Advances in lasso peptide discovery, biosynthesis, and function

Abstract

Lasso peptides are a large and sequence-diverse class of ribosomally synthesized and post-translationally modified peptide (RiPP) natural products characterized by their slip knot-like shape. These unique, highly stable peptides are produced by bacteria for various purposes. Their stability and sequence diversity make them a potentially useful scaffold for biomedically relevant folded peptides. However, many questions remain about lasso peptide biosynthesis, ecological function, and diversification potential for biomedical and agricultural applications. This review discusses new insights and open questions about lasso peptide biosynthesis and biological function. The role that genome mining has played in the development of new methodologies for discovering and diversifying lasso peptides is also discussed.

Keywords: RiPPs; biosynthesis; lasso peptides; natural products; peptide engineering.

Figure 1.

(A) Generic lasso peptide biosynthetic gene cluster (BCG) and biosynthesis pathway. The precursor peptide is first bound by the ribosomally synthesized and post-translationally modified peptide (RiPP) recognition element (RRE), proteolyzed by the leader peptidase, and then cyclized by the lasso cyclase. The core peptide undergoes presumptive adenylation and pre-folding before cyclization. (B) Lasso peptide genome mining facilitates lasso peptide discovery, diversification, and assignment of biological function. Lasso peptides are known to have antibacterial, anticancer, and antiviral activities, and can potentially facilitate resource uptake or usage in the environment.

Figure 2.. Lasso peptide biosynthesis recognition requirements.

(A) Crystal structure of the fusilassin ribosomally synthesized and post-translationally modified peptide (RiPP) recognition element (RRE; blue, WP_011291591.1) with the fusilassin leader peptide [pink, Protein Data Bank (PDB) code: 6JX3] [16]. (B) AlphaFold multimer predicted structure showing the β-sandwich interaction at the interface of the fusilassin leader peptidase (orange, WP_011291590.1) and RRE (blue). Leader peptidase catalytic residues (Cys/His) are shown as green sticks [20]. (C) Precursor peptide sequences for fusilassin [14,20] (discrete RRE), paeninodin [23] (discrete RRE, EHQ60562.1), and caulonodin V [21] (fused RRE, WP_012286029.1) annotated with residues important for RRE recognition, leader peptidolysis, and lasso peptide cyclization. The numbering convention for RiPPs is negative for leader regions and positive for core regions. The * indicates the protease cutsite. Given that the caulonodin V leader peptidase is fused to the RRE, it has not yet been established whether substitution of the blue residues only affects RRE recognition.

Figure 3.. Lasso peptide immunity mechanisms, selected bioactivities, and ecological functions.

(A) ABC transporters export lasso peptides from the producing organism [shown is McjD; protein data bank (PDB) 4PL0, WP_097313467.1]. (B) Isopeptidases linearize lasso peptides by cleaving the macrolactam bond (shown is astexin-2 isopeptidase; PDB 5TXC, ADU14095.1). The zoomed inset highlights the catalytic triad. (C) Lasso peptides have diverse bioactivities, including inhibition of RNA polymerase and lipid II biosynthesis. (D) Lasso peptides are also known inhibitors of algal photosynthesis and may even help bacteria to obtain/utilize environmental resources such as phenanthrene.

Figure 4.. Genome mining facilitates the characterization of additional post-translational modifications on lasso peptides.

Secondary modifications are shown in pink.

Figure 5.. Genome mining has led to the development of new methodologies for lasso peptide discovery.

(A) High-throughput discovery of lasso peptides involves mining new biosynthetic gene clusters (BGCs), refactoring synthetic DNA, expressing the pathways in Escherichia coli, and testing them for desired bioactivity. (B) DeepRiPP uses genomics data, deep learning, and metabolomics data to automate ribosomally synthesized and post-translationally modified peptide (RiPP) discovery.