Advancements in the Application of Ribosomally Synthesized and Post-Translationally Modified Peptides (RiPPs)

Abstract

Ribosomally synthesized and post-translationally modified peptides (RiPPs) represent a significant potential for novel therapeutic applications because of their bioactive properties, stability, and specificity. RiPPs are synthesized on ribosomes, followed by intricate post-translational modifications (PTMs), crucial for their diverse structures and functions. PTMs, such as cyclization, methylation, and proteolysis, play crucial roles in enhancing RiPP stability and bioactivity. Advances in synthetic biology and bioinformatics have significantly advanced the field, introducing new methods for RiPP production and engineering. These methods encompass strategies for heterologous expression, genetic refactoring, and exploiting the substrate tolerance of tailoring enzymes to create novel RiPP analogs with improved or entirely new functions. Furthermore, the introduction and implementation of cutting-edge screening methods, including mRNA display, surface display, and two-hybrid systems, have expedited the identification of RiPPs with significant pharmaceutical potential. This comprehensive review not only discusses the current advancements in RiPP research but also the promising opportunities that leveraging these bioactive peptides for therapeutic applications presents, illustrating the synergy between traditional biochemistry and contemporary synthetic biology and genetic engineering approaches.

Keywords: RiPPs; bioactive peptides; genetic engineering; heterologous expression; high-throughput screening; synthetic biology.

Figure 1

Representative therapeutic RiPPs and their classes. Letters in circles represent amino acids, and moieties undergoing modification are highlighted in red.

Figure 2

Schematic representation of the RiPP biosynthetic pathway. The biosynthetic gene cluster, which includes various genes responsible for RiPP synthesis, is translated into a precursor peptide and tailoring enzymes. After translation, tailoring enzymes, recruited by the leader (and/or follower) peptide, modify the core peptide. Subsequently, a protease cleaves the leader (and/or follower) peptide, resulting in the production of the mature peptide. Red stars represent the occurrence of PTM.

Figure 3

Genetic engineering for heterologous RiPP expression. (A) In DNA assembly methods such as Gibson assembly, multiple PCR products are ligated into an expression vector through a single isothermal reaction. (B) RecET facilitates homologous recombination between a lengthy linear fragment of a BGC and a vector (circular or linear) containing homologous regions. (C) ExoCET employs an exonuclease in addition to promoting recombination with longer fragments of BGC that carry non-homologous overhangs. (D) Upon transformation into yeast with fragments of BGC and a vector with homologous regions, the DNA fragments are assembled via the yeast’s native recombination system. (E) In the CAPTURE technique, the BGC fragment, isolated from genomic DNA by Cas12a, is ligated into synthetic receivers with loxP sites using DNA assembly methods, followed by circularization with the Cre enzyme. (F) Replacing native regulatory elements with uncharacterized mechanisms into well-understood systems facilitates the heterologous expression of selected BGC components, crucial for the biosynthesis of mature RiPP. (G) The expression of RiPP with bioactive properties triggers cell death in the host strain. However, by transporting a protease and a lytic protein to the periplasmic region and delaying the expression of the lytic protein, maturation of RiPP occurs, allowing the host cell to survive. (H) The addition of a fusion tag to a precursor peptide increases its stability and expression level, leading to an accumulation of mature RiPP in a heterologous cell.

Figure 4

Strategies of RiPP engineering with respect to core peptides (A–C), leader peptides (D,E), and tailoring enzymes (F,G). (A–C) Addressing core peptides, substrate flexibilities of tailoring enzymes enable site-directed mutations (A), incorporation of foreign core peptides (B), and creation of hybrid core peptides with multiple domains (C), leading to a variety of RiPPs. (D,E) Utilizing leader peptides’ properties in guiding PTMs, diverse combinations of PTMs can be introduced on a single core peptide through chimeric leader peptides with multiple domains to guide PTMs (D) and leader peptide exchange using sortase A (E). (F) Protein engineering can enhance the substrate range of tailoring enzymes, broadening their application in generating RiPP variants. (G) The regulatory mechanism in tailoring enzyme activation can be simplified by introducing a free leader peptide and by fusing a tailoring enzyme with both a leader and a precursor peptide, simplifying the RiPP biosynthesis process.

Figure 5

High-throughput screening methods utilized in RiPP research. (A) Phage display and (B) mRNA display techniques facilitate the straightforward detection of interactions between RiPPs and proteins or molecules. (C) The two-hybrid system can identify RiPPs that inhibit protein–protein interactions associated with diseases and infections. (D) Another in vivo screening method employs a genetic circuit based on intein, wherein the interaction between a RiPP and a target protein triggers the transcription of a reporter gene, allowing for the detection of RiPP–protein interactions. The antimicrobial activity of RiPPs can be assessed by their ability to inhibit the growth of (E) host cells and (F) neighboring cells. (E) Inhibition of host cell growth correlates with the concentration of RiPPs, as determined by NGS; a lower RiPP concentration signifies higher antimicrobial activity. (F) Inhibition of neighboring cell growth is evaluated using sensor cells that express a fluorescent protein; decreased fluorescence intensity indicates higher antimicrobial activity.