METHOD FOR PRODUCING HIGH VALUE-ADDED COMPOUNDS FROM POLYETHYLENE TEREPHTHALATE

Abstract

The present invention pertains to a method for producing high value-added compounds from polyethylene terephthalate. More specifically, the present invention demonstrates that a monomeric terephthalic acid obtained from the chemical hydrolysis of polyethylene terephthalate can be converted to high value-added aromatic compounds and aromatic-derived compounds, and ethylene glycol, which is another monomer of polyethylene terephthalate, can be converted to glycolic acid, which is a cosmetic material. The present invention is characterized by recycling polyethylene terephthalate waste into high value-added compounds.

Description

BACKGROUND

1. Field of the Invention

The present invention relates to a method of producing a high value-added compound from polyethylene terephthalate.

2. Discussion of Related Art

Polyethylene terephthalate (PET) is polyester of terephthalic acid (TPA) and ethylene glycol (EG). Because of its excellent physical properties, PET has been widely used in synthetic fibers and packaging materials. In 2015, annual global PET production reached 33 million tons, making PET the most commonly produced polyester in the world. Since PET does not completely decompose naturally, it causes serious environmental problems, such as the prevalence of microplastics in terrestrial ecosystems and the accumulation of waste plastics in the ocean. However, biodegradable plastics having similar physical properties and economic efficiency to PET are not yet available. Reducing PET production in the near future is unlikely, necessitating stricter PET recycling to reduce waste PET released in nature.

Among various plastics, PET and polyethylene (PE) are the only ones that are physically recyclable, and recycled plastics are produced from these waste plastics. Mechanical PET recycling has been performed for decades, but the rate of this traditional recycling is lower than approximately 21% in the United States. This low rate seems to be mainly due to the lower quality and higher costs of recycled PET (e.g., $1.3 to 1.5/kg PET) compared to virgin PET ($1.1 to 1.3/kg PET). To improve the high costs and low economic feasibility of mechanical recycling functioning as downcycling, for example, blending mechanically recycled PET with lignin for producing carbon fibers has been studied as an alternative application of mechanically recycled PET.

To overcome the problem of downcycling PET via mechanical recycling, chemical recycling in which PET is depolymerized into monomers and the monomers are repolymerized to PET was developed. However, PET production by the depolymerization and chemical recycling of PET also has no economic advantages. Therefore, it is necessary to improve the economic efficiency of PET recycling through upcycling by converting monomers to higher-value products than PET.

Recently, a method of chemically upcycling waste PET into high value-added plastics by the chemical modification of PET and reinforcement with glass fibers has been developed. In this case, the PET is biologically converted to plastic monomers such as polyhydroxyalkanoate (PHA). However, the economic sustainability of the bioconversion to PHA is still questionable.

Therefore, in the present invention, the biological valorization of PET monomers was verified for the first time to improve the economic efficiency of waste PET recycling and develop effective PET upcycling strategies. For the biological valorization of PET, PET was depolymerized by chemical hydrolysis and TPA and EG monomers were converted to various high value-added compounds using various metabolically engineered whole-cell microbial catalysts. In particular, by introducing a TPA degradation pathway into microbes, TPA is converted to high value-added aromatic compounds or aromatic-derived compounds, that is, protocatechuic acid (PCA), gallic acid (GA), pyrogallol, catechol, muconic acid (MA), and vanillic acid (VA), which are used for manufacturing pharmaceuticals, cosmetics, sanitizers, animal feeds, bioplastic monomers, and the like. Specifically, the present invention was completed by identifying key enzymes capable of catalyzing reactions required to convert TPA and microbes capable of fermenting EG into glycolic acid (GLA), and investigating their potential as key components of PET valorization.

SUMMARY OF THE INVENTION

The present invention is directed to providing a method of producing a high value-added compound from waste PET.

One aspect of the present invention provides a method of producing a high value-added compound from polyethylene terephthalate, comprising:

producing terephthalic acid and ethylene glycol through hydrolysis of polyethylene terephthalate; and

producing one or more compounds selected from the group consisting of gallic acid, pyrogallol, catechol, muconic acid, and vanillic acid through bioconversion of the terephthalic acid in the presence of a biocatalyst, wherein protocatechuic acid is an intermediate produced by the bioconversion, or

producing glycolic acid through fermentation of the ethylene glycol.

According to the present invention, by using one or a combination of the hydroxylation, decarboxylation, oxidation ring cleavage, and methylation reactions of TPA which is a PET hydrolysate, and using PCA as an intermediate, it is possible to convert PET to a variety of higher value-added compounds such as GA, pyrogallol, catechol, MA, and VA. In addition, since another PET monomer, EG, is converted to GLA using microbes capable of fermenting the same, the possibility of recycling waste PET is provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1G illustrate the depolymerization of PET into TPA and EG caused by the chemical hydrolysis of PET, and the bioconversion of TPA and EG in PET hydrolysate to PCA and GLA, respectively: FIG. 1A illustrates EG and TPA production by the chemical hydrolysis of PET and the separation of EG and TPA from the PET hydrolysate; FIG. 1B illustrates the bioconversion of TPA to PCA by Escherichia coli (E. coli) strain PCA-1; FIG. 1C illustrates the production of PCA from TPA in the PET hydrolysate by strain PCA-1; FIG. 1D is a gas chromatography-mass spectrometry (GC/MS) spectrum of PCA produced by strain PCA-1; FIG. 1E illustrates the production of GLA from EG in the PET hydrolysate by Gluconobacter oxydans (G. oxydans) KCCM 40109; FIG. 1F illustrates the time course of the whole-cell conversion of EG to GLA, wherein data is presented as the mean±standard deviation of triplicate experiments; and FIG. 1G is a GC/MS spectrum of GLA produced by G. oxydans KCCM 40109.

FIGS. 2A to 2E illustrate the production, separation, and identification of EG and TPA from PET: FIG. 2A illustrates EG and TPA production routes by the chemical hydrolysis of PET and the separation of TPA and EG from PET hydrolysate using NaOH and HCl; FIGS. 2B and 2C are ¹H NMR and ¹³C NMR spectra of TPA from the PET hydrolysate, wherein reagent-grade TPA is used as a reference; and FIGS. 2D and 2E are ¹H NMR and ¹³C NMR spectra of EG from the PET hydrolysate, wherein reagent-grade EG is used as a reference.

FIG. 3 illustrates an overall scheme for a waste PET biorefinery for PET upcycling.

FIGS. 4A to 4F are GC/MS spectra of authentic standards for PCA (FIG. 4A), GA (FIG. 4B), pyrogallol (FIG. 4C), MA (FIG. 4D), VA (FIG. 4E), and GLA (FIG. 4F).

FIGS. 5A to 5C illustrate the bioconversion of TPA to GA: FIG. 5A illustrates biosynthesis routes and whole-cell catalysts for the conversion of TPA to GA; FIG. 5B illustrates the comparison of the highest GA yields from TPA in systems GA-1, GA-2a, and GA-2b, wherein data is presented as the mean±standard deviation of triplicate experiments; and FIG. 5C is a GC/MS spectrum of GA produced from TPA by the GA-2b system.

FIGS. 6A to 6D illustrate the bioconversion of TPA to GA using whole-cell catalysts: FIG. 6A illustrates the comparison of E. coli strain HBH-1 expressing PobA and E. coli strain HBH-2 strain expressing PobA^Mut for the whole-cell conversion of PCA to GA in TG-2 buffer containing 3.4 mM PCA in conical tubes; FIG. 6B illustrates the conversion of TPA to GA by the GA-1 system consisting of E. coli strain GA-1 expressing TphAabc, TphB, and PobA^Mut at OD₆₀₀=30 in TG-2 buffer containing 2.8 mM TPA in conical tubes; FIG. 6C illustrates the conversion of TPA to GA by the GA-2a system consisting of E. coli strain PCA-1 expressing TphAabc and TphB at OD₆₀₀=20 and strain HBH-2 expressing PobA^Mut at OD₆₀₀=20 in TG-2 buffer containing 3.1 mM TPA in baffled flasks; and FIG. 6D illustrates the conversion of TPA to GA by a modified GA-2b system in which the cell densities of strains PCA-1 and HBH-2 of the GA-2b system were adjusted to OD₆₀₀=10 and 30, respectively, in TG-2 buffer containing 2.9 mM TPA, wherein all the conversions were performed at 30° C. and 250 rpm, and data is presented as the mean±standard deviation of triplicate experiments.

FIGS. 7A and 7B illustrate the docking simulation of the binding of PCA to active sites of PobAs: wild-type PobA (FIG. 7A) and PobA^Mut, which is a Y385F/T294A double mutant (FIG. 7B), wherein the flavin adenine dinucleotide (FAD)-bound structure of PobA (PDB code 6DLL) was used in the docking simulations, and the structure of PobA^Mut was constructed using MODELLER software, and molecular docking simulations were performed using AutoDockFR software.

FIGS. 8A to 8D illustrate the bioconversion of TPA to pyrogallol: FIG. 8A illustrates biosynthesis routes and whole-cell catalysts for TPA conversion to pyrogallol by systems PG-1a and PG-1b; FIG. 8B illustrates biosynthesis routes and whole-cell catalysts for TPA conversion to pyrogallol by PG-2a and PG-2b; FIG. 8C illustrates the comparison of the highest pyrogallol yields from TPA of strains PG-1a and PG-1b and systems CTL-1, PG-2a, and PG-2b, wherein data is presented as the mean±standard deviation of triplicate experiments; and FIG. 8D is a GC/MS spectrum of pyrogallol produced from TPA by strain PG-1a.

FIGS. 9A to 9E illustrate the bioconversion of TPA to pyrogallol using microbial catalysts: FIG. 9A illustrates the conversion of TPA to pyrogallol by the PG-1a system consisting of E. coli strain PG-1a expressing TphAabc, TphB, PobA^Mut, and LpdC at OD₆₀₀=30; FIG. 9B illustrates the conversion of TPA to pyrogallol by the PG-1b system consisting of E. coli strain PG-1b expressing TphAabc, TphB, PobA^Mut, LpdC, and PhKLMNOPQ at OD₆₀₀=30; FIG. 9C illustrates the conversion of TPA to catechol by E. coli strain CTL-1 expressing TphAabc, TphB, and AroY at OD₆₀₀=30 in conical tubes at 30° C. and 250 rpm; FIG. 9D illustrates the conversion of TPA to pyrogallol by the PG-2a system consisting of E. coli strain PCA-1 expressing TphAabc and TphB at OD₆₀₀=10 and E. coli strain PDC-CH-1 expressing AroY and PhKLMNOPQ at OD₆₀₀=30; and FIG. 9E illustrates the conversion of TPA to pyrogallol by the PG-2b system including strain CTL-1 expressing TphAabc, TphB, and AroY at OD₆₀₀=10 and E. coli strain CH-1 expressing PhKLMNOPQ at OD₆₀₀=30, wherein the conversion was performed in TG-2 buffer containing 3.0 mM TPA for systems CTL-1, PG-1a, and PG-1b and 3.5 mM TPA for strains PG-1 and PG-2 in baffled flasks at 30° C. and 250 rpm, and data is presented as the mean±standard deviation of triplicate experiments.

FIGS. 10A and 10B illustrate the promiscuity of GA decarboxylase LpdC toward GA: FIG. 10A illustrates the conversion of PCA to catechol by E. coli strain GDC-1 expressing LpdC; and FIG. 10B illustrates the conversion of GA to catechol by strain GDC-1 expressing LpdC, wherein the conversion was performed in TG-2 buffer containing 3.0 mM PCA or 3.0 mM GA in baffled flasks at 30° C. and 250 rpm, and data is presented as the mean±standard deviation of triplicate experiments.

FIGS. 11A to 11C illustrate enzymes used to synthesize pyrogallol and catechol: FIG. 11A illustrates catechol hydroxylase PhKLMNOPQ expressed in E. coli strain CH-1 and the production of pyrogallol from catechol; FIG. 11B illustrates PCA decarboxylase AroY expressed in E. coli strain PDC-1 and the production of catechol from PCA; and FIG. 11C illustrates PCA decarboxylase AroY and catechol hydroxylase PhKLMNOPQ coexpressed in E. coli strain PDC-CH-1 and the production of pyrogallol from PCA, wherein all the conversions were performed in TG-2 buffer in baffled flasks at 30° C. and 250 rpm, and data is presented as the mean±standard deviation of triplicate experiments.

