Small Biomolecules for Big Applications

Directed evolution has seen significant use in academia and industry for the functional optimization of peptides and proteins. By screening libraries of mutants for the most desirable phenotypes, variants of the original protein or peptide can be discovered that are optimal for any given application. Presently, directed evolution is limited to products that are directly encoded by DNA, which imparts the essential properties of replicability and mutability. As such, the approach can only be applied to large biomolecules. The current paper by the van der Donk group brings us one step closer to the goal of leveraging directed evolution for the development of novel small molecules with pharmaceutical importance. The molecules of interest to Hetrick et al. are a class of natural products known as ribosomally synthesized and posttranslationally modified peptides, or RiPPs. Unlike other classes, the substrates for RiPPs are directly encoded by a “substrate” gene, making them ideal as subjects of evolutionary methods. Once translated, these linear precursors are modified by one or more enzymes and proteolytically cleaved to produce the final, mature product of the biosynthetic pathway. The cyclization motifs commonly observed in RiPPs provide structural rigidity, impart function, and protect against proteolysis. These modifications turn otherwise unassuming linear peptides into potent small molecules. RiPPs are a particularly good class of biomolecules for the application of directed evolution because of their broad range of shapes, sizes, and bioactivities. They can be as small as the enzyme cofactor pyrroloquinoline quinone (PQQ), a mashup of two amino acids, or larger like nisin, the 34 amino acid peptidic antibiotic used in the present study. The structural organization of the precursor peptide is also favorable for in vitro evolution: the portion of the precursor peptide that is modified eventually to afford the mature RiPP is known as the “core,” usually located at the C-terminus. The N-terminal portion, or the “leader” sequence, generally serves as the recognition element for the modification enzymes. By separating the recognition element from the sequence that is modified, RiPPs are highly tolerant of mutations within the core peptide region. In a hypothetical 10mer RiPP where only two amino acids are strictly required for cyclization, randomly mutating the other eight positions can lead to a possible ∼10 RiPPs, each with a unique structure and, possibly, biological activity. The ability to generate such chemical diversity is a powerful strategy for creating new bioactive molecules.