Chassis cell growing by using ethylene glycol as unique carbon source as well as preparation method and application of chassis cell

Abstract

The invention discloses a chassis cell growing by taking ethylene glycol as a unique carbon source as well as a preparation method and application of the chassis cell. In particular discloses a chassis cell which is a recombinant cell obtained by overexpressing gcl, hyi, glxR and glxK genes or gcl, hyi, glxR, glxK and glcDEF genes in a recipient cell. The chassis cell can grow in a culture medium taking ethylene glycol as a unique carbon source and / or energy source, has good ethylene glycol metabolism performance, can greatly reduce the cost of industrial chassis cells in production of products, realizes conversion from industrial wastes to high-added-value products, and has wide application prospects. Meanwhile, the chassis cells are used for treating and / or utilizing industrial wastes containing ethylene glycol, so that the harm of ethylene glycol in the industrial wastes to the environment and human bodies can be reduced, the efficient comprehensive utilization of ethylene glycol in the industrial wastes can be realized, and the chassis cells have important economic benefits and social benefits.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a chassis cell grown using ethylene glycol as the sole carbon source, its preparation method, and its application. Background Technology

[0002] Traditional industrial chassis cells have long relied heavily on glycobase-based feedstocks such as glucose and xylose to produce high-value-added products, thereby contributing to climate change mitigation and waste pollution control. However, due to the higher price of common glycobase feedstocks compared to petroleum, traditional industrial chassis cells face challenges in competing with the traditional petrochemical industry [1,2]. To address this challenge, non-glycobase feedstocks have emerged as viable alternatives. However, it is important to note that many alternative feedstocks cannot be naturally metabolized by traditional industrial chassis cells. Therefore, substrate toxicity, biocompatibility, preparation techniques and costs, as well as the development of appropriate metabolic pathways for substrate utilization must be considered. In fact, reconstructing large metabolic pathways in traditional industrial chassis cells has proven challenging in the past [3,4].

[0003] Ethylene glycol (EDG) stands as a promising non-glycosylated feedstock candidate within the traditional industrial chassis cell due to several key factors. First, EEG is a highly promising substrate for the orthogonal production of a variety of chemicals, thanks to its ability to minimize interactions with the chemical production pathway [4,5]. Second, under equimolar conditions, EEG exhibits a superior reducing equivalence ratio of 7.5:2 compared to conventional glucose, providing ample reducing power for chemical synthesis. Furthermore, EEG is economically viable, primarily derived from ethylene through the petrochemical industry. Notably, EEG can also be produced via the electrochemical conversion of carbon dioxide, demonstrating its potential for addressing climate change through carbon sequestration [6,7]. Additionally, EEG can be obtained through the bioconversion of glycerol, a renewable bioresource and a common waste product in the biodiesel and soap industries, offering opportunities for its recycling [8,9].

[0004] In addition, ethylene glycol is also one of the major degradation products of polyethylene terephthalate (PET) plastics, which is one of the most important pollutants on Earth and in the oceans

[10] . This approach highlights the potential of developing ethylene glycol as a non-sugar feedstock for upgrading the utilization of PET pollutants. Therefore, addressing the issue of ethylene glycol metabolism in industrial chassis cells has significant economic and social benefits, both from the perspective of non-sugar feedstocks for traditional industrial chassis cells and from the perspective of solving PET waste pollution. This also represents an important step toward the ultimate goal of transforming waste into valuable resources.

[0005] Escherichia coli (E. coli) has been engineered into industrial chassis cells due to its rapid growth and ease of genetic manipulation. However, wild-type E. coli cannot utilize ethylene glycol as a raw material, despite having a theoretical pathway that is thought to metabolize ethylene glycol. Albert Boronat et al. bred spontaneous mutants of E. coli that could grow solely on ethylene glycol as their only carbon and energy source, derived from mutants that could grow on propylene glycol

[11] . Subsequent analysis revealed a significant increase in the levels of propylene glycol oxygen reductase (fucO) and glycine dehydrogenase (aldA) in these spontaneous mutants. Based on these findings, subsequent studies have focused on obtaining E. coli that can metabolize ethylene glycol by overexpressing fucO and aldA. For example, by overexpressing endogenous genes fucO and aldA in E. coli strain MG1655, it was made to efficiently utilize ethylene glycol and produce ethylene glycol acid. It is worth noting that its growth medium requires additional glycerol in addition to ethylene glycol

[12] .

[0006] In addition, Smaranika Panda et al. enhanced the utilization of ethylene glycol by E. coli chassis cells by precisely regulating the expression levels of the fucO and aldA genes and adjusting the composition of the growth medium. They successfully produced various valuable aromatic chemicals, such as L-tyrosine, L-phenylalanine, and p-coumaric acid

[13] . However, obtaining E. coli chassis cells that rely solely on ethylene glycol as the only carbon and energy source remains challenging. Notably, the research by Smaranika Panda et al. also emphasized the importance of adding low concentrations of glycerol (0.1 g / L) or supplementing with amino acids to enhance the efficient utilization of ethylene glycol

[14] . Therefore, developing an E. coli chassis cell that relies solely on ethylene glycol as the main carbon and energy source and comprehensively analyzing its ethylene glycol metabolism is crucial for advancing the use of ethylene glycol as a non-glycosylated raw material. This work also lays the foundation for using biological processes to address climate change and solve the problems of PET pollution and glycerol waste.

[0007] In the theoretical pathway of ethylene glycol metabolism in E. coli, ethylene glycol undergoes multiple oxidation reactions to produce glyoxylic acid. Glyoxylic acid can serve as the sole carbon source to maintain the growth of E. coli through ethylene glycol metabolic pathway I, in which two molecules of glyoxylic acid condense to form hydroxymalonic acid

[15] . Another hypothesis suggests that glyoxylic acid can also enter the tricarboxylic acid cycle through the glyoxylic acid shuttle branch in ethylene glycol metabolic pathway II, in which glyoxylic acid condenses with acetyl-CoA to produce malic acid

[16] . These two pathways and mechanisms require further research to verify.

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[0019]

[11] A.Boronat,E.Caballero,J.Aguilar,Experimental evolution of ametabolic pathway for ethylene glycol utilization by Escherichia coli,JBacteriol,153(1983)134-139.

[0020]

[12] A.V.Pandit,E.Harrison,R.Mahadevan,Engineering Escherichia colifor the utilization of ethylene glycol,Microbial Cell Factories,20(2021).

[0021]

[13] S.Panda,J.F.J.Zhou,M.Feigis,E.Harrison,X.Ma,V.Fung Kin Yuen,R.Mahadevan,K.Zhou,Engineering Escherichia coli to produce aromatic chemicalsfrom ethylene glycol,Metab Eng,79(2023)38-48.

[14] S.Panda,V.Y.K.Fung,J.F.J.Zhou,H.Liang,K.Zhou,Improving ethylene glycol utilization inEscherichia coli fermentation,Biochemical Engineering Journal,168(2021).

[0022]

[15] RW Hansen, JAHayashi, Glycolate metabolism in Escherichia coli, JBacteriol, 83 (1962) 679-687.

[0023]

[16] A. Bock, G. Sawers, Escherichia coli and Salmonella: Cellular and molecular biology, 2nd edition, Fermentation, (1996) 262-282. Summary of the Invention

[0024] The technical problems to be solved by this invention are how to prepare chassis cells that can grow solely on ethylene glycol as the only carbon and energy source and / or how to treat and reuse industrial waste containing ethylene glycol.

[0025] To solve the above-mentioned technical problems, the present invention first provides a chassis cell, wherein the chassis cell may be A1) or A2) as follows:

[0026] A1) Recombinant cells obtained by overexpressing the following four genes in recipient cells:

[0027] (1) Genes of ethylene glycol hydroxylase (Gcl) or its functional fragments, derivatives, homologs or isoenzymes;

[0028] (2) Genes of hydroxypyruvate isomerase (Hyi) or its functional fragments, derivatives, homologs or isoenzymes;

[0029] (3) The gene for tartrate hemialdehyde reductase 2 (GlxR) or its functional fragments, derivatives, homologs or isoenzymes;

[0030] (4) Genes of glycerokinase 2 (GlxK) or its functional fragments, derivatives, homologs or isoenzymes;

[0031] A2) Recombinant cells obtained by overexpressing the following seven genes in recipient cells: the four genes mentioned in A1) and...

[0032] (5) Genes of glycolate dehydrogenase D (GlcD) or its functional fragments, derivatives, homologs or isoenzymes;

[0033] (6) Genes of glycolate dehydrogenase E (GlcE) or its functional fragments, derivatives, homologs or isoenzymes;

[0034] (7) Genes of glycolate dehydrogenase F (GlcF) or its functional fragments, derivatives, homologs or isoenzymes.

[0035] Furthermore, the chassis cells can be grown in a culture medium with ethylene glycol as the sole carbon source.

[0036] Furthermore, the chassis cells exhibit good performance in metabolizing ethylene glycol. Specifically, the chassis cells in A2) have even better performance in metabolizing ethylene glycol than the chassis cells in A1).

[0037] Furthermore, the recipient cell may be a microbial cell.

[0038] Furthermore, the recipient cells may include, but are not limited to, Escherichia coli cells, yeast cells, Bacillus subtilis cells, and Corynebacterium glutamicum cells.

[0039] The glycolate hydroxylase (Gcl), hydroxypyruvate isomerase (Hyi), tartrate hemialdehyde reductase 2 (GlxR), glycerate kinase 2 (GlxK), glycolate dehydrogenase D (GlcD), glycolate dehydrogenase E (GlcE), or glycolate dehydrogenase F (GlcF) described in this article may be derived from bacteria or fungi.

[0040] Furthermore, the glycolate hydroxylase (Gcl), hydroxypyruvate isomerase (Hyi), tartrate semialdehyde reductase 2 (GlxR), glycerol kinase 2 (GlxK), glycolate dehydrogenase D (GlcD), glycolate dehydrogenase E (GlcE), or glycolate dehydrogenase F (GlcF) may be derived from Escherichia coli, yeast, Bacillus subtilis, or Corynebacterium glutamicum.

