Amay Ajaykumar Agrawal

Polycyclic bacterial specialized metabolites such as anthraquinones, angucyclines, and tetracyclines are predominantly produced by type II polyketide synthase (PKS) systems and represent an important source of antibiotics and anticancer agents. While type II PKS pathways are well characterized in Gram-positive bacteria, their biosynthetic potential in Gram-negative bacteria remains largely unexplored. Here, we report the discovery and activation of a cryptic type II PKS biosynthetic gene cluster from the myxobacterium Aggregicoccus edonensis MCy10622. Using a rapid PCR-based cloning and promoter-exchange strategy, the compact gene cluster was heterologously expressed in Myxococcus xanthus DK1622, leading to the production of two previously unknown specialized metabolites, aggregicyclin and oxyaggregicyclin. Structural analysis revealed an unusual polycyclic scaffold featuring a wide-spanning biaryl ether linkage. Biosynthetic analysis supports an early diversification event during first-ring cyclization as the basis for product divergence. Aggregicyclin exhibited antibacterial activity against Staphylococcus aureus and cytotoxicity against human cancer cells. Together, these findings expand the current knowledge of type II polyketide biosynthesis in Gram-negative bacteria.

Natural products remain vital sources of therapeutics, particularly anti-infectives, and members of the phylum Myxococcota constitute an especially rich reservoir for their discovery. Based on decades of microbiological efforts, we present 154 new Myxococcota genomes and propose a revised taxonomy expanding the number of described families from 11 to 28 and genera from 32 to 90. Comparison with an equivalent set from the prime source Actinomycetota shows that Myxococcota possess a comparable biosynthetic diversity, underscoring their promise for large-scale isolation and sequencing efforts. The vast untapped potential reflected in 2,387 uncharacterized gene cluster families is highlighted by genome mining efforts, yielding four validated compounds exhibiting novel chemistry, including myxolutamids and myxopentacins. We show that many Myxococcota-derived natural products, such as myxolutamid A and two new sorangicin derivatives, are conserved within taxonomic lineages. New described families thus bear high biosynthetic potential underpinning the importance of precise taxonomic classification guiding targeted drug discovery.

Microbial natural products represent a chemically diverse repertoire of small molecules with major pharmaceutical potential. Despite the increasing availability of microbial genome sequences, large-scale natural product discovery remains challenging because the existing genome mining approaches lack integrated workflows for rapid dereplication of known compounds and prioritization of novel candidates, forcing researchers to rely on multiple tools that requires extensive manual curation and expert intervention at each step. To address these limitations, we introduce LoReMINE ( Lo ng Re ad-based Microbial genome min ing pipelin e ), a fully automated end-to-end pipeline that generates high-quality assemblies, performs taxonomic classification, predicts biosynthetic gene clusters (BGCs) responsible for biosynthesis of natural products, and clusters them into gene cluster families (GCFs) directly from long-read sequencing data. By integrating state-of-the-art tools into a seamless pipeline, LoReMINE enables scalable, reproducible, and comprehensive genome mining across diverse microbial taxa. The pipeline is openly available at https://github.com/kalininalab/LoReMINE and can be installed via Conda ( https://anaconda.org/kalininalab/loremine ), facilitating broad adoption by the natural product research community. Author summary: For decades, microbial natural products have been a major source of medicines, with most of the clinically used antibiotics being their derivatives. Recent advances in DNA sequencing technologies now allow the reconstruction of more complete and continuous microbial genomes, revealing a vast and largely untapped diversity of biosynthetic gene clusters responsible for natural product biosynthesis. Despite these advances, large-scale natural product discovery remains difficult because current genome mining approaches rely on many separate tools and lack an integrated workflow to dereplicate known compounds and prioritize novel biosynthetic pathways. To address these limitations, we introduce LoReMINE, an automated pipeline designed to simplify microbial genome mining directly from long-read sequencing data. LoReMINE integrates genome assembly, taxonomic classification, identification of biosynthetic gene clusters, and their clustering into gene cluster families within a single, reproducible workflow. This streamlined approach enables scalable analysis across diverse microbial taxa and facilitates comprehensive exploration of microbial biosynthetic potential. The pipeline is designed for both experimental and computational researchers, helping to advance natural product research and contribute towards the discovery of new therapeutic drugs.

The transmission of antibiotic-resistance genes, comprising mobilization and relocation events, orchestrates the dissemination of antimicrobial resistance. Inspired by this evolutionarily successful paradigm, we developed ACTIMOT, a CRISPR-Cas9–based approach to unlock the vast chemical diversity concealed within bacterial genomes. ACTIMOT enables the efficient mobilization and relocation of large DNA fragments from the chromosome to replicative plasmids within the same bacterial cell. ACTIMOT circumvents the limitations of traditional molecular cloning methods involving handling and replicating large pieces of genomic DNA. Using ACTIMOT, we mobilized and activated four cryptic biosynthetic gene clusters from Streptomyces , leading to the discovery of 39 compounds across four distinct classes. This work highlights the potential of ACTIMOT for accelerating the exploration of biosynthetic pathways and the discovery of natural products.

The degeneration of axon terminals before the soma, referred to as “dying back”, is a feature of Parkinson’s disease (PD). Axonal assays are needed to model early PD pathogenesis as well as identify protective therapeutics. We hypothesized that defects in axon lysosomal trafficking as well as injury repair might be important contributing factors to “dying back” pathology in PD. Since primary human PD neurons are inaccessible, we developed assays to quantify axonal trafficking and injury repair using induced pluripotent stem cell (iPSC)-derived neurons with LRRK2 G2019S, which is one of the most common known PD mutations, and isogenic controls. We observed a subtle axonal trafficking phenotype that was partially rescued by a LRRK2 inhibitor. Mutant LRRK2 neurons showed increased phosphorylated Rab10-positive lysosomes, and lysosomal membrane damage increased LRRK2-dependent Rab10 phosphorylation. Neurons with mutant LRRK2 showed a transient increase in lysosomes at axotomy injury sites. This was a pilot study that used two patient-derived lines to develop its methodology; we observed subtle phenotypes that might correlate with heterogeneity in LRRK2-PD patients. Further analysis using additional iPSC lines is needed. Therefore, our axonal lysosomal assays can potentially be used to characterize early PD pathogenesis and test possible therapeutics.