of Energy Joint Genome Institute’s MycoCosm database for DMATS-containing BGCs. We found a DMATS BGC in Aspergillus homomorphus CBS 101889, which also contains a nonribosomal peptide synthetase (NRPS). This BGC appeared to have a previously unreported combination of genes, which suggested the cluster might make novel SMs. We refactored this BGC with highly inducible promoters into the model fungus Aspergillus nidulans. The expression of this refactored BGC in A. nidulans resulted in the production of eight tryptophan-containing diketopiperazines, six of which are new to science. We have named them homomorphins A-F (2, 4-8). Perhaps even more intriguingly, to our knowledge, this is the first discovery of C4-prenylated tryptophan-containing diketopiperazines and their derivatives. In addition, the NRPS from this BGC is the first described that has the ability to promiscuously combine tryptophan with either of two different amino acids, in this case, l-valine or l-allo-isoleucine.