FIGS. 12A and 12B illustrate the bioconversion of TPA to MA by the ring cleavage of catechol: FIG. 12A illustrates the ring cleavage of catechol by E. coli strain CDO-1 expressing CatA; and FIG. 12B illustrates the conversion of TPA to MA by an MA-1 system consisting of E. coli strain MA-1 expressing TphAabc, TphB, AroY, and CatA, wherein all the conversions were performed in TG-2 buffer at OD₆₀₀ =30 and 30° C. and 250 rpm in conical tubes, and data is presented as the mean±standard deviation of triplicate experiments.

FIGS. 13A to 13C illustrate the bioconversion of TPA to MA: FIG. 13A illustrates biosynthesis routes and whole-cell catalysts for TPA conversion to MA by E. coli strain MA-1 expressing TphAabc, TphB, AroY, and CatA; FIG. 13B illustrates the highest MA yields from TPA obtained in TG-2 buffer containing TPA in conical tubes at 30° C. and 250 rpm, wherein data is presented as the mean±standard deviation of triplicate experiments; and FIG. 13C is a GC/MS spectrum of MA produced from TPA by E. coli MA-1 strain.

FIGS. 14A to 14C illustrate the protein expression of O-methyltransferases (OMTs) from three eukaryotic sources and their whole-cell conversion of PCA to VA: FIG. 14A illustrates the results of SDS-PAGE of OMTs overexpressed in E. coli BL21 (DE3); FIG. 14B illustrates whole-cell conversion by E. coli OMT-1a expressing HsOMT at OD₆₀₀=20; and FIG. 14C illustrates whole-cell conversion by E. coli strain OMT-1b expressing SlOMT at OD₆₀₀=20, wherein the conversion was performed in 0.1 M sodium phosphate (pH 7.0) buffer containing 3.2 mM PCA, 10 g/L yeast extract, and 20 g/L peptone in conical tubes at 30° C. and 250 rpm, and data is presented as the mean±standard deviations of duplicate experiments. In the drawings: HsOMT, OMT from H. sapiens; SlOMT, OMT from Solanum lycopersicum (S. lycopersicum); MsOMT, OMT from Medicago sativa (M. sativa); pET28a, empty vector; T, total protein; S, soluble fraction; I, insoluble fraction; and M, marker.

FIGS. 15A to 15D illustrate the bioconversion of TPA to VA: FIG. 15A illustrates the bioconversion of TPA to VA in a VA-1 system in which E. coli strain VA-1 expressing TphAabc, TphB, and HsOMT at OD₆₀₀=30 was used in TG-1/YP buffer in conical tubes at 30° C. and 250 rpm; FIG. 15B illustrates the bioconversion of TPA to VA in a VA-2a system in which E. coli strain PCA-1 expressing TphAabc and TphB at OD₆₀₀=10 and E. coli strain OMT-2^His at OD₆₀₀=30 were simultaneously added to TG-2/YPM buffer in conical tubes at 30° C. and 250 rpm; FIG. 15C illustrates the bioconversion of TPA to VA in a modified VA-2b system in which baffled flasks were used in place of the conical tubes used in the VA-2a system; and FIG. 15D illustrates the bioconversion of TPA to VA in a modified VA-2c system in which the OD₆₀₀ values for strains PCA-1 and OMT-2^His of the VA-2b system were changed to OD₆₀₀=20 and OD₆₀₀=20, respectively, wherein data is presented as the mean±standard deviation of triplicate experiments.

FIGS. 16A to 16D illustrate glycerol and methionine consumption by various whole-cell conversion systems converting TPA to VA: FIG. 16A illustrates the comparison of glycerol consumption by systems VA-1, VA-2a, VA-2b, and VA-2c after 24 hours; FIG. 16B illustrates the comparison of methionine consumption by systems VA-1, VA-2a, VA-2b, and VA-2c after 24 hours; FIG. 16C illustrates the time course of glycerol consumption by the VA-2b system; and FIG. 16D illustrates the time course of methionine consumption by the VA-2b system, wherein glycerol and methionine were analyzed by high-performance liquid chromatography (HPLC) and GC/MS, respectively, and data is presented as the mean±standard deviation of duplicate experiments.

FIGS. 17A and 17B illustrate the effect of HsOMT protein engineering on the bioconversion of PCA to VA: FIG. 17A illustrates E. coli strain OMT-2 expressing HsOMT in TG-1/YP buffer; and FIG. 17B illustrates E. coli strain OMT-2^His expressing HsOMT^His in TG-1/YP buffer, wherein the TG-1/YP buffer refers to 50 mM Tris-HCl buffer containing 100 g/L glycerol, 10 g/L yeast extract, and 20 g/L peptone, and whole-cell conversion was performed at OD₆₀₀=30 in 50 mL conical tubes at 30° C. and 250 rpm, and data is presented as the mean±standard deviation of triplicate experiments.

FIGS. 18A and 18B illustrate the effect of methionine supplementation on the bioconversion of PCA to VA: FIG. 18A illustrates E. coli strain OMT-2^His in TG-2/YP buffer containing 20 g/L glycerol; and FIG. 18B illustrates strain OMT-2^His in TG-2/YPM buffer containing 20 g/L glycerol and 2.5 mM methionine, wherein the TG-2/YP buffer refers to 50 mM Tris-HCl buffer containing 20 g/L glycerol, 10 g/L yeast extract, and 20 g/L peptone, and the TG-2/YPM buffer was modified from the TG-2/YP buffer by supplementing 2.5 mM methionine, and the conversion was performed at OD₆₀₀=30 in 50 mL conical tubes at 30° C. and 250 rpm, and data is presented as the mean±standard deviation of triplicate experiments.

FIGS. 19A to 19C illustrate the bioconversion of TPA to VA: FIG. 19A illustrates biosynthesis routes and whole-cell catalysts for the conversion of TPA to VA; FIG. 19B illustrates the comparison of the highest VA yields from TPA, wherein data is presented as the mean±standard deviation of duplicate experiments for the VA-2a system and triplicate experiments for systems VA-1, VA-2b, and VA-2c; and FIG. 19C is a GC/MS spectrum of VA produced from TPA by the VA-2b system.

FIGS. 20A to 20C illustrate the bioconversion of EG to GLA by G. oxydans KCCM 40109: FIG. 20A illustrates the biosynthesis route of GLA from EG; and FIGS. 20B and 20C illustrate time courses of the whole-cell conversion of EG to GLA at the initial EG of 28.6 mM (FIG. 20B) and 67.6 mM (FIG. 20C), wherein in the case of FIGS. 20B and 20C, the whole-cell conversion was performed at OD₆₀₀=30 at 30° C. and 250 rpm in conical tubes containing 40 g/L sorbitol, 10 g/L yeast extract, 2.5 g/L (NH₄)₂SO₄, 1 g/L KH₂PO₄, and 2.5 g/L MgSO₄.7H₂O, and data is presented as the mean±standard deviation of triplicate experiments.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Hereinafter, the composition of the present invention will be described in detail.

One aspect of the present invention provides a method of producing a high value-added compound from polyethylene terephthalate, comprising:

producing terephthalic acid and ethylene glycol through hydrolysis of polyethylene terephthalate; and

producing one or more compounds selected from the group consisting of gallic acid, pyrogallol, catechol, muconic acid, and vanillic acid through bioconversion of the terephthalic acid in the presence of a biocatalyst, wherein protocatechuic acid is an intermediate produced by the bioconversion, or

producing glycolic acid through fermentation of the ethylene glycol.

In the method of producing a high value-added compound from polyethylene terephthalate according to the present invention, monomers terephthalic acid and ethylene glycol are produced through the chemical hydrolysis of polyethylene terephthalate, various high value-added aromatic compounds or aromatic-derived compounds such as GA, pyrogallol, catechol, MA, and VA are produced through the bioconversion of TPA which is a PET hydrolysate, and glycolic acid is produced through the fermentation of ethylene glycol.

The chemical hydrolysis of PET may be carried out through the application of microwaves at a temperature of 170 to 230° C. for 15 to 50 minutes. The PET hydrolysate may be separated into TPA solids and an EG-containing solution by filtration.

For the bioconversion of TPA, PCA is selected as a first product and a key intermediate. PCA can be a precursor compound for producing various high value-added aromatic compounds or aromatic-derived compounds such as GA, pyrogallol, catechol, MA, and VA.

As an efficient biocatalyst capable of converting TPA to PCA, TPA 1,2-dioxygenase and 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate (DCD) dehydrogenase are used, wherein TPA 1,2-dioxygenase converts TPA to DCD, and DCD dehydrogenase converts DCD to PCA. TPA 1,2-dioxygenase and DCD dehydrogenase may be derived from Comamonas sp. E6, and their respective coding gene names are TphAabc and TphB. These enzymes may utilize NADH and NADPH as cofactors. According to one embodiment of the present invention, to obtain PCA from TPA, a PET hydrolysate, microbes expressing TphAabc and TphB may be used as a biocatalyst.

Next, the bioconversion of TPA to GA may be implemented by inducing hydroxylation at the meta-position of PCA, in which case, the PCA is converted to GA. The hydroxylation may be achieved using p-hydroxybenzoate hydroxylase. The p-hydroxybenzoate hydroxylase may be derived from Pseudomonas putida (P. putida) KT2440, and its coding gene name is PobA. In addition, according to one embodiment of the present invention, a PobA mutant, that is, PobA^Mut (T294A/Y385F), may be constructed to increase GA production yield, and microbes expressing PobA^Mut may be used as a biocatalyst. Preferably, to produce GA from TPA, microbes expressing TphAabc, TphB, and PobA^Mut or a combination of microbes expressing TphAabc and TphB and microbes expressing PobA^Mut may be used as a biocatalyst.

According to one embodiment of the present invention, to improve GA production yield from TPA, microbes expressing TphAabc, TphB, and PobA^Mut (strain GA-1) and having an OD₆₀₀ value of 30 may be reacted with TPA, or microbes expressing TphAabc and TphB (strain PCA-1) and microbes expressing PobA^Mut (strain HBH-2) and having OD₆₀₀ values of 10 and 30, respectively, may be reacted with TPA, so that the GA production yield can be improved without the accumulation of PCA.

Next, the bioconversion of TPA to pyrogallol via GA may be implemented through two routes: via the decarboxylation of GA synthesized by PCA hydroxylation (first route), and via the hydroxylation of catechol that can be synthesized by PCA decarboxylation (second route).

In the case of the first route, microbes expressing TphAabc, TphB, and PobA^Mut due to including GA decarboxylase (coding gene name: LpdC) for the decarboxylation of GA synthesized by PCA hydroxylation may be used as a biocatalyst. According to one embodiment of the present invention, microbes expressing TphAabc, TphB, PobA^Mut, and LpdC (strain PG-1a) may be reacted with TPA to produce pyrogallol.

In the case of the second route, PCA decarboxylase (coding gene name: AroY) and a phenol hydroxylase (coding gene name: PhKLMNOPQ) for catechol hydroxylation may be used as a biocatalyst. According to one embodiment of the present invention, a combination of microbes expressing TphAabc, TphB, and AroY (strain CTL-1) and microbes expressing PhKLMNOPQ (strain CH-1), whose OD₆₀₀ values are 10 and 30, respectively, may be used and reacted with TPA, so that pyrogallol can be produced while minimizing the accumulation of catechol.

Next, the bioconversion from TPA to MA may be implemented by the ring cleavage of catechol synthesized from TPA via PCA, in which case, catechol is converted to MA. The ring cleavage of catechol may be implemented using catechol 1,2-dioxygenase (coding gene name: CatA) derived from P. putida KT2440. According to one embodiment of the present invention, microbes expressing TphAabc, TphB, AroY, and CatA (strain MA-1) may be reacted with TPA to produce MA.

Next, the bioconversion of TPA to VA may be implemented by converting PCA to VA by an OMT. In an O-methylation reaction catalyzed by an OMT, since adenosyl and methyl groups are supplied from ATP and methionine, S-adenosyl methionine (SAM) may be used as a co-substrate.

