ANDERSON KENNETH B (US)
WO2021145822A1 | 2021-07-22 |
US20210180007A1 | 2021-06-17 |
CLAIMS: We claim: 1. A recombinant bacterial cell comprising a heterologous DNA encoding and expressing at least one heterologous PETase-like enzyme and at least one heterologous MHETase-like enzyme, wherein the PETase-like enzyme has a secretion signal peptide linked in frame to an enzymatic activity for degrading Bis(2-hydroxyethyl) terephthalate (BHET) into Mono-(2-hydroxyethyl)terephthalic acid (MHET) and comprises Leaf-Compost Cutinase (LCC) enzyme with SEQ ID NO: 5, or a functional variant thereof having at least 85% overall sequence identity to SEQ ID NO: 5; and wherein the MHETase-like enzyme has a secretion signal peptide linked in frame to an enzymatic activity for degrading MHET into ethylene glycol and terephthalic acid and comprises a polypeptide with SEQ ID NO: 6, a functional variant thereof having at least 85% overall sequence identity to SEQ ID NO: 6, Mle046 enzyme with SEQ ID NO: 13, a functional variant thereof having at least 85% overall sequence identity to SEQ ID NO: 13, Mle046 mutant enzyme with SEQ ID NO: 14, a functional variant thereof having at least 85% overall sequence identity to SEQ ID NO: 14, or any combination thereof. 2. The recombinant bacterial cell according to claim 1, wherein the recombinant bacterial cell is Pseudomonas putida or Erwinia aphidicola. 3. The recombinant bacterial cell according to claim 1 or claim 2, wherein the PETase- like enzyme and/or the MHETase-like enzyme is thermostable and enzymatically active at a temperature ranging from about 30 °C to about 75 °C and/or at a pH ranging from about 6 to about 9. 4. The recombinant bacterial cell according to any one of claims 1-3, wherein the PETase-like enzyme and/or the MHETase-like enzyme is expressed from an inducible promoter. 5. The recombinant bacterial cell according to any one of claims 1-4, wherein the PETase-like enzyme and the MHETase-like enzyme are encoded by a plasmid and co- expressed from a single promoter. 6. The recombinant bacterial cell according to any one of claims 1-5, wherein the recombinant bacterial cell contains a mutation in its genome, the mutation eliminating expression of muconate cycloisomerase enzymatic activity with SEQ ID NO: 4, wherein the mutation is a deletion and/or insertion of at least one nucleotide or more. 7. The recombinant bacterial cell according to any one of claims 1-6, wherein the PETase-like enzyme and the MHETase-like enzyme are enzymatically active at 30°C, degrading bis(2-hydroxyethyl) terephthalate (BHET) into ethylene glycol and terephthalic acid. 8. The recombinant bacterial cell according to any one of claims 1-7, wherein the bacterial cell is capable of growing on a substrate containing biomass co-mixed with polyethylene terephthalate (PET) pretreated in oxidative hydrothermal dissolution (OHD) process. 9. The recombinant bacterial cell according to claim 8, wherein the biomass contains green tea waste, black tea waste, used green tea, used black tea, corn stover and/or coffee brewing waste; and/or wherein the substrate contains the biomass and PET in the following ratio by weight from 1:99 wt% to 50:50 wt% of PET to the biomass. 10. A method for producing an enzymatic composition for biodegradation of plastic material and/or biomass waste, the method comprising: - culturing the recombinant bacterial cell accordingly to any one of claims 1-9 in a liquid medium, wherein the recombinant bacterial cell secrets the PETase-like enzyme and the MHETase enzyme into the liquid medium; and - collecting the liquid medium containing the PETase-like enzyme and the MHETase-like enzyme. 11. The method of claim 10, wherein the method further comprises: - centrifuging a bacterial culture and producing a supernatant and a pellet; and - collecting the supernatant containing the PETase-like enzyme and the MHETase-like enzyme. 