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Escherichia coli is engineered to grow on CO2 and formic acid

Abstract

We engineered Escherichia coli to grow on CO2 and formic acid alone by introducing the synthetic CO2 and formic acid assimilation pathway, expressing two formate dehydrogenase genes, fine-tuning metabolic fluxes and optimizing the levels of cytochrome bo3 and bd-I ubiquinol oxidase. Our engineered strain can grow to an optical density at 600 nm of 7.38 in 450 h, and shows promise as a platform strain growing on CO2 and formic acid alone.

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Fig. 1: Construction of an engineered E. coli strain capable of growing on CO2 and FA alone.
Fig. 2: Growth of E. coli strain FC8 to high cell density on CO2 and FA alone.

Data availability

Data supporting our work are available in the paper, Extended Data Figs. 18 and Supplementary Information. Further information and materials related to the findings of this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank J. S. Cho at Systems Metabolic Engineering and Systems Healthcare Cross-Generation Collaborative Laboratory of KAIST for valuable advice during manuscript preparation. This work was supported by the C1 Gas Refinery Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (NRF-2016M3D3A1A01913250).

Author information

Authors and Affiliations

Authors

Contributions

S.Y.L. conceived and coordinated the project; S.Y.L. and J.B. designed research; J.B. and C.H.H. performed experiments and analysed data; C.H.H., J.H.A. and J.A.L. performed microbial cultivations. J.B., C.H.H., J.H.A., J.A.L. and S.Y.L. wrote the manuscript and all authors approved the final manuscript.

Corresponding author

Correspondence to Sang Yup Lee.

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Competing interests

The authors declare that they have competing financial interests as the work described in this paper is covered by patents filed including, but not limited to, KR1020200086811, KR102000755, US16472876, DE112017006592.5, CN201780084543.8 and IL267579, and is of commercial interest.

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Extended data

Extended Data Fig. 1 Recombinant plasmids and growth profiles of the FC1, 2, and 3 strains cultured using small amount of glucose.

a, Recombinant plasmids used to develop engineered E. coli strains. b, Growth profiles of the FC1, 2, and 3 strains when 0.1 g L−1 of glucose was supplemented every 24 h. The orange line with an open circle represents the cell growth profile of the FC1 strain. The black line with an open diamond represents the cell growth profile of the FC2 strain. The pink line with open square represents the cell growth profile of the FC3 strain. Data presented are shown in average values with error bars representing ± standard deviations obtained in triplicate experiments (n = 3).

Source data

Extended Data Fig. 2 Isotope analyses of the FC1 and FC2 strains.

a, Metabolic pathways of E. coli synthesizing amino acids from CO2, FA, and glucose. Dashed lines represent the multiple-step pathway. b, 12C ratios in proteinogenic alanine (Ala) and phenylalanine (Phe) of the FC1 and 2 strains. P values were calculated by two-tailed Student’s t-test (*P, 0.0002; **P, 0.003). c, M + 0, M + 1, and M + 2 proteinogenic tyrosine ratios of the FC1 and FC2 strains. P values were calculated by two-tailed Student’s t-test (*P, 0.0103; **P, 0.0493; ***P, 0.002). d, 12C ratios in proteinogenic valine (Val), threonine (Thr), aspartate (Asp), glutamate (Glu), glycine (Gly), and serine (Ser) of the FC1 and 2 strains. Abbreviations of metabolites are: FBP, fructose 1,6-bisphosphate; E4P, erythrose 4-phosphate PEP, phosphoenolpyruvate; PYR, pyruvate, Ac-CoA, acetyl-CoA; OXA, oxaloacetate; 2KG, α-ketoglutarate; PHE, phenylalanine; TYR, tyrosine; GLY, glycine; SER, serine; ALA, alanine; VAL, valine; ASP, aspartate; THR, threonine; GLU, glutamate. Data presented are shown in average values with error bars representing ± standard deviation obtained in triplicate experiments (n = 3). Open circles in graphs indicate individual data points.

Source data

Extended Data Fig. 3 Growth of the FC5 strain using CO2 and FA, FA only, CO2 only, and without CO2 and FA and colony forming unit profile cultured solely on CO2 and FA.

a, Images of culture broth of the FC5 strain at 0 and 100 h after cultivation. FA + and FA – represent with and without FA supplementation, respectively. CO2 + and CO2 – represent with and without CO2 supplementation, respectively. b, Colony forming unit profile of the FC5 strain cultured using CO2 and FA only. Data presented are shown in average values with error bars representing ± standard deviation obtained in triplicate experiments (n = 3). Black dots in graphs indicate individual data points.

Source data

Extended Data Fig. 4 Increase of 12C carbon ratios of the proteinogenic amino acids in the FC5 strain.

Increase of 12C ratios in proteinogenic alanine (Ala), glycine (Gly), valine (Val), serine (Ser), threonine (Thr), phenylalanine (Phe), aspartate (Asp) and glutamate (Glu) of the FC5 strain after 100 h of culture solely from CO2 and FA. Data presented are shown in average values with error bars representing ± standard deviation obtained in triplicate experiments (n = 3). Open circles in graph indicate individual data points.

