Phage display and selection of lanthipeptides on the carboxy-terminus of the gene-3 minor coat protein

Abstract

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are an emerging class of natural products with drug-like properties. To fully exploit the potential of RiPPs as peptide drug candidates, tools for their systematic engineering are required. Here we report the engineering of lanthipeptides, a subclass of RiPPs characterized by multiple thioether cycles that are enzymatically introduced in a regio- and stereospecific manner, by phage display. This was achieved by heterologous co-expression of linear lanthipeptide precursors fused to the widely neglected C-terminus of the bacteriophage M13 minor coat protein pIII, rather than the conventionally used N-terminus, along with the modifying enzymes from distantly related bacteria. We observe that C-terminal precursor peptide fusions to pIII are enzymatically modified in the cytoplasm of the producing cell and subsequently displayed as mature cyclic peptides on the phage surface. Biopanning of large C-terminal display libraries readily identifies artificial lanthipeptide ligands specific to urokinase plasminogen activator (uPA) and streptavidin.

In Vitro Biosynthesis of Peptides Containing Exotic Azoline Analogues

“…Such combinations would further expand the molecular complexity of peptides produced by the artificial in vitro biosynthesis system strategy . A significant advantage of bioengineering with RiPP‐modifying enzymes is the potential applicability for the construction of combinatorial peptide libraries and screening by display technologies . Therefore, the reprogrammed FIT‐PatD system, demonstrating the combination of reprogrammed translation and post‐translational modification, can pave the way for the development of new bioactive peptides with diverse non‐proteinogenic structures.…”

Minimal lactazole scaffold for in vitro production of pseudo-natural thiopeptides

“…Because continuous randomized epitopes can be displayed inside a thiopeptide backbone, we envision that combinatorial libraries based on this scaffold can be generated and screened akin to the recent reports on lanthipeptide bioengineering. 12–16 In those cases, lanthipeptide libraries were prepared with the use of promiscuous lanthipeptide synthases, and could be screened against a protein target of interest with the use of phage/yeast display or with the reverse two-hybrid system. These studies resulted in the discovery of lanthipeptide inhibitors of HIV budding process, 15 urokinase plasminogen activator 16 and α v β 3 integrin binders.…”

“…12–16 In those cases, lanthipeptide libraries were prepared with the use of promiscuous lanthipeptide synthases, and could be screened against a protein target of interest with the use of phage/yeast display or with the reverse two-hybrid system. These studies resulted in the discovery of lanthipeptide inhibitors of HIV budding process, 15 urokinase plasminogen activator 16 and α v β 3 integrin binders. 13 Similarly, we anticipate that the integration of the FIT-Laz system with powerful in vitro screening techniques such as mRNA display 66 will provide access to artificial thiopeptides with desirable pharmacological profiles for drug discovery purposes.…”

Genetically Encoded Libraries of Constrained Peptides

“…B) Schematic illustration of C‐terminal phage display system. Phage display of lanthipeptides on the carboxy terminus of the gene‐3 minor coat protein . C‐terminal precursor peptide fusions to pIII are enzymatically modified in the cytoplasm of the producing cell and subsequently displayed as mature cyclic peptides on the phage surface.…”