Engineering lanthipeptides by introducing a large variety of RiPP modifications to obtain new-to-nature bioactive peptides

Abstract

Natural bioactive peptide discovery is a challenging and time-consuming process. However, advances in synthetic biology are providing promising new avenues in peptide engineering that allow for the design and production of a large variety of new-to-nature peptides with enhanced or new bioactivities, using known peptides as templates. Lanthipeptides are ribosomally synthesized and post-translationally modified peptides (RiPPs). The modularity of post-translational modification (PTM) enzymes and ribosomal biosynthesis inherent to lanthipeptides enables their engineering and screening in a high-throughput manner. The field of RiPPs research is rapidly evolving, with many novel PTMs and their associated modification enzymes being identified and characterized. The modularity presented by these diverse and promiscuous modification enzymes has made them promising tools for further in vivo engineering of lanthipeptides, allowing for the diversification of their structures and activities. In this review, we explore the diverse modifications occurring in RiPPs and discuss the potential applications and feasibility of combining various modification enzymes for lanthipeptide engineering. We highlight the prospect of lanthipeptide- and RiPP-engineering to produce and screen novel peptides, including mimics of potent non-ribosomally produced antimicrobial peptides (NRPs) such as daptomycin, vancomycin, and teixobactin, which offer high therapeutic potential.

Keywords: Engineering; NRPs mimics; lanthipeptides; post-translational modification; ripps; synthetic biology.

Figure 1.

Structural representatives of diverse NRPs and RiPPs. (A) Examples of RiPPs from Lactic Acid Bacteria (LAB). Mersacidin is ribosomally produced by Bacillus sp. strain HIL Y-85, 54 728; Glycocin F (GccF) is a potent bacteriocin originally isolated from liquid culture of Lactobacillus plantarum KW30; and Nisin is a penta-cyclic antibacterial peptide produced by the bacterium L. lactis. (B) Examples of NRPs/PKS. Daptomycin is a cyclic lipopeptide antibiotic produced by Streptomyces roseosporus i.e. used for the treatment of serious Gram-positive infections; Mutanobactin A is a hybrid PKS-NRPS isolated from Streptococcus; and Turnercyclamycin, produced by Teredinibacter turnerae, is a lipopeptide antibiotic against several Gram-negative pathogens.

Figure 2.

Schematic diagram of the non-ribosomal peptide Brevicidine mimicked by ribosomal synthesis. NRPs are non-ribosomal peptides; RiPPs are ribosomally synthesized and post-translationally modified peptides.

Figure 3.

Natural combinations of diverse modifications in lanthipeptides. Cinnamycin and duramycin contain a hydroxyl group, which is essential for their antimicrobial activity. This group is marked in green in the Cinnamycin chemical structure; Lanthipeptide microbisporicin (NAI-107) contains a 5-chlorotryptophan (5-Cl-Trp) motif, which is shown in green in the corresponding structure; NAI-112, a glycosylated Class III lanthipeptide, contains a rare deoxyhexose modification N-linked to a tryptophan residue. This structural feature is highlighted in green.

Figure 4.

Schematic diagram of further modifications and combinations introduced into a brevicidine mimic. The implementation of mimicking mainly includes four parts. The fatty acid chains can be mimicked by using hydrophobic amino acids, or they can be catalysed by enzymes. The mimic of a lactone ring by a lanthionine and the fatty acid mimicking by three hydrophobic AA residues have already been achieved (Zhao et al. 2020). Further modification work mainly focuses on the incorporation of D-amino acids, the non-canonical amino acid ornithine, and the mimicking of a fatty acid chain by enzymatic methods. Piel’s group has demonstrated the possibility of introducing D-amino acids and ornithines onto similar linear peptide sequences (Mordhorst et al. 2020).

Figure 5.

Schematic diagram of various screening methods. (A) Schematic diagram of phage, bacteria, and yeast surface display systems (Bosma et al. , Urban et al. , Hetrick et al. 2018). (B) Bacterial reverse two-hybrid technology for identifying a lanthipeptide inhibitor of the p6-UEV PPI (Yang et al. 2018).

Figure 6.

A schematic diagram for nanoFleming technology application in active peptide screening (Schmitt et al. 2019).

Figure 7.

Artist’s ‘floral’ impression of combinations of diverse modifications into one peptide, putatively made possible by the use of the RiPPs enzyme.