Engineering of new-to-nature ribosomally synthesized and post-translationally modified peptide natural products

Abstract

Natural products have historically been important lead sources for drug development, particularly to combat infectious diseases. Increasingly, their structurally complex scaffolds are also envisioned as leads for applications for which they did not evolve, an approach aided by engineering of new-to-nature analogs. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are promising candidates for bioengineering because they are genetically encoded and their biosynthetic enzymes display significant substrate tolerance. This review highlights recent advances in the discovery of highly unusual new reactions by genome mining and the application of engineering approaches to generate and screen novel RiPP variants. Furthermore, through the use of synthetic biology approaches, hybrid molecules with enhanced or completely new activities have been identified, which opens the door for future advancement of RiPPs as potential next-generation therapeutics.

Figure 1.

General biosynthetic pathway of RiPPs that features a precursor peptide, modifying enzymes, protease, and transporter. The precursor peptide is composed of a leader peptide (LP), which contains enzyme recognition sites, and a core peptide (CP), which undergoes the PTMs that often include macrocyclization.

Figure 2.

Lanthipeptides discussed in this review. The parts of the structures that are Ser/Thr- and Cys-derived are shown in red and blue, respectively, and other post-translationally modified residues are shown in green. a, Structure of the characteristic PTMs in lanthipeptides. Both the chemical structure and a shorthand notation are shown; the latter is used in panel b. b, Representative examples of lanthipeptides, including members with special terminal groups. Avi, avionin; Abu, 2-aminobutyric acid.

Figure 3.

Aminoacyl-tRNA dependent RiPP biosynthetic enzymes. a, Three different architectures of aminoacyl-tRNA dependent RiPP biosynthetic enzymes. For examples of lanthipeptides, thiopeptides, and pearlins see Figures 2 and 4. b, Biosynthetic gene cluster in P. syringae that encodes the PEARL TglB, and the biosynthetic pathway towards 3-thiaglutamate. c, TglB mechanism resulting in the addition of Cys to the C-terminus of the peptide TglA.

Figure 4.

Structures of select RiPPs discussed in this review. The enzymes that install the PTMs colored in red are discussed in the text or were used for the hybrid RiPPs in Table 1. a. Structures of the pearlin ammosamide A, the borosin omphalotin A, and the cyanobactin trunkamide. b. Examples of radical SAM enzymes catalyzing spliceotide (top) and cyclophane (bottom) formation. c. Structures of the sactipeptide subtilosin, the lasso peptide citrulassin A, the linear azole containing peptide goadsporin, and the thiopeptide thiocillin. For citrulassin, the C-terminal tail threads through the macrolactam in the active fold (not shown).

Figure 5.

Summary of the flexible in vitro translation (FIT) system and its use in building nonstandard peptides and reconstituting biosynthetic pathways that result in new-to-nature RiPP analogs. Flexizymes charge tRNAs with ncAAs, in vitro translation incorporates the ncAAs into the peptide substrate, and the RiPP biosynthetic enzymes then install the PTMs in the final product.