Riboflavin Metabolism Links Metabolic State to Biofilm Development, Cell‐Envelope Homeostasis, and Antimicrobial Susceptibility in Mycobacterium tuberculosis

ABSTRACT Biofilm formation and phenotypic drug tolerance contribute to the persistence and treatment challenges associated with Mycobacterium tuberculosis ( M. tb ). Although M. tb lacks canonical quorum‐sensing systems, it exhibits coordinated metabolic and cell‐envelope adaptations during biofilm development, suggesting the existence of alternative mechanisms linking cellular metabolism to multicellular behaviour. Here, using a combination of functional genomics, metabolic perturbation and systems‐level analyses, we identify riboflavin metabolism as a regulatory axis influencing biofilm development and antimicrobial susceptibility in M. tb . CRISPRi‐mediated knockdown and targeted overexpression of riboflavin biosynthetic genes markedly altered biofilm architecture, cell morphology, envelope ultrastructure, membrane permeability and lipid composition, with ribA2 and ribF emerging as potential metabolic vulnerabilities. Exogenous riboflavin enhanced biofilm formation, whereas the riboflavin antimetabolite roseoflavin suppressed this phenotype. Notably, cell‐free supernatants from late‐stage cultures, accumulated higher levels of riboflavin and restored biofilm formation in a riboflavin‐biosynthesis‐deficient strain, supporting a diffusible regulatory function beyond the established metabolic roles of riboflavin‐derived FMN and FAD. Integrated transcriptomic and lipidomic analyses revealed coordinated reprogramming of biofilm‐associated regulatory pathways, cell‐envelope lipid metabolism and the F 420 biosynthetic network, accompanied by increased susceptibility to anti‐tuberculosis drugs. Molecular docking further identified potential interactions of riboflavin with regulatory proteins associated with biofilm and stress‐response pathways. Together, these findings establish riboflavin metabolism as a previously underappreciated regulatory layer connecting microbial metabolism, biofilm development, cell‐envelope homeostasis and drug tolerance. This metabolic axis provides a potential framework for developing microbial biotechnology‐based strategies to interfere with biofilm‐associated persistence and improve the efficacy of anti‐tuberculosis therapeutics.