As the OMT, one derived from eukaryotes may be used. For example, HsOMT from H. sapiens, SlOMT from S. lycopersicum, MsOMT from M. sativa, and the like may be used. To increase VA production yield, HsOMT from H. sapiens is preferred. In addition, to increase the protein solubility of HsOMT, HsOMT may be modified to have hexameric histidine at the N-terminus.

In addition, to improve VA production yield, aeration may be increased during the reaction of TPA and a biocatalyst. Increased aeration is correlated with the increased consumption of glycerol and methionine. That is, aeration is critical for increasing VA production from PCA because glycerol is efficiently metabolized to generate adenosine triphosphate (ATP), thus accelerating S-adenosyl methionine (SAM) synthesis from methionine by supplying S-adenosyl groups.

According to one embodiment of the present invention, to produce VA from TPA via PCA, microbes expressing TphAabc and TphB (strain PCA-1) and microbes expressing HsOMT^His (strain OMT-2^His) and having OD₆₀₀ values of 10 and 30, respectively, may be reacted with TPA in the presence of glycerol and methionine while increasing aeration to increase ATP production, and thereby VA production yield can be improved.

The method of the present invention is capable of producing GLA from EG, which is a PET hydrolysate, through fermentation. The fermentation may be performed using EG-fermenting microbes such as G. oxydans KCCM 40109, Clostridium glycolicum, P. putida, and the like.

The bioconversion of the present invention may be carried out in various reaction buffer systems. For example, the following may be used: TG-1 buffer, 50 mM Tris buffer (pH 7.0) containing 10% (w/v) glycerol; TG-2 buffer, 50 mM Tris buffer (pH 7.0) containing 2% (w/v) glycerol; TG-1/YP buffer, 50 mM Tris buffer (pH 7.0) containing 10% (w/v) glycerol, 10 g/L yeast extract, and 20 g/L peptone; TG-2/YP buffer, 50 mM Tris buffer (pH 7.0) containing 2% (w/v) glycerol, 10 g/L yeast extract, and 20 g/L peptone; TG-1/YPM buffer, TG-1/YP buffer supplemented with 2.5 mM L-methionine (Sigma-Aldrich); and TG-2/YPM buffer, TG-2/YP buffer supplemented with 2.5 mM L-methionine.

As used herein, the term “biocatalyst” refers to an enzyme involved in the bioconversion of TPA and is also used to refer to a microbe expressing the enzyme. The enzyme can be introduced into a host cell in the form of a recombinant vector containing a coding gene and expressed.

The term “recombinant vector” refers to a vector capable of expressing a target protein in an appropriate host cell and means a genetic construct including essential regulatory elements operably linked to express a gene insert in vivo or in vitro. In this specification, the terms “plasmid,” “vector,” and “expression vector” are used interchangeably.

Examples of the above-described vector include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, or viral vectors. Suitable expression vectors include expression control elements such as promoters, operators, start codons, stop codons, polyadenylation signals, and enhancers and additionally include signal sequences or leader sequences for membrane targeting or secretion, and they can be variously prepared according to the purpose. A promoter in a vector may be constitutive or inducible. In addition, the expression vector includes a selection marker for selecting a host cell including a vector, and in the case of a replicable expression vector, an origin of replication.

The term “operably linked” means that an appropriate nucleic acid molecule is linked to a regulatory sequence in such a way as to enable gene expression.

As used herein, the term “nucleic acid molecule” means any single or double-stranded nucleic acid molecule of cDNA, genomic DNA, synthetic DNA or RNA, PNAS or LNA origin, or a combination thereof. “Nucleic acid” and “polynucleotide” may be used interchangeably herein.

The recombinant vector of the present invention is preferably prepared by inserting the above-described gene into a general vector for expressing an E. coli strain. As the vector for expressing an E. coli strain, any commonly available E. coli expression vector can be used without limitation.

A host cell transformed by the recombinant vector can express an enzyme involved in the bioconversion of TPA. A method of achieving the transformation includes any method capable of introducing a nucleic acid into an organism, cell, tissue, or organ, and the transformation can be performed by selecting a standard technique suitable for the host cell, as is known in the art. Examples of such a method include, but are not limited to, electroporation, protoplast fusion, calcium phosphate (CaPO₄) precipitation, calcium chloride (CaCl₂) precipitation, agitation using silicon carbide fibers, agrobacterium-mediated transformation, and use of PEG, dextran sulfate, Lipofectamine, and the like.

In addition, since the expression level and modification of the protein are different according to the host cell, it is recommended to select and use the most suitable host cell for the purpose.

Examples of the host cell include, but are not limited to, prokaryotes such as E. coli, Zymomonas mobilis, Bacillus subtilis, Streptomyces, Pseudomonas, Proteus mirabilis, or Staphylococcus. In addition, eukaryotes such as fungi (e.g., Aspergillus) or yeast (e.g., Pichiapastoris, Saccharomyces cerevisiae, Schizosaccharomyces, Neurosporacrassa) may be used, but the present invention is not limited thereto.

Various culture methods can be applied for the (large-scale) culture of transformants, and for example, the large-scale production of expressed or overexpressed gene products from recombinant microbes can be achieved by batch or continuous culture methods. Batch and fed-batch culture methods are conventional and known in the art. Methods for controlling nutrients and growth factors for continuous culture processes and techniques for maximizing product formation rates are known in the microbial industry. In addition, as a culture medium, a medium formed of a carbon source, a nitrogen source, vitamins, and minerals may be used, and the composition of the culture medium may be configured as known in the art.

Hereinafter, the present invention will be described in more detail through Examples according to the present invention, but the scope of the present invention is not limited by the Examples presented below.

<Example 1> Bioconversion of Polyethylene Terephthalate (PET) Monomers

(1) Chemical Hydrolysis of PET

Granular PET chips (Sigma-Aldrich) were used for chemical hydrolysis experiments. PET hydrolysis reaction mixtures included 1 g granular PET in 13 mL deionized water and were input in a microwave reactor (Monowave 300, Anton Paar, Graz, Austria). PET hydrolysis was performed under microwave irradiation at various temperatures and durations: 170, 200, and 230° C. and 15, 20, 25, 30, 40, and 50 minutes. The TPA yield from hydrolysis of PET was calculated as TPA yield (% of theoretical maximum TPA=TPA produced (g)/theoretical maximum TPA produced from consumed PET (g)×100). The theoretical maximum mass of TPA to be produced from PET was calculated by multiplying PET mass by 0.864, a TPA yield coefficient for PET. Due to the high degree of polymerization of PET, the total number of cleaved ester bonds by hydrolysis was assumed to be the same as the total number of TPA and EG monomers. Therefore, the TPA yield coefficient of TPA from PET was calculated while assuming a TPA:EG:H₂O molar ratio of 1:1:2. The TPA yield coefficient was 166.13/(166.13+62.06−2×18.01)=0.864, wherein 166.13, 62.06, and 18.01 are molecular weights (MWs) of TPA, EG, and H₂O, respectively.

(2) Separation of Monomers from PET Hydrolysate

After the chemical hydrolysis of PET, TPA solids in the hydrolysate were separated from an EG-containing solution by filtration. The TPA solids in the residue were dissolved in 1 M NaOH and thereby converted to Na-TPA. After adding 2 M HCl to the Na-TPA solution, the formed TPA solids were filtered and dried in a vacuum oven at 80° C. The EG-containing solution was concentrated by evaporation and distilled to obtain purified EG. The TPA and EG purified from the PET hydrolysate were analyzed by nuclear magnetic resonance spectroscopy (NMR; Bruker 400 MHz, Billerica, Mass.) with ¹H NMR and ¹³C NMR and compared with authentic TPA (Alfa Aesar, Haverhill, Mass.) and EG (Junsei Chemical, Tokyo, Japan) standard materials.

(3) Bacterial Strains and Plasmids

E. coli DH5α was used as a host strain for plasmid construction and maintenance. E. coli BL21 (DE3) was used as a host strain for OMT enzyme screening. E. coli XL1-Blue (Stratagene, San Diego, Calif.) and E. coli MG1655 (DE3) were used as host strains for whole-cell conversion. Recombinant E. coli strains were grown in lysogeny broth (LB) or on LB agar plates (2.0% w/v) containing 10 g/L tryptone, 5 g/L NaCl, and 5 g/L yeast extract. Appropriate antibiotics (50 μg/mL ampicillin, 40 μg/mL kanamycin, or 34 μg/mL chloramphenicol) were prepared and supplemented to the medium. Plasmids pKM212, pKE112, and pKA312 were constructed as described above. All plasmids and bacterial strains used in this experiment are listed in Table 1. G. oxydans KCCM 40109 (Korean Culture Center of Microorganisms, Seoul, Korea) was used as a whole-cell biocatalyst for the bioconversion of EG to GLA.

(4) Plasmids Construction

DNA cloning was performed per standard procedures. All genes except for pobA and catA genes were synthesized by IDT or GeneArt and extracted from P. putida KT2440 by polymerase chain reactions (PCR). PCR was performed using a C1000 Thermal Cycler (Bio-Rad, Hercules, Calif.). Primers and genes used for changing restriction enzyme sites are listed in Table 2 and Table 3, respectively.

For constructing plasmids pKE112TphAabc and pKM212TphB (PCA synthesis modules), plasmids pKE112 and pKM212 were digested using restriction enzymes KpnI/HindIII and EcoRI/KpnI, respectively. Corresponding TphAabc and TphB genes were digested using KpnI/HindIII and EcoRI/KpnI, respectively, and ligated into the plasmids pKE112 and pKM212.

For constructing pET28a-based plasmids for expressing genes Sl10OMT, HsOMT, MsOMT, and HsOMT^His, pET28a was digested with NdeI/XhoI, and corresponding genes were ligated. Plasmids used to directly convert TPA to PCA were constructed by ligating HsOMT and HsOMT^His into plasmid pKE112TphB using KpnI/BamHI. To investigate the PCA hydroxylation capabilities of PobA and PobA^Mut, these genes were ligated into plasmid pET28a using NdeI/XhoI for the construction of plasmids pET28aPobA and pET28aPobA^Mut, respectively. For the direct conversion of TPA to GA, the ligation of pobA^Mut into plasmid pKE112TphB was performed using SbfI/HindIII. For the direct conversion of TPA to PG, an 1pdC gene was introduced into plasmid pKE112TphBPobA^Mut using BamHI/SbfI. To construct catechol hydroxylation module pKA312PhKLMNOPQ, a phKLMNOPQ gene fragment was ligated into plasmids pKA312, pKA312PhKLM, pKA312PhKLMNOP, and pKA312PhKLMNOPQ using EcoRI/KpnI, KpnI/BamHI, BamHI/SbfI, and SbfI/HindII, respectively.

A plasmid for catechol synthesis was constructed by the ligation of aroY into pKE112TphB using KpnI/BamHI. For experiments related to evaluating preferred substrates of enzymes LpdC and AroY, corresponding plasmids pET28aLpdC and pET28aAroY were generated by the ligation of a corresponding enzyme into pET28a using NdeI/XhoI sites. For constructing a plasmid for MA synthesis, catA was introduced into plasmids pKE112 and pKE112TphBAroY using KpnI/BamHI sites.

(5) Whole-Cell Bioconversion

Whole-cell conversion using engineered E. coli strains was performed as follows. Seed cultures were prepared overnight in 5 mL LB media using appropriate antibiotics. Subsequently, seed cultures were used to inoculate 1 L LB media in 2.8 L flasks and were incubated at 37° C. and 220 rpm. When cell densities reached an optical density of 0.4 at 600 nm (OD₆₀₀), 0.1 mM isopropyl-p-D-thiogalactopyranoside (IPTG; Sigma-Aldrich, St. Louis, Mo.) was added to the cultures. Subsequently, an incubation temperature was adjusted to 16° C. for 16 hours to facilitate the soluble expression of the introduced genes. The engineered E. coli strains were harvested by centrifugation at 4300×g for five minutes at 10° C. The harvested cells were washed and resuspended with 50 mM Tris buffer (pH 7.0) containing 2% (w/v) glycerol.

