Assimilation of formic acid and CO2 by engineered Escherichia coli equipped with reconstructed one-carbon assimilation pathways

Abstract

Gaseous one-carbon (C1) compounds or formic acid (FA) converted from CO₂ can be an attractive raw material for bio-based chemicals. Here, we report the development of Escherichia coli strains assimilating FA and CO₂ through the reconstructed tetrahydrofolate (THF) cycle and reverse glycine cleavage (gcv) pathway. The Methylobacterium extorquens formate-THF ligase, methenyl-THF cyclohydrolase, and methylene-THF dehydrogenase genes were expressed to allow FA assimilation. The gcv reaction was reversed by knocking out the repressor gene (gcvR) and overexpressing the gcvTHP genes. This engineered strain synthesized 96% and 86% of proteinogenic glycine and serine, respectively, from FA and CO₂ in a glucose-containing medium. Native serine deaminase converted serine to pyruvate, showing 4.5% of pyruvate-forming flux comes from FA and CO₂ The pyruvate-forming flux from FA and CO₂ could be increased to 14.9% by knocking out gcvR, pflB, and serA, chromosomally expressing gcvTHP under trc, and overexpressing the reconstructed THF cycle, gcvTHP, and lpd genes in one vector. To reduce glucose usage required for energy and redox generation, the Candida boidinii formate dehydrogenase (Fdh) gene was expressed. The resulting strain showed specific glucose, FA, and CO₂ consumption rates of 370.2, 145.6, and 14.9 mg⋅g dry cell weight (DCW)^-1⋅h^-1, respectively. The C1 assimilation pathway consumed 21.3 wt% of FA. Furthermore, cells sustained slight growth using only FA and CO₂ after glucose depletion, suggesting that combined use of the C1 assimilation pathway and C. boidinii Fdh will be useful for eventually developing a strain capable of utilizing FA and CO₂ without an additional carbon source such as glucose.

Keywords: CO2; formate dehydrogenase; formic acid; glycine cleavage pathway; tetrahydrofolate cycle.

Fig. 1.

The rTHF cycle and reverse gcv pathway established in E. coli. Red arrows indicate heterologous pathways, black arrows indicate native pathways, and blue arrows indicate overexpressed native pathways. The red Xs indicate genes knocked out. Carbon-labeling patterns in glycine, serine, and pyruvate based on FA and CO₂ carbons are indicated. Blue- and orange-colored carbons originated from FA and CO₂, respectively. Numbers in each carbon represent the carbon number in each compound. Recombinant plasmids designed and used in this study are also shown.

Fig. 2.

¹³C isotope analysis of serine and methionine and FA consumption in the WT, THF1, THF2, THF3, and THF4 strains. The THF1 strain is equipped with the THF cycle, the THF2 strain is equipped with the rTHF cycle, the THF3 strain is equipped with a stronger rTHF cycle, and the THF4 strain is equipped with the THF cycle with additional overexpression of the native E. coli folD gene. The MR minimal medium containing glucose was supplemented with 2.7 g⋅L^{−1 13}C-labeled sodium FA (the equivalent of 1.84 g⋅L⁻¹ FA) and 2 g⋅L⁻¹ glycine. (A and B) The number of labeled carbons and their ratios in proteinogenic methionine (A) and serine (B). (C and D) FA concentration profile (C) and growth profile (D) of the THF1, THF2, THF3, and THF4 strains during FA assimilation through the THF cycle. Data are shown as average values with error bars representing the SD obtained in duplicate experiments (n = 2).

Fig. 3.

