Adaptive Laboratory Evolution (ALE) enables carbon-negative mixotrophic fermentation and enhanced chain elongation in <i>Clostridium</i> sp. JS66

<jats:p>Improving carbon recovery during sugar fermentation remains a major challenge because a substantial fraction of substrate carbon is lost as CO2 during central metabolism. To overcome this limitation, Clostridium sp. JS66 (JS66), an acetogen producing hexanoic acid from glucose, was subjected to adaptive laboratory evolution under CO2/H2 conditions to enhance H2-assisted CO2 reassimilation during glucose fermentation. The evolved strain, ALECO2, exhibited CO2 consumption without a lag phase under autotrophic conditions and reached a 9.5-fold higher CO2 uptake rate than JS66. Under fed-batch conditions, glucose-only fermentation yielded a carbon molar yield (Cmetabolite/Csugar, CM/CS) of 0.60, whereas H2 supplementation increased CM/CS to 0.91 and redirected carbon flux toward C6 products (hexanoic acid and hexanol), which accounted for 49% of total C_output. With additional CO2 supplementation, ALECO2 further assimilated externally supplied CO2, increasing the CM/CS to 1.10 and demonstrating carbon-negative fermentation. Assimilation of externally supplied CO2 further redirected carbon flux toward chain elongation, producing 7.14 g/L hexanoic acid and increasing the C6 carbon fraction to 57% of total C_output. Constraint-based flux analysis supported increased acetyl-CoA formation through the Wood-Ljungdahl pathway and enhanced flux toward reverse β-oxidation under H2- and CO2/H2-supplemented conditions. Genome analysis identified mutations including genes encoding a putative HytB homolog and a LysR-type transcriptional regulator. These results establish ALECO2 as a promising evolved anaerobic non-photosynthetic (ANP) mixotrophy platform that links CO2 reassimilation and external CO2 assimilation with chain elongation, enabling carbon-neutral and carbon-negative production of value-added C6 products from glucose.</jats:p>