Over recent decades, the pipeline of antibiotics acting against Gram‐negative bacteria is running dry, as most discovered candidate antibiotics suffer from insufficient potency, pharmacokinetic properties, or toxicity. The darobactins, a promising new small peptide class of drug candidates, bind to novel antibiotic target BamA, an outer membrane protein. Previously, we reported that biosynthetic engineering in a heterologous host generated novel darobactins with enhanced antibacterial activity. Here we utilize an optimized purification method and present cryo‐EM structures of the Bam complex with darobactin 9 (D9), which served as a blueprint for the biotechnological generation of twenty new darobactins including halogenated analogs. The newly engineered darobactin 22 binds more tightly to BamA and outperforms the favorable activity profile of D9 against clinically relevant pathogens such as carbapenem‐resistant Acinetobacter baumannii up to 32‐fold, without observing toxic effects.

During our search for novel myxobacterial natural products, we discovered the thiamyxins: thiazole‐ and thiazoline‐rich non‐ribosomal peptide‐polyketide hybrids with potent antiviral activity. We isolated four congeners of this unprecedented natural product family with the non‐cyclized thiamyxin D fused to a glycerol unit at the C‐terminus. Alongside their structure elucidation, we present a concise biosynthesis model based on biosynthetic gene cluster analysis and isotopically labelled precursor feeding. We report incorporation of a 2‐(hydroxymethyl)‐4‐methylpent‐3‐enoic acid moiety by a GCN5‐related N‐acetyltransferase‐like decarboxylase domain featuring polyketide synthase. The thiamyxins show potent inhibition of RNA viruses in cell culture models of corona, zika and dengue virus infection. Their potency up to a half maximal inhibitory concentration of 560 nM combined with milder cytotoxic effects on human cell lines indicate the potential for further development of the thiamyxins.

Actinobacteria, the bacterial phylum most renowned for natural product discovery, has been established as a valuable source for drug discovery and biotechnology but is underrepresented within accessible genome and strain collections. Herein, we introduce the Natural Products Discovery Center (NPDC), featuring 122,449 strains assembled over eight decades, the genomes of the first 8490 NPDC strains (7142 Actinobacteria), and the online NPDC Portal making both strains and genomes publicly available. A comparative survey of RefSeq and NPDC Actinobacteria highlights the taxonomic and biosynthetic diversity within the NPDC collection, including three new genera, hundreds of new species, and ∼7000 new gene cluster families. Selected examples demonstrate how the NPDC Portal’s strain metadata, genomes, and biosynthetic gene clusters can be leveraged using genome mining approaches. Our findings underscore the ongoing significance of Actinobacteria in natural product discovery, and the NPDC serves as an unparalleled resource for both Actinobacteria strains and genomes.

Structure elucidation and total synthesis of five unprecedented terpenoid‐alkaloids, the sandacrabins, are reported, alongside with the first description of their producing organism Sandaracinus defensii MSr10575, which expands the Sandaracineae family by only its second member. The genome sequence of S. defensii as presented in this study was utilized to identify enzymes responsible for sandacrabin formation, whereby dimethylbenzimidazol, deriving from cobalamin biosynthesis, was identified as key intermediate. Biological activity profiling revealed that all sandacrabins except congener A exhibit potent antiviral activity against the human pathogenic coronavirus HCoV229E in the three digit nanomolar range. Investigation of the underlying mode of action discloses that the sandacrabins inhibit the SARS‐CoV‐2 RNA‐dependent RNA polymerase complex, highlighting them as structurally distinct non‐nucleoside RNA synthesis inhibitors. The observed segregation between cell toxicity at higher concentrations and viral inhibition opens the possibility for their medicinal chemistry optimization towards selective inhibitors.

Herein, we describe a new plasmid found in Sandaracinus sp. MSr10575 named pSa001 spanning 209.7 kbp that harbors a cryptic secondary metabolite biosynthesis gene cluster (BGC). Activation of this BGC by homologous‐recombination‐mediated exchange of the native promoter sequence against a vanillate inducible system led to the production and subsequent isolation and structure elucidation of novel secondary metabolites, the sandarazols A–G. The sandarazols contain intriguing structural features and very reactive functional groups such as an α‐chlorinated ketone, an epoxyketone, and a (2R)‐2‐amino‐3‐(N,N‐dimethylamino)‐propionic acid building block. In‐depth investigation of the underlying biosynthetic machinery led to a concise biosynthetic model for the new compound family, including several uncommon biosynthetic steps. The chlorinated congener sandarazol C shows an IC₅₀ value of 0.5 μm against HCT 116 cells and a MIC of 14 μm against Mycobacterium smegmatis, which points at the sandarazols’ potential function as defensive secondary metabolites or toxins.

Genomic information from various magnetotactic bacteria suggested that besides their common ability to form magnetosomes, they potentially also represent a source of bioactive natural products. By using targeted deletion and transcriptional activation, we connected a large biosynthetic gene cluster (BGC) of the trans-acyltransferase polyketide synthase (trans-AT PKS) type to the biosynthesis of a novel polyketide in the alphaproteobacterium Magnetospirillum gryphiswaldense. Structure elucidation by mass spectrometry and nuclear magnetic resonance spectroscopy (NMR) revealed that this secondary metabolite resembles sesbanimides, which were very recently reported from other taxa. However, sesbanimide R exhibits an additional arginine moiety the presence of which reconciles inconsistencies in the previously proposed sesbanimide biosynthesis pathway observed when comparing the chemical structure and the potential biochemistry encoded in the BGC. In contrast to the case with sesbanimides D, E, and F, we were able to assign the stereocenter of the arginine moiety experimentally and two of the remaining three stereocenters by predictive biosynthetic tools. Sesbanimide R displayed strong cytotoxic activity against several carcinoma cell lines. IMPORTANCE The findings of this study contribute a new secondary metabolite member to the glutarimide-containing polyketides. The determined structure of sesbanimide R correlates with its cytotoxic bioactivity, characteristic for members of this family. Sesbanimide R represents the first natural product isolated from magnetotactic bacteria and identifies this highly diverse group as a so-far-untapped source for the future discovery of novel secondary metabolites.