¹³C isotope analysis of glycine-, serine-, and pyruvate-forming flux in the WT, RG1, RG2, RG3, RG4, RG5, and RG6 strains which have the rTHF and/or reverse gcv pathway. (A) ¹³C isotope-labeled fraction in glycine carbon number one of the WT, RG1, and RG2 strains.¹³C-labeled NaHCO₃ (8.4 g⋅L⁻¹) was added to supply ¹³C-labeled CO₂. (B and C) Ratio of proteinogenic serine synthesized only from C1 sources (FA and CO₂) (red) or glucose (yellow) in the RG2, RG3, RG4, RG5, and RG6 strains (B). Pyruvate-forming flux from the C1 pathway in the RG2, RG3, RG4, RG5, and RG6 strains. Pyruvate-forming flux from C1 pathway represents the percentage of carbon flux of the C1 assimilation pathway in total pyruvate-forming flux from the C1 pathway and glucose (C). For the RG2, RG3, and RG4 strains, 2.7 g⋅L⁻¹ of ¹³C-labeled sodium FA (the equivalent of 1.84 g⋅L⁻¹ of FA) and 8.4 g⋅L⁻¹ of ¹³C-labeled NaHCO₃ were added to supply ¹³C-labeled FA and ¹³C-labeled CO₂, respectively. For the RG5 and RG6 strains, 2.7 g⋅L⁻¹ of ¹³C-labeled sodium FA (the equivalent of 1.84 g⋅L ¹ of FA) and 8.4 g⋅L⁻¹ of unlabeled NaHCO₃ were added. Data are shown as average values with error bars representing ± SD obtained in duplicate experiments for the WT, RG1, RG2, and RG3 strains (n = 2) and in triplicate for the RG4, RG5, and RG6 strains (n = 3).

Fig. 4.

(A and B) Cell growth and glucose and FA consumption profiles of the RG8 strain (A) and RG6 strain (B) in flask cultures. Cells were grown using M9 minimal medium initially supplemented with 5 g⋅L⁻¹ of glucose and 3.7 g⋅L⁻¹ of sodium FA (the equivalent of 2.5 g⋅L⁻¹ of FA). For the RG8 strain, the medium was supplemented with 3.7 g⋅L⁻¹ of ¹³C-labeled sodium FA (the equivalent of 2.5 g⋅L⁻¹ of FA) at the points indicated by the red arrows. Cells for ¹³C isotope analysis were taken at the P1, P2, and P3 points. (C, Center) Biosynthesis route from FA and CO₂ to various amino acids during cell growth from only FA and CO₂. Solid lines and arrows represent single-step pathways; dashed lines and arrows represent multiple-step pathways. 2KG, alpha-ketoglutarate; Ac-CoA, acetyl-CoA; Ala, alanine; E4P, erythrose 4-phosphate; FBP, fructose 1,6-bisphosphate; Glu, glutamate; Gly, glycine; Ile, isoleucine; Leu, leucine; OAA, oxaloacetate; PEP, phosphoenolpyruvate; Phe, phenylalanine; PYR, pyruvate; Ser, serine. (Left, Right, and Bottom Row) ¹³C-labeled proteinogenic amino acids ratios at three different points obtained from flask cultivation (red-outlined graphs) and bioreactor cultivation (blue-outlined graphs). Data from the flask cultivation are shown as average values with error bars representing ± SD obtained in triplicate (n = 3). (D and E) Cell growth and glucose and FA consumption profiles from the bioreactor cultivations of the RG8 strain (D) and the RG6 strain (E). Cells were grown in a bioreactor using M9 minimal medium initially supplemented with 5 g⋅L⁻¹ glucose and 3.7 g⋅L⁻¹ sodium FA (the equivalent of 2.5 g⋅L⁻¹ FA). For the RG8 strain, the medium was supplemented with a mixture of ¹³C-labeled FA and unlabeled FA (1:1 ratio; mole/mole) when the initially supplemented FA was almost depleted, and then the FA mixture was supplemented once again. After that, the medium was supplemented twice with unlabeled FA at the indicated time points. Cells for ¹³C isotope analysis were taken at the F1, F2, and F3 points. Blue lines with closed triangles indicate cell growth. Yellow lines with closed circles indicate glucose concentration. Gray lines with closed squares indicate FA concentration.