[0041] In this document, the EC number of ethylene glycol hydroxylase (Gcl) is 4.1.1.47, and its amino acid sequence may be SEQ ID No. 1. The EC number of hydroxypyruvate isomerase (Hyi) is 5.3.1.22, and its amino acid sequence may be SEQ ID No. 2. The EC number of tartrate hemialdehyde reductase 2 (GlxR) is 1.1.1.60, and its amino acid sequence may be SEQ ID No. 3. The EC number of glycerate kinase 2 (GlxK) is 2.7.1.165, and its amino acid sequence may be SEQ ID No. 4. The EC number of ethylene glycol dehydrogenase D (GlcD) is 1.1.99.14, and its amino acid sequence may be SEQ ID No. 5. The EC number of ethylene glycol dehydrogenase E (GlcE) is 1.1.99.14, and its amino acid sequence may be SEQ ID No. 6. The EC number of the glycolate dehydrogenase F (GlcF) is 1.1.99.14, and its amino acid sequence may be SEQ ID No.7.

[0042] Furthermore, the ethylene glycol hydroxylase (Gcl) derivative may be B1) or B2) as follows:

[0043] B1) A protein having more than 80% identity and the same function as the protein shown in SEQ ID No. 1, obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No. 1; B2) A fusion protein having the same function, obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID No. 1 or B1).

[0044] Furthermore, the hydroxypyruvate isomerase (Hyi) derivative may be C1) or C2) as follows:

[0045] C1) A protein having more than 80% identity and the same function as the protein shown in SEQ ID No. 2, obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No. 2; C2) A fusion protein having the same function obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID No. 2 or C1).

[0046] Furthermore, the tartrate hemialdehyde reductase 2 (GlxR) derivative may be either D1) or D2):

[0047] D1) A protein having more than 80% identity and the same function as the protein shown in SEQ ID No. 3, obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No. 3; D2) A fusion protein having the same function obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID No. 3 or D1).

[0048] Furthermore, the glycerokinase 2 (GlxK) derivative may be E1) or E2) as follows:

[0049] E1) A protein having more than 80% identity and the same function as the protein shown in SEQ ID No. 4, obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No. 4; E2) A fusion protein having the same function obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID No. 4 or E1).

[0050] Furthermore, the glycolate dehydrogenase D (GlcD) derivative may be F1) or F2) as follows:

[0051] F1) A protein having more than 80% identity and the same function as the protein shown in SEQ ID No. 5, obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No. 5; F2) A fusion protein having the same function obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID No. 5 or F1).

[0052] Furthermore, the glycolate dehydrogenase E (GlcE) derivative may be either G1) or G2):

[0053] G1) A protein having more than 80% identity and the same function as the protein shown in SEQ ID No. 6, obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No. 6; G2) A fusion protein having the same function obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID No. 6 or G1).

[0054] Furthermore, the glycolate dehydrogenase F (GlcF) derivative may be either H1) or H2):

[0055] H1) A protein having more than 80% identity and the same function as the protein shown in SEQ ID No. 7, obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No. 7; H2) A fusion protein having the same function obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID No. 7 or H1).

[0056] This invention also provides the application of biomaterials in the preparation of chassis cells capable of growing in a culture medium using ethylene glycol as the sole carbon source, said biomaterials being any of the following:

[0057] I1) Nucleic acid molecule assembly 1, wherein the nucleic acid molecule assembly 1 is composed of nucleic acid molecules encoding ethylene glycol hydroxylase (Gcl), hydroxypyruvate isomerase (Hyi), tartrate semialdehyde reductase 2 (GlxR) and glycerate kinase 2 (GlxK), respectively;

[0058] I2) Nucleic acid molecule combination 2, wherein the nucleic acid molecule combination 2 is composed of nucleic acid molecules encoding glycosidase (Gcl), hydroxypyruvate isomerase (Hyi), tartrate semialdehyde reductase 2 (GlxR), glycerol kinase 2 (GlxK), glycosidic acid dehydrogenase D (GlcD), glycosidic acid dehydrogenase E (GlcE) and glycosidic acid dehydrogenase F (GlcF), respectively;

[0059] I3) contains an expression cassette or recombinant vector of the nucleic acid molecule combination 1 described in I1);

[0060] I4) contains an expression cassette or recombinant vector of the nucleic acid molecule combination 2 described in I2).

[0061] Furthermore, the application includes overexpressing either nucleic acid molecule combination 1 or nucleic acid molecule combination 2 in recipient cells.

[0062] Furthermore, the overexpression can be achieved by introducing the nucleic acid molecule combination 1, the nucleic acid molecule combination 2, the expression cassette, or the recombinant vector into recipient cells.

[0063] In the above applications, the nucleotide sequence of the nucleic acid molecule encoding ethylene glycol hydroxylase (Gcl) (also known as the gcl gene) is as shown in positions 89-1870 of SEQ ID No. 10; the nucleotide sequence of the nucleic acid molecule encoding hydroxypyruvate isomerase (Hyi) (also known as the hyi gene) is as shown in positions 1883-2659 of SEQ ID No. 10; the nucleotide sequence of the nucleic acid molecule encoding tartrate semialdehyde reductase 2 (GlxR) (also known as the glxR gene) is as shown in positions 2759-3637 of SEQ ID No. 10; the nucleotide sequence of the nucleic acid molecule encoding glycerate kinase 2 (GlxK) (also known as the glxK gene) is as shown in positions 3726-4871 of SEQ ID No. 10; and the nucleotide sequence of the nucleic acid molecule encoding ethylene glycol dehydrogenase D (GlcD) (also known as the glcD gene) is as shown in SEQ ID No. 10. The nucleotide sequence of the nucleic acid molecule encoding glycolate dehydrogenase E (GlcE) (also known as the glcE gene) is shown in positions 3806-4858 of SEQ ID No. 9; the nucleotide sequence of the nucleic acid molecule encoding glycolate dehydrogenase F (GlcF) (also known as the glcF gene) is shown in positions 4869-6092 of SEQ ID No. 9.

[0064] The present invention also provides a method for preparing chassis cells capable of growing in a culture medium with ethylene glycol as the sole carbon source, the method comprising overexpressing the four genes or the seven genes described herein in recipient cells.

[0065] In the above method, the nucleotide sequence of the ethylene glycol hydroxylase (Gcl) gene (gcl gene) is as shown in positions 89-1870 of SEQ ID No. 10; the nucleotide sequence of the hydroxypyruvate isomerase (Hyi) gene (hyi gene) is as shown in positions 1883-2659 of SEQ ID No. 10; the nucleotide sequence of the tartrate semialdehyde reductase 2 (GlxR) gene (glxR gene) is as shown in positions 2759-3637 of SEQ ID No. 10; the nucleotide sequence of the glycerate kinase 2 (GlxK) gene (glxK gene) is as shown in positions 3726-4871 of SEQ ID No. 10; and the nucleotide sequence of the ethylene glycol dehydrogenase D (glcD) gene (glcD gene) is as shown in SEQ ID No. 10. The nucleotide sequence of the glycolate dehydrogenase E (glcE) gene (glcE gene) is shown in positions 3806-4858 of SEQ ID No. 9; the nucleotide sequence of the glycolate dehydrogenase F (glcF) gene (glcF gene) is shown in positions 4869-6092 of SEQ ID No. 9.

[0066] In the above method, the overexpression can be achieved by introducing the four genes or the seven genes described herein into recipient cells.

[0067] Further, the introduction includes any of the following methods: (1) cloning the four genes into four identical or different plasmids and introducing them into recipient cells; (2) cloning the four genes into the same plasmid and introducing them into recipient cells; (3) arbitrarily combining the four genes and then cloning them into two or three identical or different plasmids and introducing them into recipient cells; (4) cloning the seven genes into seven identical or different plasmids and introducing them into recipient cells; (5) cloning the seven genes into the same plasmid and introducing them into recipient cells; (6) arbitrarily combining the seven genes and then cloning them into two, three, four, five or six identical or different plasmids and introducing them into recipient cells.

[0068] The overexpression methods described in this invention are not limited to those described above. Expression levels can also be increased at the genomic level, such as by changing the promoter or increasing the copy number, thereby achieving the performance of metabolizing ethylene glycol.

[0069] In one embodiment of the present invention, the method for preparing chassis cells may include the following steps:

[0070] 1) The gcl, hyi, glxR, and glxK genes were cloned into a vector to obtain a recombinant vector;

[0071] 2) The recombinant vector is introduced into recipient cells to obtain recombinant cells (i.e., the chassis cells that can grow in a culture medium with ethylene glycol as the sole carbon source).

[0072] In one embodiment of the present invention, the method for preparing chassis cells may include the following steps:

[0073] 1) The gcl, hyi, glxR, and glxK genes were cloned into a vector to obtain a recombinant vector;

[0074] 2) The glcD, glcE, and glcF genes were cloned into a vector to obtain a recombinant vector;

[0075] 3) The recombinant vectors described in 1) and 2) are introduced into the recipient cells to obtain recombinant cells (i.e., the chassis cells that can grow in a culture medium with ethylene glycol as the sole carbon source).

[0076] Furthermore, the vector can be any expression vector capable of expressing the gene described in this invention.

[0077] In the above method, the recipient cells may be Escherichia coli cells, yeast cells, Bacillus subtilis cells, or Corynebacterium glutamicum cells.

[0078] Chassis cells prepared according to any of the methods described herein are also within the scope of protection of this invention.

[0079] The present invention also provides the use of the chassis cells in any of the following:

[0080] J1) Application in the metabolism of ethylene glycol or in the preparation of products for the metabolism of ethylene glycol;

[0081] J2) Application in the preparation of fermentation products using only ethylene glycol as the sole carbon source and / or energy source;

[0082] J3) Application in the treatment and / or utilization of industrial waste containing ethylene glycol;

[0083] J4) Application in the preparation of products for the treatment and / or utilization of industrial waste containing ethylene glycol.

[0084] Furthermore, the metabolized ethylene glycol may be ethylene glycol from the degradation products of metabolized PET.

[0085] Furthermore, the industrial waste containing ethylene glycol may originate from, but is not limited to, the petrochemical industry, biodiesel industry, pharmaceutical industry, soap industry, printing and dyeing industry, and agriculture, forestry and food processing industry.

