An environmentally sustainable biomimetic production of cyclic disulfide-rich peptides

Abstract

Macrocyclic, disulfide-rich peptides have found widespread applications in drug design and development. Current peptide production strategies rely heavily on solid phase peptide synthesis (SPPS) requiring large amounts of hazardous/toxic reagents and solvents which have negative environmental impacts. A possible solution is to develop a sustainable hybrid production platform incorporating recombinant production of cyclic peptide precursors in yeast followed by enzymatic maturation of these precursors into cyclic peptides using asparaginyl endopeptidases in vitro. Harnessing the efficient secretory pathway of Pichia pastoris, peptide precursors, cloned downstream of the α-mating factor secretion signal, were purified from culture supernatant mitigating the need for complex purification. To demonstrate the broad utility of the platform, three distinct classes of cyclic peptides were produced; two were structurally validated by NMR and shown to be functionally equivalent to their synthetically produced versions. Furthermore, using this platform we report the first recombinant production of any α-conotoxin in its native “globular” conformation. Using scale-up production in bioreactors, cyclic peptide yields of 85–97 mg L⁻¹ of culture were achieved, far exceeding the highest yields so far achieved for cyclic disulfide-rich peptides in any recombinant process. This platform can potentially unlock production and facilitate applications of cyclic disulfide-rich peptides previously inaccessible through large-scale chemical synthesis and reduce their environmental burden.