Discovering new molecules and chemistry from natural product biosynthesis in Clostridia

About This Grant

PROJECT SUMMARY Antimicrobial resistance represents a critical global health threat, demanding new antibiotics with novel scaffolds and mechanisms of action. Anaerobic bacteria – particularly the Clostridia, which comprise up to 40% of the human gut microbiome – represent an underexplored reservoir of bioactive natural products. These organisms have evolved unusual biosynthetic machinery and remarkable oxygen-sensitive radical enzymes to synthesize structurally unique molecules with critical physiological functions. Yet, their biosynthetic potential remains largely untapped due to significant technical challenges in their genetic manipulation. This proposal outlines our comprehensive strategy to unlock Clostridial biosynthesis, combining complementary genomics-driven and activity-guided approaches. Topic 1 focuses on characterizing a unique family of cobalamin-dependent radical S-adenosylmethionine (B12-rSAM) enzymes fused with non-ribosomal peptide synthetase (NRPS) adenylation domains that we discovered through mining of Clostridial genomes. We hypothesize these enzymes catalyze the stereoselective methylation of the carbon backbone of the incoming amino acid as it is loaded onto the assembly line – a reaction with no precedent in NRPS biochemistry, that could ultimately be harnessed to improve the stability and activity of peptide-derived antibiotics. We will characterize these enzymes both biochemically through in vitro assays of purified proteins, and through in vivo gene cluster activation and product characterization in the native producers. Topic 2 targets discovery of novel antimicrobials from cellulolytic Clostridia, which appear from bioinformatic analysis to be particularly gifted antibiotic producers. We will express promising biosynthetic gene clusters in our newly developed Clostridial chassis strain, and activate cryptic pathways in native producers through hypothesis-driven elicitor screening. My group’s unique combination of expertise in Clostridial metabolic engineering, oxygen-sensitive enzyme biochemistry, and development of state- of-the-art genetic tools positions us to overcome longstanding barriers in anaerobic natural products research. Success will yield new antimicrobial compounds and reveal novel biosynthetic chemistry, ultimately contributing new therapeutic strategies against drug-resistant pathogens and for the microbiome.