In Vivo Rate of Formaldehyde Condensation with Tetrahydrofolate
Abstract
:1. Introduction
2. Results
3. Discussion
4. Materials and Methods
4.1. Strains and Genomic Modifications
4.2. Media and Growth Conditions
4.3. Formaldehyde Concentration Measurement
4.4. Isotopic-labeling Experiments
4.5. Flux Balance Analysis
- Reactions POR5 and GLYCK were removed from the model since they are not present in E. coli cells.
- The stoichiometry of the reaction THD2pp was changed, as the translocation uses only one proton instead of two.
- PFL and OBTFL were removed, since they are catalyzed by oxygen sensitive enzymes and therefore not active in aerobic conditions.
- Based on gathered evidence, FTHFLi was removed. This reaction is especially important in our case, since it could allow charging of THF with formate directly.
- We changed the directionality of three reactions in the nucleotide biosynthesis pathway (FTHFD, GARFT, and AICART) to be irreversible, in order to avoid nucleotides degradation to charge THF, since that is very unlikely to occur.
- We changed the thymidine periplasmic symporter reaction (THYMt3pp) to be reversible, based on our experimental evidence (adding thymidine to the media rescued growth in certain conditions).
- We knocked out the glycine cleavage system reaction and the GlyA reaction (GLYCL and GHMT2r).
- We added the formaldehyde-THF spontaneous condensation (THFSPONT) reaction: thf_c + fald_c -> mlthf_c + h2o_c.
- Removed the following reactions to avoid potential carbon fixation cycles: GLXCL, GLYCL, THRD, THRA2, THRA, SERD_L.
- Added NAD-dependent formate dehydrogenase (FDH), enabling the utilization of the formate that is created from formaldehyde as source of reducing power.
- Added the crotonoyl-CoA carboxylase reaction (EtMaCoA), required for the ethylmalonyl-CoA cycle: b2coa_c + nadph_c + co2_c + q8_c + h2o_c -> glx_c + ppcoa_c + nadp_c + q8h2_c.
- Added the propionyl-CoA carboxylase reaction (MMCDr), as required for recycling the propionyl-CoA, which was created by EtMaCoA: ppcoa_c + atp_c + hco3_c -> mmcoa__S_c + adp_c + h_c + pi_c.
- Made the methylmalonyl-CoA mutase reaction (MMM) reversible so that the methylmalonyl-CoA (mmcoa__S_c) can be recycled.
- Added the reactions of the serine cycle:
- ○
- serine-hydroxypyruvate amino-transferase (SGAT1): ser__L_c + akg_c -> glu__L_c + hpyr_c.
- ○
- glycine-glyoxylate amino-transferase (SGAT2): gly_c + akg_c -> glx_c + glu__L_c.
- ○
- malate thiokinase (MTK): mal__L_c + atp_c + coa_c -> malylcoa_c + adp_c + pi_c.
- ○
- malyl-CoA lyase (MCL): malylcoa_c -> accoa_c + glx_c.
4.6. Folate Profiling
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Strain | Genotype | Source |
---|---|---|
SIJ488 | K-12 MG1655 Tn7::para-exo-beta-gam; prha-FLP; xylSpm-IsceI | [23] |
DH5α | F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG purB20 φ80dlacZΔM15 Δ(lacZYA-argF)U169, hsdR17(rK-mK+), λ- | Lab collection |
Frm | SIJ488 ΔfrmRAB | [21] |
gcv_don | K-12 MG1655 ΔgcvTHP::Km | Lab collection |
ltaE_don | K-12 BW25113 ΔltaE::Km | [24] |
panB_don | K-12 BW25113 ΔpanB::Km | [24] |
glyA_don | K-12 BW25113 ΔglyA::Km | [24] |
C1AΔ3 | SIJ488 ΔfrmRAB ΔgcvTHP ΔglyA::Km | This study |
C1AΔ5 | SIJ488 ΔfrmRAB ΔgcvTHP ΔltaE ΔpanB ΔglyA::Km | This study |
Plasmid | Genes | Source |
pZASS | p15A ori; StrepR; Ppgi-20 | Lab collection |
pZASS-soxA | pZASS::soxA; sarcosine oxidase | [21] |
pZASS-CgadhA | pZASS::CgadhA; methanol dehydrogenase | [21] |
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He, H.; Noor, E.; Ramos-Parra, P.A.; García-Valencia, L.E.; Patterson, J.A.; Díaz de la Garza, R.I.; Hanson, A.D.; Bar-Even, A. In Vivo Rate of Formaldehyde Condensation with Tetrahydrofolate. Metabolites 2020, 10, 65. https://doi.org/10.3390/metabo10020065
He H, Noor E, Ramos-Parra PA, García-Valencia LE, Patterson JA, Díaz de la Garza RI, Hanson AD, Bar-Even A. In Vivo Rate of Formaldehyde Condensation with Tetrahydrofolate. Metabolites. 2020; 10(2):65. https://doi.org/10.3390/metabo10020065
Chicago/Turabian StyleHe, Hai, Elad Noor, Perla A. Ramos-Parra, Liliana E. García-Valencia, Jenelle A. Patterson, Rocío I. Díaz de la Garza, Andrew D. Hanson, and Arren Bar-Even. 2020. "In Vivo Rate of Formaldehyde Condensation with Tetrahydrofolate" Metabolites 10, no. 2: 65. https://doi.org/10.3390/metabo10020065