CoChat
Sign Up
Cited by 69Carbon dioxide utilization in catalysisEnzyme Catalysis and ImmobilizationMicrobial Metabolic Engineering and Bioproduction

The DOI matches its Crossref record; 4 of the 4 references it cites were found in a registry or an index.

Turn air-captured CO2 with methanol into amino acid and pyruvate in an ATP/NAD(P)H-free chemoenzymatic system

Open access
Liu, Jianming, Zhang, Han, Xu, Yingying, Meng, Hao, Zeng, An-Ping·2023·Nature Communications
DOI 10.1038/s41467-023-38490-wPMC PMC10185560

Published version of a preprint

Open this paper in CoChat — free accountOpen PDF

Abstract

The use of gaseous and air-captured CO 2 for technical biosynthesis is highly desired, but elusive so far due to several obstacles including high energy (ATP, NADPH) demand, low thermodynamic driving force and limited biosynthesis rate. Here, we present an ATP and NAD(P)H-free chemoenzymatic system for amino acid and pyruvate biosynthesis by coupling methanol with CO 2 . It relies on a re-engineered glycine cleavage system with the NAD(P)H-dependent L protein replaced by biocompatible chemical reduction of protein H with dithiothreitol. The latter provides a higher thermodynamic driving force, determines the reaction direction, and avoids protein polymerization of the rate-limiting enzyme carboxylase. Engineering of H protein to effectively release the lipoamide arm from a protected state further enhanced the system performance, achieving the synthesis of glycine, serine and pyruvate at g/L level from methanol and air-captured CO 2 . This work opens up the door for biosynthesis of amino acids and derived products from air.