[0086] Furthermore, the industrial waste containing ethylene glycol may be derived from PET waste.

[0087] Furthermore, the industrial waste containing ethylene glycol may be a PET degradation product.

[0088] Further, the application described in J2) includes: fermenting any of the chassis cells described herein in a culture medium in which ethylene glycol is the sole carbon source and / or energy source to obtain fermentation products.

[0089] Furthermore, the application described in J3) or J4) includes co-culturing the chassis cells described in any of the present invention with the industrial waste containing ethylene glycol. This not only reduces the harm of ethylene glycol in industrial waste to the environment and human body, but also reuses the ethylene glycol in industrial waste, which can greatly reduce the cost of industrial chassis cells in product production and realize the transformation of industrial waste into high value-added products.

[0090] The PET degradation products described in this article can be obtained by degrading PET or PET-containing waste through methods such as hydrolysis, enzymatic hydrolysis, or chemical depolymerization. The main PET degradation products include terephthalic acid (TPA) and ethylene glycol (EG).

[0091] The present invention also provides the application of the nucleic acid molecule combination 2 or an expression cassette or recombinant vector containing the nucleic acid molecule combination 2 in improving the performance of recipient cells in metabolizing ethylene glycol.

[0092] Furthermore, the application includes overexpressing the nucleic acid molecule combination 2 in recipient cells.

[0093] Furthermore, the overexpression can be achieved by introducing the nucleic acid molecule combination 2 or an expression cassette or recombinant vector containing the nucleic acid molecule combination 2 into recipient cells.

[0094] The recipient cells described herein may be Escherichia coli cells, yeast cells, Bacillus subtilis cells, Corynebacterium glutamicum cells, or any of the chassis cells described herein.

[0095] This invention first utilizes ultraviolet radiation mutagenesis to treat the wild-type E. coli MG1655 strain, enabling it to grow using ethylene glycol as the sole carbon source. Subsequently, laboratory adaptive evolutionary techniques were used to further optimize the ethylene glycol metabolism capacity of E. coli. Transcriptome analysis elucidated the mechanism of ethylene glycol metabolism in the mutant strains. Based on the results of comparative transcriptome analysis, this invention successfully engineered four wild-type E. coli strains through reverse metabolic engineering, enabling them to grow using ethylene glycol as the sole carbon source. The inventors of this invention elucidate the mechanism of ethylene glycol metabolism for the first time and discovered that overexpression of downstream genes (gcl, hyi, glxR, and glxK) is sufficient to enable recipient cells to grow using ethylene glycol as the sole carbon source. Furthermore, simultaneous overexpression of both downstream genes (gcl, hyi, glxR, and glxK) and midstream genes (glcD, glcE, and glcF) enhances the ethylene glycol metabolism performance of recipient cells.

[0096] This invention bred strains EG01 and EG02 from E. coli MG1655 through ultraviolet radiation mutagenesis and laboratory adaptive evolution. Both strains are capable of growth using ethylene glycol as the sole carbon source. The domesticated EG02 strain can metabolize ethylene glycol independently for growth at a rate of 8.10 ± 1.31 mmol / g DW.h. Overexpression of downstream genes (gcl, hyi, glcR, and glxK) in four wild-type E. coli strains (MG1655, DH5α, BL21(DE3), and ATCC8739) enables them to grow independently using ethylene glycol as both the sole carbon and energy source. Simultaneous overexpression of both downstream genes (gcl, hyi, glxR, and glxK) and midstream genes (glcD, glcE, and glcF) enhances their ethylene glycol metabolism performance. Among them, the chassis cell EG-BL21(DE3)(Down+Mid), which simultaneously overexpresses downstream genes (gcl, hyi, glxR and glxK) and midstream genes (glcD, glcE and glcF), has the best performance in metabolizing ethylene glycol. It can completely metabolize 87 mM ethylene glycol in PET degradation products within 96 hours.

[0097] This invention provides a practical solution to the problem of PET plastic waste pollution. Simply adding M9 salt to PET degradation products converts them into a non-glycosylated carbon source for E. coli chassis cells. Our invention also identifies the importance of downstream genes in the metabolism of ethylene glycol, an aspect often overlooked in traditional research.

[0098] This invention not only deepens the understanding of E. coli's metabolism of ethylene glycol, but also positions ethylene glycol as a highly promising non-glycosylated feedstock candidate for E. coli chassis cells. The chassis cells of this invention can grow in media using ethylene glycol as the sole carbon and / or energy source and exhibit excellent ethylene glycol metabolism performance. This can significantly reduce the cost of industrial chassis cells in product manufacturing, enabling the conversion of industrial waste into high-value-added products. Furthermore, using the chassis cells of this invention to treat and / or utilize industrial waste containing ethylene glycol can not only reduce the environmental and human health hazards of ethylene glycol in industrial waste, but also achieve efficient and comprehensive utilization of ethylene glycol in industrial waste, resulting in significant economic and social benefits.

[0099] Terminology Definition

[0100] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, to better understand this invention, definitions and explanations of relevant terms are provided below.

[0101] The term "chassis cell" generally refers to artificially modified cells that have been incorporated into certain functional biological system modules and possess specific functions. Chassis cells are host cells for synthetic biological reactions; commonly used chassis cells include yeast, *Escherichia coli*, *Bacillus subtilis*, and *Corynebacterium glutamicum*. Chassis cell culture can be performed according to conventional methods in the art, including but not limited to plate culture, shake flask culture, batch culture, continuous culture, and fed-batch culture, and various culture conditions such as temperature, time, and pH of the culture medium can be appropriately adjusted according to actual conditions. In one or more embodiments of the present invention, the chassis cell refers to artificially modified *Escherichia coli* cells that can grow in a culture medium using ethylene glycol as the sole carbon source and / or energy source, while exhibiting good ethylene glycol metabolism performance. In this document, the term "chassis cell" has the same meaning as "engineered bacteria" and "recombinant cell" and can be used interchangeably.

[0102] The term "recipient cell," also known as a host cell, generally refers to any type of cell that can be used to introduce a vector, such as microbial cells, animal cells, or plant cells. The recipient cell described in this invention can be a microbial cell, including bacteria, fungi, actinomycetes, and algae. The bacteria can be derived from genera such as *Escherichia* sp., *Erwinia* sp., *Agrobacterium* sp., *Flavobacterium* sp., *Alcaligenes* sp., *Pseudomonas* sp., and *Bacillus* sp. The fungi can be derived from genera such as *Saccharomyces* sp., *Fusarium* sp., *Rhizoctonia* sp., *Verticillium* sp., *Penicillium* sp., *Aspergillus* sp., and *Cephalosporium* sp. The algae can be derived from genera such as *Fucus* sp., *Achnanthes* sp., *Amphiprora* sp., *Amphora* sp., *Ankistrodesmus* sp., *Aste romonas* sp., and *Boekelovia* sp. The recipient cells are preferably *Escherichia coli* cells, yeast cells, *Bacillus subtilis* cells, or *Corynebacterium glutamicum* cells.

[0103] The term “functional fragment” in this document can be understood as a fragment or part of a full-length functional enzyme that still has the same or similar activity and / or function as the full-length functional enzyme.

[0104] The term "derivative" is intended to encompass enzyme variants obtained by substitution and / or deletion and / or addition of amino acid residues, fusion proteins obtained by tagging, and other amino acid modifications known to those skilled in the art that do not affect enzyme activity (such as capping, N-terminal acetylation, and C-terminal amidation). "Derivative" may have the same meaning herein as "variant" or "fusion protein".

[0105] The term "homologous" in this document can refer to different nucleic acids or proteins that are homologous or identical. Homologous sequences include homologous sequences from the same species and other species, as well as orthologous sequences from the same species and other species.

[0106] The term "isozyme" usually refers to enzymes in a living organism that catalyze the same reaction but have different molecular structures. In this article, it can be understood as enzymes that have the same or similar enzymatic activity and / or function but different primary structures.

[0107] The term "vector" generally refers to a vector capable of delivering exogenous DNA or a target gene into a host cell for amplification and / or expression. This vector can be a cloning vector or an expression vector. The vector can be introduced into a host cell through transformation, transduction, or transfection, allowing the genetic material it carries to be amplified and / or expressed within the host cell. Those skilled in the art can select a suitable vector based on the purpose of the genetic engineering and the nature of the recipient cell. The vectors include, but are not limited to, plasmids, phages (such as λ phage or M13 phage), cosmids (i.e., Cosmids), phagemids, and shuttle vectors (such as yeast expression vectors). The vectors may include, but are not limited to, the pET series, Duet series, pGEX series, pHY300, pHY300PLK, pPIC3K, pPIC9K, or pGAPZα series vectors. In one or more embodiments of the present invention, the vector is pRSFDuet, pET 32aDuet, or pACYCDuet vector.

[0108] The term "recombinant vector" generally refers to a recombinant DNA molecule constructed by linking a foreign target gene to a vector in vitro. It can be constructed in any suitable way, as long as the constructed recombinant vector can carry the foreign target gene into the recipient cell and provide the foreign target gene with the ability to replicate, integrate, amplify and / or express in the recipient cell.

[0109] The term "expression cassette" generally refers to a nucleic acid construct containing sufficient nucleic acid elements to express a target gene. A typical expression cassette includes a promoter, a multiple cloning site (MCS), and a terminator. Expression cassettes may also include the target gene, marker genes (such as TK, DHFR, CAT, and NEO genes), ribosome recognition and binding sites (SDs), transcription factor binding sites (TFBSs), enhancers, silencers, repressors, introns, poly(A) signal sequences, and / or mRNA splicing signal sequences. Elements within an expression cassette can be directly linked or indirectly linked through adapters.

[0110] The term "overexpression" generally refers to increasing or upregulating the level and / or activity of a target protein or gene. Overexpression can be achieved through regulation at the gene level (such as gene replication, transcription, translation, post-transcriptional modification, and / or post-translational modification) or by promoting or increasing the content, activity, and / or function of the target protein at the protein level. There are no particular limitations on the means of overexpression, and many methods for achieving overexpression are well known to those skilled in the art. For example, the nucleic acid molecule to be overexpressed or the nucleic acid molecule encoding the enzyme to be overexpressed can be placed under the control of a strong promoter; the copy number of one or more genes encoding the enzyme described in this invention can be increased; or the strength of the ribosome binding site or Kozak sequence can be increased, the stability of the mRNA can be improved, codon usage can be altered, or the stability of the enzyme can be increased, etc.

