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Aqueous microdroplets promote C–C bond formation and sequences in the reverse tricarboxylic acid cycle

Nature Ecology & Evolution volume 7, pages 1892–1902 (2023)Cite this article

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Abstract

The reverse tricarboxylic acid cycle (rTCA) is a central anabolic network that uses carbon dioxide (CO2) and may have provided complex carbon substrates for life before the advent of RNA or enzymes. However, non-enzymatic promotion of the rTCA cycle, in particular carbon fixation, remains challenging, even with primordial metal catalysis. Here, we report that the fixation of CO2 by reductive carboxylation of succinate and α-ketoglutarate was achieved in aqueous microdroplets under ambient conditions without the use of catalysts. Under identical conditions, the aqueous microdroplets also facilitated the sequences in the rTCA cycle, including reduction, hydration, dehydration and retro-aldol cleavage and linked with the glyoxylate cycle. These reactions of the rTCA cycle were compatible with the aqueous microdroplets, as demonstrated with two-reaction and four-reaction sequences. A higher selectivity giving higher product yields was also observed. Our results suggest that the microdroplets provide an energetically favourable microenvironment and facilitate a non-enzymatic version of the rTCA cycle in prebiotic carbon anabolism.

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Fig. 1: Hypothetical rTCA cycle pathway consisting of the AcCoA pathway and the rTCA cycle.
Fig. 2: Reductive carboxylation of α-ketoglutarate to isocitrate and citrate in aqueous microdroplets.
Fig. 3: Reductive carboxylation of succinate (0.1 mM) to α-ketoglutarate in microdroplets.
Fig. 4: Characterization of the reductive carboxylations of succinate and α-ketoglutarate.
Fig. 5: Reductive mechanism for carboxylation of succinate and α-ketoglutarate.

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All the results supporting the findings are in Supplementary Information. Source data are provided with this paper.

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Acknowledgements

We acknowledge financially support from National Natural Science Foundation of China grant no. 21904029 to H.Z. and grant no. 22074026 to J.J. We acknowledge assistance from R. N. Zare in providing key suggestions on manuscript organization and acknowledge C. Gao for discussing the reductive carboxylation mechanism.

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Authors and Affiliations

  1. School of Marine Science and Technology, Harbin Institute of Technology at Weihai, Weihai, PR China

    Yun Ju, Hong Zhang, Yanxiao Jiang, Wenxin Wang, Guangfeng Kan, Kai Yu, Xiaofei Wang, Jilin Liu & Jie Jiang

  2. State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin, PR China

    Yun Ju, Hong Zhang, Yanxiao Jiang, Wenxin Wang, Kai Yu, Jilin Liu & Jie Jiang

  3. School of Chemistry and Chemical Engineering, Harbin Institute of Technology, Harbin, PR China

    Yun Ju, Jilin Liu & Jie Jiang

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Contributions

H.Z. and J.J. developed the original ideas presented in the manuscript and supervised the research. H.Z., J.J. and Y. Ju designed the experiments and Y. Ju completed the experiments with assistance from W.W., J.L., G.K., K.Y. and X.W. The reductive carboxylation mechanism was developed by J.J., H.Z., Y. Jiang and J.L. and the data analyses were mainly performed by H.Z., Y. Ju and Y. Jiang. The HPLC analysis was completed by Y. Ju and Y. Jiang. The first draft of the paper was written by Y. Ju, H.Z. and J.J. All authors contributed to finalizing the manuscript. All authors have jointly revised the manuscript and agreed to the published version of the paper.

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Correspondence to Hong Zhang or Jie Jiang.

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Extended data

Extended Data Fig. 1 Comparison of succinate 7 in CO2- and 13CO2-saturated aqueous microdroplets.

a, Experimental setup; the use of the tube between the sprayer and the MS inlet aimed to avoid the interference of atmosphere. b, Potential intermediates and products from nebulization of 7 in CO2- and 13CO2-saturated water solution.

Extended Data Fig. 2 Comparison of propionic acid in CO2- and 13CO2-saturated aqueous microdroplets.

a, Mass spectra of potential intermediates and products in CO2- and 13CO2-saturated aqueous microdroplets. b, Tandem mass spectra of m/z 145 in the case of CO2-saturated solution. c, Tandem mass spectra of m/z 147 in the case of 13CO2-saturated solution. Nebulization of propionic acid in 13CO2-saturated water solution clearly showed peaks of m/z 102 and m/z 147, which was 1 Da and 2 Da shift compared to those with CO2. This was also consisted with that from nebulization of 7 in CO2- and 13CO2-saturated water solution (Extended Data Fig. 1). The tandem mass spectrum of m/z 147 showed a similar pattern compared to that of m/z 145, which was consistent with the standard 9 (Fig. 3a). This suggested that nebulization of propionic acid could also generate 9 and further supported the assumption that initial decarboxylation of 7 generated propionic acid.

Extended Data Fig. 3 Reductive carboxylation of succinate 7 and α-ketoglutarate 9 in acetonitrile microdroplets.

a, Succinate 7. b, α-ketoglutarate 9. c, Expansion showing spectra for m/z 142-148 and m/z 114-120 from nebulization of 7 and 9 using acetonitrile. d, Expansion showing spectra for m/z 142-148 and m/z 114-120 from nebulization of 7 and 9 using water. When acetonitrile was used as solvent, no corresponding products of m/z 145 and 191, originated from 7 and 9, were observed. This suggested that the reductive carboxylation reactions in aprotic solvent was unfavourable. This supported that the important role of water during the reductive carboxylation. When either water or acetonitrile were used as solvents, the reagent radical anions (7•-, m/z 118; 9•-, m/z 146) were not observed and at least that were lower than the detection limitation. Peaks of m/z 118 and 146 in c and d corresponded to the isotope peaks of reagents because the simulation abundance of these peaks were about 5.4 %. Combing with these results and OH• identification63 suggested that the electrons may be mainly derived from OH− generated by water splitting. In addition, the formation of OH• at the microdroplet surface was also confirmed by other groups66,71.

Extended Data Fig. 4 Effect of high voltage on reductive carboxylation of α-ketoglutarate 9.

a, Reaction with high voltage of −4.5 kV. b, Reaction without high voltage. c, Reaction with the stainless-steel tube grounded. d, Reaction with peek tube instead of the stainless-steel tube. No voltage was used in c and d. The concentration of 9 was 0.1 mM. The product abundances showed no substantial difference.

Extended Data Fig. 5 Effect of high voltage on reductive carboxylation of succinate 7.

a, Reaction with high voltage of −4.5 kV. b, Reaction without high voltage. c, Reaction with the stainless-steel tube grounded. d, Reaction with peek tube instead of the stainless-steel tube. No voltage was used in c and d. The concentration of 7 was 0.1 mM. From Extended Data Fig. 4 and Fig. 5, the abundances for the corresponding products (m/z 191 and m/z 145) showed no substantial difference. This suggested that in absence of high voltage, the products could be also generated when the reagent solution was nebulized.

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Ju, Y., Zhang, H., Jiang, Y. et al. Aqueous microdroplets promote C–C bond formation and sequences in the reverse tricarboxylic acid cycle. Nat Ecol Evol 7, 1892–1902 (2023). https://doi.org/10.1038/s41559-023-02193-8

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