Overview

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1038/s41467-023-38490-w

Cites

4 of 4 references verified0 citation errors · 0 not checkable

Statistics

Figures

Fig. 1 Thermodynamic characterization of different glycine biosynthetic pathways and corresponding reactions. a The six metabolic pathways leading to the synthesis of glycine. b Thermodynamic profiles of the Gibbs free energy change for individual steps of the pathways under physiological conditions (pH 7.5, ionic strength I = 0.20, substrates = 1 mM). c Thermodynamic and kinetic features of individual reaction steps in the sixth pathway from methanol to glycine. [6-1], methanol oxidation. [6-2], formaldehyde (HCHO) and THF condensation. [6-3], glycine synthesis. [6-3-x], lipoic acid reduction by different reducing agents. DTT Dithiothreitol, TCEP Tris(2-carboxylethyl)phosphine, β-ME β-mercaptoethanol, THF tetrahydrofolate, Formyl-P formyl phosphate, rGCS reverse glycine cleavage system. 1ΔrG′=-RTln(J+/J−), J−, the forward flux in glycine synthesis, J−, the backward flux in glycine cleavage, the fraction in the enzymatic flux is indicated.
Fig. 1 Thermodynamic characterization of different glycine biosynthetic pathways and corresponding reactions. a The six metabolic pathways leading to the synthesis of glycine. b Thermodynamic profiles of the Gibbs free energy change for individual steps of the pathways under physiological conditions (pH 7.5, ionic strength I = 0.20, substrates = 1 mM). c Thermodynamic and kinetic features of individual reaction steps in the sixth pathway from methanol to glycine. [6-1], methanol oxidation. [6-2], formaldehyde (HCHO) and THF condensation. [6-3], glycine synthesis. [6-3-x], lipoic acid reduction by different reducing agents. DTT Dithiothreitol, TCEP Tris(2-carboxylethyl)phosphine, β-ME β-mercaptoethanol, THF tetrahydrofolate, Formyl-P formyl phosphate, rGCS reverse glycine cleavage system. 1ΔrG′=-RTln(J+/J−), J−, the forward flux in glycine synthesis, J−, the backward flux in glycine cleavage, the fraction in the enzymatic flux is indicated.CC BY · PubMed Central
Fig. 2 Design and implementation of an ATP and NAD(P)H-free CO2-fixation pathway via biocatalysis and biocompatible non-enzymatic chemistry. a The reaction system to convert CO2 and methanol into C2 glycine, C3 serine and pyruvate. DTT is used to replace the function of L protein (dotted line). b, c DTT and TCEP as potential reducing agents to reduce Hox to Hred as a non-enzymatic reaction step. d Measurement of NADH generation coupled with the disulfide-reducing chemistry. The dynamic change of Hox to Hred in the reaction catalyzed by reducing agents (R) is measured using NADH generation in the presence of L protein and NAD+. e Glycine production rate underlined (mM/min) with the components listed. Data represent mean values ± SEM of 3 independent experiments. 5,10-CH2-THF, N5, N10-methylene-THF; A, alcohol oxidase, EC 1.1.3.13; Hint, aminomethylated form of the lipoylated H protein; Hox and Hred, oxidized and reduced form of the lipoylated H protein, respectively; PLP, pyridoxal 5-phosphate monohydrate; P, glycine decarboxylase, EC 1.4.4.2; T, aminomethyltransferase, EC 2.1.2.10; L, dihydrolipoyl dehydrogenase, EC 1.8.1.4; S, serine hydroxymethyltransferase, EC 2.1.2.1; D, L-serine deaminase, EC 4.3.1.17.
Fig. 2 Design and implementation of an ATP and NAD(P)H-free CO2-fixation pathway via biocatalysis and biocompatible non-enzymatic chemistry. a The reaction system to convert CO2 and methanol into C2 glycine, C3 serine and pyruvate. DTT is used to replace the function of L protein (dotted line). b, c DTT and TCEP as potential reducing agents to reduce Hox to Hred as a non-enzymatic reaction step. d Measurement of NADH generation coupled with the disulfide-reducing chemistry. The dynamic change of Hox to Hred in the reaction catalyzed by reducing agents (R) is measured using NADH generation in the presence of L protein and NAD+. e Glycine production rate underlined (mM/min) with the components listed. Data represent mean values ± SEM of 3 independent experiments. 5,10-CH2-THF, N5, N10-methylene-THF; A, alcohol oxidase, EC 1.1.3.13; Hint, aminomethylated form of the lipoylated H protein; Hox and Hred, oxidized and reduced form of the lipoylated H protein, respectively; PLP, pyridoxal 5-phosphate monohydrate; P, glycine decarboxylase, EC 1.4.4.2; T, aminomethyltransferase, EC 2.1.2.10; L, dihydrolipoyl dehydrogenase, EC 1.8.1.4; S, serine hydroxymethyltransferase, EC 2.1.2.1; D, L-serine deaminase, EC 4.3.1.17.CC BY · PubMed Central
Fig. 3 Enhanced glycine synthesis through the change of redox state of P protein. a The oxidized state of P protein with the formation of disulfide bond between C335 and C949 (or its neighboring C951) and its reduced state, when the disulfide bond was open. b Quantification of P protein using HPLC. c Glycine production rate using P protein or different variants in the absence of DTT. d in the presence of DTT. Bars represent the mean change ± SEM of 3 independent experiments. e The disulfide bond-crosslinked network in P proteins. f Native PAGE analysis of P, T protein with (without) DTT and P variants (One representative figure is shown in 3 independent experiments).