For whole-cell conversion, microbial cell pellets were resuspended in 4 mL or 20 mL of reaction buffer with appropriate concentrations of substrates and incubated at 250 rpm and 30° C. The compositions of reaction buffers used for bioconversion in this experiment were as follows: TG-1 buffer, 50 mM Tris buffer (pH 7.0) containing 10% (w/v) glycerol; TG-2 buffer, 50 mM Tris buffer (pH 7.0) containing 2% (w/v) glycerol; TG-1/YP buffer, 50 mM Tris buffer (pH 7.0) containing 10% (w/v) glycerol, 10 g/L yeast extract, and 20 g/L peptone; TG-2/YP buffer, 50 mM Tris buffer (pH 7.0) containing 2% (w/v) glycerol, 10 g/L yeast extract, and 20 g/L peptone; TG-1/YPM buffer, TG-1/YP buffer supplemented with 2.5 mM L-methionine (Sigma-Aldrich); and TG-2/YPM buffer, TG-2/YP buffer supplemented with 2.5 mM L-methionine. All experiments were performed in triplicate unless otherwise indicated. The whole-cell conversion using the VA-2a system was performed in duplicate. TPA, PCA, GA, pyrogallol, catechol, MA, and VA standard materials were purchased from Sigma-Aldrich.

Whole-cell bioconversion by G. oxydans KCCM 40109 was performed as follows. Seed cultures were prepared overnight in 5 mL media in 50 mL conical tubes. The media contained 80 g/L sorbitol, 20 g/L yeast extract, 5 g/L (NH₄)₂SO₄, 2 g/L KH₂PO₄, and 5 g/L MgSO₄.7H₂. The seed cultures were inoculated 1 L media in 2.8 L flasks and were incubated at 30° C. and 220 rpm. The cells were collected by centrifugation at 6500×g for eight minutes at 10° C. and were washed and resuspended in phosphate buffer (pH 7.0). Whole-cell bioconversion mixtures were prepared by appropriate resuspending concentrations of whole-cell catalysts in 4 or 20 mL buffers and were incubated at 30° C. and 250 rpm for 12 hours. Bioconversion buffers were composed of 40 g/L sorbitol, 10 g/L yeast extract, 2.5 g/L (NH₄)₂SO₄, 1 g/L KH₂PO₄, and 2.5 g/L MgSO₄.7H₂O. The bioconversion buffers were supplemented with EG at different concentrations: 11.3, 28.6, and 67.6 mM.

(6) SDS-PAGE Analysis

The expression of eukaryotic OMT enzymes SlOMT, HsOMT, and MsOMT in E. coli BL21 (DE3) cells was verified using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Recombinant E. coli BL21 (DE3) cells harboring the respective plasmids were cultivated in 100 mL LB media in 500 mL flasks at 37° C. and 220 rpm. The cultures were supplemented with 0.1 mM IPTG upon reaching an OD₆₀₀ of 0.4 and were cultivated for 16 hours at 16° C. and 180 rpm. Cell pellets were collected by centrifugation at 6,500×g for 10 minutes at 4° C. and washed with 16 mL of 100 mM sodium phosphate buffer (pH 7.0).

Aliquots were prepared from the cell suspension and were used as total protein samples for SDS-PAGE. Cell lysates of recombinant E. coli were obtained by sonication (Branson 450, Marshall Scientific, Hampton, N.H.). Solid and liquid fractions containing insoluble and soluble proteins, respectively, were separated by centrifugation at 16,000×g for 20 minutes at 4° C. The separated solid fraction was resuspended in 16 mL of 100 mM sodium phosphate buffer (pH 7.0). The aliquoted cell suspension and the liquid and solid fractions were mixed with 5×SDS buffer (Biosesang, Seongnam, Korea) and boiled at 100° C. for 10 minutes. Protein samples were separated by 12% (w/v) SDS-PAGE with a pre-stained SDS standard marker (Bio-Rad).

(7) Analytical Methods

The OD₆₀₀ was measured using a spectrophotometer (xMark™, Bio-Rad). TPA and products converted from TPA were analyzed using HPLC (Agilent 1100, Agilent Technologies, Santa Clara, Calif.) equipped with an OptimaPak C18 column (RS tech, Daejeon, Korea) at a flow rate of 1.0 mL/min while maintaining a column temperature at 30° C.

A mobile phase was composed of 10% (v/v) acetonitrile in 0.1% (v/v) trifluoroacetic acid (Sigma-Aldrich) in deionized water. The injection volume was 5 μL, and UV detection was performed at 254 nm. Concentrations of EG, GLA, and glycerol were measured by HPLC (Agilent 1100) equipped with a refractive index (RI) detector and an Aminex HPX-87H column (Bio-Rad) at 65° C. with a 0.01 N H₂SO₄ mobile phase at a flow rate of 0.5 mL/min.

GC/MS analysis was used to verify the conversion of TPA to PCA, GA, pyrogallol, catechol, MA, and VA and the conversion of EG to GLA and quantify L-methionine. The GC/MS analysis was performed using Agilent 7890A GC/5975C MSD (Agilent Technologies) equipped with an RTX-5Sil MS capillary column (30 m×0.25 mm, 0.25 μm film thickness; Restek, Bellefonte, Pa.) with an additional 10 m integrated guard column. One microliter of a sample was injected in a splitless mode with an inlet temperature of 250° C. The oven temperature was initially maintained at 50° C. for one minute and then increased to 320° C. at a rate of 20° C./min and then maintained for 25 minutes. Helium was used as a carrier gas at a 1 mL/min flow rate, and mass spectra were recorded by scanning from 50 to 700 m/z. Temperatures of a transfer line and an ion source were set at 280 and 230° C., respectively.

(8) Computer Docking Simulation

The computational modeling of a protein structure PobA derived from P. putida KT2440 was performed using the Discovery Studio software (BIOVIA, San Diego, Calif.). The FAD-bound structure of PobA (PDB code: 6DLL) was used in computational docking simulations. A wild-type PobA structure does not have 4-hydroxybenzoic acid (4-HBA) in its active site, indicating that the crystal structure of wild-type PobA may not represent the complex conformation of 4-HBA and FAD. For the docking simulations, the binding conformation of FAD in the active site of P. putida KT2440 PobA was modeled using MODELER by comparing its active site with that of Pseudomonas fluorescence PobA complexed with 4-HBA and FAD (PDB codes: 1PBE and 1BGN). The structure of PobA^Mut (T294A/Y385F) was constructed by MODELER. The flexible docking of substrate PCA was performed using AutodockFR, and nine residues (Y386, Y201, T294, L210, S212, R220, W185, Y222, and I43) were selected as flexible residues. All parameters were set at default values for docking simulations, and the resulting binding mode was analyzed using the PyMOL software (PyMOL Molecular Graphics System, ver. 1.4.1; Schrodinger, New York, N.Y.).

TABLE 1
Strains, strategies, and plasmids used in present invention
Strain, strategy, or plasmid	Relevant characteristics	Reference
Strains
E. coli XL1-Blue	recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [FA1proAB	Stratagene
	lacIqZΔM15 Tn10 (TetR)]
E. coli MG1655 (DE3)	K-12 F− λ− ilvG− rfb-50 rph−1 (DE3)	Korea Research
		Institute of
		Bioscience &
		Biotechnology
G. oxydans KCCM 40109	GLA produced from EG	KCCM
Recombinant strain for PCA synthesis
PCA-1 (TPA→PCA)	E. coli XL1-Blue with pKM212TphAabc and pKE112TphB	Present invention
Recombinant strains for GA synthesis
HBH-1 (PCA→GA)	E. coli MG1655 (DE3) with pET28aPobA	Present invention
HBH-29 (PCA→GA)	E. coli MG1655 (DE3) with pET28aPobAMut	Present invention
GA-1 (TPA→GA)	E. coli XL1-Blue with pKM212TphAabc and
	pKE112TphBPobAMut	Present invention
Recombinant strains for pyrogallol synthesis
GDC-1 (GA→pyrogallol)	E. coli MG1655 (DE3) with pET28aLpdC	Present invention
CH-1 (catechol→pyrogallol)	E. coli MG1655 (DE3) with pKA312PhKLMNOPQ	Present invention
PDC-CH-1 (PCA→pyrogallol)	E. coli MG1655 (DE3) with pET28aAroY and	Present invention
	pKA312PhKLMNOPQ
PG-1 (TPA→pyrogallol)	E. coli XL1-Blue with pKM212TphAabc and	Present invention
	pKE112TphBLpdCPobAMut
PG-2 (TPA→pyrogallol)	E. coli XL1-Blue with pKM212TphAabc,	Present invention
	pKE112TphBLpdCPobAMut and pKA312PhKLMNOPQ
Recombinant strains for catechol synthesis
PDC-1 (PCA→catechol)	E. coli MG1655 (DE3) with pET28aAroY	Present invention
CTL-1 (TPA→catechol)	E. coli XL1-Blue with pKM212TphAabc and pKE112TphBaroY	Present invention
Recombinant strains for MA synthesis
CDO-1 (catechol→MA)	E. coli XL1-Blue with pKE112CatA	Present invention
MA-1 (TPA→MA)	E. coli XL1-Blue with pKM212TphAabc and	Present invention
	pKE112TphBAroYCatA
Recombinant strains for VA synthesis
OMT-1a (PCA→VA)	E. coli BL21 (DE3) with pET28aHsOMT	Present invention
OMT-1b (PCA→VA)	E. coli BL21 (DE3) with pET28aSlOMT	Present invention
OMT-2 (PCA→VA)	E. coli MG1655 (DE3) with pET28aHsOMT	Present invention
OMT-2His (PCA→VA)	E. coli MG1655 (DE3) with pET28aHsOMTHis	Present invention
VA-1 (TPA→VA)	E. coli XL1-Blue with pKM212TphAabc and	Present invention
	pKE112TphBHsOMT
Whole-cell conversion systems
GA-1 system	Strain GA-1 as a single catalyst under increased aeration (OD600	Present invention
	of strain GA-1 = 30)
GA-2a system	Simultaneous addition of strains PCA-1 and HBH-2 under	Present invention
	increased aeration (OD600 of strain PCA-1 = 20, OD600 of strain
	HBH-2 = 20)
GA-2b system	Simultaneous addition of strains PCA-1 and HBH-2 under	Present invention
	increased aeration (OD600 of PCA-1 strain = 10, OD600 of
	HBH-2 strain = 30)
PG-1a system	Strain PG-1a as a single catalyst under increased aeration (OD600	Present invention
	of strain PG-1a strain = 30)
PG-1b system	Strain PG-1b as a single catalyst under increased aeration (OD600	Present invention
	of strain PG-1b = 30)
PG-2a system	Simultaneous addition of strains PCA-1 and PDC-CH-1 under	Present invention
	increased aeration (OD600 of strain PCA-1 = 10, OD600 of strain
	PDC-CH-1 = 30)
PG-2b system	Simultaneous addition of strains CTL-1 and CH-1 under
	increased aeration (OD600 of strain CTL-1 = 10, OD600 of strain	Present invention
	CH-1 = 30)
MA-1 system	Strain MA-1 as a single catalyst (OD600 of strain MA-1 = 30)	Present invention
VA-1 system	Strain VA-1 as a single catalyst (OD600 of strain VA-1 = 30)	Present invention
VA-2a system	Simultaneous addition of strains PCA-1 and OMT-2His (OD600 of	Present invention
	strain PCA-1 = 10, OD600 of strain OMT-2His = 30)
VA-2b system	Simultaneous addition of strains PCA-1 and OMT-2His under	Present invention
	increased aeration (OD600 of strain PCA-1 = 10, OD600 of strain
	OMT-2 = 30)
VA-2c system	Simultaneous addition of strains PCA-1 and OMT-2His under	Present invention
	increased aeration (OD600 of strain PCA-1 = 20, OD600 of strain
	OMT-2His = 20)
Plasmids
pKM212TphAabc	pKM212; Ptac promoter, Comamonas sp. strain E6 tphAIIabc	Present invention
	genes, KmR
pKE112TphB	pKE112; Ptac promoter, Comamonas sp. strain E6 tphB gene,	Present invention
	AmpR
pET28aHsOMT	pET28a; T7 promoter, H. sapiens OMT gene, KmR	Present invention
pET28aSlOMT	pET28a; T7 promoter, S. lycopersicum OMT gene, KmR	Present invention
pET28aMsOMT	pET28a; T7 promoter, M. sativa OMT gene, KmR	Present invention
pET28aHsOMTHis	pET28a; T7 promoter, H. sapiens OMT' gene, KmR	Present invention
pKE112TphBHsOMT	pKE112; Ptac promoter, OMT gene of H. sapiens inserted into	Present invention
	pKE112TphB, AmpR
pKE112TphBPobAMut	pKE112; Ptac promoter, pobAMut gene of P. putida KT2440	Present invention
	inserted into pKE112TphB, AmpR
pET28aPobA	pET28a; T7 promoter, P. putida KT2440pobA gene, KmR	Present invention
pET28aPobAm'	pET28a; T7 promoter, P. putida KT2440pobAMut gene, KmR	Present invention
pET28aAroY	pET28a; T7 promoter, E. cloacae aroY gene, KmR	Present invention
pET28aLpdC	pET28a; T7 promoter, L. plantarum lpdC gene, KmR	Present invention
pKE112TphBaroY	pKE112; Ptac promoter, aroY gene of E. cloacae inserted into	Present invention
	pKE112TphB, AmpR
pKE112TphBAroYCatA	pKE112; Ptac promoter, catA gene of P. putida KT2440 inserted	Present invention
	into pKE112TphBAroY, AmpR
pKE112TphBPobAMutLpdC	pKE112; Ptac promoter, lpdC gene of L. plantarum inserted into	Present invention
	pKE112TphBPobAMut, AmpR
pKA312PhKLMNOPQ	pKE112; Ptac promoter, PhKLMNOPQ gene of P. stutzeri OX1,	Present invention
	CmR
pKE112CatA	pKE112; Ptac promoter, catA gene of P. putida KT2440, AmpR	Present invention