[0111] The term "introduction" generally refers to the transfer of a foreign gene into recipient cells, such as eukaryotic or prokaryotic recipient cells. There are no particular limitations on the method of introduction. The introduction can be achieved by transforming a vector carrying the DNA molecule of the present invention into host bacteria (recipient cells) using any known transformation method, such as chemical transformation (e.g., Ca2+-induced transformation, polyethylene glycol-mediated transformation, or metal cation-mediated transformation) or electroporation transformation; it can also be achieved by transducing the DNA molecule of the present invention into host bacteria (recipient cells) using bacteriophage transduction. The introduction can also be achieved by transfecting a vector carrying the DNA molecule of the present invention into host cells (recipient cells) using any known transfection method, such as calcium phosphate co-precipitation, liposome-mediated transfection, electroporation, or viral vector transfection.

[0112] The term "linkage" generally refers to the association of two or more molecules. Linkages can be covalent or non-covalent. The linkages described herein can be direct peptide bonds or linkages via linkers.

[0113] The term "tag" generally refers to a tagged protein fused to a target protein for the purpose of purification, detection, or tracing. Such tags include, but are not limited to: GST (glutathione thiotransferase) tagged protein, His-tag protein, MBP (maltose-binding protein) tagged protein, Flag tagged protein, SUMO tagged protein, HA tagged protein, Myc tagged protein, LacZ tagged protein, CBD (cellulose-binding domain) tagged protein, GFP (green fluorescent protein), CFP (cyan fluorescent protein), YFP (yellow-green fluorescent protein), mCherry (monomer red fluorescent protein), or AviTag tagged protein. Those skilled in the art know how to select a suitable tagged protein according to the desired purpose (e.g., purification, detection, or tracing). The use of a tag does not alter the function of the target protein; therefore, the tagged proteins applicable to this application are not limited to a specific type. The tag can be separated from the target protein by chemical lysis methods or enzymatic methods known in the art.

[0114] The term "identity" generally refers to the degree to which two (nucleotide or amino acid) sequences have identical residues at the same position in an alignment, and is usually expressed as a percentage. The identity described herein can refer to the identity of an amino acid sequence or a nucleotide sequence. Two copies having completely identical sequences have 100% identity. Those skilled in the art will recognize that the identity of an amino acid or nucleotide sequence can be determined using identity search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, the identity of an amino acid sequence can be calculated by using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Perresidue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and then performing a search to obtain the identity value (%). Alternatively, it can be determined using sequence analysis software such as CLC Main Workbench and MegAlign™, for example, using the computer program BLAST with default parameters, especially BLASTP or TBLASTN. In this document, the 80% or more of identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more of identity. Attached Figure Description

[0115] Figure 1To optimize the intensity of ultraviolet radiation-induced mutagenesis, E. coli chassis cells capable of metabolizing ethylene glycol were cultured. Specifically, the OD of E. coli MG1655 cells in the exponential growth phase was adjusted. 600 =1.0, diluted 10 to 10 3 Double 10 6 The solution was diluted 10 times and spread onto LB and M9(EG) medium plates, respectively. 3 Dilute 10⁻⁵ times, open the petri dish, and place it in a clean bench for UV irradiation for 2, 4, and 6 minutes. Since some UV light is blocked by the side walls of the petri dish, only the colony count within the elliptical areas of the petri dish is performed. 6 Single colonies were cultured directly in a 37°C incubator without UV radiation on M9 (EG) agar plates. Results showed that a single colony (marked with a yellow circle) was observed on the M9 (EG) agar plate after 6 minutes of irradiation. The red / green bar graph on the right represents the survival rate of *E. coli* under different UV radiation intensities.

[0116] Figure 2 High-performance liquid chromatography (HPLC) was used for the quantitative analysis of EG, TPA, MHET, and BHET. Figure 2 A: Ethylene glycol (EG); Figure 2 B: Terephthalic acid (TPA); Figure 2 C: Mono(2-hydroxyethyl) terephthalate (MHET); Figure 2 D: (2-Hydroxyethyl) terephthalate (BHET). The left side shows the HPLC peak chromatograms of the standard substance at different concentrations, and the right side shows the peak area and standard curve of the standard substance. Error bars represent the standard error (n=3).

[0117] Figure 3 This is a characteristic of the ethylene glycol metabolism of the UV-induced mutant bacterium EG01. Among them, Figure 3 In LB medium, the doubling times of EG01 (blue) and E. coli MG1655 (green) were 1.26 h and 1.58 h, respectively. In M9 (EG) medium with ethylene glycol as the sole carbon source, the doubling time of EG01 (black) was 5.22 h, while E. coli MG1655 showed no growth. The red line indicates the change in ethylene glycol consumption of EG01 in M9 medium. The initial addition of ethylene glycol as the carbon source was 10 g / L. Figure 3 B: Effects of different ethylene glycol concentrations on UV radiation-induced mutant EG01; Figure 3 C: Effect of culture temperature on UV-induced mutant EG01; error bars represent standard error (n=3).

[0118] Figure 4Laboratory adaptive evolution of UV-induced mutant EG01 was performed. EG01 was cultured continuously for 24 days, subcultured every two days, and passaged 12 times to obtain strain EG02. Compared to EG01, in M9(EG) medium with ethylene glycol as the sole carbon source, after 48 hours of incubation, the final biomass increased from 0.21±0.43 OD to 2.09±0.64 OD, and the ethylene glycol consumption rate also increased from 4.82±0.85 mmol / g DW.h to 8.10±1.31 mmol / g DW.h. The initial addition of ethylene glycol as the carbon source was 10 g / L. Error bars represent standard error (n=3).

[0119] Figure 5 To compare the transcriptome analysis of the ethylene glycol metabolism mechanism in *E. coli* chassis cells EG02, the theoretical metabolic pathway of ethylene glycol in *E. coli* can be divided into three stages: upstream (blue), midstream (red), and downstream (yellow), regulated by upstream genes (fucO and aldA), midstream genes (glcDEF), and downstream genes (gcl, hyi, glcR, glxK, or glcB), respectively. Notably, except for the upstream genes, the midstream and downstream genes belong to the same operon structure. fucO, lactalaldehyde reductase; aldA, glycaldehyde dehydrogenase A; glcDEF, ethylene glycol dehydrogenase; glcB, malate synthase G; gcl, ethylene glycol hydroxylase; hyi, hydroxypyruvate isomerase; glxK, glycerate kinase 2; glxR, tartrate hemialdehyde reductase 2. EG, ethylene glycol; GLA, ethylene glycol aldehyde; GA, ethylene glycolic acid; GLO, ethylene glycolic acid; TSA, (2R)-tartaric acid semialdehyde; (OH)-PYR, hydroxypyruvate; GLR, D-glyceric acid; 2PG, 2-phospho-D-glyceric acid; MAL, malic acid. The black dashed boxes illustrate the data presentation style, and the FPKM values ​​reflect the expression level per kb of sequence length for each gene. The black solid boxes compare the reducing equivalents produced when glucose and ethylene glycol are used as a single carbon source.

[0120] Figure 6 To rationally construct E. coli substrate cells that rely solely on ethylene glycol for growth. Among them, Figure 6 A: Schematic diagram of plasmids used to construct the ethylene glycol metabolic pathway. Upstream plasmid: pRSFDuet-fucO / aldA; Midstream plasmid: pET 32aDuet-glcDEF; Downstream plasmid I: pACYCDuet-gcl / hyi / glxR / glxK; Downstream plasmid II: pACYCDuet-glcB. Figure 6B: Reverse metabolic engineering of *E. coli* MG1655 for growth using ethylene glycol alone. Up, Mid, DownI, DownII, DownI+up, and DownI+Mid represent the upstream, midstream, and downstream plasmids, respectively, transformed into *E. coli* MG1655 strain alone or in combination. The OD of the strain after 96 hours of culture... 600 The values ​​are represented by blue bars. The residual percentage of ethylene glycol is represented by a pink pie chart. Figure 6 C: Rational construction of E. coli chassis cells using ethylene glycol as the sole carbon and energy source. Four E. coli strains (MG1655, ATCC8739, DH5α, and BL21(DE3)) were individually or co-transformed with the Mid and DownI plasmids. The blue bars depict the OD of the cultured strains after 96 hours. 600 The values ​​are shown in red, while the red bars indicate the residual ethylene glycol content (EG, g / L). All ethylene glycol carbon source media were initially added at 10 g / L. Error bars represent the standard error (n = 3).

[0121] Figure 7 The effect of TPA on the growth of Escherichia coli chassis cells was investigated. E. coli strains EG-BL21(DE3)(Down) and EG-BL21(DE3)(Down+Mid), capable of metabolizing ethylene glycol, were inoculated into M9 medium containing different concentrations of ethylene glycol (16mM, 80mM, 161mM, 484mM, 806mM, and 161mM), and supplemented with an equimolar concentration of sodium terephthalate (TPA-Na2). Red columns represent the initial amount of ethylene glycol added to M9 medium; light red columns represent the residual amount of ethylene glycol after 96 hours of fermentation; green columns represent the initial amount of TPA added to M9 medium; light green columns represent the residual amount of TPA after 96 hours of fermentation; and blue columns represent the OD of the strain after 96 hours of fermentation. 600 Value. The error bar represents the standard error (n=3).

