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.

The bioactivity of vancomycin is enabled by three aromatic crosslinks, the biosynthesis of which has been an active area of investigation for two decades. Two cytochrome P450 enzymes, OxyB and OxyA, have been shown to introduce bisaryl ether linkages with the help of a so-called X-domain. The final crosslink, however, a biaryl bond thought to be installed by OxyC, has remained elusive. We report the in vitro reconstitution of the OxyC reaction and formation of the first carbon-carbon crosslink in any glycopeptide antibiotic. Using a cascade sequence, in which the peptide substrate was incubated with the Oxy enzymes in turn, we completed the chemoenzymatic synthesis of a vancomycin aglycone variant. This approach was also used to generate a new analogue carrying a thioamide linkage at residue 4, a precursor to the amidine derivative, which is effective against vancomycin-resistant pathogens. Our results set the stage for creating therapeutic vancomycin derivatives by using the native metalloenzymes.

Studies on the biosynthesis of glycopeptide antibiotics have provided many insights into the strategies that Nature employs to build architecturally strained molecules. A key structural feature of vancomycin, the founding member of this class, is a set of three aromatic cross-links that are introduced via yet unknown mechanisms. Previous reports have identified three cytochrome P450 enzymes involved in this process and demonstrated enzymatic activity for OxyB, which installs the first aromatic cross-link. However, the activities of the remaining two P450 enzymes have not been recapitulated. Herein, we show that OxyA generates the second bis-aryl ether bond in vancomycin and that it exhibits strict substrate specificity toward the chlorinated, OxyB-cross-linked product. No OxyA product is detected with the unchlorinated substrate. Together with previous results, these data suggest that chlorination occurs after OxyB- but before OxyA-catalyzed cross-link formation. Our results have important implications for the chemo-enzymatic synthesis of vancomycin and its analogs.

Streptide is a ribosomally synthesized and post-translationally modified peptide with a unique cyclization motif consisting of an intramolecular lysine-tryptophan cross-link. Three radical S-adenosylmethionine enzymes, StrB, AgaB, and SuiB from different species of Streptococcus, have been shown to install this modification onto their respective precursor peptides in a leader-dependent fashion. Herein, we conduct detailed investigations to differentiate among several plausible mechanistic proposals, specifically addressing radical versus electrophilic addition to the indole during cross-link formation, the role of substrate side chains in binding in the enzyme active site, and the identity of the catalytic base in the reaction cycle. Our results are consistent with a radical electrophilic aromatic substitution mechanism for the key carbon-carbon bond-forming step. They also elaborate on other mechanistic features that underpin this unique and synthetically challenging post-translational modification.

We report the largest actinomycete genome to date, which encodes >30 secondary metabolites, including the kistamicin biosynthetic gene cluster.

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Microbes are phylogenetically and metabolically diverse. Yet capturing this diversity, assigning functions to host organisms and exploring the biosynthetic potential in natural environments remains challenging. We reconstructed >25,000 draft genomes, including from >2,500 uncharacterized species, from globally-distributed ocean microbial communities, and combined them with ~10,000 genomes from cultivated and single cells. Mining this resource revealed ~40,000 putative biosynthetic gene clusters (BGCs), many from unknown phylogenetic groups. Among these, we discovered Candidatus Eudoremicrobiaceae as one of the most biosynthetically diverse microbes detected to date. Discrete transcriptional states structuring natural populations were associated with a potentially niche-partitioning role for BGC products. Together with the characterization of the first Eudoremicrobiaceae natural product, this study demonstrates how microbiomics enables prospecting for candidate bioactive compounds in underexplored microbes and environments.

Vancomycin is one of the most important clinical antibiotics in the fight against infectious disease. Its biological activity relies on three aromatic cross-links, which create a cup-shaped topology and allow tight binding to nascent peptidoglycan chains. The cytochrome P450 enzymes OxyB, OxyA, and OxyC have been shown to introduce these synthetically challenging aromatic linkages. The ability to utilize the P450 enzymes in a chemo-enzymatic scheme to generate vancomycin derivatives is appealing but requires a thorough understanding of their reactivities and mechanisms. Herein, we systematically explore the scope of OxyB biocatalysis and report installation of diverse diaryl ether and biaryl cross-links with varying macrocycle sizes and compositions, when the enzyme is presented with modified vancomycin precursor peptides. The structures of the resulting products were determined using one-dimensional/two-dimensional nuclear magnetic resonance spectroscopy, high-resolution mass spectrometry (HR-MS), tandem HR-MS, and isotopic labeling, as well as ultraviolet–visible light absorption and fluorescence emission spectroscopies. An exploration of the biological activities of these alternative OxyB products surprisingly revealed antifungal properties. Taking advantage of the promiscuity of OxyB, we chemo-enzymatically generated a vancomycin aglycone variant containing an expanded macrocycle. Mechanistic implications for OxyB and future directions for creating vancomycin analogue libraries are discussed.