Synergistic substrate cofeeding stimulates reductive metabolism

Advanced bioproduct synthesis via reductive metabolism requires coordinating carbons, ATP and reducing agents, which are generated with varying efficiencies depending on metabolic pathways. Substrate mixtures with direct access to multiple pathways may optimally satisfy these biosynthetic requirements. However, native regulation favouring preferential use precludes cells from co-metabolizing multiple substrates. Here we explore mixed substrate metabolism and tailor pathway usage to synergistically stimulate carbon reduction. By controlled cofeeding of superior ATP and NADPH generators as 'dopant' substrates to cells primarily using inferior substrates, we circumvent catabolite repression and drive synergy in two divergent organisms. Glucose doping in Moorella thermoacetica stimulates CO₂ reduction (2.3 g gCDW^-1 h^-1) into acetate by augmenting ATP synthesis via pyruvate kinase. Gluconate doping in Yarrowia lipolytica accelerates acetate-driven lipogenesis (0.046 g gCDW^-1 h^-1) by obligatory NADPH synthesis through the pentose cycle. Together, synergistic cofeeding produces CO₂-derived lipids with 38% energy yield and demonstrates the potential to convert CO₂ into advanced bioproducts. This work advances the systems-level control of metabolic networks and CO₂ use, the most pressing and difficult reduction challenge.