[0122] Figure 8 To completely metabolize ethylene glycol in PET degradation products. Among them, Figure 8 A: A schematic diagram of developing ethylene glycol, a product of PET enzymatic hydrolysis, into a non-glycosylated raw material. Figure 8 B: EG-BL21(DE3)(Down) Chassis cells completely metabolize PET enzymatic hydrolysis product ethylene glycol. Figure 8In the C group, EG-BL21(DE3)(Down+Mid) chassis cells completely metabolized PET enzymatic hydrolysate ethylene glycol. Serial dilutions of the PET hydrolysate were performed using M9(EG) medium containing 87 mM equimolar ethylene glycol at ratios of 0:5, 1:4, 4:1, and 5:0. Equal volumes of EG-BL21(DE3)(Down) and EG-BL21(DE3)(Down+Mid) strains were inoculated into each culture medium. Red bars represent the amount of PET hydrolysate added to each medium. Green bars represent the amount of M9(EG) component added to each medium. Light red bars represent the residual ethylene glycol after 96 hours of fermentation. Blue bars represent the OD of the strain after 96 hours of fermentation. 600 Value. The error bar represents the standard error (n=3). Detailed Implementation

[0123] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0124] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0125] In the following examples, the composition of the LB liquid medium was: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, with the remainder being water. The LB plate medium was prepared by adding agar to the LB liquid medium to bring the final agar concentration to 15 g / L.

[0126] The M9 liquid culture medium in the following examples consisted of: 64 g / L Na₂HPO₄·7H₂O, 32 g / L KH₂PO₄, 2.5 g / L NaCl, 5 g / L NH₄Cl, 2 mM MgSO₄, 0.1 mM CaCl₂, and 1 mL / L trace element solution (1.6 g / L FeCl₃, 0.2 g / L CoCl₂·6H₂O, 0.1 g / L (NH₄)₂CuCl₄·2H₂O, 0.2 g / L ZnCl₂, 0.2 g / L Na₂MoO₄, 0.05 g / L H₃BO₃, 100 mM HCl). The M9 plate culture medium was prepared by adding agar to the M9 liquid culture medium to a final agar concentration of 15 g / L.

[0127] The method for preparing M9 liquid (or plate) culture medium containing 10 g / L ethylene glycol in the following examples is as follows: Ethylene glycol is added to the M9 liquid (or plate) culture medium to make the final concentration of ethylene glycol 10 g / L.

[0128] In the following examples, the M9 liquid culture medium containing 10 g / L ethylene glycol is referred to as M9(EG) liquid culture medium.

[0129] Example 1: Ultraviolet radiation-induced mutagenesis of E. coli MG1655, grown solely in ethylene glycol.

[0130] Studies have shown that wild-type *E. coli* cannot metabolize ethylene glycol. While *E. coli* strains constructed by overexpressing propylene glycol oxidoreductase (fucO) and glycolaldehyde dehydrogenase (aldA) can metabolize ethylene glycol, they cannot grow using ethylene glycol as their sole carbon and energy source. Only *E. coli* mutants capable of growing on propylene glycol can acquire the ability to utilize ethylene glycol as their sole carbon and energy source through adaptive evolution. Therefore, this invention employs ultraviolet radiation mutagenesis to treat *E. coli*, followed by screening for mutant strains using a culture medium containing only ethylene glycol as the sole carbon and energy source. To improve the success rate of obtaining *E. coli* mutants capable of metabolizing ethylene glycol as their sole carbon and energy source, this invention optimizes the intensity of ultraviolet radiation mutagenesis.

[0131] (1) Optimize the intensity of ultraviolet radiation-induced mutation

[0132] On day 1, a single colony of wild E. coli MG1655 was picked from the plate and inoculated into 3 mL of antibiotic-free LB liquid medium and incubated overnight at 37°C (250 rpm).

[0133] The next day, in a sterile laminar flow hood, E. coli MG1655 cells were collected by centrifugation, washed three times with sterile water to remove residual LB liquid medium, and then prepared into OD medium. 600 =1 bacterial suspension. 100 μL of bacterial suspensions at different dilution gradients were spread onto LB agar plates. 10 of these dilutions... 3 The diluted agar plates were placed in a clean bench, the lids opened, and subjected to UV radiation mutagenesis for 2, 4, and 6 minutes, then transferred to a 37°C incubation chamber for single colonies (N1); 10 6 After dilution, the plates were directly incubated at 37°C. Single colonies were used for viability counting (N2). The UV radiation survival rate (SR) of wild-type E. coli MG1655 strain was calculated using the following formula, thus completing a semi-quantitative assessment of the intensity of UV radiation mutagenesis. N1 and N2 represent the number of single colonies with and without UV irradiation, respectively.

[0134] SR = N1 / N2 × 10 -4 %

[0135] The results are as follows Figure 1 The results showed that the survival rates of wild-type E. coli MG1655 strain after 2, 4, and 6 minutes of ultraviolet radiation were 33.90 × 10⁻⁶. -4 %, 4.12×10 -4 % and 2.71×10 -4 The intensity of ultraviolet radiation-induced mutagenesis is positively correlated with the mutagenesis time, but the correlation is not a simple linear one. This completes the semi-quantitative determination of the intensity of ultraviolet radiation-induced mutagenesis.

[0136] (2) Ultraviolet radiation-induced mutagenesis of E. coli MG1655 in ethylene glycol-dependent growth alone

[0137] The above 10 3 The diluted E. coli MG1655 was spread on M9 agar plates containing 10 g / L ethylene glycol as the carbon source. The plates were opened and subjected to UV radiation for 4 and 6 minutes, respectively, and then transferred to 37°C for single colony incubation.

[0138] The results are as follows Figure 1 The results showed that after three days of continuous culture, a single colony of mutant bacteria, named EG01, grew on one of the plates that had been subjected to UV radiation mutagenesis for six minutes.

[0139] (3) Doubling time of UV-induced mutant EG01

[0140] On the first day, single colonies of E. coli MG1655 and UV-induced mutant EG01 were picked from the plates and inoculated into LB liquid medium and incubated overnight at 37°C (250 rpm).

[0141] The next day, in a sterile laminar flow hood, E. coli MG1655 and UV-induced mutant EG01 were collected by centrifugation. Residual LB liquid culture medium was removed by washing three times with sterile water, and then prepared as OD medium. 600 =1 bacterial suspension. Then, E. coli MG1655 and UV-induced mutant EG01 were transferred to 30 mL of antibiotic-free LB liquid medium and M9 liquid medium containing 10 g / L ethylene glycol, respectively, at an inoculation rate of 1‰, and cultured at 37℃ (250 rpm). Three parallel experiments were designed for each experimental group.

[0142] Samples of the bacterial cultures from the four experimental groups were taken at 0.5-hour intervals, and the concentrations were measured using a spectrophotometer. Growth curves for each strain were plotted, and the cell doubling time was calculated using the following formula:

[0143]

[0144] DT: Doubling Time; c jand c i It is time point t j and t i Bacterial OD measured at time 600 value.

[0145] The results are as follows Figure 3 The results showed that, under the same conditions, the doubling time of UV-induced mutant EG01 in LB medium was 1.26 h, slightly faster than that of E. coli MG1655 (1.58 h); the doubling time of UV-induced mutant EG01 in M9 medium with ethylene glycol as the carbon source was 5.22 h, while E. coli MG1655 showed no growth even after 84 h of culture in M9 medium with ethylene glycol as the carbon source.

[0146] (4) Metabolic consumption of ethylene glycol by ultraviolet radiation-induced mutant EG01

[0147] HPLC analysis method: The high performance liquid chromatograph was an Agilent 1290; the analytical column was a Bio-Rad Aminex HPX-87H column (300mm × 7.8mm); the column temperature was 60℃; the mobile phase was 5mM H2SO4 aqueous solution, the flow rate was 0.8ml / min; and the ultraviolet detection wavelength was 240nm.

[0148] Ethylene glycol standard curve: A stock solution of 10 g / L ethylene glycol was prepared. This stock solution was then diluted to concentrations of 0.05 g / L, 0.1 g / L, 0.5 g / L, 1 g / L, and 2 g / L, respectively, and analyzed by HPLC. The peak areas were linearly fitted to known concentrations to plot a standard curve. The results are as follows: Figure 2 As shown in Figure A, the ethylene glycol concentration X (g / L) and peak area Y (10) are... 3 The standard curve equation for Y is: Y = 234.5X + 2.740(R) 2 =0.998)

[0149] The culture medium of UV-induced mutant EG01 before transfer and the cultures after different fermentation times at 1‰ inoculum were centrifuged at 6000g for 10 min at 4℃, filtered through a 0.22μm sterile filter membrane, and detected by high-performance liquid chromatography (HPLC). The results are as follows: Figure 3 As shown in Figure A, approximately 50% of the ethylene glycol in the M9 medium was metabolized and consumed.

[0150] (5) Effect of raw material ethylene glycol concentration on UV radiation-induced mutant EG01

[0151] On the first day, a single colony of UV-induced mutant EG01 was picked from the plate and inoculated into LB liquid medium and incubated overnight at 37°C (250 rpm).

[0152] The next day, in a sterile laminar flow hood, the UV-induced mutant bacteria EG01 were collected by centrifugation, washed three times with sterile water to remove residual LB liquid culture medium, and then prepared into OD medium. 600 =1 bacterial suspension. Then, the UV-induced mutant EG01 was transferred to 30 mL of M9 liquid medium containing 5 g / L, 10 g / L, 20 g / L, 30 g / L and 40 g / L ethylene glycol at an inoculation rate of 1‰, and cultured at 37℃ (250 rpm). Three parallel experiments were designed for each experimental group.

[0153] The results are as follows Figure 3 Figure B shows that the growth of the UV-induced mutant strain EG01 was enhanced when the ethylene glycol concentration increased from 5 g / L to 10 g / L. However, the growth of EG01 was inhibited at concentrations of 20 g / L and 30 g / L. At 40 g / L, the growth of EG01 was almost completely inhibited.

[0154] (6) Effect of culture temperature on UV-induced mutant EG01

[0155] On the first day, a single colony of UV-induced mutant EG01 was picked from the plate and inoculated into LB liquid medium and incubated overnight at 37°C (250 rpm).

[0156] The next day, in a sterile laminar flow hood, the UV-induced mutant bacteria EG01 were collected by centrifugation, washed three times with sterile water to remove residual LB liquid culture medium, and then prepared into OD medium. 600 =1 bacterial suspension. Then, the UV radiation-induced mutant EG01 was transferred to 30 mL of antibiotic-free M9 liquid medium containing 10 g / L ethylene glycol at an inoculation rate of 1‰. The culture was carried out at 30℃, 37℃ and 42℃ (250 rpm) respectively. Three parallel experiments were designed for each experimental group.

