Engineering of Microbial Cell Factories for Enhanced Acetic Acid and Ethanol Production Via Heterologous Overexpression of the CODH Gene in CO₂ Fermentation

This study evaluates the performance of carbon monoxide dehydrogenase (<i>codh</i>)-embedded strains in bench-scale microbial electrochemical systems (MES) for CO₂ reduction to biofuels and biochemicals. CO₂ fermentation efficiency was evaluated by comparing the wild-type <i>Clostridium acetobutylicum</i> (Wild), a negative control <i>E. coli</i> strain lacking the <i>codh</i> gene (NC-BL21), and engineered <i>E. coli</i> strain (Eng) alone and with IPTG induction (Eng+IPTG). Four electrochemical systems were used, viz. Wild+E, NC-BL21+E, Eng+E, and Eng+IPTG+E, with a poised potential of -0.6 V applied to the working electrode. CO₂ and bicarbonate were supplemented to a total inorganic carbon (IC) concentration of 40 g/L, with a retention time of 60 h. The engineered strain demonstrated enhanced metabolic performance compared to the wild-type and negative control strains, yielding maximum formic acid (2.1 g/L) and acetic acid (9.3 g/L) under the Eng+IPTG+E condition. Solventogenesis also influenced positively in the same system with the ethanol yield of 3.9 g/L, substantially exceeding biochemicals yield observed in the wild-type strain (2.4 g/L, acetic acid). The engineered strains exhibited superior cumulative yields (0.40 g/g), enhanced CODH-mediated charge flux stability (60 vs 5 in the wild type), and upregulated expression of key genes in the Wood-Ljungdahl pathway (WLP). Bioelectrochemical performance analysis demonstrated elevated reductive catalytic currents, enhanced CO₂ reduction, and optimal charge transfer kinetics. This study highlights the synergistic potential of genetic engineering, specifically CODH overexpression, combined with electro-fermentation for enhanced biofuel and biochemical production from C1 gases.