Exploring a general multi-pronged activation strategy for natural product discovery in Actinomycetes.

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.

Natural Products from Singapore Soil-Derived Streptomycetaceae Family and Evaluation of Their Biological Activities.

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.

Cost-effective hybrid long-short read assembly delineates alternative GC-rich Streptomyces hosts for natural product discovery.

With the advent of rapid automated in silico identification of biosynthetic gene clusters (BGCs), genomics presents vast opportunities to accelerate natural product (NP) discovery. However, prolific NP producers, Streptomyces, are exceptionally GC-rich (>80%) and highly repetitive within BGCs. These pose challenges in sequencing and high-quality genome assembly which are currently circumvented via intensive sequencing. Here, we outline a more cost-effective workflow using multiplex Illumina and Oxford Nanopore sequencing with hybrid long-short read assembly algorithms to generate high quality genomes. Our protocol involves subjecting long read-derived assemblies to up to 4 rounds of polishing with short reads to yield accurate BGC predictions. We successfully sequenced and assembled 8 GC-rich Streptomyces genomes whose lengths range from 7.1 to 12.1 Mb with a median N50 of 8.2 Mb. Taxonomic analysis revealed previous misrepresentation among these strains and allowed us to propose a potentially new species, Streptomyces sydneybrenneri. Further comprehensive characterization of their biosynthetic, pan-genomic and antibiotic resistance features especially for molecules derived from type I polyketide synthase (PKS) BGCs reflected their potential as alternative NP hosts. Thus, the genome assemblies and insights presented here are envisioned to serve as gateway for the scientific community to expand their avenues in NP discovery.