RiPP antibiotics: biosynthesis and engineering potential

Abstract

The threat of antibiotic resistant bacterial infections continues to underscore the need for new treatment options. Historically, small molecule metabolites from microbes have provided a rich source of antibiotic compounds, and as a result, significant effort has been invested in engineering the responsible biosynthetic pathways to generate novel analogs with attractive pharmacological properties. Unfortunately, biosynthetic stringency has limited the capacity of non-ribosomal peptide synthetases and polyketide synthases from producing substantially different analogs in large numbers. Another class of natural products, the ribosomally synthesized and post-translationally modified peptides (RiPPs), have rapidly expanded in recent years with many natively displaying potent antibiotic activity. RiPP biosynthetic pathways are modular and intrinsically tolerant to alternative substrates. Several prominent RiPPs with antibiotic activity will be covered in this review with a focus on their biosynthetic plasticity. While only a few RiPP enzymes have been thoroughly investigated mechanistically, this knowledge has already been harnessed to generate new-to-nature compounds. Through the use of synthetic biology approaches, on-going efforts in RiPP engineering hold great promise in unlocking the potential of this natural product class.

Figure 1

A generalized RiPP biosynthetic gene cluster complete with a precursor peptide, modifying enzymes, and proteolysis/export enzymes. RiPP precursor peptides feature leader and core regions that separate substrate recognition residues from residues that undergo modification.

Figure 2

Representative members of several RiPP classes. RiPPs display an array of structural diversity and antimicrobial activity. Sites of post-translational modifications are indicated in red.

Figure 3

Post-translational modifications (PTMs), mode of substrate recognition, and substrate tolerance of various RiPP enzymes covered in this review. RiPP biosynthetic enzymes display a spectrum of substrate tolerances with the most tolerant enzymes likely being the best-suited for applications in synthetic biology. Az = azole.

Figure 4

New-to-nature hybrid RiPPs generated through RiPP engineering. By leveraging knowledge of substrate recognition from various RiPP classes, chimeric precursor peptides have been designed to recruit biosynthetic enzymes not naturally found in the same context. The resulting hybrid RiPPs have functional groups not currently found together in any natural product such as thiazolines/lanthionines, thiazolines/sactionines, and thiazolines/lanthionines/D-Ala; the latter is generated by enzymatic reduction of Dha.