Ramos-Aboites, H. E. | bioRxiv Science

bioRxiv2022

Specialized metabolic convergence in the gut microbiomes of cycad-feeding insects tolerant to β-methylamino-L-alanine (BMAA)

Ingestion of the cycad toxins {beta}-methylamino-L-alanine (BMAA) and azoxyglycosides is harmful to diverse organisms. However, some insects are specialized to feed on toxin-rich cycads with apparent immunity. Some cycad-feeding insects possess a common set of gut bacteria, which might play a role in detoxifying cycad toxins. Here, we investigated the composition of gut microbiota from a worldwide sample of cycadivorous insects and characterized the biosynthetic potential of bacteria isolated as putative keystone taxa. Cycadivorous insects shared a core gut microbiome consisting of six bacterial taxa, mainly belonging to the Proteobacteria, which we were able to isolate. To further investigate these potential keystone taxa from diverging lineages, we performed shotgun metagenomic sequencing of co-cultured bacterial sub-communities. We postulate and characterize four putative keystone bacteria from Serratia, Pantoea, and two different Stenotrophomonas lineages. The biosynthetic potential of these microorganisms includes a suite of biosynthetic gene clusters notably rich in siderophores and carotenoid-like aryl polyene pathways. Siderophore semi-untargeted metabolomics revealed a broad range of chemically related yet diverse iron-chelating metabolites, indicating a complex evolutionary landscape in which siderophores may have converged within the guts of cycadivorous insects. Among these, we provide evidence of the occurrence of an unprecedent desferrioxamine-like biosynthetic pathway that remains to be identified. These results provide a foundation for future investigations into how cycadivorous insects tolerate diets rich in azoxyglycosides, BMAA, and other cycad toxins, and highlight convergent evolution underlying chemical diversity.

microbiology↗

bioRxiv2020

Convergent evolution of Streptomyces protease inhibitors involving a tRNA-mediated condensation-minus NRPS

Small peptide aldehydes (SPAs) with protease inhibitory activity are natural products typically synthesized by nonribosomal peptide synthetases (NRPS). SPAs are widely used in biotechnology, as therapeutic agents, they are physiologically relevant and regulate development of the natural hosts. During genome evolutionary analysis of Streptomyces lividans 66 we identified an NRPS-like biosynthetic gene cluster (BGC) that lacked a condensation (C) domain but included a tRNA-Utilizing Enzyme (tRUE) belonging to the leucyl/phenylalanyl (L/F) transferase family. This system was predicted to direct the synthesis of a novel SPA with protease inhibitory activity, called livipeptin. Following genome mining and phylogenomic analyses we confirmed the presence of tRUEs within diverse Streptomyces genomes, including fusions with a C-minus NRPS-like protein. We further demonstrate functional cooperation between these enzymes and provide the biosynthetic rules for the synthesis of livipeptin, expanding the known universe of acetyl-leu/phe-arginal SPAs. The L/F-transferase C-minus NRPS productive interaction was shown to be tRNA-dependent after semisynthetic assays in the presence of RNAse, which contrasts with leupeptin, an acetyl-leu-arginal SPA that we show to be produced by Streptomyces roseous ATCC 31245 via a tRUE-minus BGC with multiple complete NRPSs. Thus, livipeptin and leupeptin are the result of convergent evolution, which has driven the appearance of unprecedented biosynthetic logics directing the synthesis of protease inhibitors thought to be at the core of Streptomyces colony biology. Our results pave the way for understanding this Streptomyces trait, as well as for the discovery of novel natural products following evolutionary genome mining approaches. Abstract importanceConvergent evolution in microbiology is believed to be highly recurrent yet examples that have been comprehensively characterized are scarce. Proteases inhibition by small peptide aldehydes is at the core of many microbiological processes, both within the cell and during colony development, and in microbial ecology. Here we report the biosynthetic foundations of leupeptin, the main Streptomyces protease inhibitor, and of livipeptin, a protease inhibitor produced by Streptomyces lividans. Although these peptides belong to the same chemical class, here we show that their biosynthetic routes result from convergent evolution, as they involve unrelated biosynthetic mechanisms, including the recruitment of a tRNA-utilizing enzyme that functionally replaces the condensation domain of a nonribosomal peptide synthetase during livipeptin biosynthesis. Thus, these results pave the way for understanding Streptomyces protease inhibitors as a trait and provide unprecedented knowledge for genome mining of natural products and synthetic biology where proteases inhibition is desirable.

microbiology↗