coli strains, lacking the adaptive mutations, cannot thrive on ethylene glycol. This phenomenon highlights the crucial role of adaptive mutations in enabling E. coli to utilize ethylene glycol as a sole carbon and energy source. These mutations are not merely beneficial; they are essential for survival.
coli MG1655, which had a doubling time of 1.5 h. However, in M9 medium, the mutant showed a significantly faster doubling time of 1.0 h compared to E. coli MG1655’s 1.5 h.
The figure shows the metabolic pathway of ethylene glycol (EG) in E. coli MG1655 and a UV-induced mutant strain. The mutant strain exhibits a significant change in the pathway, highlighting the importance of UV exposure in altering metabolic pathways. **Detailed Text:**
The figure presents a comparative analysis of ethylene glycol (EG) metabolism in two distinct strains of *E.
Additionally, we further elucidated the ethylene glycol metabolism capability of EG01. Increasing the feedstock ethylene glycol concentration from 5 to 10 g/L resulted in enhanced growth of EG01. However, growth inhibition occurred at concentrations of 20 g/L and 30 g/L. At 40 g/L, the growth of EG01 was nearly completely suppressed (Figure S3A). We also assessed EG01’s growth performance at temperatures of 30 ℃, 37 ℃, and 42 ℃, with the optimal growth temperature aligning with typical E. coli strains at 37 ℃ (Figure S3B). Enhancing E. coli chassis cells for ethylene glycol as a non-sugar feedstock Adaptive laboratory evolution produces evolved microbial strains with desired traits through the application of natural selection principles in a lab setting [22]. To address the limited growth of EG01 in liquid M9 (10 g/L EG) medium, we implemented laboratory adaptive evolution to enhance E. coli’s exclusive ability to grow on ethylene glycol as the sole source of carbon and energy. Additionally, to increase the mutation rate and expedite the acquisition of strains with enhanced ethylene glycol metabolism, we introduced DNAB-AID, a novel tool for random genomic base editing. Consequently, EG01, equipped with ptrcDnaB-AID, underwent continuous cultivation for 24 days, with transfers to fresh medium every two days, covering a span of 12 generations [23]. As depicted in Fig. 2, strain EG02 exhibited substantial growth improvement, achieving an OD value of 2.1 ± 0.6 compared to EG01’s 0.2 ± 0.1 after 48 h of cultivation. Moreover, EG02 demonstrated an ethylene glycol uptake rate of 8.1 ± 1.3 mmol/gDW·h, surpassing EG01’s 4.8 ± 0.8 mmol/gDW h in shake-flasks. Therefore, the growth performance of E. coli chassis cells relying solely on ethylene glycol as a non-sugar feedstock showed remarkable enhancement.
The summary provided focuses on the enhancement of Escherichia coli (E. coli) bioavailability in M9 minimal medium supplemented with 2% glucose (EG) medium. This enhancement is achieved through the addition of a specific compound, which is not explicitly named in the figure. This compound is believed to improve the growth and metabolic activity of E. coli, leading to increased bioavailability.
However, these methods often lead to undesirable side effects, such as reduced growth rates and altered metabolic profiles. Comparative transcriptome analysis of E. coli cells for metabolic utilization of ethylene glycol has been employed to identify novel genes involved in ethylene glycol metabolism.
coli, the primary pathway is the Embden-Meyerhof-Parnas (EMP) pathway. This pathway is responsible for the production of ATP, the energy currency of the cell. The EMP pathway is a central metabolic pathway that is involved in the breakdown of glucose and other carbohydrates. The EMP pathway is a ten-step process that involves the conversion of glucose to pyruvate. This process is highly regulated and involves several key enzymes.
Comparative transcriptome analysis revealed that, compared to the initial genes fucO and aldA, the subsequent genes (glcDEF, gcl, hyi, and glxR), except for glxK, exhibited approximately twice the fold changes. Additionally, their expression levels, measured by FPKM (Fig. 3), showed a notable increase of at least tenfold. This disparity provides insights into EG02’s capability to utilize ethylene glycol as the sole carbon and energy source for growth. This finding contrasts with conventional approaches for constructing ethylene glycol-utilizing strains, which primarily rely on the overexpression of fucO and aldA [11, 14]. 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 log 2 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 Full size image
This approach aims to develop a sustainable and efficient bioprocess for producing ethylene glycol, a valuable chemical compound used in various industries. The study investigated the metabolic pathways of E. coli cells under ethylene glycol as the sole carbon source. It was found that E. coli cells can utilize ethylene glycol for growth and biosynthesis, demonstrating its potential as a sustainable bioprocess. The study also explored the engineering of E.
