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The newly identified BioE\u2010BioL module constitutes a new biotin biosynthesis pathway, yet its mechanisms remain incompletely characterized. Phylogenetic analyses reveal widespread distribution of the\n                    <jats:italic>bioE<\/jats:italic>\n                    , including obligate intracellular\n                    <jats:italic>Chlamydia<\/jats:italic>\n                    , despite the genus lacking its cognate repressor BioL. Structural modeling and biochemical characterization of\n                    <jats:italic>Elizabethkingia meningoseptica<\/jats:italic>\n                    BioE (EmBioE) and\n                    <jats:italic>Chlamydia psittaci<\/jats:italic>\n                    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\u2010chain acyl\u2010ACP and acyl\u2010CoA, whereas CpBioE functions as a monomer restricted to acyl\u2010ACP. Heterologous overexpression of EmBioE, but not CpBioE, induces a fitness cost in\n                    <jats:italic>Escherichia coli<\/jats:italic>\n                    . Genetic ablation of\n                    <jats:italic>bioL<\/jats:italic>\n                    leads to biotin auxotrophy in\n                    <jats:italic>Elizabethkingia<\/jats:italic>\n                    , mainly attributed to the unregulated EmBioE pathway exhausting long\u2010chain 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\u2010dependent antibacterial activity against\n                    <jats:italic>Elizabethkingia<\/jats:italic>\n                    . Balanced BioE expression is critical for bacterial viability, positioning BioE as a druggable target for antimicrobial discovery against multidrug\u2010resistant pathogens.\n                  <\/jats:p>","DOI":"10.1002\/advs.202510491","type":"journal-article","created":{"date-parts":[[2025,12,13]],"date-time":"2025-12-13T01:44:26Z","timestamp":1765590266000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Balanced Expression of the Diiron Oxygenase BioE Is Essential for Biotin Homeostasis in\n                    <i>Elizabethkingia meningoseptica<\/i>"],"prefix":"10.1002","volume":"13","author":[{"given":"Meng","family":"Zhang","sequence":"first","affiliation":[{"name":"Zhejiang Key Laboratory of Medical Epigenetics Department of Immunology and Pathogen Biology School of Basic Medical Sciences Hangzhou Normal University  Hangzhou Zhejiang 311121 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