Identification of MscS as a Key L‐Glutamate Exporter in <i>Bacillus methanolicus</i>

Small-conductance mechanosensitive channels (MscS) are established l-glutamate exporters in industrially relevant bacteria, yet their role in the methylotrophic bacterium Bacillus methanolicus, a promising platform for sustainable methanol-based l-glutamate production, remains unexplored. Our research on B. methanolicus MGA3 identifies its MscS as the sole mechanosensitive channel in this organism and a key exporter of the amino acid l-glutamate, providing valuable insights into its potential for industrial applications. Transcriptomic analysis of B. methanolicus wild type cultured on an l-glutamate production medium revealed downregulation of the fatty acid biosynthesis genes fadR, fadF, mutB2, and acdA, suggesting that fatty acid metabolism is influenced by l-glutamate overproduction, with consequent changes in membrane fluidity likely driving mechanosensitive channel-mediated l-glutamate efflux. The MscS-like channel in B. methanolicus shares structural and functional similarities with MscS in Escherichia coli and with MscCG in Corynebacterium glutamicum. In silico structural predictions show that MGA3 MscS forms a homoheptameric structure with a transmembrane TM-barrel, resembling that of E. coli MscS. The opening mechanism of the channel, driven by membrane dynamics, involves coordinated rotation and flipping of its transmembrane helices, with variations in lipid composition potentially influencing the channel's activity. Additionally, under biotin-replete conditions, where this essential coenzyme supports carboxylases involved in fatty acid biosynthesis, l-glutamate overproduction was suppressed in MGA3. Finally, metabolic engineering experiments inducing MscS gain- and loss-of-function further confirmed the channel's critical role in B. methanolicus amino acid production, proportionally enhancing and reducing l-glutamate efflux, respectively. These findings open doors to novel strategies for engineering B. methanolicus and related methylotrophic organisms for sustainable amino acid production.