Adaptive laboratory evolution enables autotrophic growth of Eubacterium limosum under defined medium conditions

Eubacterium limosum is an acetogen capable of metabolizing one-carbon (C1) substrates, such as carbon monoxide (CO), carbon dioxide (CO₂), and methanol, via the Wood-Ljungdahl pathway. However, most acetogens require complex media containing undefined components, such as yeast extract, to support their autotrophic growth, which hinders a precise understanding of their fundamental autotrophic metabolism and impedes cost-effective bioprocess development. In this study, E. limosum was subjected to adaptive laboratory evolution through the stepwise reduction of yeast extract over 653 generations under 66 % CO syngas conditions. The resulting evolved strain, designated ECO_X, exhibited robust growth in a chemically defined medium devoid of yeast extract, achieving a specific CO consumption rate of 0.081 ± 0.014 mmol h^-1 and a specific growth rate of 0.103 ± 0.009 h^-1. ECO_X also exhibited a 1.9-fold reduction in cell size, markedly reduced biofilm formation, and a greater than 2-fold improvement in electroporation efficiency relative to the parental strain, establishing it as a tractable platform for metabolic engineering. Whole-genome resequencing identified a key mutation (Gly395Ser) within a type VII ABC transporter (B2M23_RS08970) as the genetic basis of this phenotype. Furthermore, transcriptomic analysis revealed a significant upregulation of the tryptophan biosynthesis pathway in ECO_X within the yeast extract-free defined medium, indicating that limited tryptophan availability is a key constraint underlying the yeast extract dependence of the parental strain under autotrophic conditions. Collectively, this study establishes a practical approach for the chemically defined cultivation of E. limosum and provides insight into the metabolic basis of its yeast extract dependence under autotrophic conditions.