12. A method for decomposing a plastic material containing PET (polyethylene terephthalate) or PBAT (poly(butylene adipate-co-terephthalate), the method comprising: contacting the plastic material with the recombinant bacterial cell according to any one of claims 1-9 and/or an enzymatic compositioncomprising at least one PETase-like enzyme and at least one MHETase-like enzyme, the enzymatic composition being produced or producible by the recombinant bacterial cell. 13. The method of claim 12, wherein the method further comprises prior to contacting the plastic material with the recombinant bacterial cell, co-mixing the plastic material with a biomass and processing the co-mixture by oxidative hydrothermal dissolution (OHD) in the presence of oxygen in subcritical water at a temperature in the range 100- 374°C and a pressure in the range 1500 to 3500 psi. 14. The method of claim 13, wherein the biomass and PET are present in a ratio by weight ranging from 1:99 wt% to 50:50 wt% of PET to the biomass. 15. The method of claim 13 or 14, wherein the plastic material is contacted at ambient temperature and a pH in the range from about 6 to about 9. 16. A method for converting a biomass into carbon-containing substrate for synthesizing polymeric products, the method comprising treating the biomass in a hydrothermal dissolution (OHD) process in the presence of oxygen in subcritical water at a temperature in the range 100 -374 °C and a pressure in the range 1500 to 3500 psi, and contacting the OHD-treated biomass with a recombinant bacterium according to any one of claims 1 to 9, or an enzymatic composition comprising at least one PETase-like enzyme and at least one MHETase-like enzyme, the enzymatic composition being produced or being producible by the recombinant bacterial cell. 17. The method of claim 16, wherein the biomass includes tea waste, coffee waste and/or corn stover. 18. A dual enzyme composition comprising at least one PETase-like enzyme and at least one MHETase-like enzyme, the composition being produced and secreted by the recombinant bacterial cell or being producible by the recombinant bacterial cell according to any one of claims 1-9. 19. A dual enzyme composition comprising at least one PETase-like enzyme and at least one MHETase-like enzyme, wherein the PETase-like enzyme comprises at least amino acids 28-320 of the polypeptide with SEQ ID NO: 5 and/or a functional variant therefore having at least 80% overall sequence identity to amino acids 28-320 of SEQ ID NO: 5, or any combination thereof, wherein the PETase-like enzyme having an enzymatic activity for degrading bis(2-hydroxyethyl) terephthalate (BHET) to mono-(2- hydroxyethyl)terephthalic acid (MHET); and wherein the MHETase-like enzyme comprises at least amino acids 18-613 of the polypeptide with SEQ ID NO: 6 and/or a functional variant thereof having at least 80% overall sequence identity to amino acids 28-320 of SEQ ID NO: 5, or any combination thereof, and/or Mle046 enzyme comprising SEQ ID NO: 13 and/or a functional variant therefore having at least 80% overall sequence identity to SEQ ID NO: 13, and/or mutant Mle046 enzyme comprising SEQ ID NO: 14, or a functional variant thereof having at least 80 % overall sequence identity to SEQ ID NO: 14, and wherein the MHETase-like enzyme has an enzymatic activity for degrading MHET to terephthalic acid (TPA) and ethylene glycol (EG). 20. The dual enzyme composition according to claim 19, wherein the at least one PETase- like enzyme and/or the at least one MHETase-like enzyme is linked in frame with at least one signal peptide and/or at least one purification tag. 21. The dual enzyme composition according to any one of claims 19-20, wherein the molar ratio of the PETase-like enzyme to the MHETase-like enzyme is in a range from about 1:99 to about 99:1. 22. A method for producing cis-cis muconate, the method comprising contacting a substrate comprising terephthalic acid (TPA) with a recombinant bacterial cell containing a mutation in its genome, the mutation eliminating expression of muconate cycloisomerase enzymatic activity with SEQ ID NO: 4, wherein the mutation is a deletion and/or insertion of at least one nucleotide or more and wherein the substrate is produced by degradation of biomass waste and/or plastic material. 23. The method of claim 22, wherein the degradation includes an oxidative hydrothermal dissolution (OHD) process and/or reacting the substrate with the dual enzyme composition according to claim 18 or 19. |
