The rise of multidrug-resistant pathogens, such as Staphylococcus aureus and Mycobacterium tuberculosis, underscores an urgent need for therapeutic innovation. The antibiotic development pipeline targeting these bacteria is critically limited, with most discovered candidates exhibiting structurally similar features of prominent chemical entities and with well-established molecular targets or binding modes. The myxobacterial α-pyrone antibiotics, myxopyronins, represent a highly promising compound class due to their ability to inhibit RNA polymerase by binding to the "switch region", a distinct binding site to that of standard-of-care antibiotics. Mutasynthesis, leveraging engineered microorganisms and tailored precursors, provides a viable alternative to total synthesis for generating novel derivatives. This study utilized a heterologous expression system in Myxococcus xanthus DK1622 to generate analogs. Two carrier protein domain mutants were engineered to facilitate mutasynthesis-based production of structurally diverse derivatives. A trifluoromethyl-modified analog, once accessible only through total synthesis but now obtained via mutasynthesis, exhibits potent antimicrobial activity against Gram-positive pathogens including Mycobacterium tuberculosis and favorable in vitro absorption, distribution, metabolism, excretion and toxicity properties. These findings highlight a promising pathway for developing optimized α-pyrone antibiotics to address the global antimicrobial-resistance crisis.

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.

DNA polymerase III sliding clamp (DnaN) was recently validated as a new anti-tuberculosis target employing griselimycins. Three (2 S ,4 R )−4-methylproline moieties of methylgriselimycin play significant roles in target binding and metabolic stability. Here, we identify the mycoplanecin biosynthetic gene cluster by genome mining using bait genes from the 4-methylproline pathway. We isolate and structurally elucidate four mycoplanecins comprising scarce homo-amino acids and 4-alkylprolines. Evaluating mycoplanecin E against Mycobacterium tuberculosis surprisingly reveals an excitingly low minimum inhibition concentration at 83 ng/mL, thus outcompeting griselimycin by approximately 24-fold. We show that mycoplanecins bind DnaN with nanomolar affinity and provide a co-crystal structure of mycoplanecin A-bound DnaN. Additionally, we reconstitute the biosyntheses of the unusual l -homoleucine, l -homonorleucine, and (2 S ,4 R )−4-ethylproline building blocks by characterizing in vitro the full set of eight enzymes involved. The biosynthetic study, bioactivity evaluation, and drug target validation of mycoplanecins pave the way for their further development to tackle multidrug-resistant mycobacterial infections.

Supercritical fluid extraction (SFE) is widely used for the isolation of natural products from plants, but its application in efforts to identify structurally and physicochemically often dissimilar microbial natural products is limited to date. In this study we evaluated the impact of SFE on the extractability of myxobacterial secondary metabolites aiming to improve the prospects of discovering novel natural products. We investigated the influence of different co-solvents on the extraction efficiency of secondary metabolites from three myxobacterial strains as well as the antimicrobial activity profiles of the corresponding extracts. For each known secondary metabolite we found extraction conditions using SFE leading to superior yields in the extracts compared to conventional solvent extraction. Compounds with a logP higher than 3 showed best extraction efficiency using 20% EtOAc as a co-solvent, whereas compounds with logP values lower than 3 were better extractable using more polar co-solvents like MeOH. Extracts generated with SFE showed increased antimicrobial activities including the presence of activities not explained by known myxobacterial secondary metabolites, highlighting the advantage of SFE for bioactivity-guided isolation. Moreover, non-targeted metabolomics analysis revealed a group of chlorinated metabolites produced by the well-studied model myxobacterium Myxococcus xanthus DK1622 which were not accessible previously due to their low concentration in conventional extracts. The enriched SF extracts were used for isolation and subsequent structure elucidation of chloroxanthic acid A as founding member of a novel secondary metabolite family. Our findings encourage the increased utilization of SFE as part of future microbial natural products screening workflows.