Fig. 3 Enhanced glycine synthesis through the change of redox state of P protein. a The oxidized state of P protein with the formation of disulfide bond between C335 and C949 (or its neighboring C951) and its reduced state, when the disulfide bond was open. b Quantification of P protein using HPLC. c Glycine production rate using P protein or different variants in the absence of DTT. d in the presence of DTT. Bars represent the mean change ± SEM of 3 independent experiments. e The disulfide bond-crosslinked network in P proteins. f Native PAGE analysis of P, T protein with (without) DTT and P variants (One representative figure is shown in 3 independent experiments).CC BY · PubMed Central
Fig. 4 Self-protection of H protein and increased glycine synthesis by breaking it. a The lipoamide arm is engaged as a mobile substrate that commutes successively between the other two enzymes and breaking its self-protection in the original Hint can bypass the process in which the swinging arm shuttles inside and outside of the H cavity. HM, H mutants. b Key amino acids important to maintain the aminomethyl arm in the H cavity. c The glycine production rate using different H variants (10 mM) and their corresponding native PAGE analysis. Bars represent the mean change ± SEM of 3 independent experiments. d Molecular dynamics simulations for Hwt and Y70A. The distance between Ca at E12 of the H protein and the N atom of the lipoamide, shown in Fig. 4b, is measured for the first 400 ns in the simulations. e The representative structures in MD trajectory for Hwt and Y70A as labeled in Fig. 4d.
Fig. 4 Self-protection of H protein and increased glycine synthesis by breaking it. a The lipoamide arm is engaged as a mobile substrate that commutes successively between the other two enzymes and breaking its self-protection in the original Hint can bypass the process in which the swinging arm shuttles inside and outside of the H cavity. HM, H mutants. b Key amino acids important to maintain the aminomethyl arm in the H cavity. c The glycine production rate using different H variants (10 mM) and their corresponding native PAGE analysis. Bars represent the mean change ± SEM of 3 independent experiments. d Molecular dynamics simulations for Hwt and Y70A. The distance between Ca at E12 of the H protein and the N atom of the lipoamide, shown in Fig. 4b, is measured for the first 400 ns in the simulations. e The representative structures in MD trajectory for Hwt and Y70A as labeled in Fig. 4d.CC BY · PubMed Central
Fig. 5 Glycine synthesis from formaldehyde and CO2. a The combination of non-enzymatic and enzymatic chemistry to synthesize glycine. b The effect of H concentrations on glycine production rate. c Glycine production from formaldehyde and bicarbonate. d Glycine production from formaldehyde and gaseous CO2. e Glycine production from formaldehyde and air-derived CO2. 10 mM formaldehyde was added in the beginning followed by another 10 mM formaldehyde after 1 h to avoid high levels of precipitation caused by the reaction between a high concentration of formaldehyde and ammonium. All the Data represent mean values ± SEM of 2 independent experiments.
Fig. 5 Glycine synthesis from formaldehyde and CO2. a The combination of non-enzymatic and enzymatic chemistry to synthesize glycine. b The effect of H concentrations on glycine production rate. c Glycine production from formaldehyde and bicarbonate. d Glycine production from formaldehyde and gaseous CO2. e Glycine production from formaldehyde and air-derived CO2. 10 mM formaldehyde was added in the beginning followed by another 10 mM formaldehyde after 1 h to avoid high levels of precipitation caused by the reaction between a high concentration of formaldehyde and ammonium. All the Data represent mean values ± SEM of 2 independent experiments.CC BY · PubMed Central
Fig. 6 Turn methanol and CO2 into C2 (glycine) and C3 (serine and pyruvate). a The process of converting C1 to C2 and C3. b Methanol and air-captured CO2 as carbon source for glycine production with the components listed in the embedded table. c The biosynthesis of serine using the same components as listed in (b) with 50 mM methanol, 1 mM THF and 20 mM SHMT. d The production of pyruvate using the same components in (c) with 5 mM LSD. All the Data represent mean values ± SEM of 2 independent experiments.
Fig. 6 Turn methanol and CO2 into C2 (glycine) and C3 (serine and pyruvate). a The process of converting C1 to C2 and C3. b Methanol and air-captured CO2 as carbon source for glycine production with the components listed in the embedded table. c The biosynthesis of serine using the same components as listed in (b) with 50 mM methanol, 1 mM THF and 20 mM SHMT. d The production of pyruvate using the same components in (c) with 5 mM LSD. All the Data represent mean values ± SEM of 2 independent experiments.CC BY · PubMed Central

