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Synergistic substrate cofeeding stimulates reductive metabolism

Nature Metabolism volume 1, pages 643–651 (2019)Cite this article

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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.

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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.

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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 https://github.com/jopark/moorella_yarrowia. 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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Acknowledgements

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.

Author information

Author notes

  1. Junyoung O. Park

    Present address: Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, Los Angeles, CA, USA

  2. M. Ahsanul Islam

    Present address: Department of Chemical Engineering, Loughborough University, Loughborough, UK

  3. These authors contributed equally: Junyoung O. Park, Nian Liu.

Authors and Affiliations

  1. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

    Junyoung O. Park, Nian Liu, Kara M. Holinski, David F. Emerson, Kangjian Qiao, Benjamin M. Woolston, Jingyang Xu, Zbigniew Lazar, M. Ahsanul Islam & Gregory Stephanopoulos

  2. Key Laboratory of Drug Prevention and Control Technology of Zhejiang Province, Zhejiang Police College, Hangzhou, China

    Jingyang Xu

  3. Department of Biotechnology and Food Microbiology, Wroclaw University of Environmental and Life Sciences, Monskiego, Wroclaw, Poland

    Zbigniew Lazar

  4. Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA, USA

    Charles Vidoudez & Peter R. Girguis

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Contributions

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). https://doi.org/10.1038/s42255-019-0077-0

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