Investigating the Specificity of the Dehydration and Cyclization Reactions in Engineered Lanthipeptides by Synechococcal SyncM

ProcM-like enzymes are class II promiscuous lanthipeptide synthetases that are an attractive tool in synthetic biology for producing lanthipeptides with biotechnological or clinically desired properties. SyncM is a recently described modification enzyme from this family used to develop a versatile expression platform for engineering lanthipeptides. Most remarkably, SyncM can modify up to 79 SyncA substrates in a single strain. Six SyncAs were previously characterized from this pool of substrates. They showed particular characteristics, such as the presence of one or two lanthionine rings, different flanking residues influencing ring formation, and different ring directions, demonstrating the relaxed specificity of SyncM toward its precursor peptides. To gain a deeper understanding of the potential of SyncM as a biosynthetic tool, we further explored the enzyme's capabilities and limits in dehydration and ring formation. We used different SyncA scaffolds for peptide engineering, including changes in the ring's directionality (relative position of Ser/Thr to Cys in the peptide) and size. We further aimed to rationally design mimetics of cyclic antimicrobials and introduce macrocycles in prochlorosin-related and nonrelated substrates. This study highlights the largest lanthionine ring with 15 amino acids (ring-forming residues included) described to date. Taking advantage of the amino acid substrate tolerance of SyncM, we designed the first single-SyncA-based antimicrobial. The insights gained from this work will aid future bioengineering studies. Additionally, it broadens SyncM's application scope for introducing macrocycles in other bioactive molecules.