Embleporicin: A Novel Class I Lanthipeptide from the Actinobacteria Embleya sp. NF3

Abstract

Genome mining has emerged as a revolutionary tool for discovering new ribosomally synthesized and post-translationally modified peptides (RiPPs) in various genomes. Recently, these approaches have been used to detect and explore unique environments as sources of RiPP-producing microorganisms, particularly focusing on endophytic microorganisms found in medicinal plants. Some endophytic actinobacteria, especially strains of Streptomyces, are notable examples of peptide producers, as specific biosynthetic clusters encode them. To uncover the genetic potential of these organisms, we analyzed the genome of the endophytic actinobacterium Embleya sp. NF3 using genome mining and bioinformatics tools. Our analysis led to the identification of a putative class I lanthipeptide. We cloned the core biosynthetic genes of this putative lanthipeptide, named embleporicin, and expressed them in vitro using a cell-free protein system (CFPS). The resulting product demonstrated antimicrobial activity against Micrococcus luteus ATCC 9341. This represents the first RiPP reported in the genus Embleya and the first actinobacterial lanthipeptide produced through cell-free technology.

Keywords: actinobacterium Embleya sp. NF3; antimicrobials; cell-free expression; genome mining; lanthipeptides.

Figure 1

Schematic representation of embleporicin cluster and EmpA sequence. (A) Organization of the main genes of embleporicin cluster in Embleya sp. NF3 and (B) alignment of homologous sequences to embleporicin is marked with a red line for the conserved motifs and putative cutting sites. In contrast, the red star shows the additional cysteine residue of embleporicin. The accession number of alignment sequences is shown in Table S3.

Figure 2

Phylogenetic tree of homologous proteins of embleporicin. In orange, SCO0268 members are indicated, while homolog peptides of embleporicin are shown in blue. The accession sequences are shown in Table S3.

Figure 3

Reconstitution of embleporicin biosynthesis using the CFPS system. (A) Extract preparation and addition of other extract components for the synthesis of embleporicin. (B) SDS-PAGE electrophoresis of the crude extract to visualize the post-translational modifying enzymes (EmpB and EmpC). The red arrows indicate the expected size of the enzymes; PC means positive control, and NC means negative control. (C) Western-blot analysis of the post-translational modifying enzymes (EmpB and EmpC). (D) Tricine SDS-PAGE to visualize the modified peptide (mEmpA). The red arrow indicates the expected size of the mEmpA, and NC means negative control. (E) Addition of Furin to the in vitro reaction and an antimicrobial assay of the modified peptide (mEmpA) and the mature peptide (embleporicin). Nisin (Thermo Scientific, Waltham, MA, USA) was used as a positive control, and empty plasmids with and without the addition of Furin were used as negative controls.