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 CO 2 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 CO 2 -derived lipids with 38% energy yield and demonstrates the potential to convert CO 2 into advanced bioproducts. This work advances the systems-level control of metabolic networks and CO 2 use, the most pressing and difficult reduction challenge. Bioproduct synthesis via reductive metabolism occurs with different efficiencies according to the availability of carbons, ATP and reducing agents. To maximize overall product synthesis efficiency, the authors develop a substrate cofeeding strategy, which circumvents catabolite repression and drives synergy in lipid synthesis from CO 2 using two microbes.