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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.

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Fig. 1: Continuous glucose cofeeding relieves repression of acetate in Y. lipolytica.
Fig. 2: Cofeeding substrates near the oxidative PPP accelerates cell growth and lipogenesis from acetate.
Fig. 3: Gluconate generates NADPH via the pentose cycle.
Fig. 4: Glucose generates ATP for CO2 fixation but leads to decarboxylation in M. thermoacetica.
Fig. 5: Continuous glucose cofeeding accelerates acetogenesis from CO2 fixation at the autotrophic limit.
Fig. 6: Synergy and coordination of substrate cofeeding accelerate the conversion of CO2 and H2 into lipids.

Data availability

The data that support the findings of this study are available in the Supplementary Files and from the corresponding author upon request.

Code availability

The code for the metabolic flux and free energy analysis is available on the GitHub public repository at The data that support the findings of this study are available in the Supplementary Files and from the corresponding author upon request.


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The authors would like to thank C. Lewis and E. Freinkman for their help with the LC–MS. This research was supported by the U.S. Department of Energy grant nos. DE-AR0000433, DE-SC0008744 and DE-SC0012377, as well as a Mobility Plus Fellowship no. 1284/MOB/IV/2015/0.

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Authors and Affiliations



J.O.P, N.L. and G.S. designed the study and wrote the paper. J.O.P., N.L. and K.M.H performed the experiments and flux analysis. J.O.P., N.L., B.M.W. and C.V. developed the methods for the LC–MS and gas chromatography–mass spectrometry. J.O.P., N.L., D.F.E. and J.X. designed the bioreactors. J.O.P. and M.A.I. developed the updated metabolic model. J.O.P., N.L., K.Q., Z.L., P.R.G. and G.S. analysed the data.

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Correspondence to Gregory Stephanopoulos.

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Supplementary Information

Supplementary Figs. 1–9, Supplementary Tables 1–7 and Supplementary Note

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Supplementary Dataset 1

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Park, J.O., Liu, N., Holinski, K.M. et al. Synergistic substrate cofeeding stimulates reductive metabolism. Nat Metab 1, 643–651 (2019).

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