World Health Organization (WHO) has declared antimicrobial resistance as one of the top 10 global public health threats facing humanity. Thus, there is an urgent need to restock our dwindling drug pipeline to combat rising antimicrobial resistance. Natural products (NPs) have been an unparalleled source of bioactive compounds with numerous success stories, offering compounds with highly desirable qualities: exploiting novel targets and/or possessing unique chemical scaffolds. This proposal aims to deliver bioactive NPs by: 1. Deciphering NP biosynthesis: Non-ribosomal peptide synthetases (NRPSs) are megaenzymes with many moving parts and reactions centres producing a plethora of bioactive NPs. Despite being extensively studied NRPS gatekeeping function, substrate transfer and reactive intermediate chaperoning are unclear. To fill these gaps in our understanding we plan to structurally characterise NRPS in action in their native subcellular environment using cryogenic electron tomography (Cryo-ET) - a technique that to date has not been used for NRPS structural studies. This coupled with deep learning framework will allow us to visualize how structural heterogeneity effects domain-domain interactions in situ, while reactive intermediates are channelled across the assembly line with high catalytic efficiency and precision. The in-depth mechanistic understanding would catapult NRPS reengineering efforts to generate peptides with new or improved biological activities. 2. Isolation of novel NP: Myxobacteria are well-established factories of NPs, including ribosomally synthesized and post-translationally modified peptides (RiPPs). We plan to exploit as yet under-explored myxobacterial RiPP potential by integrating genome-mining tools with streamlined biosynthetic pathway refactoring and heterologous expression of RiPPs in a semi high-throughput fashion. Subsequently, the isolated NPs and the bioengineered derivatives would be evaluated for antimicrobial activity.