Bioinformatic and Reactivity-Based Discovery of Linaridins

Abstract

Linaridins are members of the ribosomally synthesized and post-translationally modified peptide (RiPP) family of natural products. Five linaridins have been reported, which are defined by the presence of dehydrobutyrine, a dehydrated, alkene-containing amino acid derived from threonine. This work describes the development of a linaridin-specific scoring module for Rapid ORF Description and Evaluation Online (RODEO), a genome-mining tool tailored toward RiPP discovery. Upon mining publicly accessible genomes available in the NCBI database, RODEO identified 561 (382 nonredundant) linaridin biosynthetic gene clusters. Linaridin BGCs with unique gene architectures and precursor sequences markedly different from previous predictions were uncovered during these efforts. To aid in data set validation, two new linaridins, pegvadin A and B, were detected through reactivity-based screening and isolated from Streptomyces noursei and Streptomyces auratus, respectively. Reactivity-based screening involves the use of a probe that chemoselectively modifies an organic functional group present in the natural product. The dehydrated amino acids present in linaridins as α/β-unsaturated carbonyls were appropriate electrophiles for nucleophilic 1,4-addition using a thiol-functionalized probe. The data presented within significantly expand the number of predicted linaridin biosynthetic gene clusters and serve as a roadmap for future work in the area. The combination of bioinformatics and reactivity-based screening is a powerful approach to accelerate natural product discovery.

Figure 1.. Biosynthetic gene clusters and structures of linaridins.

(A) BGCs responsible for cypemycin and legonaridin. The “Lin” protein naming scheme unify the nomenclature for all linaridins with known functions indicated. (B) Abbreviated structures of cypemycin and legonaridin. Purple, N-terminal demethylation; Blue, Dhb; Red, AviCys; a-Ile, allo-isoleucine.

Figure 2.. Sequence similarity network of linaridin precursors.

Nodes within the SSN are color based on the co-occurrence of a LinM (methyltransferase, purple), LinD (decarboxylase, red), or both (yellow). Groups containing >5 members are numbered. Nodes representing known linaridins are shown as triangles and labeled. This SSN was generated using EFI-EST and visualized with Cytoscape. Protein sequences are conflated at 100% identity (i.e. identical sequences are only represented once), resulting in 457 nodes. Edges indicate an alignment score of 7 (expectation value of <10⁻⁷). Phylogenetic tree information for LinE homologs is available in Supplemental Dataset 5.

Figure 3.. Reactivity-based screening of novel linaridins.

MALDI-TOF mass spectra of unreacted (top) and DTT-labeled (bottom) for (A) S. noursei and (B) S. auratus extract.

Figure 4.. BGC and structure of pegvadin A and B.

(A) BGCs for pegvadin A and B. Specific gene names are noted, derived from Pegvadin A (Pva) and Pegvadin B (Pvb) respectively. (B) Structures of pegvadin A and B with the C-alpha stereochemical configuration for pegvadin A being determined by Marfey’s analysis. Post-translational modifications are color-coded: purple, dimethylation (n=1); blue, Dhb (n=6), orange, epimerization (n=22).