TABLE 2
Primers used in present invention
		SEQ ID
Primer name	Sequence (5′-3′)	NO:	Gene source
pKM212-TphAabc-	Gene synthesis at IDT		Comamonas sp.
F/R			strain E6
pKE112-TphB-F/R	Gene synthesis at IDT		Comamonas sp.
			strain E6
pET28a-HsOMT	Gene synthesis at IDT		H. sapiens
pET28a-SlOMT	Gene synthesis at IDT		S. lycopersicum
pET28a-MsOMT	Gene synthesis at IDT		M. sativa
pKE112-HsOMT-F	GGTACCTTTCACACAGGAAACAGACCATGGGCGATACCAAAG	20	pET28a-HsOMT
	AACAG
pKE112-HsOMT-R	GGATCC TTAAGTTACGGACCTGCTTCG	21
pKE112-	GGTACC		pET28a-HsOMT
HsOMTHis-F	TTTCACACAGGAAACAGACCATGCATCACCATCACCATCATGG	22
	CGATACCAAAGAACAGCG
pKE112-	Same as that of HsOMT-R	23
HsOMTHis-R	(GGATCC TTAAGTTACGGACCTGCTTCG)
pET28a- HsOMTHis-	CATATG	24	pET28a-HsOMT
F	CATCACCATCACCATCATGGCGATACCAAAGAACAGCG
pET28a-	CTCGAG TTAAGTTACGGACCTGCTTCG	25
HsOMTHis-R
pET28a-PobA-F	CATATG AAAACTCAGGTTGCAATTATTG	26	P. putida KT2440
pET28a-PobA-R	CTCGAG TCAGGCAACTTCCTCGAACG	27
pKE112-PobA-F	CCTGCAGG TTTCACACAGGAAACAGACC	28	P. putida KT2440
	ATGAAAACTCAGGTTGCAATTATTG
pKE112-PobA-R	AAGCTT TCAGGCAACTTCCTCGAACG	29
pKE112-PobAMut-	Gene synthesis at IDT		Mutant of P. putida
F/R			KT2440 pobA gene
pET28a-PobAMut-F	Same as that of pET28a-PobA-F	26	pKE112-PobAMut-
			F/R
pET28a-PobAMut-R	Same as that of pET28a-PobA-R	27
pKE112-LpdC-F/R	Gene synthesis at Gene Art		P. putida KT2440
pET28a-LpdC-F	CATATG GCAGAACAACCATGGGATT	30	pKE112-LpdC-FIR
pET28a-LpdC-R	CTCGAG TTACTTCAAATACTTCTCCCAGTC	31
pKE112-AroY-F/R	Gene synthesis at Gene Art		L. plantarum
			WCFS1
pET28a-AroY-F	CATATG CAGAACCCGATCAACGAC	32	pKE112-AroY-F/R
pET28a-AroY-R	CTCGAG TTACTTCTTGTCGCTGAACAGC	33
pKA312-	Gene synthesis at Gene Art		P. stutzeri OX1
PhKLMOPQ-F/R
pKE112-CatA-F	GGTACCTTTCACACAGGAAACAGACC	34	P. putida KT2440
	ATGACCGTGAAAATTTCCCACAC
pKE112-CatA-R	GGATCC TCAGCCCTCCTGCAACGC	35