Verification

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Citations

Cites

Research works printed but not linked: 44

  1. Cambridge University Press. Climate Change 2022: Impacts, adaptation and vulnerability. contribution of working group II to the sixth assessment report of the intergovernmental panel on climate change (2022).
  2. Qiu, Y. et al. Environmental trade-offs of direct air capture technologies in climate change mitigation toward 2100. Nat. Commun. 13, 3635 (2022). · DOI
  3. Scown, C. Prospects for carbon-negative biomanufacturing. Trends Biotechnol. 40, 1415–1424 (2022). · DOI
  4. Clomburg, J. M., Crumbley, A. M. & Gonzalez, R. Industrial biomanufacturing: the future of chemical production. Science 355, aag0804 (2017). · DOI
  5. Rasor, B. J. et al. Toward sustainable, cell-free biomanufacturing. Curr. Opin. Biotechnol. 69, 136–144 (2021). · DOI
  6. Lee, S. Y. & Kim, H. U. Systems strategies for developing industrial microbial strains. Nat. Biotechnol. 33, 1061–1072 (2015). · DOI
  7. Kim, S. et al. Growth of E. coli on formate and methanol via the reductive glycine pathway. Nat. Chem. Biol. 16, 538–545 (2020). · DOI
  8. Bang, J., Hwang, C. H., Ahn, J. H., Lee, J. A. & Lee, S. Y. Escherichia coli is engineered to grow on CO2 and formic acid. Nat. Microbiol. 5, 1459–1463 (2020). · DOI
  9. Tashiro, Y., Hirano, S., Matson, M. M., Atsumi, S. & Kondo, A. Electrical-biological hybrid system for CO2 reduction. Metab. Eng. 47, 211–218 (2018). · DOI
  10. He, H., Höper, R., Dodenhöft, M., Marlière, P. & Bar-Even, A. An optimized methanol assimilation pathway relying on promiscuous formaldehyde-condensing aldolases in E. coli. Metab. Eng. 60, 1–13 (2020). · DOI
  11. Chen, F. Y.-H., Jung, H.-W., Tsuei, C.-Y. & Liao, J. C. Converting Escherichia coli to a synthetic methylotroph growing solely on methanol. Cell 182, 933–946.e14 (2020). · DOI
  12. Yu, H. & Liao, J. C. A modified serine cycle in Escherichia coli converts methanol and CO2 to two-carbon compounds. Nat. Commun. 9, 3992 (2018). · DOI
  13. Bar-Even, A. Formate assimilation: the metabolic architecture of natural and synthetic pathways. Biochemistry 55, 3851–3863 (2016). · DOI
  14. Cai, T. et al. Cell-free chemoenzymatic starch synthesis from carbon dioxide. Science 373, 1523–1527 (2021). · DOI
  15. Luo, S. et al. A cell-free self-replenishing CO2-fixing system. Nat. Catal. 5, 154–162 (2022). · DOI
  16. Schwander, T., Schada von Borzyskowski, L., Burgener, S., Cortina, N. S. & Erb, T. J. A synthetic pathway for the fixation of carbon dioxide in vitro. Science 354, 900–904 (2016). · DOI
  17. Liu, Z., Wang, K., Chen, Y., Tan, T. & Nielsen, J. Third-generation biorefineries as the means to produce fuels and chemicals from CO2. Nat. Catal. 3, 274–288 (2020). · DOI
  18. Cotton, C. et al. Renewable methanol and formate as microbial feedstocks. Curr. Opin. Biotechnol. 62, 168–180 (2020). · DOI
  19. Beber, M. et al. eQuilibrator 3.0: a database solution for thermodynamic constant estimation. Nucleic Acids Res. 50, D603–D609 (2022). · DOI
  20. Caspi, R. et al. The MetaCyc database of metabolic pathways and enzymes - a 2019 update Nucleic Acids Res. 48(D1) D445-D453 (2020). · DOI
  21. Noor, E. et al. Pathway thermodynamics highlights kinetic obstacles in central metabolism. PLoS Comput. Biol. 10, e1003483 (2014). · DOI
  22. Hasse, D. et al. Structure of the homodimeric glycine decarboxylase P-protein from Synechocystis sp. PCC 6803 suggests a mechanism for redox regulation. J. Biol. Chem. 288, 35333–35345 (2013). · DOI
  23. Cohen-Addad, C., Pares, S., Sieker, L., Neuburger, M. & Douce, R. The lipoamide arm in the glycine decarboxylase complex is not freely swinging. Nat. Struct. Mol. Biol. 2, 63–68 (1995). · DOI
  24. Roche, O., Hinsen, K. & Field, M. J. Theoretical study of the conformation of the H-protein lipoamide arm as a function of its terminal group. Proteins: Struct. Funct. Genet. 36, 228–237 (1999). · DOI
  25. Zhang, X., Nie, J., Zheng, Y., Ren, J. & Zeng, A.-P. Activation and competition of lipoylation of H protein and its hydrolysis in a reaction cascade catalyzed by the multifunctional enzyme lipoate-protein ligase A. Biotechnol. Bioeng. 117, 3677–3687 (2020). · DOI
  26. Xu, Y., Meng, H., Ren, J. & Zeng, A.-P. Formaldehyde formation in the glycine cleavage system and its use for an aldolase-based biosynthesis of 1,3-propanediol. J. Biol. Eng. 14, 15 (2020). · DOI
