Direct CO₂ Transformation to Malate via Bioelectrosynthesis upon Engineered <i>Shewanella oneidensis</i>

Microbial electrosynthesis (MES) offers a sustainable and low-carbon approach for CO₂ valorization, with <i>Shewanella oneidensis</i> (<i>S. oneidensis</i>) MR-1 identified as an ideal microbe for MES. However, no prior research has demonstrated that <i>S. oneidensis</i> MR-1 can directly metabolize CO₂ into multicarbon (C₂₊) products due to its inability to perform the intracellular formate assimilation pathway. Here, we provide initial proof-of-concept evidence of direct bioelectrochemical CO₂ reduction to the C₄ product of malate. Specifically, the transformation of CO₂ to malate attains a notable production concentration of 1.18 mmol·L^-1, marking the first instance of direct C₄ compound bioelectrosynthesis. Such remarkable CO₂-to-C₄ conversion performances are attributed to the successful implementation of dual-plasmid systems in <i>S. oneidensis</i> MR-1, which facilitate the overexpression of the reductive glycine pathway (Plasmid I) for assimilating CO₂-derived formate and the alternative malate biosynthetic pathway (Plasmid II) to channel metabolic intermediates toward the biosynthesis of malate. Advancing CO₂ valorization toward carbon-negative C₂₊ bioproducts, our sophisticated dual-plasmid systems engineered in microbes can be further refined for scalable CO₂ bioelectrolysis with the objective of facilitating industrial applications.