Promiscuity of Omphalotin A Biosynthetic Enzymes Allows de novo Production of Non‐Natural Multiply Backbone N‐Methylated Peptide Macrocycles in Yeast

Multiple backbone N‐methylation and macrocyclization improve the proteolytic stability and oral availability of therapeutic peptides. Chemical synthesis of such peptides is challenging, in particular for the generation of peptide libraries for screening purposes. Enzymatic backbone N‐methylation and macrocyclization occur as part of both non‐ribosomal and ribosomal peptide biosynthesis, exemplified by the fungal natural products cyclosporin A and omphalotin A, respectively. Omphalotin A, a 9fold backbone N‐methylated dodecamer isolated from the agaricomycete Omphalotus olearius, can be produced in Pichia pastoris by coexpression of the ophMA and ophP genes coding for the peptide precursor protein harbouring an autocatalytic peptide α‐N‐methyltransferase domain, and a peptide macrocyclase, respectively. Since both OphMA and OphP were previously shown to be relatively promiscuous in terms of peptide substrates, we expressed mutant versions of ophMA, encoding OphMA variants with altered core peptide sequences, along with wildtype ophP and assessed the production of the respective peptide macrocycles by the platform by high‐performance liquid chromatography, coupled with tandem mass spectrometry (HPLC–MS/MS). Our results demonstrate the successful production of fifteen non‐natural omphalotin‐derived macrocycles, containing polar, aromatic and charged residues, and, thus, suggest that the system may be used as biotechnological platform to generate libraries of non‐natural multiply backbone N‐methylated peptide macrocycles. The biosynthetic pathway for the peptide natural product omphalotin A of the agaricomycete Omphalotus olearius can be exploited for the production of non‐natural, multiply backbone N‐methylated peptide macrocycles in the yeast Pichia pastoris (Komagataella phaffii).