[0157] The results are as follows Figure 3 The results show that the optimal temperature for UV-induced mutagenesis EG01 is 37℃.

[0158] Example 2: Laboratory-adaptive ultraviolet radiation-induced mutant bacteria EG01

[0159] (1) Preparation of competent cells of EG01 strain

[0160] On day 1, EG01 glycerol bacteria were streaked onto LB agar plates. The plates were then incubated overnight at 37°C to obtain single colonies.

[0161] The next day, a single colony was picked and inoculated into a 30 mL sterile LB broth culture base and placed in a 250 mL Erlenmeyer flask, and incubated at 37°C with shaking at 250 rpm. When the OD of EG01... 600When the value approaches 0.6, remove the Erlenmeyer flask and place it in an ice box for 30 minutes. The following method follows the procedure for preparing the competent cell preparation kit: Pour the culture medium into a 50 mL centrifuge tube (Nest), centrifuge at 1,500 g for 5 minutes, and discard the supernatant. Add 3 mL of Solution A (stored at 4°C) to the centrifuge tube, resuspend the cells using a pipette, centrifuge at 1,500 g for 5 minutes, and discard the supernatant. Add 3 mL of Solution B (stored at 4°C) to each microcentrifuge tube, resuspend the cells using a pipette, then aliquot into 100 μL portions and freeze at -80°C for later use.

[0162] (2) Transformation tool plasmid ptrcDnaB-AID for EG01 strain

[0163] Remove the prepared EG01 competent cells from the -80℃ freezer and immediately place them in an ice box. Allow them to thaw slowly, then add 1 μL of the ptrc DnaB-AID tool plasmid (this plasmid is described in the following literature: Wang J, Zhao D, Li J, et al. Helicase-AID: A novel molecular device for base editing at random genomicloci[J]. Metabolic engineering, 67:396-402[2024-04-17]. DOI:10.1016 / j.ymben.2021.08.005. The function of this plasmid is to increase the random mutation rate of the whole genome of EG01 bacteria and accelerate domestication efficiency). Return the cells to the ice box and let them stand for about 30 minutes. Then, heat shock them in a 42℃ water bath for 90 seconds, quickly remove them, and place them on ice for 2 minutes. Then add 1 ml of LB liquid medium, place on a shaker, and revive at 250 rpm and 37°C for 45 min; take out a portion of the revived bacterial solution, spread it on an LB plate containing chloramphenicol, and place it in a 37°C incubator for 12 to 18 h.

[0164] (3) Laboratory adaptive evolution of UV-induced mutant EG01

[0165] Single colonies of EG01 transformed with the tool plasmid ptrcDnaB-AID were picked and activated in LB broth containing chloramphenicol. After centrifugation at 9,000g for 1 min, the bacterial sludge was washed three times with sterile water. The washed strain was then inoculated into Erlenmeyer flasks containing M9 basal medium (i.e., M9 broth containing 10 g / L ethylene glycol) with ethylene glycol as the sole carbon source for subculturing. The initial OD of each generation was... 600 Adjust to 0.1. Incubate at 37℃ and 250 rpm for 48 h, and measure OD daily using a microplate reader (Tecanlinded M200). 600Value. The bacteria were subcultured every two days for 12 consecutive generations. Afterward, the growth rate and condition of the bacteria were observed, and the domesticated strain was named EG02. Results are as follows: Figure 4 As shown, strain EG02 showed significantly improved growth after 48 hours of cultivation, with OD... 600 The value was 2.09±0.64, while that of EG01 was 0.21±0.03. Furthermore, the ethylene glycol uptake rate of EG02 was 8.10±1.31 mmol / g DW.h, exceeding that of EG01 (4.82±0.85 mmol / g DW.h). Therefore, the growth performance of *E. coli* chassis cells relying solely on ethylene glycol as a non-sugar feedstock was significantly improved.

[0166] Example 3: Comparative transcriptomic analysis of the mechanism of ethylene glycol metabolism in E. coli EG02 chassis cells

[0167] The EG02 glycerol bacteria, stored at -80℃, were removed and inoculated onto LB liquid medium and M9(EG) liquid medium for activation. The activated strains were then transferred to 200mL Erlenmeyer flasks for further culture. Cultured until OD... 600 At a concentration of 3, bacterial cells were collected by centrifugation at 9000 rpm for 5 min. Total RNA of EG02 was extracted from both bacterial strains (triple replicates for each sample). Strand-specific RNA-seq libraries were then prepared, and sequencing was performed on an Illumina NovaSeq 6000 platform, generating 150 bp conjoint reads (Genewiz, Tianjin, China). To estimate gene expression, the reads were aligned with a genome assembled using Bowtie2 (2.3.4.3). The number of reads for each gene was then calculated using HTSeq (v0.9.1). Differential expression of EG02 in LB liquid medium and M9(EG) liquid medium was analyzed using DESeq2R software (1.20.0). Thresholds for significant differential expression were defined as padj < 0.05 and |log2(fold change)| > 0.

[0168] The results are as follows Figure 5As shown, comparative analysis of differential gene expression along the ethylene glycol metabolic pathway in *E. coli* revealed that in the upstream pathway, enzymes responsible for the conversion of ethylene glycol to glycolaldehyde (fucO) and glycolaldehyde to glycolic acid (aldA) are located in different operons, exhibiting fold changes of 4.02 and 5.19, respectively. However, in the midstream pathway, the enzyme promoting the conversion of glycolic acid to glyoxylate (glcDEF) is located in the same operon, with fold changes of 9.40, 9.46, and 7.73, respectively. Furthermore, in the downstream pathway, glyoxylate can be converted to 2-phosphoglycerate via ethylene glycol degradation pathway I, involving four consecutive catalytic steps: gcl, hyi, glxR, and glxK, all located within the same operon. The fold changes of these four enzymes were 8.48, 9.38, 9.76, and 4.26, respectively. Additionally, glyoxylate can be converted to malate via the glyoxylate branch, as part of ethylene glycol degradation pathway II, catalyzed by glcB, with a fold change of 7.34. glcB and glcDEF (glcD, glcE, glcF) are located within the same midstream gene operon. Comparative transcriptome analysis, measured by FPKM, showed that, except for glxK, the fold change of the midstream and downstream genes was approximately twice that of the upstream gene. This difference helps explain why UV-induced mutant *E. coli* can utilize ethylene glycol as its sole carbon and energy source. Conversely, this also reflects a difference from traditional methods for constructing *E. coli* that metabolize ethylene glycol.

[0169] Example 4: Construction of Escherichia coli MG1655, which grows solely in dependence of ethylene glycol, using reverse metabolic engineering.

[0170] Comparative transcriptome analysis revealed that among the significantly upregulated genes were not only the upstream genes fucO and aldA, previously highlighted in previous studies, but also midstream genes (glcDEF, i.e., glcD, glcE, and glcF) and downstream genes (gcl, hyi, glxR, and glxK). Furthermore, the latter two were upregulated by twice the fold of the former, potentially making them more significant for further research. To verify the effects of midstream and downstream genes on ethylene glycol metabolism, we overexpressed the midstream gene (glcDEF) and the downstream genes (gcl, hyi, glxR, and glxK) in wild-type Escherichia coli MG1655.

[0171] (1) Cloning and constructing plasmids

[0172] The implementation of this invention requires the construction of four plasmids: pRSFDuet-fucO / aldA, pET 32aDuet-glcDEF, pACYCDuet-gcl / hyi / glxR / glxK, and pACYCDuet-glcB, as shown in Table 1. The construction steps are as follows:

[0173] Sequence analysis of the *E. coli* MG1655 genome was performed to determine the sequences of upstream genes fucO and aldA, midstream genes (glcDEF), and downstream genes (gcl, hyi, glxR, and glxK). Primers were designed and optimized using the J5 Device Editor online software, as shown in Table 2. These primers were then sent to Beijing Qingke Biotechnology Co., Ltd. for synthesis. PCR amplification was performed using pRSFDuet, pET32aDuet, pACYCDuet, and the *E. coli* MG1655 genome as templates.

[0174] The amplification system consisted of: 10 μl New England Biolabs Phusion 5× buffer, 1 μl dNTPs (10 mM each), 20 ng DNA template, 2 μl primers (10 μM each), 0.5 μl Phusion High-Fidelity DNA polymerase (2.5 U / μl), and 33.5 μl distilled water, for a total volume of 50 μl.

[0175] The amplification conditions were: 98℃ pre-denaturation for 2 minutes (1 cycle); 98℃ denaturation for 10 seconds, Tm value -5℃ annealing for 10 seconds, 72℃ extension for 30 seconds (30 cycles); 72℃ extension for 5 minutes (1 cycle).

[0176] The PCR products were assembled using the GoldenGate method. The assembly reaction system consisted of: 100 ng of PCR fragment, 1.5 μL of 10× buffer, 1.5 μL of 10× BSA, 1 μL of BsaI, 1 μL of T4 ligase, 1 μL of DpnI, and water added to bring the total volume to 15 μL.

[0177] The GoldenGate assembly reaction conditions are: 37℃ digestion for 3 minutes, 16℃ T4 ligase ligation for 4 minutes (25 cycles); 50℃ digestion for 5 minutes (1 cycle); 80℃ enzyme inactivation for 5 minutes (1 cycle).

[0178] Transformation of Trans10 competent cells. Calcium chloride transformation method: Add 50 μl of Trans10 competent cells (purchased from Beijing TransGen Biotech Co., Ltd.) and incubate on ice for 30 minutes. Heat shock at 42°C for 30 seconds, then immediately place on ice for 2 minutes. Add 250 μl of LB liquid medium and incubate at 30°C for 1 hour at 200 rpm. Transformed cells are plated on Str (100 mg / L), Amp (100 mg / L), and Cm (30 mg / L) antibiotic plates and cultured overnight at 37°C. Single clones are picked, plasmid DNA is extracted, and sequenced for verification. The correct plasmids are named pRSFDuet-fucO / aldA, pET 32aDuet-glcDEF, pACYCDuet-gcl / hyi / glxR / glxK, and pACYCDuet-glcB, respectively.