TABLE 3
Nucleic acid sequences of genes used in present invention
	UniProtKB		SEQ ID
Gene	Accession Number	Sequence (5′-3′)	NO:
tphAa	Q3C1D2	ATGAACCACCAGATCCATATCCACGACTCCGATATCGCGTTCCCCTGCGC	1
		GCCCGGGCAATCCGTACTGGATGCAGCTCTGCAGGCCGGCATCGAGCTG
		CCCTATTCCTGCCGCAAAGGTAGCTGTGGCAACTGTGCGAGTACGCTGC
		TCGACGGAAATATTGCCTCCTTCAATGGCATGGCCGTGCGAAACGAACT
		CTGCGCCTCGGAACAAGTGCTGCTGTGCGGCTGCACTGCAGCCAGCGAT
		ATACGTATCCACCCGAGCTCCTTTCGCCGTCTCGACCCGGAAGCCCGAA
		AACGTTTTACGGCCAAGGTGTACAGCAATACACTGGCGGCACCCGATGT
		CTCGCTGCTGCGCCTGCGCCTGCCTGTGGGCAAGCGCGCCAAATTTGAA
		GCCGGCCAATACCTGCTGATTCACCTCGACGACGGGGAAAGCCGCAGCT
		ACTCTATGGCCAATCCACCCCATGAGAGCGATGGCATCACATTGCATGTC
		AGGCATGTACCTGGTGGTCGCTTCAGCACTATCGTTCAGCAGTTGAAGT
		CTGGTGACACATTGGATATCGAACTGCCATTCGGCAGCATCGCACTGAA
		GCCTGATGACGCAAGGCCCCTGATTTGCGTTGCGGGTGGCACGGGATTT
		GCGCCCATTAAATCCGTTCTTGATGACTTAGCCAAACGCAAGGTTCAGC
		GCGACATCACGCTGATCTGGGGGGCTCGCAACCCCTCGGGCCTGTATCT
		TCCTAGCGCCATCGACAAGTGGCGCAAAGTCTGGCCACAGTTTCGCTAC
		ATTGCAGCCATCACCGACCTAGGCGATATGCCTGCGGATGCTCACGCAG
		GTCGGGTGGATGACGCGCTACGCACTCACTTTGGCAACCTGCACGATCA
		TGTGGTGCACTGCTGTGGCTCACCAGCTCTGGTTCAATCAGTGCGCACA
		GCCGCTTCCGATATGGGCCTGCTTGCACAGGACTTCCACGCGGATGTTTT
		TGCGACAGGCCCGACTGGTCACCACTAG
tphAb	Q3C1D5	AACAGGCCAACCTTATCGGCCCGGCCGGATTCATTTCCATGGAAGACG	2
		GAGCTGTCGGTGGATTCGTGCAGCGTGGCATCGCAGGCGCTGCCAACC
		TTGATGCAGTCATCGAGATGGGCGGAGACCACGAAGGCTCTAGCGAGG
		GCCGCGCCACGGAAACCTCGGTACGCGGCTTTTGGAAGGCCTACCGCA
		AGCATATGGGACAGGAGATGCAAGCATGA
tphAc	Q3C1D4	ATGATCAATGAAATTCAAATCGCGGCCTTCAATGCCGCCTACGCGAAG	3
		ACCATAGACAGTGATGCAATGGAGCAATGGCCAACCTTTTTCACCAAG
		GATTGCCACTATTGCGTCACCAATGTCGACAACCATGATGAGGGACTT
		GCTGCCGGCATTGTCTGGGCGGATTCGCAGGACATGCTCACCGACCGA
		ATTTCTGCGCTGCGCGAAGCCAATATCTACGAGCGCCACCGCTATCGCC
		ATATCCTGGGTCTGCCTTCGATCCAGTCAGGCGATGCAACACAGGCCA
		GCGCTTCCACTCCGTTCATGGTGCTGCGCATCATGCATACAGGGGAAA
		CAGAGGTCTTTGCCAGCGGTGAGTACCTCGACAAATTCACCACGATCG
		ATGGCAAGTTACGTCTGCAAGAACGCATCGCGGTTTGCGACAGCACGG
		TGACGGACACGCTGATGGCATTGCCGCTATGA
tphB	Q3C1D3	ATGACAATAGTGCACCGTAGATTGGCTTTGGCCATCGGCGATCCCCAC	4
		GGTATTGGCCCAGAAATCGCACTGAAAGCTCTCCAGCAGCTGTCTGTC
		ACCGAAAGGTCTCTTATCAAGGTCTATGGACCTTGGAGCGCTCTCGAG
		CAAGCCGCACGGGTTTGCGAAATGGAGCCGCTTCTTCAAGACATCGTT
		CACGAGGAAGCCGGCACACTTACACAACCAGTTCAATGGGGAGAAATC
		ACCCCGCAGGCTGGTCTATCTACGGTGCAATCCGCAACAGCGGCTATC
		CGAGCGTGCGAAAACGGCGAAGTCGATGCCGTCATTGCCTGCCCTCAC
		CATGAAACGGCCATTCACCGCGCAGGCATAGCGTTCAGCGGCTACCCA
		TCTTTGCTCGCCAATGTTCTTGGCATGAACGAAGACCAGGTATTCCTGA
		TGCTGGTGGGGGCTGGCCTGCGCATAGTGCATGTCACTTTGCATGAAA
		GCGTGCGCAGCGCATTGGAGCGGCTCTCTCCTCAGTTGGTGGTCAACG
		CGGCGCAGGCTGCCGTGCAGACATGCACCTTACTCGGAGTGCCTAAAC
		CAAAAGTCGCTGTATTCGGGATCAACCCTCATGCATCTGAAGGACAGT
		TGTTCGGCCTGGAGGACTCCCAGATCACCGTTCCCGCTGTCGAGACACT
		GCGCAAGCGCGGCCTAGCAGTAGACGGCCCCATGGGAGCTGACATGGT
		TCTGGCACAGCGCAAGCACGACCTGTATGTAGCCATGCTGCACGATCA
		GGGCCATATCCCCATCAAGCTGCTGGCACCTAACGGAGCCAGCGCACT
		ATCTATCGGTGGCAGGGTGGTGCTTTCCAGCGTGGGCCATGGCAGCGC
		CATGGACATTGCCGGCCGTGGCGTGGCTGACGCCACGGCCCTCCTACG
		CACAATAGCCCTACTCGGAGCCCAACCGGTCTGA
SlOMT	K4CX40	GTGAAGCTGGATTCAAAGGTGTTAACCTAATATGTTGTGTCTGTAATTT	5
		TTGGGTCATGGAATTTTACAAGTAG
HsOMT	P21964	ATGGGCGATACCAAAGAACAGCGTATTCTGAATCATGTTCTGCAGCAT	6
		GCCGAACCGGGTAATGCACAGAGCGTTCTGGAAGCAATTGATACCTAT
		TGTGAACAGAAAGAATGGGCCATGAATGTGGGTGATAAAAAAGGCAA
		AATTGTGGATGCCGTGATCCAAGAACATCAGCCGAGCGTGCTGCTGGA
		ACTGGGTGCATATTGTGGTTATAGCGCAGTTCGTATGGCACGTCTGCTG
		AGTCCGGGTGCACGTCTGATTACCATTGAAATTAACCCGGATTGTGCA
		GCAATTACCCAGCGTATGGTTGATTTTGCCGGTGTTAAAGATAAAGTTA
		CCCTGGTTGTTGGTGCAAGCCAGGATATTATTCCGCAGCTGAAAAAAA
		AATATGACGTGGATACCCTGGATATGGTGTTTCTGGATCATTGGAAAG
		ATCGTTATCTGCCGGATACCCTGCTGCTGGAAGAATGTGGTCTGCTGCG
		TAAAGGCACCGTTCTGCTGGCAGATAATGTTATTTGTCCTGGTGCACCG
		GATTTTCTGGCACATGTTCGTGGTAGCAGCTGTTTTGAATGTACCCATT
		ATCAGTCCTTTCTGGAATATCGTGAAGTTGTTGATGGTCTGGAAAAAGC
		CATCTATAAAGGTCCGGGTAGCGAAGCAGGTCCGTAACTTAA
MsOMT	P28002	GTGCTGGATTCCAAGGTTTCAAAGTCCATTGTAATGCTTTCAACACATA	7
		CATCATGGAGTTTCTTAAGAAGGTTTAA
pobA	Q88H28	GCTTCAGCTGGTTCATGACCCAACTGCTGCATGACTTCGGTAGCCACAA	8
		GGACGCCTGGGACCAGAAGATGCAGGAAGCTGACCGCGAGTACTTCCT
		GACCTCGCCGGCGGGCCTGGTGAACATTGCCGAGAACTATGTGGGGCT
		GCCGTTCGAGGAAGTTGCCTGA
pobAMut		GCTTCAGCTGGTTCATGACCCAACTGCTGCATGACTTCGGTAGCCACAA	9
		GGACGCCTGGGACCAGAAGATGCAGGAAGCTGACCGCGAGTACTTCCT
		GACCTCGCCGGCGGGCCTGGTGAACATTGCCGAGAACTTTGTGGGGCT
		GCCGTTCGAGGAAGTTGCCTGA
aroY	B2DCZ6	ACGCCGTGGAAGAGGCGATCCCAGGCTTCCTGCAGAACGTGTACGCCC	10
		ACACCGCCGGTGGCGGTAAGTTCCTGGGCATCCTGCAGGTCAAGAAGC
		GCCAGCCGAGCGACGAAGGCCGTCAGGGCCAAGCCGCCCTGATCGCCC
		TGGCCACCTACAGCGAGCTGAAGAACATCATCCTGGTGGACGAGGACG
		TGGACATCTTCGACAGCGACGACATCCTGTGGGCCATGACCACCCGCA
		TGCAGGGCGACGTGAGCATCACCACCCTGCCAGGCATCCGTGGCCATC
		AGCTGGACCCGAGCCAGAGCCCAGACTACAGCACCAGCATCCGTGGCA
		ACGGCATCAGCTGCAAGACCATCTTCGACTGCACCGTGCCGTGGGCCC
		TGAAAGCCCGTTTCGAGCGTGCCCCATTCATGGAAGTGGACCCGACCC
		CGTGGGCCCCAGAGCTGTTCAGCGACAAGAAGTAA
lpdC	F9US27	AATTGGTTAACCGTGCCATTCCTGGTAAAGTGACGAATGTTTATAATCC	11
		GCCGGCTGGTGGTGGTAAGTTGATGACCATCATGCAGATTCACAAGGA
		TAATGAAGCGGATGAAGGCATTCAACGGCAAGCTGCCTTGCTTGCGTT
		CTCAGCCTTTAAGGAATTGAAGACTGTTATCCTGGTTGATGAAGATGTT
		GATATTTTTGATATGAATGATGTGATTTGGACGATGAATACCCGTTTCC
		AAGCCGATCAGGACTTGATGGTCTTATCAGGCATGCGGAATCATCCGT
		TGGACCCATCGGAACGCCCACAATATGATCCAAAGTCGATTCGTTTCC
		GTGGGATGAGTTCTAAACTAGTGATTGATGGCACCGTACCATTCGATAT
		GAAGGACCAATTTGAACGGGCCCAATTCATGAAAGTGGCTGACTGGGA
		GAAGTATTTGAAGTAA
phK	A0A0S2UP50	ATGACAACTCAACCGGAAACCAAATCCTTTGAAGAGCTGACCCGATAC	12
		ATCCGAGTGCGCAGTGAGCCGGGCGACAAGTTCGTGGAATTCGACTTC
		GCCATTGCTTACCCCGAGCTCTTCGTTGAGCTCGTGCTGCCTCACGAGG
		CCTTCGAGATTTTCTGCAAACATAACAAAGTCGTCCACATGGACTCCAA
		CATAATCCGCAAAATTGACGAAGACATGGTCAAGTGGCGGTTCGGAGA
		GCATGGCAAGCGCTACTGA
phL	Q84AQ4	ATGAGTATTGAAATCAAGACCAATTCGGTGGAACCTATCCGCCATACT	13
		TATGGCCACATCGCCCGTCGCTTCGGTGATAAGCCGGCTACCCGTTATC
		AGGAGGCCAGCTACGACATTGAGGCAAAGACCAATTTCCATTACCGGC
		CCCAGTGGGATTCCGAGCACACCCTGAACGATCCCACGCGTACCGCCA
		TCCGCATGGAAGACTGGTGCGCCGTTTCCGATCCCCGCCAGTTTTACTA
		TGGCGCCTATGTCGGCAACCGGGCCAAGATGCAGGAGTCGGCCGAGAC
		CAGCTTTGGCTTCTGCGAAAAGCGTAATCTGCTGACCCGCCTTTCCGAA
		GAAACCCAGAAGCAATTGTTGCGGCTGCTGGTGCCCCTGCGTCATGTC
		GAGCTTGGCGCCAACATGAACAACGCCAAGATCGCCGGTGATGCCACC
		GCCACGACCGTCTCCCAGATGCACATCTACACTGGGATGGATCGCTTG
		GGCATTGGCCAGTACCTGTCCCGTATTGCATTGATGATTGATGGCAGCA
		CCGGTGCCGCTCTGGATGAGTCCAAGGCCTACTGGATGGATGACGAAA
		TGTGGCAACCCATGCGCAAGCTGGTCGAAGACACGCTTGTGGTCGATG
		ATTGGTTTGAGCTGACTCTGGTTCAGAACATTCTTATCGACGGAATGAT
		GTACCCGCTGGTCTACGACAAGATGGACCAGTGGTTCGAAAGCCAGGG
		TGCTGAAGATGTGTCCATGCTCACGGAGTTCATGCGTGACTGGTACAA
		GGAATCCCTACGCTGGACTAATGCCATGATGAAAGCCGTGGCCGGTGA
		AAGTGAGACTAACCGTGAGTTGCTTCAAAAATGGATCGATCACTGGGA
		ACCGCAGGCCTACGAAGCCCTGAAACCTCTGGCCGAAGCCTCCGTTGG
		CATCGACGGGCTGAATGAAGCCCGGGCGGAACTCTCTGCCCGCCTGAA
		GAAATTCGAACTGCAGAGCCGGGGAGTCTCAGCATGA
phM	Q84AQ3	ATGAGCCAGCTTGTATTTATTGTATTCCAGGACAACGACGACTCCCGCT	14
		ACCTCGCGGAAGCCGTTATGGAAGATAACCCCGACGCCGAAATGCAGC
		ACCAGCCGGCCATGATCCGGATCCAGGCGGAAAAACGTCTGGTGATCA
		ACCGCGAAACCATGGAAGAAAAGCTGGGGCGAGACTGGGATGTTCAG
		GAAATGCTCATAAATGTTATCAGCATCGCCGGCAACGTCGATGAAGAC
		GATGATCACTTCATTCTTGAATGGAATTAA
phN	Q84AQ2	AGTACTGCCAGGCCACCAACTTCCATACTTGGATTCCGGAGAAGGAAG	15
		AGATGGACTGGATGTCCGAGAAGTATCCGGACACTTTCGACAAGTACT
		ACCGTCCGCGTTACGAGTACCTGGCGAAAGAGGCTGCCGCTGGCCGTC
		GCTTCTACAACAACACCCTGCCGCAGCTGTGCCAAGTGTGTCAGATCCC
		GACCATTTTCACCGAGAAAGATGCCCCAACCATGCTCAGCCATCGGCA
		GATAGAACATGAGGGCGAACGCTATCACTTCTGCTCTGACGGCTGCTG
		CGACATCTTCAAACACGAGCCGGAGAAGTACATACAGGCCTGGCTGCC
		GGTGCACCAGATCTACCAGGGCAACTGTGAAGGCGGGGATCTCGAGAC
		CGTGGTGCAGAAGTATTACCACATCAATATCGGAGAGGACAATTTCGA
		CTACGTTGGATCGCCCGACCAGAAACACTGGCTGTCGATCAAGGGCCG
		GAAGCCTGCAGACAAGAACCAGGACGCCGCCTGA
phO	Q84AQ1	ATGAGTGTAAACGCACTTTACGACTACAAGTTTGAACCTAAAGACAAG	16
		GTCGAGAACTTCCACGGCATGCAGCTGCTGTATGTCTACTGGCCCGATC
		ACCTGCTGTTCTGCGCGCCCTTCGCGCTGCTGGTGCAGCCGGGTATGAC
		CTTCAGTGCCCTGGTGGACGAGATTCTCAAGCCGGCTACCGCCGCGCA
		CCCGGACTCTGCCAAGGCGGACTTCCTGAATGCCGAGTGGTTGCTGAA
		CGATGAACCGTTCACACCCAAGGCTGACGCCAGCCTGAAAGAGCAGGG
		TATTGATCACAAGAGCATGCTGACGGTGACCACGCCGGGCCTGAAGGG
		CATGGCGAACGCCGGTTACTGA
phP	Q84AQ0	CTGAAGACACCCAGCGTTCGGCCCTGTTCAAGAAGATATAG	17
phQ	A0A0S2UPA7	ATGGGCATGGGTTTCCTAGTGTTCAACCGCACAACGGGAGGTCACTTT	18
		ACCTGCCAGGAGGGCCAGAGTGTGCTCAAGGCCATGGAGCAGAGGGG
		CCTGAAGTGTGTCCCCGTGGGCTGCCGGGGTGGTGGTTGCGGATTTTGT
		AAGATCCGGGTTCTGGAAGGGTATTTCGAGTGCGGCAAGATGAGCAAG
		CGGCACGCCCCGCCTGAAGCCGTTGAAAAAGGGGAAGTTCTGGCCTGC
		CGGATCTACCCACTGACTGATCTGATCATTGAGTGTCCGCCGCAACCGG
		CGGCGGACTTTGCGAGCTAG
catA	Q88GK8	ATGACCGTGAAAATTTCCCACACTGCCGACATTCAAGCCTTCTTCAACC	19
		GGGTAGCTGGCCTGGACCATGCCGAAGGAAACCCGCGCTTCAAGCAGA
		TCATTCTGCGCGTGCTGCAAGACACCGCCCGCCTGATCGAAGACCTGG
		AGATTACCGAGGACGAGTTCTGGCACGCCGTCGACTACCTCAACCGCC
		TGGGCGGCCGTAACGAGGCAGGCCTGCTGGCTGCTGGCCTGGGTATCG
		AGCACTTCCTCGACCTGCTGCAGGATGCCAAGGATGCCGAAGCCGGCC
		TTGGCGGCGGCACCCCGCGCACCATCGAAGGCCCGTTGTACGTTGCCG
		GGGCGCCGCTGGCCCAGGGCGAAGCGCGCATGGACGACGGCACTGAC
		CCAGGCGTGGTGATGTTCCTTCAGGGCCAGGTGTTCGATGCCGACGGC
		AAGCCGTTGGCCGGTGCCACCGTCGACCTGTGGCACGCCAATACCCAG
		GGCACCTATTCGTACTTCGATTCGACCCAGTCCGAGTTCAACCTGCGTC
		GGCGTATCATCACCGATGCCGAGGGCCGCTACCGCGCGCGCTCGATCG
		TGCCGTCCGGGTATGGCTGCGACCCGCAGGGCCCAACCCAGGAATGCC
		TGGACCTGCTCGGCCGCCACGGCCAGCGCCCGGCGCACGTGCACTTCT
		TCATCTCGGCACCGGGGCACCGCCACCTGACCACGCAGATCAACTTTG
		CTGGCGACAAGTACCTGTGGGACGACTTTGCCTATGCCACCCGCGACG
		GGCTGATCGGCGAACTGCGTTTTGTCGAGGATGCGGCGGCGGCGCGCG
		ACCGCGGTGTGCAAGGCGAGCGCTTTGCCGAGCTGTCATTCGACTTCC
		GCTTGCAGGGTGCCAAGTCGCCTGACGCCGAGGCGCGAAGCCATCGGC
		CGCGGGCGTTGCAGGAGGGCTGA

Experimental Example 1

For the depolymerization of PET into monomers TPA and EG, 1 g of PET in 13 mL of water was reacted, and thus the depolymerization of PET was carried out at various temperatures of 170, 200, and 230° C. using microwaves for various reaction times of 15 to 50 minutes (FIG. 1A). During the initial hydrolysis periods, the amount of TPA slowly increased due to the random chain cleavage of PET into TPA and EG (FIG. 1A). After these periods, PET depolymerization rapidly increased by autocatalysis induced by the reaction product TPA. Among the various reaction conditions, the highest TPA yield was obtained after 50 minutes at 230° C. (FIG. 1A). This highest yield was determined to be 99.9% of the theoretical maximum TPA yield calculated from the PET consumed during the reaction, in which 24.1% (w/w) of the initial input PET was consumed after 50 minutes at 230° C. by the reaction. These results indicate that a large amount of TPA in monomeric form can be obtained from PET hydrolysis without overdegradation.

