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An orthogonal metabolic framework for one-carbon utilization

Abstract

Metabolic engineering often entails concurrent engineering of substrate utilization, central metabolism and product synthesis pathways, inevitably creating interdependency with native metabolism. Here we report an alternative approach using synthetic pathways for C1 bioconversion that generate multicarbon products directly from C1 units and hence are orthogonal to the host metabolic network. The engineered pathways are based on formyl-CoA elongation (FORCE) reactions catalysed by the enzyme 2-hydroxyacyl-CoA lyase. We use thermodynamic and stoichiometric analyses to evaluate FORCE pathway variants, including aldose elongation, α-reduction and aldehyde elongation. Promising variants were prototyped in vitro and in vivo using the non-methylotrophic bacterium Escherichia coli. We demonstrate the conversion of formate, formaldehyde and methanol into various products including glycolate, ethylene glycol, ethanol and glycerate. FORCE pathways also have the potential to be integrated with the host metabolism for synthetic methylotrophy by the production of native growth substrates as demonstrated in a two-strain co-culture system.

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Fig. 1: FORCE pathways for product synthesis from C1 substrates.
Fig. 2: Thermodynamic analysis of FORCE pathways.
Fig. 3: In vitro assessment of the core module of the FORCE pathway using purified enzymes.
Fig. 4: Cell-free prototyping the α-reduction variant of the FORCE product synthesis pathway.
Fig. 5: Resting-cell bioconversions of C1 substrate formaldehyde using the aldose elongation and α-reduction variants of the FORCE pathways.
Fig. 6: FORCE pathway implementation in growing cell cultures using methanol as the C1 substrate.
Fig. 7: Simulated flux maps from genome-scale E. coli models for growth using FORCE pathways variants.
Fig. 8: Two-strain system for evaluating the ability of FORCE pathways to enable growth on C1 substrates.

Data availability

All data supporting the findings of this study are included in the paper and its supplementary information as well as the following public databases: MetaCyc (https://metacyc.org/), eQuilibrator (https://equilibrator.weizmann.ac.il/) and Uniprot (https://www.uniprot.org/). Uniprot accession numbers for enzymes involved in the study are available in Supplementary Table 1.

Code availability

The scripts used to perform the analyses in the study are available at https://github.com/ahc7/FORCE_manuscript/.

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Acknowledgements

We thank S. Qian for assistance with genetic methods. We thank M. Nattermann, S. Burgener and T. J. Erb (Max Planck Institute for Terrestrial Microbiology) for the suggestion and sequence of wild-type CaAbfT. We thank N. Paczia at the Max Planck Institute for Terrestrial Microbiology for informative suggestions on LC–MS analysis. We thank the University of South Florida Chemical Purification Analysis and Screening core facility for access to the Agilent 6540 Q-TOF LC–MS system with the help of L. Calcul. BsmHACL was identified and the DNA was provided as part of a grant from the US Department of Energy Joint Genome Institute DNA Synthesis programme. This work was supported by grants from the US National Science Foundation (CBET-1605999), the US Department of Energy (DE-EE0008499), and BASF Corporation. The funders had no role in study design, data collection and analysis, or preparation of the manuscript.

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Contributions

R.G. conceptualized the research and supervised the project. A.C., J.M.C., S.H.L., F.Z. and R.G. designed the methodology. A.C. performed thermodynamic analysis, flux balance analysis and cell-free experiments. A.C., F.Z. and J.M.C. performed resting-cell experiments. S.H.L. and J.M.C. performed methanol experiments. F.Z. performed purified enzyme experiments. S.H.L., F.Z. and J.M.C. performed the two-strain growth experiments. A.C., S.H.L. and F.Z. constructed E. coli strains. A.C., J.M.C., F.Z. and S.H.L. analysed the data. A.C., S.H.L., J.M.C., F.Z. and R.G. prepared the manuscript with feedback from all authors.

Corresponding author

Correspondence to Ramon Gonzalez.

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Competing interests

A.C., J.M.C. and R.G. are co-inventors and assignees on a patent application (PCT/US2015/058121), which relates to the reported research. The remaining authors declare no competing interests.

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Peer review information Nature Metabolism thanks Marina Kalyuzhnaya, Steven Singer and the other, anonymous, reviewers for their contribution to the peer review of this work. Primary Handling Editor: Isabella Samuelson.

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Extended data

Extended Data Fig. 1 Canonical (a) and orthogonal, synthetic (b) architectures for biological C1 utilization.

a, ‘Bowtie’ architecture of metabolism in which carbon substrates are consolidated into central metabolites from which a host of products can be produced through fermentative and biosynthetic pathways. Metabolic engineering typically operates within this framework by manipulating either one or all of the three components of the bowtie. b, The orthogonal FORCE pathways serve as a platform for both product synthesis and for providing substrates/metabolites for growth. This is an alternative framework to the traditional approach, which feeds all carbon through central metabolism, and from which both products and biomass are derived.

