Rewiring a methanol-responsive regulatory system improves glucose-methanol co-utilization in Eubacterium limosum

Methanol is a promising one-carbon (C1) feedstock for sustainable bioproduction, and its mixotrophic co-utilization with other substrates can improve product formation. However, mixotrophy often leads to sequential substrate utilization that delays methanol assimilation, and the regulatory basis underlying this phenotype remains unclear. In this study, we aimed to elucidate the regulatory mechanism governing methanol utilization in a methylotrophic acetogen and to determine whether rewiring this system could improve methanol co-utilization.

Here, we identify a dual-layer regulatory circuit centered on P_mtaR, the promoter driving the mta operon in Eubacterium limosum, as the key regulatory locus where methanol-responsive activation and carbon catabolite repression are integrated to govern the onset of methanol utilization. We show that robust P_mtaR activation requires the AraC-type regulator MtaR along with an upstream activation region within the promoter, whereas this activation is counteracted by a catabolite-responsive element (cre) embedded in P_mtaR, consistent with CcpA-mediated repression. This dual-layer regulatory architecture explains the delayed induction of the mta operon and the sequential utilization of glucose and methanol in E. limosum. Rewiring mta expression with a cre-free methanol-responsive promoter relieved repression enabled improved glucose-methanol co-utilization with enhanced methanol assimilation during glucose consumption. This achieved up to 3-fold increases in growth, substrate uptake, and product formation rates, accompanied by a metabolic shift towards butyrate production.

This study reveals a dual regulatory mechanism governing methanol utilization in E. limosum by integrating local methanol-responsive activation with global carbon catabolite repression. This mechanism explains sequential substrate utilization during glucose-methanol mixotrophy and provides a practical engineering strategy to improve methanol co-utilization and product formation in acetogenic bioprocesses.