The PET hydrolysate was separated into solid and liquid fractions by filtration. First, to obtain TPA, the solid fraction containing TPA was dissolved in 1 M NaOH, and Na-TPA was then precipitated as TPA by 2 M HCl at room temperature. The precipitated TPA was filtered and vacuum-dried at 80° C. (FIG. 2A). To confirm the identity of the TPA samples, ¹H and ¹³C NMR analyses were performed (FIGS. 2B and 2C). Chemical shifts of the two spectra were identical to those of reagent-grade TPA.

For the separation of EG from the PET hydrolysate obtained by chemical hydrolysis, the liquid fraction was distilled, and ¹H and ¹³C NMR analyses were performed to confirm the identity of the EG samples (FIGS. 2D and 2E). In this case, chemical shifts were identical to those of reagent-grade EG.

<Experimental Example 2> Bioconversion of TPA to PCA

To experimentally validate the applicability of TPA obtained from waste PET as a feedstock for producing higher-value compounds than PET, PCA was selected as a first product as well as a key intermediate. PCA can serve as a precursor compound for producing various high value-added aromatic compounds or aromatic-derived compounds such as GA, pyrogallol, catechol, MA, and VA (FIG. 3). Therefore, it is important to establish an efficient biocatalyst capable of converting TPA to PCA. The bioconversion of TPA to PCA via DCD has been revealed only in vitro from the TPA degradation pathway of several bacteria that metabolize TPA as a sole carbon source, such as Comamonas sp. E6, Delftia tsuruhatensis T7, and Rhodococcus sp. DK17. The TPA degradation pathway includes two enzymes, TPA 1,2-dioxygenase and DCD dehydrogenase, wherein TPA 1,2-dioxygenase converts TPA to DCD, and DCD dehydrogenase converts DCD to PCA.

In this experiment, TphAabc which is TPA 1,2-dioxygenase and TphB which is DCD dehydrogenase, both derived from Comamonas sp. E6, were used for a biosynthesis route from TPA to PCA in E. coli. Unlike other corresponding enzymes of other microbes, these two enzymes have the advantage of possessing dual cofactor utilization capability for both NADH and NADPH. To dissolve TPA in a buffer solution, NaOH was added to adjust pH to 7, and then a 50 g/L TPA solution was prepared for further conversion reactions wherein TPA can be dissolved at a concentration of more than 0.5 g/L. When TPA from the PET hydrolysate was incubated with E. coli strain PCA-1 expressing TphAabc and TphB in TG-1 buffer (FIG. 1B), 2.8 mM PCA was produced at a molar yield of 81.4% after three hours, and this was similar to that from reagent-grade TPA (FIG. 1C). PCA production was confirmed by GC/MS (FIGS. 1D and 4A). Since the in vivo production of compounds from TPA has not been reported yet, this experiment is the first experimental demonstration of in vivo PCA production from TPA. TPA from the PET hydrolysate can be used as a feedstock for producing PCA, a key precursor for aromatic compounds or aromatic-derived compounds. Since PCA is a key intermediate compound in lignin refineries, PCA itself has value as a substrate in other bioconversions.

<Experimental Example 3> Bioconversion of TPA to GA

GA is currently used in the pharmaceutical industry to produce trimethoprim, an antibacterial agent, and propyl gallate, an antioxidant. When a PCA hydroxylase having hydroxylating activity at the meta-position of PCA is identified, TPA can be converted to GA via PCA (FIG. 5A). Although a wild-type p-hydroxybenzoate hydroxylase (PobA from Pseudomonas aeruginosa) hydroxylated 4-HBA but not PCA (i.e., 3,4-dihydroxybenzoic acid), structure-based engineered PobA hydroxylated both 4-HBA and PCA to GA.

In this experiment, PobA from P. putida KT2440 was expressed in E. coli (Strain HBH-1) to test the ability of PobA from P. putida KT2440 to hydroxylate the meta-position of PCA. As a result, strain HBH-1 produced 1.4 mM GA from PCA at a molar yield of 40.1% after 12 hours in TG-2 buffer at 30° C. and 250 rpm (FIG. 6A). To enhance the hydroxylation by PobA, structure-based enzyme engineering following a previous approach was performed. According to molecular docking simulations, Tyr201 formed two hydrogen bonds with Tyr386 and PCA in active sites of wild-type PobA (FIG. 7A). However, PCA formed two hydrogen bonds with Tyr201 and Ala294 at the active site of modeled double mutant PobA^Mut (T294A/Y385F), thus leading to a shorter binding distance between a substrate and a FAD cofactor (FIG. 7B). To validate the modeling results, double mutant PobA^Mut (T294A/Y385F) was constructed and was expressed in E. coli strain HBH-2. Using the HBH-2 strain expressing PobA^Mut, 2.5 mM GA was produced from PCA at a molar yield of 74.3% after 12 hours (FIG. 6A), which was an 83.7% increase compared to that of wild-type PobA.

First, the single-catalyst GA-1 system consisting of E. coli strain GA-1 expressing TphAabc, TphB, and PobA^Mut was tested to produce GA from TPA. The GA-1 system produced only 1.3 mM GA at a molar yield of 46.6% from TPA after 12 hours in TG-2 buffer, but 1.1 mM PCA remained (FIG. 6B). This may be due to the redox imbalance caused by the biosynthesis of GA from PCA by utilizing NAD(P)H in the single-strain system GA-1. To improve the GA yield from TPA, the GA-2a system was constructed in which strains PCA-1 and HBH-2 were simultaneously added to catalyze the synthesis of PCA from TPA and the synthesis of GA from PCA, respectively. However, the GA synthesis catalyst produced only 0.5 mM GA from 3.1 mM TPA with a molar yield of 15.9% (FIG. 6C). Since 2.1 mM of intermediate PCA was accumulated without being converted to GA, the second reaction by the GA synthesis catalyst was a rate-limiting reaction in the GA-2a system. To promote the second conversion step, the OD₆₀₀ values between PCA and GA synthesis catalysts were changed from 20 and 20 in the GA-2a system to 10 and 30 in the GA-2b system (FIG. 6D). As a result, the production and molar yield of GA from TPA increased to 2.7 mM GA and 92.5%, respectively, after 24 hours without accumulating PCA (FIGS. 5B and 6D). GA production by the GA-2b system was confirmed by GC/MS (FIGS. 5C and 4B).

Experimental Example 4> Bioconversion of TPA to Pyrogallol via GA

Pyrogallol is another high value-added compound that can be produced from TPA via PCA. Pyrogallol is currently used as an antioxidant in the oil industry. Pyrogallol can be biosynthesized through two routes: via the decarboxylation of GA synthesized by PCA hydroxylation (FIG. 8A), and via the hydroxylation of catechol that can be synthesized by PCA decarboxylation (FIG. 8B).

To develop biosynthesis routes for pyrogallol via GA, LpdC, which was found to be a GA decarboxylase in vitro, was introduced as a GA decarboxylation module into the GA biosynthesis route. As a result, E. coli strain PG-1a expressing TphAabc, TphB, PobA^Mut, and LpdC was constructed (FIG. 8A). The PG-1a strain produced 1.1 mM pyrogallol from TPA at a molar yield of 32.7% in TG-2 buffer at 30° C. and 250 rpm after six hours (FIGS. 8C and 9A), and pyrogallol production was confirmed by GC/MS (FIGS. 8D and 4C). However, a substantial amount of catechol, 1.6 mM, was also produced as a byproduct after six hours; this was caused by the promiscuity of GA decarboxylase, LpdC, toward PCA. LpdC converted not only GA to pyrogallol but also PCA to catechol. For example, E. coli strain GDC-1 expressing LpdC converted 3.0 mM PCA to 2.9 mM catechol after eight hours and then converted 3.0 mM GA to 2.8 mM pyrogallol after 18 hours (FIGS. 10A and 10B). These results indicate that LpdC has promiscuous activity toward PCA because LpdC was previously reported as a GA decarboxylase. Therefore, catechol production was inevitable when using the GA biosynthesis route with PCA as an intermediate.

For relieving catechol accumulation due to LpdC promiscuity, it was necessary to convert accumulated catechol to pyrogallol. Although a catechol hydroxylase capable of converting catechol to pyrogallol has not yet been reported, a PhKLMNOPQ operon encoding a phenol hydroxylase from Pseudomonas stutzeri OX1 was recently found to exhibit promiscuous activity in converting catechol to pyrogallol. E. coli strain CH-1 expressing PhKLMNOPQ produced 2.6 mM pyrogallol from catechol at a molar yield of 67.1% after 24 hours (FIG. 11A). The catechol hydroxylation module of PhKLMNOPQ was added to E. coli strain PG-1a to construct E. coli strain PG-1b (FIG. 8A). However, even when the PG-1b system harboring the catechol hydroxylation module of PhKLMNOPQ was applied to pyrogallol production from TPA, catechol accumulation slightly increased, and the pyrogallol production of 0.7 mM after 12 hours was slightly lower (FIG. 9B) than 1.1 mM produced by the PG-1a system after six hours (FIG. 9B). Comparing GA accumulation and its conversion to pyrogallol between the PG-1a and PG-1b systems, PG-1b accumulated less GA but produced less pyrogallol (FIG. 9B) than PG-1a (FIG. 9A). In PG-1b, the catechol hydroxylation module seemed inactive without converting catechol but accumulated a larger amount of catechol (FIG. 9B) than in PG-1a (FIG. 9A). These results imply that pyrogallol synthesis via the two biosynthesis routes involving two different hydroxylation reactions may be inefficient. This may be because both hydroxylation modules require one or more NAD(P)H molecules.

<Experimental Example 5> Bioconversion of TPA to Pyrogallol via Catechol

To synthesize pyrogallol without forming a catechol byproduct caused by the promiscuity of LpdC, an alternative pyrogallol synthesis route via catechol was adopted. Based on the PCA synthesis module for TPA conversion to PCA (i.e., the PCA-1 system), the pyrogallol synthesis route was constructed by integrating the PCA decarboxylation module for PCA conversion to catechol and the catechol hydroxylation module for catechol conversion by PhKLMNOPQ in either a single- or double-strain system (i.e., the PG-2a and PG-2b systems, respectively) (FIG. 8B). An AroY enzyme, which was identified as a PCA decarboxylase in several microbes, was adopted as the PCA decarboxylation module. First, the conversion of PCA to catechol by E. coli strain PDC-1 expressing AroY was verified, in which PCA was converted to 2.9 mM catechol at a molar yield of 97.8% in TG-2 buffer after five hours (FIG. 11B). The function of the catechol hydroxylation module for catechol conversion to pyrogallol by PhKLMNOPQ had been verified using the CH-1 strain (FIG. 11A). To further evaluate the capability of the PCA decarboxylation module to produce catechol and AroY in conjunction with the PCA synthesis module for TPA conversion to PCA, E. coli strain CTL-1 expressing TphAabc, TphB, and AroY was tested, and strain CTL-1 produced 2.7 mM catechol from TPA at a molar yield of 90.1% without PCA accumulation after four hours (FIG. 9C).

