Genome mining- and synthetic biology-enabled production of hypermodified peptides

Abstract

The polytheonamides are among the most complex and biosynthetically distinctive natural products known to date. These potent peptide cytotoxins are derived from a ribosomal precursor processed by 49 mostly non-canonical posttranslational modifications. Since the producer is a "microbial dark matter" bacterium only distantly related to any cultivated organism, >70-step chemical syntheses have been developed to access these unique compounds. Here we mined prokaryotic diversity to establish a synthetic platform based on the new host Microvirgula aerodenitrificans that produces hypermodified peptides within two days. Using this system, we generated the aeronamides, new polytheonamide-type compounds with near-picomolar cytotoxicity. Aeronamides, as well as the polygeonamides produced from deep-rock biosphere DNA, contain the highest numbers of D-amino acids in known biomolecules. With increasing bacterial genomes being sequenced, similar host mining strategies might become feasible to access further elusive natural products from uncultivated life.

A multiproducer microbiome generates chemical diversity in the marine sponge Mycale hentscheli

“…In M. hentscheli, a multiproducer consortium of variable composition is the likely reason for the diverse chemotypes. With knowledge on producers and their BGCs available, further development of M. hentscheli compounds could be expedited by permitting rapid PCR profiling of specimens, targeted isolation of producers from the host or alternative sources (66,74), or the establishment of heterologous expression systems.…”

“…ODIS Experiments. E. coli Tuner (DE3) was cotransformed with Nhis-ganA precursor in pACYCDuet-1 and poyD or aerD in pCDFBAD-Myc-HisA (66), which is derived from pBAD/Myc-His A with the native origin of replication replaced by that of pCDFDuet. Cells were plated on Luria-Bertani (LB) agar containing chloramphenicol (25 μg/mL) and ampicillin (100 μg/mL).…”

“…Since we previously showed that the core sequence largely dictates the D-amino acid pattern introduced by rSAM epimerases (65), we performed individual coexpression experiments with PoyD and AerD to obtain clues about D-residues in gananamide. AerD is a recently identified epimerase with improved processitvity in E. coli from the polytheonamidetype aeronamide pathway in Microvirgula aerodenitrificans (66). For both epimerase homologs, additional peaks with different retention times appeared in the HPLC-MS data (SI Appendix, Fig.…”

Structure Prediction and Synthesis of a New Class of Macrocyclic Peptide Natural Products

“…Together, these advances have led to the discovery of numerous new natural products [24][25][26][27][28] and, in the case of RiPPs, new post-translational modifications. 14,[29][30][31][32][33][34][35] Bioinformatics recently has been utilized to discover new RiPP classes, such as the α-keto β-amino acid-containing peptides, 36 RiPPs generating thiaglutamate by peptide-amino acyl tRNA ligases (PEARLs), 33 aliphatic ether-containing rotapeptides 31 and non-α thioether-containing ranthipeptides, 37 as well as the pyritides, described herein.…”

Posttranslationally Acting Arginases Provide a Ribosomal Route to Non‐proteinogenic Ornithine Residues in Diverse Peptide Sequences

“…Common features are large nitrile hydratase‐like leader peptides (NHLP) and diverse modification reactions, often catalysed by radical S ‐adenosylmethionine (rSAM) enzymes [1] . The first characterised proteusins were the hypermodified, cytotoxic polytheonamides and related compounds, which carry a large number of d ‐amino acids, methylated residues, and other modifications and are mostly known from uncultivated bacteria [3–5] . However, the genetic variability of orphan proteusin gene clusters present in bacterial genomes suggests a much higher diversity of undiscovered peptide types with rich maturation enzymology, suggesting great potential for drug discovery and synthetic biology alike.…”