Example 2. Analysis of BHET Hydrolysis Products. [00135] Shake flask experiments were performed using M9 minimal medium (Fisher Scientific) containing 33.9 g/L disodium phosphate (anhydrous), 15.0 g/L monopotassium phosphate, 2.5 g/L sodium chloride, 5.0 g/L ammonium chloride, 4 mM magnesium sulphate, 36 μM ferrous sulphate, 200 μM calcium chloride supplemented with 20 mM glucose (Fisher Scientific) and 5 mM bis (2-hydroxyethyl) terephthalate (BHET) (Fisher Scientific). Overnight cultures of strains in LB medium supplemented with the antibiotics were harvested and washed with M9 medium. A fresh culture of 25 mL in 250 mL flasks were prepared with a starting OD600 value of 1.0 and shake flasks were incubated at 30 °C shaking at 225 rpm. All the shake flask experiments were performed in 3 replicates. The concentrations of BHET and terephthalic acid (TPA) filtered standard solutions were measured using high performance liquid chromatography (HPLC) using Agilent 1100 system using Eclipse Plus C18 column (4.6X100 mm, 3.5 μm) (Agilent,USA). Acetonitrile (C) and 0.5 % formic acid (D) were used as the mobile phase with a flow rate of 0.5 ml/min and maximum pressure of 4000.00 psi and the method parameters are as given in TABLE 2. The compounds were detected using diode array detector (DAD) and fluorescence detectors (FLD). All instrument control, data analysis and data processing were performed with Agilent OpenLAB control Panel software. Compounds were identified based on the retention times compared with that of the standards and the concentrations were calculated based on calibration curve generated for each sample by the software. HPLC analysis was done at time intervals 0, 6, 12, 24, 36, and 48 hours. Growth of the cultures was determined by measuring OD600 value at each time point by using spectrophotometer (GENESYS 30, Thermo Scientific, USA). TABLE 2. HPLC solvent gradient for 20 min run. Example 3. CRISPR-Cas9 Based-Gene Deletion in Erwinia aphidicola LJJL-01. [00136] Erwinia aphidicola LJJL01 gene deletion was done using CRISPR-Cas9 genome editing system. All electrocompetent competent cells used in this experiment were prepared using 0.3M sucrose. Overnight cultures of 5 mL LB with the antibiotics were used to make fresh LB cultures in the following day with OD600 value of 0.1. Strains containing λ-Red plasmid (modified by our lab) and pX2-Cas9 plasmids (Addgene: Cat# 85811) were induced with 10 mM arabinose and the culture was allowed to grow until it reaches an OD600 value of 0.4. Competent cells were prepared as described by Franden et al., 2018.5 μL of plasmids were transformed into 50 μL of electrocompetent cells in chilled electroporation cuvettes and electroporated at 1600V using Eppendorf Electroporator 2510. Five hundred microliters of SOC medium was used for recovery at 30°C shaking at 225 rpm for 3 hours. Transformation tubes were centrifuged at 14000 rpm for 1 minute and the cell pellet was dissolved in 1 mL LB broth with the appropriate antibiotics. 200 μL of the mixture is added to LB agar selection plates with the appropriate antibiotic and incubated at 30°C overnight. Example 4. Deletion of Muconate Cycloisomerase Gene in E. aphidicola LJJL01(2752379084). [00137] The above-mentioned CRISPR-Cas9 based method was adopted to delete the gene. The gRNA (with tetracycline resistance) carrying the spacer sequence of “TCTGGCGCAGTTGATATGTA” was constructed using Q5 mutagenesis, the SS9 gRNA plasmid was used as the template (Addgene Cat#71656). The sequenced verified gRNA and the repair DNA (attached the FASTA file) to delete the gene were used for the construction. The gene knockout colonies were identified on a 10 mg/mL tetracycline-containing LB plate. The deleted colony were verified with the diagnostic colony PCR with the primers oLJLJ038: ACGAATTCGAGCTCGGTACCCGGGGATCCTATATGTGCCGGACGCCG (SEQ ID NO: 47), oLJLJ041: CGGCCAGTGCCAAGCTTGCATGCCTGCAGGTTGGCAGGCCGATGCCAAG (SEQ ID NO: 48). The plasmids used for the CRISPR-Cas9 genome editing were cured by passing the strain on LB medium without antibiotics, and the glycerol stock of clean (without plasmids), E. aphidicola LJJL01lacking muconate cycloisomerase was prepare and stored in -80 °C freezer. [00138] The gRNA plasmid sequence is shown as SEQ ID NO: 1 in the