Natural microbial communities are phylogenetically and metabolically diverse. In addition to underexplored organismal groups1, this diversity encompasses a rich discovery potential for ecologically and biotechnologically relevant enzymes and biochemical compounds2,3. However, studying this diversity to identify genomic pathways for the synthesis of such compounds4 and assigning them to their respective hosts remains challenging. The biosynthetic potential of microorganisms in the open ocean remains largely uncharted owing to limitations in the analysis of genome-resolved data at the global scale. Here we investigated the diversity and novelty of biosynthetic gene clusters in the ocean by integrating around 10,000 microbial genomes from cultivated and single cells with more than 25,000 newly reconstructed draft genomes from more than 1,000 seawater samples. These efforts revealed approximately 40,000 putative mostly new biosynthetic gene clusters, several of which were found in previously unsuspected phylogenetic groups. Among these groups, we identified a lineage rich in biosynthetic gene clusters (‘Candidatus Eudoremicrobiaceae’) that belongs to an uncultivated bacterial phylum and includes some of the most biosynthetically diverse microorganisms in this environment. From these, we characterized the phospeptin and pythonamide pathways, revealing cases of unusual bioactive compound structure and enzymology, respectively. Together, this research demonstrates how microbiomics-driven strategies can enable the investigation of previously undescribed enzymes and natural products in underexplored microbial groups and environments.

Chorismatases are a class of chorismate-converting enzymes involved in the biosynthetic pathways of different natural products, many of them with interesting pharmaceutical characteristics. So far, three subfamilies of chorismatases are described that convert chorismate into different (dihydro-)benzoate derivatives (CH-FkbO, CH-Hyg5, and CH-XanB2). Until now, the detailed enzyme mechanism and the molecular basis for the different reaction products were unknown. Here we show that the CH-FkbO and CH-Hyg5 subfamilies share the same protein fold, but employ fundamentally different reaction mechanisms. While the FkbO reaction is a typical hydrolysis, the Hyg5 reaction proceeds intramolecularly, most likely via an arene oxide intermediate. Two nonconserved active site residues were identified that are responsible for the different reaction mechanisms in CH-FkbO and CH-Hyg5. Further, we propose an additional amino acid residue to be responsible for the discrimination of the CH-XanB2 subfamily, which catalyzes the formation of two different hydroxybenzoate regioisomers, likely in a single active site. A multiple sequence alignment shows that these three crucial amino acid positions are located in conserved motifs and can therefore be used to assign unknown chorismatases to the corresponding subfamily.

Lipopeptides represent a large group of microbial natural products that include important antibacterial and antifungal drugs and some of the most-powerful known biosurfactants. The vast majority of lipopeptides comprise cyclic peptide backbones N-terminally equipped with various fatty acyl moieties. The known compounds of this type are biosynthesized by nonribosomal peptide synthetases, giant enzyme complexes that assemble their products in a non–gene-encoded manner. Here, we report the genome-guided discovery of ribosomally derived, fatty-acylated lipopeptides, termed selidamides. Heterologous reconstitution of three pathways, two from cyanobacteria and one from an arctic, ocean-derived alphaproteobacterium, allowed structural characterization of the probable natural products and suggest that selidamides are widespread over various bacterial phyla. The identified representatives feature cyclic peptide moieties and fatty acyl units attached to (hydroxy)ornithine or lysine side chains by maturases of the GCN5-related N-acetyltransferase superfamily. In contrast to nonribosomal lipopeptides that are usually produced as congener mixtures, the three selidamides are selectively fatty acylated with C10, C12, or C16 fatty acids, respectively. These results highlight the ability of ribosomal pathways to emulate products with diverse, nonribosomal-like features and add to the biocatalytic toolbox for peptide drug improvement and targeted discovery.

Chorismate-converting enzymes are involved in many biosynthetic pathways leading to natural products and can often be used as tools for the synthesis of chemical building blocks. Chorismatases such as FkbO from Streptomyces species catalyse the hydrolysis of chorismate yielding (dihydro)benzoic acid derivatives. In contrast to many other chorismate-converting enzymes, the structure and catalytic mechanism of a chorismatase had not been previously elucidated. Here we present the crystal structure of the chorismatase FkbO in complex with a competitive inhibitor at 1.08Å resolution. FkbO is a monomer in solution and exhibits pseudo-3-fold symmetry; the structure of the individual domains indicates a possible connection to the trimeric RidA/YjgF family and related enzymes. The co-crystallised inhibitor led to the identification of FkbO's active site in the cleft between the central and the C-terminal domains. A mechanism for FkbO is proposed based on both interactions between the inhibitor and the surrounding amino acids and an FkbO structure with chorismate modelled in the active site. We suggest that the methylene group of the chorismate enol ether takes up a proton from an active-site glutamic acid residue, thereby initiating chorismate hydrolysis. A similar chemistry has been described for isochorismatases, albeit implemented in an entirely different protein scaffold. This reaction model is supported by kinetic data from active-site variants of FkbO derived by site-directed mutagenesis.