Abstract
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.
Highlights
One of the greatest feats of metabolism is the ability to synthesize reduced compounds from input substrates with varying oxidation states
We explored mixed substrate metabolism and therein observed enhanced metabolic productivity that exceeds the sum of individual-substrate productivities
Current engineering efforts often focus on funneling metabolic fluxes through product synthesis pathways via assembling various gene pools and knocking out competing pathways with existing genetic tools[32]
Summary
One of the greatest feats of metabolism is the ability to synthesize reduced compounds from input substrates with varying oxidation states. Cells reassemble the output of substrate catabolism for energy-dense bioproduct synthesis[1]. This process is often implemented in both laboratory and industry with single organic carbon sources (e.g., sugars) as inputs due to simplicity[2,3]. Single substrates naturally impose stoichiometric constraints on available carbons, energy, and redox cofactors, leading to biosynthetic imbalance and suboptimal product yield. On the other hand, present the potential to alleviate such stoichiometric constraints in reductive metabolism without genetic rewiring. Despite the recent success of substrate mixture batch fermentation using limited substrate pairs (that do not trigger catabolite repression)[10,11], genetic engineering[12,13], and directed evolution[14,15,16], the full mixture spectrum remains inaccessible and unexplored
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