Data from: Robust cellular transformations of PET deconstruction products by import of glycol esters
Data files
Jul 30, 2026 version files 186.32 KB
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Dryad_data_file_for_SMv2.xlsx
104.63 KB
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Dryad_Date_file.xlsx
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README.md
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Abstract
Efforts to transform polyethylene terephthalate (PET) deconstruction products using live cells have been limited by terephthalic acid (TPA) uptake. Here, we used an intracellular carboxylate reduction assay to show that apparent TPA uptake in E. coli cells that lack a dedicated TPA transporter sharply increases between pH 5-6. Furthermore, we discovered that glycol ester deconstruction products, mono(2-hydroxyethyl) terephthalate (MHET) and bis(2-hydroxyethyl) terephthalate (BHET), surprisingly each result in rapid pH-independent uptake. We exploited glycol ester uptake along with deletion of 22 cellular oxidoreductases to design intracellular hydrolysis routes for synthesis of upcycled reduction products from BHET at >90% yields, and from real PET wastes after tandem catalytic glycolysis and cell-based valorization at >80% combined yields. Our work has important ramifications for PET utilization by cells and adds new perspectives on the evolution of the PETase/MHETase system.
Dataset DOI: 10.5061/dryad.z612jm6qr
Description of the data and file structure
Files and variables
File: Dryad_Date_file.xlsx
Description: Data found in the main text figures
Fig 2. (B) Endpoint assay showing the conversion of acid substrates to their corresponding aldehydes after 4 h. (C) Time course of the relationship between pH and TPAL production from supplemented TPA. (D) Variation of initial pH, which led to conversion of TPA to TPAL at 4 h. Starting pH was measured before the addition of 5 mM TPA. (F) Time course of supplemented TPAL oxidation in resting RARE.Δ16 cells. (G) The newly engineered E. coli ROAR.Δ22 strain limits oxidation and reduction of TPAL compared to wild-type (MG1655) and previously engineered strains (RARE.Δ16 and ROAR.Δ12). ROAR.Δ22 cells with MaCAR enable efficient generation and stability of aldehydes (TPAL from TPA and MHET-ald from MHET) to determine uptake of (I) TPA and (J) MHET at pH ranges from 4.8-7.5. Sample sizes are N = 3 using biological replicates. Data shown are mean ± s.d.
Fig 3. Intercellular expression of ETases could allow for TPA to be produced within the cell, preventing the need for active transport. Supplementation of 5 mM (B) BHET (C) MHET or (E) MHET-ald to RARE.Δ16 cells expressing ETase variants in MOPS media. Endpoint concentrations were measured after 24 h. (F) Effect of ETase expression (induction indicated by dotted line at 3 h) on cellular growth. Evaluation of coupling FAST and MaCAR in growing cells in either separate strains (Strain A and B, respectively) or in the same strain (Strain C) for the production of TPAL from (H) TPA, (I) MHET or (J) BHET. Sample sizes are N = 3 using biological replicates. Data shown are mean ± s.d.
Fig 4. Endogenous oxidation can limit pXYL production in resting cells. (B) pXYL biosynthesis from TPAL at varying pH ranges. One-pot two-step production of pXYL from TPA, MHET, and BHET. TPA was added to reaction mixtures of Strain B at a starting pH of (D) 7.5 or (E) 5.2. After 2 h, the reaction mixture was titrated if needed to 7.5, and Strain D was added. (F) MHET and (G) BHET were added to Strains A, Strain B, and Strain C, as well as the optimum ratio of Strain A and B found previously. After 2 h, Strain D was added. Sample sizes are N = 3 using biological replicates. Data shown are mean ± s.d.
Fig 5. Catalytic glycolysis was done on polyester textile waste as well as PET plastic bottles. These products were then coupled with the proposed whole-cell cascade for the biosynthesis of pXYL. Strain C was added to resuspended PET deconstruction products from (B) polyester textile waste (red shirt) and (C) PET bottles. At 8 h, Strain D was added, and the reaction was conducted for an additional 8 h. Sample sizes are N = 2, and the data shown are mean ± s.d.
