Functional Expression and Characterization of the Highly Promiscuous Lanthipeptide Synthetase SyncM, Enabling the Production of Lanthipeptides with a Broad Range of Ring Topologies

Abstract

Lanthipeptides are ribosomally synthesized and post-translationally modified peptides characterized by the presence of lanthionine rings that provide stability and functionality. Genome mining techniques have shown their huge diversity and potential for the discovery of novel active molecules. However, in many cases, they are not easily produced under laboratory conditions. The heterologous expression of these molecules using well-characterized lanthipeptide biosynthetic enzymes is rising as an alternative system for the design and production of new lanthipeptides with biotechnological or clinical properties. Nevertheless, the substrate-enzyme specificity limits the complete modification of the desired peptides and hence, their full stability and/or biological activity. New low substrate-selective biosynthetic enzymes are therefore necessary for the heterologous production of new-to-nature peptides. Here, we have identified, cloned, and heterologously expressed in Lactococcus lactis the most promiscuous lanthipeptide synthetase described to date, i.e., SyncM from the marine cyanobacteria Synechococcus MITS9509. We have characterized the functionality of SyncM by the successful expression of 15 out of 18 different SyncA substrates, subsequently determining the dehydration and cyclization processes in six representatives of them. This characterization highlights the very relaxed substrate specificity of SyncM toward its precursors and the ability to catalyze the formation of exceptionally large rings in a variety of topologies. Our results suggest that SyncM could be an attractive enzyme to design and produce a wide variety of new-to-nature lanthipeptides with a broad range of ring topologies.

Lanthipeptides from the Same Core Sequence: Characterization of a Class II Lanthipeptide Synthetase from Microcystis aeruginosa NIES-88

“…5−8 Recently, another highly promiscuous ProcM-like enzyme, SyncM, has been discovered as the only LanM enzyme from the cyanobacterium Synechococcus MITS9509, whose genome encodes 79 precursors with diverse cores. 9 18 of them were coexpressed with SyncM, wherein 15 were successfully produced and 6 structurally determined to have various ring sizes and topologies. 9 Those studies highlight the extremely high tolerance of ProcM-like enzymes for the core sequences, indicating the tremendous potential of applying ProcM-like enzymes to combinatorial biosynthesis.…”

“…9 18 of them were coexpressed with SyncM, wherein 15 were successfully produced and 6 structurally determined to have various ring sizes and topologies. 9 Those studies highlight the extremely high tolerance of ProcM-like enzymes for the core sequences, indicating the tremendous potential of applying ProcM-like enzymes to combinatorial biosynthesis. 10−12 These biosynthetic features and the catalytic promiscuity of ProcM-like enzymes make lanthipeptides wellsuited for genome mining and structural engineering.…”

“…ProcM has been experimentally evaluated to convert 18 potential precursors into products with diverse ring topologies. Additionally, ProcM has been explored to produce noncognate lanthipeptides by coexpression of ProcM and chimeric precursors which contain a ProcA leader or ProcM recognition segment and the noncognate cores. − Recently, another highly promiscuous ProcM-like enzyme, SyncM, has been discovered as the only LanM enzyme from the cyanobacterium Synechococcus MITS9509, whose genome encodes 79 precursors with diverse cores . 18 of them were coexpressed with SyncM, wherein 15 were successfully produced and 6 structurally determined to have various ring sizes and topologies .…”

Mutational Studies of the Mersacidin Leader Reveal the Function of Its Unique Two-Step Leader Processing Mechanism

“…Additional processing after export might occur under natural conditions, leading to inactive peptides upon heterologous expression. 23 Additionally, it is common practice to substitute the leader cleavage site with that of a well-established lanthipeptide, like the cleavage site of NisP, 38 LahT150, 16 or that of a commercially available protease. 39 While this approach is versatile and convenient, it might replace residues in the original leader that are essential for a specific modification, like the substitution of D-5 and E-3 in the mersacidin leader prevents a specific modification from occurring while the rest of the peptide is still modified.…”

A Silent Biosynthetic Gene Cluster from a Methanotrophic Bacterium Potentiates Discovery of a Substrate Promiscuous Proteusin Cyclodehydratase

“…Here, we posit that RiPP biosynthetic enzymes present in biosynthetic gene clusters (BGCs) containing multiple substrate peptide-encoding genes will inherently be substrate promiscuous as they need to modify different substrates in their native physiological context. This rationalization is supported by the discovery of broadly substrate-tolerant lanthipeptide synthetases that modify numerous different substrate peptides encoded in cyanobacterial genomes. − Also reminiscent are the substrate promiscuous peptide macrocyclases that modify multiple different core regions present in a single substrate peptide for the biosynthesis of cyanobactins, microviridins, and fungal and plant macrocyclic RiPPs . These substrate promiscuous RiPP biosynthetic enzymes can then be used as peptide-modifying biotechnology tools , and also used combinatorically with other RiPP biosynthetic enzymes to build expansive natural product libraries. − …”