Sequence Listing section below. The repair DNA sequence is shown as SEQ ID NO: 2 in the Sequence Listing section below. [00139] Deletion of pcaIJ gene in P. putida KT2440: The gene was deleted using pK18mobsacB-based plasmid via SacB-based gene deletion method [Jha et al., 2018]. Example 5. OHD Process of Waste Tea, Coffee, Corn Stover, and PET. [00140] Two hundred grams of commercially available green and black tea leaves or coffee purchased from the store (IG, Carbondale, IL, USA). Then, leaves were rinsed thoroughly with water, and then 1.0 Liter of boiling water was poured over 5 grams of dry leaves or coffee until water ran clear, the obtained solid waste Tea and Coffee were then subjected to grinding. 2 Liters of slurry was prepared by grinding the leaves in water at 1.0% dry solids. For the Corn Stover, or Corn Stover and plastic mix condition, the Corn Stover obtained from field (Carbondale, IL) were subjected to grinding as the method mentioned above. The Polyethylene terephthalate powder (crystallinity > 45%) obtain from GoodFellow, USA were mixed with 95 % (w/w) Corn Stover to obtain Corn Stover-plastic OHD mix. An OHD experiment was performed at 260 °C, 2000 psi, with an Oxygen: Carbon mass ratio of 0.3: 1.0, and residence time of 22 seconds in the reactor. 3 replicates were performed, collecting ~900 mL each, and the product was combined after filtering to 0.8 micron using Millipore ATTP poly filters. All the samples were produced under the same set of conditions, no mass balance closure was performed. Conversion was roughly 80-90%, unconverted solids were filtered off. No char or solid carbon was produced. For the microbial testing, pH of the OHD was adjusted to 7.0 by adding NaOH, and the samples were filtered with 0.2 micron filters to sterilize the medium. Example 6. Identification and Quantification of the OHD Products. [00141] Identification and quantification of individual compounds contained in the OHD were undertaken based on the suite of methods developed by Dr. Anderson’s laboratory, and the analytical methods adopted in thermochemical wastewater characterization to achieved 100% mass closure. Briefly, organic products of corn stover OHD were extracted from the raw liquor with ethyl acetate and analyzed by with in-situ derivatization using Tetramethylammonium hydroxide for methylation of acidic oxygen functional groups.(Sanders, 2017) GC-MS analyses of OHD products were performed on an Agilent Technologies 7890A GC system equipped with a 5975C inert XL MS detector and a 7683B series injector. The GC system contains a 60 m Zebron ZB-1701 column, 0.25 mm internal diameter, and 0.25 μm film thickness. Analyses were performed using He carrier gas flow controlled to 1ml/min in constant flow mode and the GC oven was temperature programmed as follows: initial temperature of 40°C held for 4 min, increased at 4°C/min to 280°C, held for 15 min. The MS and transfer line will be programmed to 150°C and 250°C, respectively. The MS scanned a range of m/z 10 to 400 to record full spectra. Data analysis were performed using Agilent software. Identification was based on a comparison of spectra with the Wiley and National Institute of Standards and Technology (NIST) mass spectral libraries, literature data, comparison with standards and interpretation. The method was successfully used to identify and quantify the ~35 organic compounds from cane bagasse OHD. All analyses were performed in triplicate experiments, and all quantitative standard curves were maintained with an R 2 value of ≥0.995 with five or more points of reference concentrations. Individual analytical grade standards were used to construct the calibration curve, and select internal standards will be added to adjust for chromatographic and detector response shift. Example 7. Monitoring Growth and Conversion of OHD Substrates by Microbes. [00142] To assess the growth both 96 well-plate and 50 mL shake flask experiments were performed using 50 mL baffled flasks containing 50 mL modified M9 media supplemented with 1% (v/v) FPF (pH 7) and inoculated to OD 600 0.2 with cells prepared as above but resuspended in M9 medium containing 1% (v/v) FPF. Cultures were incubated shaking at 225 rpm, 30 °C.2 mL samples