Natural product-based drugs account for roughly half of all current pharmaceuticals. The diversity of bacterial natural products is vast, with some estimates of more than one billion unique compounds in bacteria. However, less than 1% of known bacteria have been cultured in laboratory conditions. Despite this, metagenomic data can be used to identify natural product biosynthetic gene clusters (BGCs) which can be reassembled to access these unknown natural products.
Polytheonamides are a class of peptide natural products derived from sea sponge microbiomes. Polytheonamides are notable for including many non-proteinogenic amino acids, including numerous D-amino acid residues. Surprisingly, polytheonamides are peptides of ribosomal origin and attain their final structure through extensive post-translational modification. These complex natural products have great potential for bioactivity, but chemical syntheses have required more than 70 steps. The polytheonamide BGC can access novel chemistry in peptide natural products and offers immense biosynthetic potential. Using a metagenomic approach, Bhushan et al. were able to create a biosynthetic platform for polytheonamide-like natural products in Microvirgula aerodenitrificans.
Beginning with the known polytheonamide pathway, they were able to mine metagenomic data from uncultivated marine organisms and identify pathways with similar genetic features. The AerA pathway, identified in Microvirgula aerodenitrificans, was selected for further study. Through heterologous expression of the AerA pathway in E. coli and subsequent LC-MS analysis, Bhushan et al. were able to identify many L-amino acid to D-amino acid epimerizations that suggested the AerA pathway produced a polytheonamide-like product, dubbed an aeronamide. Motivated by these results, Bhushan et al. were able to optimize growth conditions for Microvirgula aerodenitrificans in laboratory culture so that the AerA pathway could be expressed in its native host. The team was able to develop a production platform for aeronamides: the penultimate hypermodified peptide product could be expressed efficiently in Microvirgula aerodenitrificans, then final cleavage from the leader peptide could be accomplished through in vitro incubation with purified proteinase. Pushing this expression platform further, Bhushan et al. were able to characterize more hypermodified peptides by swapping the core peptide for others that were identified in the metagenomic search of marine deep rock.
Production of aeronamides and similar hypermodified peptide natural products was formerly possible via excruciatingly long chemical synthesis. The new platform described here allows for biosynthesis of diverse products in merely two days. One of the products isolated using this strategy was found to contain 23 D-amino acids, the largest number reported in a natural peptide to date. This extensive L to D epimerization of amino acids could offer great utility in the biosynthesis of D-amino acid containing peptides. Additionally, this pathway demonstrated biosynthetic potential for C-H functionalization through methylation of unactivated carbons. These post-translational modifications, amongst others described in this study, can access peptides with extensive non-canonical residues, something of great interest to us at C-GEM and I am excited to see how this production platform can be utilized in the future.
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