  27. Zhang, H., Li, Y., Nie, J., Ren, J. & Zeng, A.-P. Structure-based dynamic analysis of the glycine cleavage system suggests key residues for control of a key reaction step. Commun. Biol. 3, 1–12 (2020). · DOI
  28. Kallen, R. G. & Jencks, W. P. The mechanism of the condensation of formaldehyde with tetrahydrofolic acid. J. Biol. Chem. 241, 5851–5863 (1966). · DOI
  29. Xu, Y., Ren, J., Wang, W. & Zeng, A.-P. Improvement of glycine biosynthesis from one-carbon compounds and ammonia catalyzed by the glycine cleavage system in vitro. Eng. Life Sci. 22, 40–53 (2022). · DOI
  30. Richmond, H. H., Myers, G. S. & Wright, G. F. The Reaction between formaldehyde and ammonia. J. Am. Chem. Soc. 70, 3659–3664 (1948). · DOI
  31. Rybniker, J. et al. The cytotoxic early protein 77 of mycobacteriophage L5 interacts with MSMEG_3532, an L-serine dehydratase of Mycobacterium smegmatis. J. Basic Microbiol. 51, 515–522 (2011). · DOI
  32. Bruska, M. K., Stiebritz, M. T. & Reiher, M. Analysis of differences in oxygen sensitivity of Fe–S clusters. Dalton Trans. 42, 8729–8735 (2013). · DOI
  33. Wang, X. et al. Cofactor NAD(P)H regeneration inspired by heterogeneous pathways. Chem 2, 621–654 (2017). · DOI
  34. Shaked, Z., Barber, J. J. & Whitesides, G. M. Combined electrochemical/enzymic method for in situ regeneration of NADH based on cathodic reduction of cyclic disulfides. J. Org. Chem. 46, 4100–4101 (1981). · DOI
  35. Zhang, S. et al. Pyruvate metabolism redirection for biological production of commodity chemicals in aerobic fungus Aspergillus oryzae. Metab. Eng. 61, 225–237 (2020). · DOI
  36. Zhang, X. et al. Quantitative study of H protein lipoylation of the glycine cleavage system and a strategy to increase its activity by co-expression of LplA. J. Biol. Eng. 13, 32 (2019). · DOI
  37. Liu, J. et al. Combining metabolic engineering and biocompatible chemistry for high-yield production of homo-diacetyl and homo-(S,S)−2,3-butanediol. Metab. Eng. 36, 57–67 (2016). · DOI
  38. Long, C. P. & Antoniewicz, M. R. High-resolution 13C metabolic flux analysis. Nat. Protoc. 14, 2856–2877 (2019). · DOI
  39. Salomon-Ferrer, R., Case, D. & Walker, R. An overview of the amber biomolecular simulation package. WIREs Comput. Mol. Sci. 3, 198–210 (2013). · DOI
  40. Kräutler, V., van Gunsteren, W. F. & Hünenberger, P. H. A fast SHAKE algorithm to solve distance constraint equations for small molecules in molecular dynamics simulations. J. Comput. Chem. 22, 501–508 (2001). · DOI
  41. Bordoli, L. et al. Protein structure homology modeling using SWISS-MODEL workspace. Nat. Protoc. 4, 1–13 (2009). · DOI
  42. Jurrus, E. et al. Improvements to the APBS biomolecular solvation software suite. Protein Sci. 27, 112–128 (2018). · DOI
  43. Siegel, J. B. et al. Computational protein design enables a novel one-carbon assimilation pathway. Proc. Natl Acad. Sci. USA 112, 3704–3709 (2015). · DOI
  44. Xiao, L. et al. A minimized synthetic carbon fixation cycle. Acs. Catal. 12, 799–808 (2022). · DOI

Metadata

Authors
Liu, J., Zhang, H., Xu, Y., Meng, H., & Zeng, A.-P.
Affiliations
  • Westlake University (CN)
  • Beijing University of Chemical Technology (CN)
Corresponding author
Zeng, An-Ping
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2023-05-15
Volume
14
Issue
1
Pages
2772-2772
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-023-38490-w
PMC
PMC10185560
OpenAlex
W4376641097
Cited by
69
References
4
Citations by year
2026: 21 · 2025: 24 · 2024: 14 · 2023: 10
FWCI
5.29
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • Research Square (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
Westlake University
Topics
  • Physical Sciences › Chemical Engineering › Process Chemistry and Technology › Carbon dioxide utilization in catalysis
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Enzyme Catalysis and Immobilization
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
Keywords
methanol, amino acid biosynthesis, chemoenzymatic synthesis, glycine cleavage system, L protein, dithiothreitol reduction, carboxylase, lipoamide, serine biosynthesis, thermodynamic driving force, protein polymerization
MeSH terms
Adenosine Triphosphate, Methanol, Amino Acids, Carbon Dioxide, NAD, Pyruvic Acid

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review