[0179] A schematic diagram of the plasmid constructing the ethylene glycol metabolic pathway is shown below. Figure 6 As shown in SEQ ID A. The nucleotide sequence of plasmid pRSFDuet-fucO / aldA is shown in SEQ ID No. 8; the nucleotide sequence of plasmid pET 32aDuet-glcDEF is shown in SEQ ID No. 9; the nucleotide sequence of plasmid pACYCDuet-gcl / hyi / glxR / glxK is shown in SEQ ID No. 10; and the nucleotide sequence of plasmid pACYCDuet-glcB is shown in SEQ ID No. 11.

[0180] (2) Preparation of E. coli MG1655 competent cells

[0181] The method for preparing E. coli MG1655 competent cells is the same as that in Example 2, “Preparation of competent cells of EG01 strain”.

[0182] (3) Transform E. coli MG1655 competent cells

[0183] The method for transforming E. coli MG1655 competent cells is as described in Example 2, using the "EG01 strain transformation tool plasmid ptrcDnaB-AID". The plasmids pRSFDuet-fucO / aldA, pET 32aDuet-glcDEF, pACYCDuet-gcl / hyi / glxR / glxK, and pACYCDuet-glcB were transformed into E. coli MG1655 competent cells, either individually or in combination.

[0184] (4) Reverse metabolic engineering to construct E. coli MG1655 that metabolizes ethylene glycol alone

[0185] To verify the effects of upstream, midstream, and downstream genes on ethylene glycol metabolism, we inoculated wild-type *E. coli* MG1655 transformed with pRSFDuet-fucO / aldA, pET32aDuet-glcDEF, pACYCDuet-gcl / hyi / glxR / glxK, and pACYCDuet-glcB, respectively, into M9 medium containing only 10 g / L ethylene glycol as a carbon source and incubated at 37°C for 48 h. The results are as follows: Figure 6 As shown in Figure B, overexpression of only downstream genes (gcl, hyi, glxR, and glxK) was sufficient to enable *E. coli* MG1655 to grow using ethylene glycol as the sole carbon source. In contrast, the midstream gene glcDEF and another downstream gene glcB did not enable *E. coli* MG1655 to grow solely on ethylene glycol. Similarly, consistent with previous studies, overexpression of only upstream genes fucO and aldA also failed to enable *E. coli* MG1655 to grow in media with ethylene glycol as the sole carbon source.

[0186] In addition, we co-transformed wild-type *E. coli* MG1655 with pRSFDuet-fucO / aldA and pACYCDuet-gcl / hyi / glxR / glxK; and co-transformed wild-type *E. coli* MG1655 with pET 32aDuet-glcDEF and pACYCDuet-gcl / hyi / glxR / glxK. The results showed that co-transformation with pET 32aDuet-glcDEF and pACYCDuet-gcl / hyi / glxR / glxK improved the ethylene glycol metabolism performance of wild-type *E. coli* MG1655, while co-transformation with pRSFDuet-fucO / aldA and pACYCDuet-gcl / hyi / glxR / glxK did not show a significant enhancement effect. This indicates that downstream and midstream gene clusters play a crucial and decisive role in ethylene glycol metabolism.

[0187] Example 5: Rational construction of engineered bacteria that metabolize ethylene glycol independently.

[0188] (1) Preparation of Escherichia coli DH5α, BL21(DE3) and ATCC8739 competent cells

[0189] The method for preparing competent Escherichia coli cells is the same as that in Example 2, “Preparation of competent cells of strain EG01”.

[0190] (2) Transformation of Escherichia coli DH5α, BL21(DE3) and ATCC8739 competent cells

[0191] The method for transforming E. coli competent cells is as described in Example 2, using the "EG01 strain transformation tool plasmid ptrcDnaB-AID". The plasmid pACYCDuet-gcl / hyi / glxR / glxK was transformed alone or together with pET 32aDuet-glcDEF into DH5α, BL21(DE3), and ATCC8739 competent cells.

[0192] The engineered bacteria obtained by transforming plasmid pACYCDuet-gcl / hyi / glxR / glxK into DH5α, BL21(DE3) and ATCC8739 respectively were named EG-DH5α(Down), EG-BL21(DE3)(Down) and EG-8739(Down).

[0193] The engineered bacteria obtained by co-transforming DH5α, BL21(DE3) and ATCC8739 with plasmids pACYCDuet-gcl / hyi / glxR / glxK and pET 32aDuet-glcDEF were named EG-DH5α(Down+Mid), EG-BL21(DE3)(Down+Mid) and EG-8739(Down+Mid), respectively.

[0194] (3) Rationally construct engineered bacteria that can metabolize and utilize ethylene glycol independently.

[0195] The results are as follows Figure 6 As shown in Figure C, transforming E. coli DH5α, BL21(DE3), and ATCC8739 with pACYCDuet-gcl / hyi / glxR / glxK alone enabled them to metabolize ethylene glycol and grow independently. Co-transforming pACYCDuet-gcl / hyi / glxR / glxK and pET 32aDuet-glcDEF further enhanced their ethylene glycol metabolism. Among them, strain BL21(DE3), co-transformed with plasmids pACYCDuet-gcl / hyi / glxR / glxK and pET 32aDuet-glcDEF, exhibited the best ethylene glycol metabolism ability and was named EG-BL21(DE3)(Down+Mid).

[0196] Example 6: Effect of PET degradation product TPA on E. coli chassis cells that metabolize ethylene glycol

[0197] Since both complete enzymatic hydrolysis and chemical depolymerization of PET plastic produce equimolar amounts of ethylene glycol and terephthalic acid (TPA), it is crucial to investigate the effect of TPA on the growth of ethylene glycol-metabolizing E. coli basal cells. E. coli EG-BL21(DE3)(Down) and EG-BL21(DE3)(Down+Mid) that metabolize ethylene glycol were cultured in M9 medium containing different concentrations of ethylene glycol, with equimolar levels of TPA-Na2 added. This was primarily due to the high solubility of TPA-Na2 in water, while TPA is almost insoluble in water.

[0198] (1) The method for high performance liquid chromatography (HPLC) detection of TPA is as follows:

[0199] The high-performance liquid chromatograph was an Agilent 1290; the analytical column was a Bio-Rad Aminex HPX-87H column (300 mm × 7.8 mm); the column temperature was 30 °C; the mobile phase was an aqueous solution of 40% methanol and 0.06% formic acid; the flow rate was 0.6 ml / min; and the UV detection wavelength was 240 nm.

[0200] TPA Standard Curve: Terephthalic acid was prepared into a 1 mM stock solution using DMSO. The stock solution was then diluted to 10 μM, 50 μM, 100 μM, 200 μM, and 500 μM for HPLC analysis. The peak areas were linearly fitted against known concentrations to plot a standard curve. Results are as follows: Figure 2 The diagram shows that TPA concentration X (μM) and peak area Y (10) are related to the concentration of TPA in μM and the peak area in μM. 3 The standard curve equation for is: Y = 31.43X - 27.35(R) 2 =0.9983).

[0201] (2) Effects of PET degradation product TPA on E. coli basal cells that metabolize ethylene glycol

[0202] On the first day, single colonies of EG-BL21(DE3)(Down) and EG-BL21(DE3)(Down+Mid) were picked from the plate and inoculated into LB liquid medium and incubated overnight at 37°C (250 rpm).

[0203] The next day, in a sterile laminar flow hood, EG-BL21(DE3)(Down) and EG-BL21(DE3)(Down+Mid) were collected by centrifugation. The samples were washed three times with sterile water to remove residual LB liquid culture medium and then prepared as OD. 600=1 bacterial suspension. Then, EG-BL21(DE3)(Down) and EG-BL21(DE3)(Down+Mid) were transferred to 30 ml of M9 medium containing 16 mM, 80 mM, 161 mM, 484 mM and 806 mM ethylene glycol at an inoculation rate of 1‰, respectively. An equimolar amount of TPANa2 (Aldrich product, cat: 932566) was added to the above medium, and the culture was carried out at 37°C for 96 hours (250 rpm). Three parallel experiments were designed for each experimental group.

[0204] The results are as follows Figure 7 The results showed that when the initial concentrations of ethylene glycol and TPA were 30 g / L (484 mM) or lower, the biomass of EG-BL21(DE3)(Down) and EG-BL21(DE3)(Down+Mid) at 37°C increased with increasing concentration. HPLC analysis revealed that when the concentrations of ethylene glycol and TPA were 16 mM, 80 mM, and 161 mM, ethylene glycol was almost completely consumed after 96 hours of fermentation, while a significant amount of ethylene glycol remained at a concentration of 484 mM. The TPA content remained essentially unchanged before and after fermentation in each experimental group. When the initial concentrations of ethylene glycol and TPA reached 806 mM, almost no ethylene glycol was metabolized after 96 hours of fermentation, and the growth of EG-BL21(DE3)(Down) and EG-BL21(DE3)(Down+Mid) strains was significantly inhibited. To investigate whether ethylene glycol or TPA inhibited the growth of EG-BL21(DE3)(Down) and EG-BL21(DE3)(Down+Mid), this invention designed two strains to be cultured in combination with 161 mM EG and 484 mM TPA. After culturing at 37°C for 96 h, their biomass was 3.01 OD and 3.27 OD, respectively. This result indicates that when the molar concentration of ethylene glycol does not inhibit the growth of *E. coli* chakra cells, an equimolar amount of TPA also does not impede their growth. Therefore, ethylene glycol from PET degradation products can be developed as a non-glycosylated carbon source for the growth of *E. coli* chakra cells.

[0205] Example 7: Ethylene glycol in the complete metabolic PET degradation products

[0206] Ethylene glycol, as a non-glycosylated carbon source for microbial cell factories, is widely available, typically obtained from the oxidation of ethylene in the petrochemical industry and also from the degradation products of PET plastics. Using ethylene glycol as a glycosylated feedstock helps reduce production costs by utilizing inexpensive waste, improves the competitiveness of bio-industrial products, and promotes sustainable development. In this invention, we discovered that simply adding M9 basic salt (i.e., M9 liquid culture medium) to the enzymatic hydrolysis products of PET can create a non-glycosylated carbon source for E. coli cell factories. A schematic diagram illustrating the development of ethylene glycol from PET enzymatic hydrolysis products into a non-glycosylated feedstock is shown below. Figure 8 As shown in Figure A.

