Natural products possess significant therapeutic potential but remain underutilized despite advances in genomics and bioinformatics. While there are approaches to activate and upregulate natural product biosynthesis in both native and heterologous microbial strains, a comprehensive strategy to elicit production of natural products as well as a generalizable and efficient method to interrogate diverse native strains collection, remains lacking. Here, we explore a flexible and robust integrase-mediated multi-pronged activation approach to reliably perturb and globally trigger antibiotics production in actinobacteria. Across 54 actinobacterial strains, our approach yielded 124 distinct activator-strain combinations which consistently outperform wild type. Our approach expands accessible metabolite space by nearly two-fold and increases selected metabolite yields by up to >200-fold, enabling discovery of Gram-negative bioactivity in tetramic acid analogs. We envision these findings as a gateway towards a more streamlined, accelerated, and scalable strategy to unlock the full potential of Nature’s chemical repertoire.

In recent years, CRISPR-Cas toolboxes for Streptomyces editing have rapidly accelerated natural product discovery and engineering. However, Cas efficiencies are oftentimes strain-dependent, and the commonly used Streptococcus pyogenes Cas9 (SpCas9) is notorious for having high levels of off-target toxicity effects. Thus, a variety of Cas proteins is required for greater flexibility of genetic manipulation within a wider range of Streptomyces strains. This study explored the first use of Acidaminococcus sp. Cas12j, a hypercompact Cas12 subfamily, for genome editing in Streptomyces and its potential in activating silent biosynthetic gene clusters (BGCs) to enhance natural product synthesis. While the editing efficiencies of Cas12j were not as high as previously reported efficiencies of Cas12a and Cas9, Cas12j exhibited higher transformation efficiencies compared to SpCas9. Furthermore, Cas12j demonstrated significantly improved editing efficiencies compared to Cas12a in activating BGCs in Streptomyces sp. A34053, a strain wherein both SpCas9 and Cas12a faced limitations in accessing the genome. Overall, this study expanded the repertoire of Cas proteins for genome editing in actinomycetes and highlighted not only the potential of recently characterized Cas12j in Streptomyces but also the importance of having an extensive genetic toolbox for improving the editing success of these beneficial microbes.

Large scale cultivation and chemical investigation of an extract obtained from Actimonadura sp. resulted in the identification of six previously undescribed spirotetronates (pyrrolosporin B and decatromicins C–G; 7–12), along with six known congeners, namely decatromicins A–B (1–2), BE-45722B–D (3–5), and pyrrolosporin A (6). The chemical structures of compounds 1–12 were characterized via comparison with previously reported data and analysis of 1D/2D NMR and MS data. The structures of all new compounds were highly related to the spirotetronate type compounds, decatromicin and pyrrolosporin, with variations in the substituents on the pyrrole and aglycone moieties. All compounds were evaluated for antibacterial activity against the Gram-negative bacteria, Acinetobacter baumannii and Gram-positive bacteria, Staphylococcus aureus and were investigated for their cytotoxicity against the human cancer cell line A549. Of these, decatromicin B (2), BE-45722B (3), and pyrrolosporin B (7) exhibited potent antibacterial activities against both Gram-positive (MIC₉₀ between 1–3 μM) and Gram-negative bacteria (MIC₉₀ values ranging from 12–36 μM) with weak or no cytotoxic activity against A549 cells.

Molecular biodiversity results from branched metabolic pathways driven by enzymatic regioselectivities. An additional complexity occurs in metabolites with an internal structural symmetry, offering identical extremities to the enzymes. For example, in the terpene family, β-carotene presents two identical terminal closed-ring structures. Theses cycles can be hydroxylated by cytochrome P450s from the CYP97 family. Two sequential hydroxylations lead first to the formation of monohydroxylated β-cryptoxanthin and subsequently to that of dihydroxylated zeaxanthin. Among the CYP97 dihydroxylases, CYP97H1 from Euglena gracilis has been described as the only monohydroxylase. This study aims to determine which enzymatic domains are involved in this regioselectivity, conferring unique monohydroxylase activity on a substrate offering two identical sites for hydroxylation. We explored the effect of truncations, substitutions and domain swapping with other CYP97 members and found that CYP97H1 harbours a unique N-terminal globular domain. This CYP97H1 N-terminal domain harbours a hydrophobic patch at the entrance of the substrate channel, which is involved in the monohydroxylase activity of CYP97H1. This domain, at the surface of the enzyme, highlights the role of distal and non-catalytic domains in regulating enzyme specificity.

Natural products have long been used as a source of antimicrobial agents against various microorganisms. Actinobacteria are a group of bacteria best known to produce a wide variety of bioactive secondary metabolites, including many antimicrobial agents. In this study, four actinobacterial strains found in Singapore terrestrial soil were investigated as potential sources of new antimicrobial compounds. Large-scale cultivation, chemical, and biological investigation led to the isolation of a previously undescribed tetronomycin A (1) that demonstrated inhibitory activities against both Gram-positive bacteria Staphylococcus aureus (SA) and methicillin-resistant Staphylococcus aureus (MRSA) (i.e., MIC₉₀ of 2–4 μM and MBC₉₀ of 9–12 μM), and several known antimicrobial compounds, namely nonactin, monactin, dinactin, 4E-deacetylchromomycin A3, chromomycin A2, soyasaponin II, lysolipin I, tetronomycin, and naphthomevalin. Tetronomycin showed a two- to six-fold increase in antibacterial activity (i.e., MIC₉₀ and MBC₉₀ of 1–2 μM) as compared to tetronomycin A (1), indicating the presence of an oxy-methyl group at the C-27 position is important for antibacterial activity.

Natural products are a family of diverse compounds with multiple impactful applications, especially in therapeutics. Recent advances in genomics and bioinformatics have also hinted at vast untapped chemical potential within Nature. However, despite the many strategies available for activation and upregulation of natural product biosyntheses in native and heterologous microbial strains, there is yet to be a generalizable and efficient approach for interrogating diverse native strain collections. Here, we describe and demonstrate a flexible and robust one-step integrase-mediated genetic- and cultivation-based approach to perturb and activate antibiotics production in a set of 54 actinobacterial strains. Our multi-pronged strategy significantly increases accessible metabolite space by two-fold, resulting in the discovery of the first example of Gram-negative bioactivity in new tetramic acid analogs. We envision these results to serve as the first step toward a more streamlined, accelerated, and scalable strategy to unlock the full potential of Nature’s chemical repertoire.