Next, in the PG-2a system, when the PCA synthesis strain PCA-1 and the PCA-to-pyrogallol conversion strain PDC-CH-1 expressing AroY and PhKLMNOPQ were simultaneously incubated with 3.1 mM TPA, only 0.2 mM pyrogallol was produced, but 2.4 mM catechol remained unconverted after 20 hours in TG-2 buffer (FIG. 9D). To troubleshoot the PG-2a system, when strain PDC-CH-1 was tested alone with 3.2 mM PCA as a substrate, PCA seemed to be completely converted to catechol after 20 hours, but only 1.2 mM pyrogallol was produced from catechol at a molar yield of approximately 39.0% (FIG. 11C). This pyrogallol yield from catechol by the PDC-CH-1 strain was 1.7 times lower than the 2.6 mM pyrogallol produced by E. coli strain CH-1 expressing only PhKLMNOPQ after 24 hours (FIGS. 11A and 11C). These results indicate that the expression of PhKLMNOPQ alone was advantageous for the conversion of catechol to pyrogallol. This may have been due to the uncertainty in generating the correct form of multiunit PhKLMNOPQ composed of PhK, Ph(LNO)₂, PhP, PhQ, and PhM subunits when co-expressed with AroY. Therefore, the PGA-2a system consisting of strains PCA-1 and PDC-CH-1 was replaced with the PG-2b system (FIG. 8B) consisting of E. coli strain CTL-1 expressing AroY, TphAabc, and TphB and E. coli strain CH-1 expressing PhKLMNOPQ alone (FIG. 9E). Pyrogallol production by the PG-2b system (i.e., 0.6 mM) was three times higher than that by the PG-2a system (i.e., 0.2 mM); this is likely attributed to the singular expression of PhKLMNOPQ. However, a substantial amount of catechol, 1.6 mM, remained unconverted (FIG. 9E).

<Experimental Example 6> Bioconversion of TPA to MA

Catechol synthesized from TPA via PCA can be converted to MA by the ring cleavage of catechol. MA is currently used in the chemical industry to produce adipic acid, which is widely used to produce plastics. To develop an MA biosynthesis route from TPA, CatA, a catechol 1,2-dioxygenase originating from P. putida KT2440, was tested as the ring-cleavage module. When 4.5 mM catechol was incubated with E. coli strain CDO-1 expressing CatA, its complete conversion to MA occurred after 10 minutes (FIG. 12A). This MA synthesis module was combined with the catechol biosynthesis route of strain CTL-1 expressing TphAabc, TphB, and AroY (FIG. 9C) to construct E. coli strain MA-1 harboring the MA biosynthesis route beginning with TPA (FIG. 13C). The MA-1 system containing the MA-1 strain converted 3.2 mM TPA to 2.7 mM MA at a molar conversion yield of 85.4% after six hours without accumulating intermediates (FIGS. 12B and 13B). The biosynthesis route of TPA to PCA exhibited no redox imbalances (FIG. 13A). GC/MS confirmed that Ma was produced by strain MA-1 (FIGS. 4D and 13C).

<Experimental Example 7> Bioconversion of TPA to VA using Single-Catalyst Systems

VA is used as the direct precursor of vanillin in the pharmaceutical industry. PCA is converted to VA by an OMT both in vitro and in vivo. To supply a methyl group to this O-methylation reaction catalyzed by an OMT, SAM is used as a co-substrate, and the adenosyl and methyl groups are supplied from ATP and methionine, respectively. Currently known OMTs are derived from eukaryotes; however, in the present invention, the expression of OMTs from various sources was tested in E. coli BL21 (DE3) to construct a VA synthesis module. Among the three OMTs examined in the present invention, HsOMT from H. sapiens, SlOMT from Solanum lycopersicum, and MsOMT from M. sativa, only HsOMT and SlOMT were expressed in active forms (FIG. 14A). Whole-cell conversions by E. coli strains OMT-1a and OMT-1b expressing HsOMT and SlOMT, respectively, were compared. By strain OMT-1a, 3.2 mM PCA was converted to 1.0 mM VA at a molar yield of 29.4% (FIG. 14B) in 0.1 M phosphate buffer supplemented with 20 and 10 g/L peptone containing 0.94 and 0.54 mM methionine, respectively; wherein a yield by strain OMT-1a was 1.4 times higher than that by strain OMT-1b (FIG. 14C). Therefore, HsOMT was selected for the synthesis module for PCA conversion to VA in further experiments.

To produce VA directly from TPA via PCA, the biosynthesis route of TPA to PCA was connected to the PCA to VA route using HPAOM by constructing E. coli strain VA-1 expressing TphAabc, TphB, and HsOMT (FIG. 19A). When 3.3 mM TPA was incubated with strain VA-1, TPA was completely consumed; however, only 0.02 mM VA was produced, and 2.3 mM PCA accumulated after 72 hours in TG-1/YP buffer (FIG. 15A). This low conversion of PCA to VA was confirmed by the poor consumption of glycerol (FIG. 16A) and methionine (FIG. 16B). This low conversion yield may be attributed to the low soluble expression of HsOMT (FIG. 14A).

To improve the low PCA conversion to VA by strain VA-1 (FIG. 15A), the protein solubility of HsOMT was increased by the attachment of hexameric histidine to the N-terminus of HsOMT, which is known to increase protein solubility. Although strain OMT-2 expressing wild-type HsOMT produced 0.65 mM VA (FIG. 17A), strain OMT-2^His expressing HsOMT^His with the N-terminal hexameric histidine exhibited 10.7% higher VA production than the strain wild-type HsOMT (FIGS. 17A and 17B). Therefore, HsOMT^His was used in further experiments.

To further improve the low conversion of PCA to VA, conversion conditions were optimized using strain OMT-2^His. In particular, since endogenous SAM regeneration may be inefficient, it was promoted by supplementing methionine in TG-2/YPM buffer. When strain OMT-2^His was incubated in TG-2/YP buffer lacking supplemented methionine, only 0.9 mM VA was produced from 2.9 mM PCA after 48 hours (FIG. 18A). Since TG-2/YP buffer contains only 0.8 mM free methionine from yeast extract and peptone, 2.5 mM methionine was added to the TG-2/YPM buffer to balance the methionine molar concentration with that of PCA. As a result, the molar yield of VA from 2.9 mM PCA by strain OMT-2^His in TG-2/YPM buffer was 44.5%, which was 40.0% higher than that in TG-2/YP buffer (FIG. 18B).

<Experimental Example 8> Bioconversion of TPA to VA using Double-Catalyst Systems

In the single-catalyst system, PCA was accumulated due to the negligible conversion of PCA to VA. To bolster the conversion of PCA to VA, the present inventors developed the double-catalyst VA-2a system in which strain PCA-1 expressing TphAabc and TphB and strain OMT-2^His expressing HsOMT^His (FIG. 15B) and having different OD₆₀₀ values of 10 and 30, respectively, were simultaneously added into TG-2/YPM buffer containing TPA (FIG. 19A). As a result, VA produced from 3.4 mM TPA by the VA-2a system increased to 0.3 mM after 48 hours (FIGS. 15A and 15B), but the molar yield of VA from PCA converted from TPA remained at 6.4% (FIG. 19B). To further increase the conversion of PCA to VA, O-methylation catalyzed by HsOMT^His was enhanced by increasing oxygen supply because adenosyl groups necessary for O-methylation may be supplied from ATP. To increase ATP generation by improving aeration, the VA-2b system used baffled flasks in place of the conical tubes used for the VA-2a system (FIG. 19A). As a result, VA produced by VA-2b after 48 hours increased to 1.4 mM VA at a molar yield of 41.6% (FIG. 15C), which was 4.7 times higher than that produced by the VA-2a system using conical tubes (FIGS. 15C and 15B). This enhanced VA production due to increased aeration was correlated with increased glycerol and methionine consumption (FIGS. 16A to 16D). Therefore, aeration is critical for increasing VA consumption from PCA because glycerol is efficiently metabolized to generate ATP, thus accelerating SAM synthesis from methionine by supplying S-adenosyl groups. VA production by the VA-2b system was confirmed by GC/MS (FIGS. 19C and 4E). These results imply that SAM regeneration by the improved supply of methionine and adenosyl groups from ATP is critical for O-methylation of PCA by an OMT.

In the VA-2b system, however, 1.4 mM TPA remained unconverted (FIG. 15C). TPA conversion to PCA was increased to address this issue by adjusting the OD₆₀₀ values of strains PCA-1 and OMT-2^His in the VA-2b system from 10 to 20 and 30 to 20, respectively. However, VA production after 48 hours decreased to 0.4 mM VA while TPA was completely consumed (FIG. 15D). To increase the conversion of TPA to VA, the flux of conversion of TPA to PCA and PCA to VA requires further optimization.

<Experimental Example 9> Bioconversion of EG to GLA

To experimentally validate the applicability of EG from waste PET as a feedstock, an EG sample obtained from the PET hydrolysate was tested using G. oxydans KCCM 40109 to produce GLA (FIGS. 1E and 20). GLA is used as an exfoliant in cosmetics. Reagent-grade samples containing 11.3, 28.6, and 67.6 mM EG were converted to GLA after 12 hours with molar yields of 95.3, 99.7, and 89.4%, respectively (FIGS. 20B and 20C). The sample containing 10.7 mM EG from the PET hydrolysate was converted to GLA with a molar yield of 98.6% after 12 hours (FIG. 1F). GLA production by G. oxydans was confirmed by GC/MS (FIGS. 1G and 4F).

The present invention is applicable to the field of PET upcycling.

Claims

1. A method of producing a high value-added compound from polyethylene terephthalate, comprising: producing terephthalic acid and ethylene glycol through hydrolysis of polyethylene terephthalate; andproducing one or more compounds selected from the group consisting of gallic acid, pyrogallol, catechol, muconic acid, and vanillic acid through bioconversion of the terephthalic acid in the presence of a biocatalyst, wherein protocatechuic acid is an intermediate produced by the bioconversion, orproducing glycolic acid through fermentation of the ethylene glycol.

2. The method of claim 1, wherein the hydrolysis of polyethylene terephthalate is performed by applying microwaves.

3. The method of claim 1, wherein the bioconversion of terephthalic acid to protocatechuic acid is performed using a microbe expressing terephthalic acid 1,2-dioxygenase and 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate dehydrogenase as a biocatalyst.

4. The method of claim 1, wherein the bioconversion of terephthalic acid to gallic acid is performed using a microbe expressing terephthalic acid 1,2-dioxygenase, 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate dehydrogenase, and p-hydroxybenzoate hydroxylase as a biocatalyst, or using a combination of a microbe expressing terephthalic acid 1,2-dioxygenase and 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate dehydrogenase and a microbe expressing p-hydroxybenzoate hydroxylase as a biocatalyst.

5. The method of claim 1, wherein the bioconversion of terephthalic acid to pyrogallol is performed using a microbe expressing terephthalic acid 1,2-dioxygenase, 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate dehydrogenase, p-hydroxybenzoate hydroxylase, and gallic acid decarboxylase as a biocatalyst, or using a combination of a microbe expressing terephthalic acid 1,2-dioxygenase, 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate dehydrogenase, and protocatechuic acid decarboxylase and a microbe expressing a phenol hydroxylase as a biocatalyst.

6. The method of claim 1, wherein the bioconversion of terephthalic acid to muconic acid is performed using a microbe expressing terephthalic acid 1,2-dioxygenase, 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate dehydrogenase, protocatechuic acid decarboxylase, and catechol 1,2-dioxygenase as a biocatalyst.

7. The method of claim 1, wherein the bioconversion of terephthalic acid to vanillic acid is performed in a medium containing glycerol and methionine while using a combination of a microbe expressing terephthalic acid 1,2-dioxygenase and 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate dehydrogenase and a microbe expressing human-derived O-methyltransferase as a biocatalyst.

8. The method of claim 1, wherein the fermentation of ethylene glycol is performed using one or more ethylene glycol-fermenting microbes selected from the group consisting of Gluconobacter oxydans KCCM 40109, Clostridium glycolicum, and Pseudomonas putida.