Bacterial natural products and their analogues constitute more than half of the new small molecule drugs developed over the last several decades. Despite this success, interest in natural products from major pharmaceutical companies has decreased even as genomics has uncovered the large number of biosynthetic gene clusters (BGCs) that encode for novel natural products. To date though, there is still a lack of universal strategies and enabling technologies to discover natural products at scale and speed. This review highlights several of the opportunities provided by genome sequencing and bioinformatics, challenges associated with translating genomes into natural products, and examples of successful strain prioritization and BGC activation strategies that have been used in the genomic era for natural product discovery from cultivatable bacteria.

Nonheme diiron monooxygenases make up a rapidly growing family of oxygenases that are rarely identified in secondary metabolism. Herein, we report the in vivo, in vitro, and structural characterizations of a nonheme diiron monooxygenase, PtmU3, that installs a C-5 β-hydroxyl group in the unified biosynthesis of platensimycin and platencin, two highly functionalized diterpenoids that act as potent and selective inhibitors of bacterial and mammalian fatty acid synthases. This hydroxylation sets the stage for the subsequent A-ring cleavage step key to the unique diterpene-derived scaffolds of platensimycin and platencin. PtmU3 adopts an unprecedented triosephosphate isomerase (TIM)-barrel structural fold for this class of enzymes and possesses a noncanonical diiron active site architecture with a saturated six-coordinate iron center lacking a μ-oxo bridge. This study reveals the first member of a previously unidentified superfamily of TIM-barrel fold enzymes for metal-dependent dioxygen activation, with the majority predicted to act on CoA-linked substrates, thus expanding our knowledge of nature's repertoire of nonheme diiron monooxygenases and TIM-barrel fold enzymes.

Platensimycin (PTM) and platencin (PTN) are highly functionalized bacterial diterpenoids of ent-kauranol and ent-atiserene biosynthetic origin. C7 oxidation in the B-ring plays a key biosynthetic role in generating structural complexity known for ent-kaurane and ent-atisane derived diterpenoids. While all three oxidation patterns, α-hydroxyl, β-hydroxyl, and ketone, at C7 are seen in both the ent-kaurane and ent-atisane derived diterpenoids, their biosynthetic origins remain largely unknown. We previously established that PTM and PTN are produced by a single biosynthetic machinery, featuring cryptic C7 oxidations at the B-rings that transform the ent-kauranol and ent-atiserene derived precursors into the characteristic PTM and PTN scaffolds. Here, we report a three-enzyme cascade affording C7 α-hydroxylation in PTM and PTN biosynthesis. Combining in vitro and in vivo studies, we show that PtmO3 and PtmO6 are two functionally redundant α-ketoglutarate-dependent dioxygenases that generate a cryptic C7 β-hydroxyl on each of the ent-kauranol and ent-atiserene scaffolds and PtmO8 and PtmO1, a pair of NAD+/NADPH-dependent dehydrogenases, subsequently work in concert to invert the C7 β-hydroxyl to α-hydroxyl via a C7 ketone intermediate. PtmO3 and PtmO6 represent the first dedicated C7 β-hydroxylases characterized to date, and, together with PtmO8 and PtmO1, provide an account for the biosynthetic origins of all three C7 oxidation patterns that may shed light on other B-ring modifications in bacterial, plant, and fungal diterpenoid biosynthesis. Given their unprecedented activities in C7 oxidations, PtmO3, PtmO6, PtmO8, and PtmO1 enrich the growing toolbox of novel enzymes that could be exploited as biocatalysts to rapidly access complex diterpenoid natural products.

Reversible NIR-active nanoparticle clusters with controlled size from 20 to 100 nm were assembled from 5 nm gold nanoparticles (Au NP), with either citrate (CIT) or various binary ligands on the surface, by tuning the electrostatic repulsion and the hydrogen bonding via pH. The nanoclusters were bound together by vdW forces between the cores and the hydrogen bonds between the surface ligands and dissociated to primary nanoparticles over a period of 20 days at pH 5 and at pH 7. When high levels of citrate ligands were used on the primary particle surfaces, the large particle spacings in the nanoclusters led to only modest NIR extinction. However, a NIR extinction (E) ratio of up to ∼0.4 was obtained for nanoclusters with binary ligand mixtures composed of citrate and either cysteine (CYS), glutathione (GSH), or thioctic acid zwitterion (TAZ) while maintaining full reversibility to primary particles. The optimum ligand ratio for both an E of ∼0.4 and full reversibility decreased with increasing length of the secondary ligand (1.5/1 for CYS/CIT, 0.75/1 for GSH/CIT, and 0.5/1 for TAZ/CIT) because a longer secondary ligand maintains a sufficient interparticle spacing required for dissociation more effectively. Interestingly, the zeta potential and the first-order rate constant for nanocluster dissociation were similar for all three systems at the optimum ligand ratios. After incubation in 10 mM GSH solution (intracellular concentration), only the TAZ/CIT primary nanoparticles were resistant to protein opsonization in 100% fetal bovine serum, as the bidentate binding and zwitterion tips of TAZ resisted GSH exchange and protein opsonization, respectively.

trans-Acyltransferase assembly lines possess enzymatic domains often not observed in their better characterized cisacyltransferase counterparts. Within this repertoire of largely unexplored biosynthetic machinery is a class of enzymes called the pyran synthases that catalyze the formation of five- and sixmembered cyclic ethers from diverse polyketide chains. The 1.55 Å resolution crystal structure of a pyran synthase domain excised from the ninth module of the sorangicin assembly line highlights the similarity of this enzyme to the ubiquitous dehydratase domain and provides insight into the mechanism of ring formation. Functional assays of point mutants reveal the central importance of the active site histidine that is shared with the dehydratases as well as the supporting role of a neighboring semiconserved asparagine.