The second module focuses on the adaptation of glycolate to the cellular environment, with the glyoxylate cycle being a key player. The third module involves the conversion of glycolate to glycine, catalyzed by the enzyme glyA. This module is crucial for the synthesis of essential amino acids. The fourth module focuses on the synthesis of ethylene glycol from glycine, a process that is essential for the regeneration of the initial substrate.
This finding suggests that the third module, containing genes involved in the utilization of ethylene glycol, plays a crucial role in this metabolic adaptation. The third module, as highlighted in Fig. 4B, consists of genes encoding for enzymes involved in the breakdown of ethylene glycol.
This paper explores the rational engineering of *Escherichia coli* MG1655 cells to utilize ethylene glycol as a sole carbon source. The authors present a detailed analysis of the metabolic pathways involved in ethylene glycol metabolism and propose a rational approach to engineer the bacteria for efficient ethylene glycol utilization. The study focuses on two key aspects:
coli’s resistance to the antibiotic ciprofloxacin. This finding suggests that the second module genes play a crucial role in mediating the antibiotic resistance response. The study also investigated the role of the third module genes in antibiotic resistance. The researchers found that co-overexpression of the third module genes with either the first or second module genes did not significantly affect E.
Complete metabolic utilization of ethylene glycol in PET degradation product The current effective approach to tackling PET plastic pollution involves the enzymatic or chemical depolymerization of the polymer into ethylene glycol and terephthalic acid (TPA) monomers [23,24,25,26]. However, the newly generated ethylene glycol poses environmental pollution concerns. From an environmental perspective, transforming ethylene glycol within PET degradation products into a non-sugar feedstock for an E. coli chassis cell emerges as the most efficient strategy to holistically address PET plastic pollution. Given that the complete enzymatic or chemical depolymerization of PET plastic results in equimolar proportions of ethylene glycol and TPA, it is crucial to explore the impact of TPA on the growth of ethylene glycol-utilizing E. coli chassis cells.
The ethylene glycol-utilizing E. coli strains, EG-BL21(DE3) and EG02 were cultured separately in M9 medium supplemented with varying concentrations of ethylene glycol (16 mM, 80 mM, 161 mM, 484 mM, 806 mM), along with equimolar levels of TPA-Na 2 . Ethylene glycol was nearly completely metabolized after 72 h at initial concentrations of 16 mM, 80 mM, and 161 mM. However, at an initial concentration of 30 g/L (484 mM), a significant amount of ethylene glycol remained post-fermentation. The TPA content in the culture media remained unchanged before and after fermentation across all groups. At an initial concentration of 50 g/L (806 mM) of ethylene glycol and TPA-Na 2 , ethylene glycol metabolism was notably hindered after 72 h, resulting in reduced growth of the ethylene glycol-utilizing E. coli strains (Fig. 5).
coli cells were grown in minimal media supplemented with 0.5% glucose and 0.5% ethylene glycol. The growth curves were plotted over time. The growth curves show that TPA significantly inhibits the growth of ethylene glycol-utilizing E. coli cells. This inhibition is more pronounced in the EG-BL21(DE3) strain, which is a high-copy-number plasmid strain.
To investigate the inhibitory effects of high concentrations of ethylene glycol or TPA-Na 2 on EG-BL21(DE3) and EG02 (Fig. 5), both strains were cultured using a combination of 10 g/L (161 mM) ethylene glycol and 484 mM TPA-Na 2 . Their biomass reached 3.01 OD and 3.27 OD after 72 h of culture at 37 °C. These findings, combined with those shown in Figure S3A, confirm that high concentrations of ethylene glycol primarily inhibit the growth of these E. coli chassis cells, rather than TPA-Na 2 . Therefore, ethylene glycol in PET degradation products can be developed as a non-sugar feedstock for ethylene glycol-utilizing E. coli chassis cells.
The current study focuses on the enzymatic degradation of ethylene glycol, a key component of PET, using DepoPETase. This approach aims to break down PET into its constituent monomers, ultimately leading to the development of a sustainable and environmentally friendly recycling process. **Key Points:**
* **DepoPETase’s Role:** DepoPETase is a highly efficient enzyme that plays a crucial role in the enzymatic degradation of PET.
