Balanced Expression of the Diiron Oxygenase BioE Is Essential for Biotin Homeostasis in Elizabethkingia meningoseptica

Biotin is an essential cofactor for central metabolic pathways in all organisms. The newly identified BioE‐BioL module constitutes a new biotin biosynthesis pathway, yet its mechanisms remain incompletely characterized. Phylogenetic analyses reveal widespread distribution of the bioE , including obligate intracellular Chlamydia , despite the genus lacking its cognate repressor BioL. Structural modeling and biochemical characterization of Elizabethkingia meningoseptica BioE (EmBioE) and Chlamydia psittaci BioE (CpBioE) reveal a conserved diiron oxygenase catalytic core but divergent oligomeric structure state and substrate preferences. EmBioE forms a homodimer capable of recognizing both long‐chain acyl‐ACP and acyl‐CoA, whereas CpBioE functions as a monomer restricted to acyl‐ACP. Heterologous overexpression of EmBioE, but not CpBioE, induces a fitness cost in Escherichia coli . Genetic ablation of bioL leads to biotin auxotrophy in Elizabethkingia , mainly attributed to the unregulated EmBioE pathway exhausting long‐chain fatty acids and depleting ATP/SAM metabolic pools. This highlights EmBioE's biphasic role: initiating biotin synthesis to sustain viability while inducing stress upon overexpression, requiring BioL regulation for metabolic homeostasis. Virtual screening uncovers compound 466982 as a selective BioE inhibitor with dose‐dependent antibacterial activity against Elizabethkingia . Balanced BioE expression is critical for bacterial viability, positioning BioE as a druggable target for antimicrobial discovery against multidrug‐resistant pathogens.