Extended Data Fig. 2 An alternative FORCE pathway based on dehydration of the 2-hydroxyacyl-CoA and α-reduction.

The pathway resembles β-oxidation reversal (β-reduction)39. This pathway is also a potential route for the production of unsaturated products. HACL: 2-hydroxyacyl-CoA lyase; HACD: 2-hydroxyacyl-CoA dehydratase; TER: trans-2-enoyl-CoA reductase; ACR: acyl-CoA reductase.

Extended Data Fig. 3 The impact of NADH/NAD+ ratio on formaldehyde (top) and methanol (bottom) conversion to glycolate or acetate via FORCE pathways.

Vertical dotted lines indicate the physiological range of NADH/NAD+ ratios.

Extended Data Fig. 4 The impact of termination on the iterative aldose elongation pathway.

Termination by hydrolysis of the acyl-CoA to produce sugar acids increases the driving force of the pathway for low numbers of iterations, but the driving forces converge as the number of iterations increase.

Extended Data Fig. 5 Production of glycolate from formate by E. coli engineered with a formate-activating pathway.

Resting cell experiments were performed with a strain expressing CaAbfT and BsmHACL (blue bars) and the corresponding control lacking BsmHACL (orange bars). Cultures (2.5 OD600 = 2.5*109 CFU/mL) were incubated at 30 °C for 24 hours in 25 mL flasks shaking at 200 rpm using 10 mM formate (plus 1 mM formaldehyde) as carbon source (control cultures with 1 mM formaldehyde and no formate also shown). All data points are shown for n = 6 replicates. Bars are drawn to the mean values.

Extended Data Fig. 6 Predicted biomass electron and carbon yields from various C1 substrates by the implementation of select pathways enabling methylotrophy.

Abbreviations: Formald: formaldehyde, FORCE-Glycerald: FORCE pathway with reactions enabling glyceraldehyde production, RuMP: Ribulose monophosphate pathway, FORCE-Ac: FORCE pathway with reactions enabling acetate production, SACA: Synthetic Acetyl-CoA pathway, FORCE-Glycolate: FORCE pathway with reactions enabling glycolate production. The scenarios in bold correspond to the predicted flux maps illustrated in Fig. 8.

Extended Data Fig. 7 Paraformaldehyde solubilization rate and resting cell bioconversion with paraformaldehyde.

a, Solubilization rate of commercially available paraformaldehyde (pFALD) with different particle sizes. Solubilization rates are measured in 10 mL M9 media in a 25 mL flask at 30 °C shaking at 200 rpm. b, Resting cell bioconversion of strains expressing BsmHACL, LmACR and AldA induced with 40 μM cumate and 100 μM IPTG. 3 mg prilled paraformaldehyde is added to 20 mL M9 media (2.5 mM formaldehyde equivalent) in a 25 mL flask at 30 °C shaking at 200 rpm. Formaldehyde accumulates only at sub-millimolar concentrations under these conditions.

Extended Data Fig. 8 Time course profiles for glycolate, formate, and formaldehyde concentration and cell-growth of the sensor strain in the two-strain system with 5 mM paraformaldehyde.

a, Time course in which the producer strain did not express an HACL. b, Plates from a representative experiment of the time course shown in panel a. c, Time course in which the producer strain expresses HACL. d, Plates from a representative experiment of the time course shown in panel c. 50 μL of cultures (5 × 10−3 dilution) at various time points plated on minimal media plates containing 2.5 g/L glycolate. All data is shown for duplicate replicates (n = 2). Lines are drawn to the mean values.

Extended Data Fig. 9 Time course profiles for glycolate, formate, and formaldehyde concentration and cell-growth of the sensor strain in the two-strain system with 500 mM methanol.

a, Time course in which the producer strain did not express an HACL. b, Plates from a representative experiment of the time course shown in panel a. c, Time course in which the producer strain expresses HACL. d, Plates from a representative experiment of the time course shown in panel c. 50 μL of cultures (5×10−3 dilution) at various time points plated on minimal media plates containing 2.5 g/L glycolate. All data is shown for duplicate replicates (n = 2). Lines are drawn to the mean values.

Extended Data Fig. 10 Time course profiles for glycolate, and formaldehyde concentration in the two-strain system with 1 mM formaldehyde and 10 mM formate.

a, Time course in which the producer strain expresses BsmHACL. b, Plates from a representative experiment of the time course shown in panel a. 50 μL of cultures (5 × 10−3 dilution) at various time points plated on minimal media plates containing 2.5 g/L glycolate. All data is shown for duplicate replicates (n = 2). Lines are drawn to the mean values.

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Chou, A., Lee, S.H., Zhu, F. et al. An orthogonal metabolic framework for one-carbon utilization. Nat Metab 3, 1385–1399 (2021). https://doi.org/10.1038/s42255-021-00453-0

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