File: Dryad_data_file_for_SMv2.xlsx
Description: Data found in the supplemental material figures
Fig S1. RARE.Δ16 cells expressing MaCAR were cultured in MOPS media with 100 mM disodium phosphate at pH 7.4. Time course of analysis of the relationship between pH and TPAL production. Sample sizes are N = 3 using biological replicates. Data shown are mean ± s.d.
Fig S2. E. coli resting cells of MG1655, RARE.Δ16, ROAR.Δ12 and ROAR.Δ22 were supplemented in 200 mM HEPES (B, D) without or (C, E) with 25 mM glucose at pH 7.5. MHET-ald stability was measured at (B, C) 2 h and (D, E) 4 h. Samples sizes are N = 3 using biological replicates. Data shown are mean ± s.d.
Fig S3. Growth of wild-type E. coli MG1655, RARE.Δ16, ROAR.Δ12 and ROAR.Δ22 was monitored via OD600 in 96-well plate for 12 h in (A) LB media and (B) MOPS media with 2 % glucose. (C) Growth of the previously mentioned strains in 250 mL shake flasks with 50 mL of LB media. Growth was tracked every 0.5 h in duplicate via spectrophotometer measurements to measure optical density at 600 nm (OD600). Sample sizes are N = 3 using biological replicates. Data shown are mean ± s.d.
Fig S4. E. coli resting cells were cultured in LB media at 37 °C until mid-exponential phase, then dropped to 18 °C overnight for 18 h. The cells were washed in 200 mM HEPES without (B, D) or with 25 mM glucose (C, E) at pH 7.5 and then resuspended in the same buffer at 50 mg wet cell weight per mL. We measured TPAL stability at 2 h (B, C) and 4 h (D, E). Sample sizes are N = 3 using biological replicates. Data shown are mean ± s.d.
Fig S5. E. coli resting cells were cultured in LB media at 37 °C until mid-exponential phase, then dropped to 18 °C overnight for 18 h. The cells were washed and then resuspended in 200 mM HEPES at pH 5.8 (A, C) and pH 7.5 (B, D) at 50 mg wet cell weight per mL. We measured TPAL stability at 2 h (A, B) and 4 h (C, D). Sample sizes are N = 3 using biological replicates. Data shown are mean ± s.d.
Fig S7. Reaction concentrations starting from (B, C) MHET or (E, F) MHET-ald were measured after (B, E) 8 h and (C, F) 20 h. Sample sizes are N = 3 using biological replicates. Data shown are mean ± s.d.
Fig S8. Reaction diagram of a potential CAR and ETase coupled reaction with the brown arrow indicating a cascade branching point that could be utilized to be used to funnel reaction intermediates towards TPA or MHET-ald. Supplementation of (B, D) MHET or (C, E) MHET-ald to RARE.Δ16 cell expressing ICCG (B, C) or FAST (D, E) in MOPS media at pH of 7.4. Concentrations were measured for 20 h, with time points taken at 0, 1, 2, 4, 8, and 20 h. Sample sizes are N = 3 using biological replicates. Data shown are mean ± s.d.
Fig S10. TPAL biosynthesis using resting cells of FAST (strain A), MaCAR (strain B), and FAST + MaCAR (strain C) with (B, C) MHET and (E, F) BHET as starting substrates. Samples were taken at (B, E) 2 h and (C, F) 4 h. Samples sizes are N = 3 using biological replicates. Data shown are mean ± s.d.
Fig S11. pAMBA was targeted as a potential alternative valorization target. (C) Resting cells of FAST (strain A), MaCAR (strain B), and CvTA (strain D) were added to reaction mixtures with MHET independently and at varying cellular ratios. (D) Resting cells of FAST and MaCAR (strain C) and CvTA (strain D) were added to reaction mixtures with MHET independently and at varying cellular ratios. Sample sizes are N = 3 using biological replicates. Data shown are mean ± s.d.
Fig S12. Resting ROAR.Δ22 cells containing overexpressed FAST and MaCAR (strain C) at higher substrate loading (10, 20 and 40 mM) of MHET or BHET were incubated at 30 °C with 400 mM HEPES, 10 mM MgCl2, and 75 mM glucose. Reactions were sampled at (B, C) 4 h and (E, F) 8 h. Samples sizes are N = 3 using biological replicates. Data shown are mean ± s.d.
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