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

Abstract

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.

Seeing the Invisibles: Detection of Peptide Enantiomers, Diastereomers, and Isobaric Ring Formation in Lanthipeptides Using Nanopores

“…We then tested SyncA2, a 17-residue peptide with an 11 amino acid ring (Figure B), a remarkable aspect in lanthipeptide research . The dehydration and cyclization of the peptide are both catalyzed by SyncM, a newly characterized enzyme.…”

Proteases Involved in Leader Peptide Removal during RiPP Biosynthesis

“…The LahT PCAT is encoded in a BGC that contains as many as nine putative precursor peptides with conserved leader peptides but very diverse core peptides, explaining why it is able to remove the LP from many noncognate substrates . Indeed, LahT147 (and LahT150) has become a useful tool for removing LPs for many different RiPPs from a wide variety of families. ,,− Another interesting approach has been the covalent attachment of the C39 protease domain of BovT to the lanthipeptide synthetase BovM to generate the class II lanthipeptide bovicin HJ50 . The excised LahT150 protease domain and the full length transporter LtnT involved in lacticin 3147 biosynthesis (Figure A) are quite tolerant in terms of both LP and CP residues as well as the form of the CP (modified or unmodified) as long as the recognition motif is present in the LP.…”

Promiscuous Enzymes for Residue‐Specific Peptide and Protein Late‐Stage Functionalization

“…[99] Enzymes that belong to the LanM family such as ProcM [100] from the cyanobacterium Prochlorococcus sp. and SyncM [101] from the cyanobacterium Synechococcus sp. are bifunctional enzymes involved in the biosynthetic gene clusters (BGCs) of the cyclic ribosomal peptides, class II lanthipeptides.…”

“…[98] As mentioned in section 5 (Figure 3D), LanM enzymes are bifunctional and can dehydrate Ser and Thr and form dehydroalanine and dehydrobutyrine, respectively, before forming thiol ether bridge. [100,101] These enzymes have low substrate specificity and can modify Ser/Thr present in different positions in peptides (Figure 9E).…”