Source data

Extended Data Fig. 5 Optimization of IPTG concentration and aeration condition.

a, Growth profiles of the FC5 strain cultured with different concentrations of isopropyl β-D-1-thiogalactopyranoside (IPTG) as an inducer. The light gray line with an open triangle represents cell growth profile of the FC5 strain when 1 mM of IPTG was supplemented. The gray line with an open square represents the cell growth profile of the FC5 strain when 0.1 mM of IPTG was supplemented. The black line with an open circle represents the cell growth profile of the FC5 strain when 0.05 mM of IPTG was supplemented. b, Growth profiles of the FC5 strain cultured in different aeration conditions. The FC5 strain was cultured using 300 mL baffled flask with a working volume of 50 mL (FC5 High aeration, dark blue line with open circle), 100 mL baffled flask with a working volume of 30 mL (FC5 Medium aeration, blue line with open square), and 100 mL Erlenmeyer flask with a working volume of 30 mL (FC5 Low aeration, light blue line with open triangle), respectively. c, Relative messenger RNA (mRNA) ratios of the ppsA and gcvT genes in the FC5 strain cultured supplementing FA, gas mixture and different concentrations of IPTG (1, 0.1, and 0.05 mM). Relative mRNA ratios were calculated by setting ppsA mRNA to rrsA mRNA and gcvT mRNA to rrsA mRNA ratios as 1, respectively, when cells were cultured supplementing 1 mM of IPTG. Data presented are shown in average values with error bars representing ± standard deviation obtained in triplicate experiments (n = 3). Open circles in graph indicate individual data points.

Source data

Extended Data Fig. 6 CO2 and FA assimilation and FA consumption by the FC8 strain.

a, Increase of 12C ratios in proteinogenic alanine (Ala), glycine (Gly), valine (Val), serine (Ser), threonine (Thr), phenylalanine (Phe), aspartate (Asp), and glutamate (Glu) of the FC8 strain after 100 h of culture solely from CO2 and FA. b, Consumed FA by the FC5 and 8 strains after 100 h of cultivation. Red bars represent the consumed FA of the FC5 and FC8 strains. Data presented are shown in average values with error bars representing ± standard deviation obtained in triplicate experiments (n = 3). Open circles indicate individual data points.

Source data

Extended Data Fig. 7 Growth of the FC8 strain solely on CO2 and FA at lower culture temperatures.

a, Cell growth profiles of the FC8 strain cultured solely from FA and CO2 at 30 and 33 °C. The light blue line with an open circle represents the cell growth profile of the FC8 strain at 30 °C. The purple line with an open diamond represents the cell growth profile of the FC8 strain at 33 °C. The dashed line is the growth profile of the FC8 strain at 32 °C (Fig. 2d) and was provided for better comparison. b, Images of culture broths of the FC8 strain at 0 and 200 h after cultivation under different conditions. Cells were cultured at 32 °C. FA+ and FA– indicate with and without FA supplementation, respectively. CO2+ and CO2– indicate with and without CO2 supplementation, respectively. c, 12C ratios in proteinogenic alanine (Ala), glycine (Gly), valine (Val), serine (Ser), threonine (Thr), phenylalanine (Phe), aspartate (Asp), and glutamate (Glu) of the FC8 strain at 0, 140, and 260 h. Cells were first cultured using 13C labeled FA and U-13C-glucose. Afterward, cells were cultured at 32 °C using CO2 and FA only. d, Relative messenger RNA (mRNA) ratios of cyoA, cyoB, cydA, and cydB in the FC8 strain cultured at 32 °C compared with those in the FC8 strain cultured at 37 °C. Relative mRNA ratios were calculated by setting those four mRNA to rrsA ratios as 1, when cells were cultured at 37 °C. Data presented are shown in average values with error bars representing ± standard deviation obtained in triplicate experiments (n = 3). Black dots and open circles in graph indicate individual data points.

Source data

Extended Data Fig. 8 Growth properties of the FC8 strain in flask and bioreactor cultures.

a, Doubling time and specific growth rate profiles of the FC8 strain cultured at low initial inoculum (OD600 of 0.018). Data presented are shown in average values obtained in duplicate experiments (n = 2). b, Cell growth, FA concentration, and consumed FA profiles of a single bioreactor cultivation of the FC8 strain using agitation speed of 500 rpm. c, Doubling time and specific growth rate profiles of the Fig. 2g bioreactor culture. df, Reproduced bioreactor culture profiles of the FC8 strain solely on CO2 and FA. Data shown in d, e, and f are obtained from the second, third, and fourth bioreactor culture trials of the FC8 strain solely on CO2 and FA, respectively.

Source data

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2, Notes 1–10 and discussion.

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Source data

Source Data Fig. 1

Data presented in Fig. 1.

Source Data Fig. 2

Data presented in Fig. 2.

Source Data Extended Data Fig. 1

Data presented in Extended Data Fig. 1.

Source Data Extended Data Fig. 2

Data presented in Extended Data Fig. 2.

Source Data Extended Data Fig. 3

Data presented in Extended Data Fig. 3.

Source Data Extended Data Fig. 4

Data presented in Extended Data Fig. 4.

Source Data Extended Data Fig. 5

Data presented in Extended Data Fig. 5.

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Data presented in Extended Data Fig. 6.

Source Data Extended Data Fig. 7

Data presented in Extended Data Fig. 7.

Source Data Extended Data Fig. 8

Data presented in Extended Data Fig. 8.

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Bang, J., Hwang, C.H., Ahn, J.H. et al. Escherichia coli is engineered to grow on CO2 and formic acid. Nat Microbiol 5, 1459–1463 (2020). https://doi.org/10.1038/s41564-020-00793-9

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