Engineering Escherichia coli for utilization of PET degraded ethylene glycol as sole feedstock

Abstract

From both economic and environmental perspectives, ethylene glycol, the principal constituent in the degradation of PET, emerges as an optimal feedstock for microbial cell factories. Traditional methods for constructing Escherichia coli chassis cells capable of utilizing ethylene glycol as a non-sugar feedstock typically involve overexpressing the genes fucO and aldA. However, these approaches have not succeeded in enabling the exclusive use of ethylene glycol as the sole source of carbon and energy for growth. Through ultraviolet radiation-induced mutagenesis and subsequent laboratory adaptive evolution, an EG02 strain emerged from E. coli MG1655 capable of utilizing ethylene glycol as its sole carbon and energy source, demonstrating an uptake rate of 8.1 ± 1.3 mmol/gDW h. Comparative transcriptome analysis guided reverse metabolic engineering, successfully enabling four wild-type E. coli strains to metabolize ethylene glycol exclusively. This was achieved through overexpression of the gcl, hyi, glxR, and glxK genes. Notably, the engineered E. coli chassis cells efficiently metabolized the 87 mM ethylene glycol found in PET enzymatic degradation products following 72 h of fermentation. This work presents a practical solution for recycling ethylene glycol from PET waste degradation products, demonstrating that simply adding M9 salts can effectively convert them into viable raw materials for E. coli cell factories. Our findings also emphasize the significant roles of genes associated with the glycolate and glyoxylate degradation I pathway in the metabolic utilization of ethylene glycol, an aspect frequently overlooked in previous research.

Keywords: Escherichia coli; Ethylene glycol; Laboratory adaptive evolution; Metabolic engineering; Non-sugar feedstock; Polyethylene terephthalate (PET); Transcriptome analysis.

Fig. 1

Characterization of ethylene glycol metabolism in E. coli MG1655 and UV-induced mutant E. coli. In LB medium, both EG01 (green) and E. coli MG1655 (blue) exhibited doubling times of 1.58 h⁻¹ and 1.26 h⁻¹, respectively. In M9 medium with ethylene glycol as the sole carbon source, EG01 (black) had a doubling time of 5.22 h.⁻¹, whereas the growth of E. coli MG1655, MG1655 (fucO) overexpressing fucO, and MG1655 (fucO/aldA) overexpressing both fucO and aldA was undetectable. The red line illustrates the change in ethylene glycol consumption by EG01 in M9 (10 g/L EG) medium. Error bars indicated standard error (n = 3)

Fig. 2

Enhancing E. coli chassis cells for ethylene glycol as a non-sugar feedstock. EG01 underwent continuous cultivation for 24 days, with transfers to fresh medium every two days, covering a span of 12 generations, resulting in the strain EG02. Compared to EG01, after 48 h of cultivation in M9 (10 g/L EG) medium, the final biomass increased from 0.2 ± 0.1 OD to 2.1 ± 0.6 OD, and the uptake rate of ethylene glycol (uptake rate of EG) also rose from 4.8 ± 0.8 mmol/gDW.h to 8.1 ± 1.3 mmol/gDW.h. Error bars indicated standard error (n = 3)

Fig. 3

Comparative transcriptome analysis of E. coli chassis cells for metabolic utilization of ethylene glycol. Whole transcriptome sequencing (RNA-seq) was performed to quantify gene expression in EG02 under two distinct conditions: LB liquid medium and M9 (10 g/L EG) medium. The analysis results are presented in the proposed pathways for ethylene glycol metabolism in E. coli. The gray dotted box provides an explanation of the data presentation style legend. FPKM (fragments per kilobase of exon model per million reads) indicates the expression level of each gene. The log₂ fold change represents the fold increase in gene expression of EG02 in M9 (10 g/L EG) medium compared to LB medium. The solid black box compares the production of reducing equivalents when using glucose and ethylene glycol as sole carbon sources. fucO, lactaldehyde reductase; aldA, aldehyde dehydrogenase A; glcDEF, glycolate dehydrogenase; glcB, malate synthase G; gcl, glyoxylate carboligase; hyi, hydroxypyruvate isomerase; glxK, glycerate 2-kinase 2; glxR, tartronate semialdehyde reductase 2. EG, Ethylene glycol; GLA, glycolaldehyde; GA, Glycolate; GLO, Glyoxylate; TSA, (2R)-tartronate semialdehyde; (OH)-PYR, hydroxypyruvate; GLR, D-glycerate; 2PG, 2-phospho-D-glycerate; MAL, malate

Fig. 4

Rational engineering of E. coli chassis cells utilizing ethylene glycol as the sole carbon source. A Schematic diagram illustrating plasmid construction for the pathways enabling ethylene glycol metabolism. Module I: PM93-fucO/aldA; Module II: PM93-glcD/glcE/glcF; Module III: PM93-gcl/hyi/glxK/glxR; Module IV: PM93-glcB. B Reverse metabolic engineering of E. coli MG1655 for ethylene glycol utilization. Individual or co-transformation of Module I, II, III, and IV plasmids into E. coli MG1655 strains. The OD₆₀₀ value of the strain after 72 h is shown in the blue bar graph, while the residual percentage of ethylene glycol (%) is depicted in the red pie chart. C Rational engineering of six E. coli chassis cells (E. coli MG1655, ATCC8739, E. coli DH5α, E. coli BL21(DE3), EG01, and EG02) utilizing ethylene glycol as the sole carbon source. The OD₆₀₀ value of the strains after 72 h is represented in the blue bar graph, while the residual amount of ethylene glycol (g/L) is shown in the red bar graph. M9 (10 g/L EG) medium was employed for culturing all E. coli strains mentioned above. Error bars indicate standard error (n = 3)

Fig. 5

The influence of TPA on the growth of ethylene glycol-utilizing E. coli chassis cells. A EG02; (B) EG-BL21(DE3). The E. coli strains, EG02 and EG-BL21(DE3), capable of utilizing ethylene glycol, were separately introduced into M9 medium containing varying concentrations of ethylene glycol, supplemented with equimolar doses of TPA-Na₂ (TPA). 1: 16 mM EG, 16 mM TPA; 2: 80 mM EG, 80 mM TPA; 3: 161 mM EG, 161 mM TPA; 4: 484 mM EG, 484 mM TPA; 5: 806 mM EG, 806 mM TPA; 6: 161 mM EG, 484 mM TPA. The red bars show the initial addition of ethylene glycol in M9 medium, with light red indicating the residual amounts after 72 h of fermentation. The green bars show the initial addition of TPA, with light green indicating the residual amounts after 72 h. The blue bars represent the OD₆₀₀ values of the strain after 72 h of fermentation. Error bars indicated standard error (n = 3)

Fig. 6

Complete metabolic utilization of ethylene glycol in PET degradation product. A Schematic illustration of developing PET enzymatic degradation products as a non-sugar feedstock. B EG02; C EG-BL21(DE3). The Impact of other components, such as MHET, in PET enzymatic degradation products on the growth of EG02 and EG-BL21(DE3). A gradient dilution of the PET degradation product was conducted with M9 medium containing 87 mM equimolar ethylene glycol. Four sets of media were prepared in ratios of 0:5, 1:4, 4:1, and 5:0, and equal amounts of EG02 and EG-BL21(DE3) were inoculated into each. Red bars indicated the addition of PET enzymatic degradation product components in each culture medium. Green bars indicated the addition of M9 components in each culture medium. Light red bars indicated the residual ethylene glycol after 72 h of fermentation. The blue bars represented the OD₆₀₀ value of the strain after 72 h of fermentation. Error bars indicated standard error (n = 3)