Ouchene, R. | bioRxiv Science

bioRxiv2026

An Integrated Miniaturized Metabologenomics Workflow for High-Throughput Actinomycetes Prioritization

Antibiotic resistance is a growing global health threat, while the discovery of new antimicrobial agents remains constrained by labor-intensive cultivation, extraction, and screening workflows. We developed a miniaturized, high-throughput metabologenomics workflow integrating Duetz system-based microfermentation, extraction-free UHPLC-HRMS/MS profiling, antibacterial screening, and genome mining. A collection of 80 soil-derived actinomycetes was screened against clinically relevant pathogens, identifying seven isolates active against methicillin-resistant Staphylococcus aureus (MRSA), Enterococcus faecium, or both. All isolates were cultivated in Duetz system-based microfermentation plates, and cell-free culture filtrates were analyzed directly by UHPLC-HRMS/MS without solvent extraction. Compared with a conventional workflow, the approach reduced culture-medium consumption by approximately 99%, decreased recurring consumable costs by approximately 96%, and reduced the time required to obtain LC-MS-ready samples from typically 5 days to approximately 20 min. Integration of antibacterial, metabolomic, and genomic datasets supported the annotation of diverse metabolite families, including actinomycins, polyether ionophores, ansamycins, angucyclines, hydroxamate siderophores, ectoines, tambromycin-associated metabolites, and putative lydicamycin metabolites. Selected annotations were validated using commercial standards. Overall, this study demonstrates that integrating miniaturized Duetz system-based cultivation, extraction-free metabolomics, antibacterial screening, and genome mining provides a scalable, robust, and cost-effective platform for high-throughput strain prioritization and natural product discovery.

microbiology↗

bioRxiv2021

Integrated metabolomic, molecular networking and genome mining analyses uncover novel angucyclines from Streptomyces sp. RO-S4 isolated from Bejaia Bay, Algeria

Multi-omic approaches have recently made big strides towards the effective exploration of microorganisms and accelerating the discovery of new bioactive compounds. We combined metabolomic, molecular networking, and genomic-based approaches to investigate the metabolic potential of the Streptomyces sp. RO-S4 strain isolated from the polluted waters of Bejaia Bay in Algeria. Antagonistic assays against methicillin-resistant Staphylococcus aureus with RO-S4 organic extracts showed an inhibition zone of 20 mm by the agar diffusion method, and its minimum inhibitory concentration was 16 g/mL. A molecular network was created using GNPS and annotated through the comparison of MS/MS spectra against several databases. The predominant compounds in the RO-S4 extract belonged to the angucyclines family. Three compounds were annotated as known metabolites, while all the others were putatively new to Science. Notably, all compounds had fridamycin-like aglycones, and several of them had a lactonized D ring analogous to that of urdamycin L. The whole genome of Streptomyces RO-S4 was sequenced to identify the biosynthetic gene cluster (BGC) encoding for these angucyclines, which yielded a draft genome of 7,497,846 bp with 72.4% G+C content. Subsequently, a genome mining analysis revealed 19 putative biosynthetic gene clusters, including a grincamycin-like BGC with a high similarity to that of Streptomyces sp. CZN-748 previously reported to also produce mostly open fridamycin-like aglycones. As the ring-opening process leading to these compounds is still not defined, we performed comparative analysis with other angucycline BGCs and advanced some hypotheses to explain the ring-opening and lactone formation, possibly linked to the uncoupling between the activity of GcnE and GcnM homologues in the RO-S4 strain. The combination of metabolomic and genomic approaches greatly improved the interpretation of the metabolic potential of the RO-S4 strain.

microbiology↗