[0207] (1) Preparation of PET enzymatic hydrolysate

[0208] Untreated PET waste (crystallinity less than 10%) was added to a 3L bioreactor (New Brunswick BioFlo 115, Eppendorf, Germany) containing 2.5L of glycine-sodium hydroxide buffer (pH 9.0, 100mM), and the solution was added at 0.4% W. enzyme / W PET Add purified DepoPETase enzyme in the specified proportion. Maintain the temperature at 50°C in a water bath and continuously stir at 500 rpm using a single impeller. Add 0.5M sodium hydroxide via a peristaltic pump to control the reaction pH at 8.65 ± 0.20. After 120 hours, add M9 basic salts according to the M9 medium formulation to prepare the medium for PETase degradation products.

[0209] (2) Detection of components in PET enzymatic hydrolysate

[0210] Since the products of PET enzymatic hydrolysis include not only monomeric ethylene glycol and terephthalic acid, but also potentially incompletely depolymerized mono(2-hydroxyethyl) terephthalate (MHET) and bis(2-hydroxyethyl) terephthalate (BHET), the components of the enzymatic hydrolysate were detected using high-performance liquid chromatography (HPLC).

[0211] The method for high-performance liquid chromatography (HPLC) detection of ethylene glycol is the same as that in step (4) of Example 1 above. The method for HPLC detection of TPA is the same as that in step (1) of Example 6 above. The methods for HPLC detection of mono(2-hydroxyethyl) terephthalate (MHET) and bis(2-hydroxyethyl) terephthalate (BHET) are as follows:

[0212] The high-performance liquid chromatograph was an Agilent 1290; the analytical column was a Bio-Rad Aminex HPX-87H column (300 mm × 7.8 mm); the column temperature was 30 °C; the mobile phase was an aqueous solution of 40% methanol and 0.06% formic acid; the flow rate was 0.6 ml / min; and the UV detection wavelength was 240 nm.

[0213] Preparation of MHET and BHET standard curves: Mono(2-hydroxyethyl) terephthalate and bis(2-hydroxyethyl) terephthalate were prepared into 1 mM stock solutions using DMSO. These stock solutions were then diluted to 5 μM, 25 μM, 50 μM, 100 μM, and 250 μM for HPLC analysis. The peak areas were linearly fitted against known concentrations to plot standard curves. Results are as follows: Figure 2 C and Figure 2 The standard curve equation for MHET concentration X (μM) and peak area Y, as shown in the D diagram, is: Y = 10.29X + 16.68(R²). 2 =0.9999); The standard curve equation for BHET concentration X (μM) and peak area Y is: Y = 6.01X - 6.61(R) 2 =0.9998).

[0214] HPLC analysis revealed that the enzymatic hydrolysis products of PET contained 87 mM EG, 87 mM TPA, and 32 mM MHET (PET hydrolysis intermediate). In addition, there was 100 mM glycine-sodium hydroxide buffer and trace amounts of residual DepoPETase.

[0215] (3) Metabolism of ethylene glycol, a product of PET enzymatic hydrolysis, by EG-BL21(DE3)(Down) and EG-BL21(DE3)(Down+Mid)

[0216] On the first day, single colonies of EG-BL21(DE3)(Down) and EG-BL21(DE3)(Down+Mid) were picked from the plate and inoculated into LB liquid medium and incubated overnight at 37°C (250 rpm).

[0217] The next day, in a sterile laminar flow hood, EG-BL21(DE3)(Down) and EG-BL21(DE3)(Down+Mid) were collected by centrifugation. The samples were washed three times with sterile water to remove residual LB liquid culture medium and then prepared as OD. 600=1 bacterial suspension. Then, to mitigate the influence of other components in the PET enzyme degradation products, such as MHET, on the growth of engineered E. coli chassis cells, the PET degradation product raw materials were serially diluted with M9 medium containing 87 mM equimolar ethylene glycol. Four mixed culture media were prepared at ratios of 0:5; 1:4; 4:1; 5:0 (volume ratio). EG-BL21(DE3)(Down) and EG-BL21(DE3)(Down+Mid) were transferred to 30 ml of the above four mixed culture media at an inoculation rate of 1‰, and cultured at 37℃ for 96 hours (250 rpm). Three parallel experiments were designed for each experimental group.

[0218] The results are as follows Figure 8 China B and Figure 8 As shown in Figure C, strains EG-BL21(DE3)(Down) and EG-BL21(DE3)(Down+Mid) can completely consume 87 mM of ethylene glycol in PET degradation products, achieving complete metabolic utilization of ethylene glycol in PET degradation products. This provides application value for engineered E. coli chassis cells and offers an innovative and promising solution to the problem of plastic pollution.

[0219] Table 1. Plasmids constructed and used in this invention

[0220]

[0221]

[0222] Table 2. Primers used in this invention

[0223]

[0224] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A chassis cell, characterized in that, The chassis cell is either A1) or A2) below: A1) Recombinant cells obtained by overexpressing the following four genes in recipient cells: (1) Genes of ethylene glycol hydroxylase or its functional fragments, derivatives, homologs or isoenzymes; (2) Genes of hydroxypyruvate isomerase or its functional fragments, derivatives, homologs or isoenzymes; (3) The gene for tartrate hemialdehyde reductase 2 or its functional fragments, derivatives, homologs or isoenzymes; (4) Genes of glycerokinase 2 or its functional fragments, derivatives, homologs or isoenzymes; A2) Recombinant cells obtained by overexpressing the following seven genes in recipient cells: the four genes mentioned in A1) and... (5) Genes of glycolate dehydrogenase D or its functional fragments, derivatives, homologs or isoenzymes; (6) Genes of glycolate dehydrogenase E or its functional fragments, derivatives, homologs or isoenzymes; (7) Genes of glycolate dehydrogenase F or its functional fragments, derivatives, homologs or isoenzymes.

2. The application of biomaterials in the preparation of chassis cells capable of growing in a culture medium using ethylene glycol as the sole carbon source, wherein the biomaterial is any one of the following: I1) Nucleic acid molecule combination 1, wherein the nucleic acid molecule combination 1 is composed of nucleic acid molecules encoding ethylene glycol hydroxylase, hydroxypyruvate isomerase, tartrate semialdehyde reductase 2 and glycerate kinase 2 respectively; I2) Nucleic acid molecule combination 2, wherein the nucleic acid molecule combination 2 is composed of nucleic acid molecules that respectively encode ethylene glycol hydroxylase, hydroxypyruvate isomerase, tartrate semialdehyde reductase 2, glycerol kinase 2, ethylene glycol dehydrogenase D, ethylene glycol dehydrogenase E and ethylene glycol dehydrogenase F. I3) contains an expression cassette or recombinant vector of the nucleic acid molecule combination 1 described in I1); I4) contains an expression cassette or recombinant vector of the nucleic acid molecule combination 2 described in I2).

3. The application according to claim 2, characterized in that, The nucleotide sequences of the nucleic acid molecules encoding glycolate hydroxylase are shown in positions 89-1870 of SEQ ID No. 10; the nucleotide sequences of the nucleic acid molecules encoding hydroxypyruvate isomerase are shown in positions 1883-2659 of SEQ ID No. 10; the nucleotide sequences of the nucleic acid molecules encoding tartrate semialdehyde reductase 2 are shown in positions 2759-3637 of SEQ ID No. 10; the nucleotide sequences of the nucleic acid molecules encoding glycerate kinase 2 are shown in positions 3726-4871 of SEQ ID No. 10; the nucleotide sequences of the nucleic acid molecules encoding glycolate dehydrogenase D are shown in positions 2307-3806 of SEQ ID No. 9; the nucleotide sequences of the nucleic acid molecules encoding glycolate dehydrogenase E are shown in positions 3806-4858 of SEQ ID No. 9; and the nucleotide sequences of the nucleic acid molecules encoding glycolate dehydrogenase F are shown in positions 4869-6092 of SEQ ID No.

9.

4. A method for preparing chassis cells capable of growing in a culture medium using ethylene glycol as the sole carbon source, characterized in that, The method includes overexpressing the four genes or the seven genes described in claim 1 in recipient cells.

5. The method according to claim 4, characterized in that, The nucleotide sequence of the glycolate hydroxylase gene is shown in positions 89-1870 of SEQ ID No. 10; the nucleotide sequence of the hydroxypyruvate isomerase gene is shown in positions 1883-2659 of SEQ ID No. 10; the nucleotide sequence of the tartrate semialdehyde reductase 2 gene is shown in positions 2759-3637 of SEQ ID No. 10; the nucleotide sequence of the glycerol kinase 2 gene is shown in positions 3726-4871 of SEQ ID No. 10; the nucleotide sequence of the glycolate dehydrogenase D gene is shown in positions 2307-3806 of SEQ ID No. 9; the nucleotide sequence of the glycolate dehydrogenase E gene is shown in positions 3806-4858 of SEQ ID No. 9; and the nucleotide sequence of the glycolate dehydrogenase F gene is shown in positions 4869-6092 of SEQ ID No.

9.

6. The method according to claim 4 or 5, characterized in that, The overexpression is achieved by introducing the four genes or the seven genes described in claim 1 into recipient cells.

7. The method according to any one of claims 4-6, characterized in that, The recipient cells are Escherichia coli cells, yeast cells, Bacillus subtilis cells, or Corynebacterium glutamicum cells.

8. Chassis cells prepared according to any one of claims 4-7.

9. The use of the chassis cell according to claim 1 or 8 in any of the following: J1) Application in the metabolism of ethylene glycol or in the preparation of products for the metabolism of ethylene glycol; J2) Application in the preparation of fermentation products using only ethylene glycol as the sole carbon source and / or energy source; J3) Application in the treatment and / or utilization of industrial waste containing ethylene glycol; J4) Application in the preparation of products for the treatment and / or utilization of industrial waste containing ethylene glycol.

10. The use of the nucleic acid molecule combination 2 as described in claim 2 or 3, or an expression cassette or recombinant vector containing the nucleic acid molecule combination 2, in improving the performance of recipient cells in metabolizing ethylene glycol.

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