Refactoring and Heterologous Expression of Class III Lanthipeptide Biosynthetic Gene Clusters Lead to the Discovery of N,N-Dimethylated Lantibiotics from Firmicutes

Abstract

Class III lanthipeptides are an emerging subclass of lanthipeptides, representing an underexplored trove of new natural products with potentially broad chemical diversity and important biological activity. Bioinformatic analysis of class III lanthipeptide biosynthetic gene cluster (BGC) distribution has revealed their high abundance in the phylum Firmicutes. Many of these clusters also feature methyltransferase (MT) genes, which likely encode uncommon class III lanthipeptides. However, two hurdles, silent BGCs and low-yielding pathways, have hindered the discovery of class III lanthipeptides from Firmicutes. Here, we report the design and construction of a biosynthetic pathway refactoring and heterologous overexpression strategy which seeks to overcome these hurdles, simultaneously activating and increasing the production of these Firmicutes class III lanthipeptides. Applying our strategy to MT-containing BGCs, we report the discovery of new class III lanthipeptides from Firmicutes bearing rare N,N-dimethylations. We reveal the importance of the first two amino acids in the N-terminus of the core peptide in controlling the MT dimethylation activity. Leveraging this feature, we engineer class III lanthipeptides to enable N,N-dimethylation, resulting in significantly increased antibacterial activity. Furthermore, the refactoring and heterologous overexpression strategy showcased in this study is potentially applicable to other ribosomally synthesized and post-translationally modified peptide BGCs from Firmicutes, unlocking the genetic potential of Firmicutes for producing peptide natural products.

Insights into the production and evolution of lantibiotics from a computational analysis of peptides associated with the lanthipeptide cyclase domain

“…However, functional assessment of non-lantibiotic lanthipeptides can be challenging: the prochlorosins were identified in 2010, but no function has yet been assigned to them [87]. While culturing strains under appropriate conditions to produce lantibiotics or lanthipeptides may be challenging, heterologous expression systems [88][89][90] can help overcome these issues. Until the single-cysteine peptides identified in this study have been validated experimentally, we cannot rule out that they may include false positives.…”

Bacillus subtilis as a host for natural product discovery and engineering of biosynthetic gene clusters

“…This dimethylation, found in andalusicin 137 and variants of paenithopeptin, 136 was found to increase their antibacterial activity 136,137 and is dependent on specic amino acids present at the rst two positions of the core peptide. 136 RiPPs show a remarkable biosynthetic malleability thanks to the tolerance of many RiPP modifying enzymes to amino acid changes in the core peptide sequence. The organization of precursor peptides in two distinctive parts (leader and core peptide) greatly facilitates this promiscuity as it allows leader peptide-dependent tailoring enzymes to remain substrate-specic while processing a wide variety of core sequences.…”

“…134 The heterologous expression of RiPP BGCs in Bacillus has also led to the discovery of novel class III lanthipeptides from Firmicutes. [135][136][137] Most of these lanthipeptide BGCs lack a protease gene. Through a combination of correlation network and co-expression analysis, possible proteases for these lanthipeptides were predicted.…”

“…As a proof of concept, two previously unreported class III lanthipeptide BGCs were heterologously expressed in Bacillus, together with their predicted protease genes. This approach resulted in the discovery of bacinapeptins A and B, and paenithopeptins A-E 136 . Additionally, heterologous expression in Bacillus revealed an uncommon N-terminal dimethylation in a subset of class III lanthipeptides.…”

Facile Method for Determining Lanthipeptide Stereochemistry

“…However, the stereochemistry of the Lan and MeLan residues of the great majority of newly reported lanthipeptides is not determined. Therefore, as more and more lanthipeptides are discovered by genome mining, − a convenient method for determining their stereochemistry that is accessible to most laboratories would be valuable. We report here such a method including access to standards that use biochemical approaches that are complementary to chemical synthesis and methods available in most if not all laboratories studying lanthipeptides or RiPPs.…”