biostudies-literatureWang XPharmaceutical Research and Manufacturers of America Foundation (PhRMA Foundation)U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)U.S. Department of Health & Human Services | NIH | NIH Office of the Director (OD)4198https://www.ebi.ac.uk/biostudies/studies/S-EPMC12056006biostudies-literatureUnknown16(1)Nearly 10,000 plant species are represented by RNA-seq datasets in the NCBI sequence read archive, which are difficult to search in unassembled format due to database size. Here, we optimize RNA-seq assembly to transform most of this public RNA-seq data to a searchable database for biosynthetic gene discovery. We test our transcriptome mining pipeline towards the diversification of moroidins, which are plant ribosomally-synthesized and posttranslationally-modified peptides (RiPPs) biosynthesized from copper-dependent peptide cyclases. Moroidins are bicyclic compounds with a conserved stephanotic acid scaffold, which becomes cytotoxic to non-small cell lung adenocarcinoma cells with an additional C-terminal macrocycle. We discover moroidin analogs with second ring structures diversified at Nature communicationsLarge-scale transcriptome mining enables macrocyclic diversification and improved bioactivity of the stephanotic acid scaffold.PMC12056006T32GM140223S10OD034245R35GM146934R01GM152417F32GM146395Predoctoral FellowshipF31GM155959Sexton JZShafiq KMydy LSChigumba DNDavis DKersten RDWang XMcDonough KMOusley DAfalseLarge-scale transcriptome mining enables macrocyclic diversification and improved bioactivity of the stephanotic acid scaffold.Nearly 10,000 plant species are represented by RNA-seq datasets in the NCBI sequence read archive, which are difficult to search in unassembled format due to database size. Here, we optimize RNA-seq assembly to transform most of this public RNA-seq data to a searchable database for biosynthetic gene discovery. We test our transcriptome mining pipeline towards the diversification of moroidins, which are plant ribosomally-synthesized and posttranslationally-modified peptides (RiPPs) biosynthesized from copper-dependent peptide cyclases. Moroidins are bicyclic compounds with a conserved stephanotic acid scaffold, which becomes cytotoxic to non-small cell lung adenocarcinoma cells with an additional C-terminal macrocycle. We discover moroidin analogs with second ring structures diversified at 2025-01-01T00:00:00Z2025 May2026-06-02T21:00:24.378Z2026-04-20T03:14:34.821ZS-EPMC120560064032879710.1038/s41467-025-59428-4