were collected periodically and subjected to HPLC analysis, and OD600 growth measurement using a Beckman DU640 spectrophotometer (Beckman Coulter, Brea CA) or MPlex plate reader (TECAN, USA). The above-mentioned the HPLC method (Section 2) was adopted to detect the compounds in OHD during the microbial conversion. We monitored the β-ketoadipate production with the biosensor previously developed by Jha and coworkers [Jha et al., 2018]. Example 8. Monitoring Growth on Bacterial OHD. [00143] To evaluate the potential of using Bacterial-OHD as a growth media, we grow the P. putida KT2440 and E. aphidicola LJJL01 bacterial cells on 1 L LB medium, 30°, 225 rpm, and overnight. Cells were harvested by centrifugation (4000 rpm at 10 min), and subjected to the OHD process as described in Example 5. Next, we used different concentrations of Bacterial OHD and tested the growth of P. putida KT2440 and E. aphidicola LJJL01 with/without supplement M9 medium. Two hundred milliliters of the medium in a 96-well plate were inoculated with the strain (OD600=0.1), and incubated at maximum shaking, 30 °C, using a Mplex plate reader (TECAN, USA) to monitor the growth. Example 9. Engineered E. aphidicola LJJL01to Produce Muconate from OHD. [00144] Biological production of cis, cis-muconate was demonstrated with purified sugars or lignin-derived aromatics via engineered microbes. Fundamentally, the CatA/B deletion enables the accumulation of muconate [Johnson et al., 2019; Bentley et al., 2020]. Also, microbial systems have been engineered to produce muconate by introducing heterologous genes [Leavitt et al., 2017]. [00145] This is the first report of the utilization of E. aphidicola LJJL01to produce muconate from OHD substrates (unpurified heterogeneous compounds). The potent genes, 2752379084 (sequence given, only 56 similarities to CatA, muconate cycloisomerase of P. putida [see FASTA the sequence]). We knocked out and developed the strain to produce cis,cis- muconate from aromatics molecules (FIG.1). The production of cis-cis muconate from Catechol was demonstrated. The potent pathways of production of muconate from OHD-plastic & biomass described. (FIG.1). [00146] Given that E. aphidicola LJJL01exhibits high tolerance to most of the toxic chemicals, the engineered organism will serve as one of the fundamental chassis or developing high-value bioproducts such as muconate from OHD substrates or aromatics. [00147] FIG. 2 shows the developed integrated process of OHD and engineered monoculture to funnel heterogeneous OHD products (aromatics, sugars, acids) to a single product (i.e. beta-ketoadipate). (~90% yield from OHD aromatics). Example 10. Engineered Microbes to Selectively Degrade Plastic. [00148] Microbes have been engineered to degrade plastic into original monomers. However, substrates, intermediates, and product toxicity hamper the efforts. In addition, the limitation of secretion of the plastic degradation enzymes and selectivity of degradation are key hurdles to developing efficient cell factories [Jayakody & Dissanayake, 2021]. [00149] The French company Carbois, created the efficient enzyme, Leaf-branch compost cutinase (LCC), to degrade plastic at high temperature (80 °C) [Tournier et al., 2020]. [00150] Previous teams have developed P. putida KT2440 to degrade the PET selectively by secreting the PETase and MHETase using I. sakainesis secretion peptides. [00151] We demonstrated the E. aphidicola L a better platform organism relative to P. putida for selectively degrade PET. For instance, BHET can be 100% selectively degraded by expressing the codon-optimized engineered LCC enzyme (see the sequences) and the MHETase enzyme at 30 °C. (FIGS.3 - 6). [00152] For the first time, we developed E. aphidicola LJJL01 to secrete the plastic degradation enzymes using secretion signal peptides originated from I. sakaiensis. (FIGS. 3 and 7) We simultaneously secret the enzymes efficiently, and enable efficient degradation (FIG.4). [00153] We discovered the synergistic effect of LCC and MHETase to degrade BHET. (FIG.6). [00154] We developed the whole-cell biocatalyst (E. aphidicola LJJL01) to degrade PET and other plastics efficiently. [00155] In addition to PET degradation enzymes, we successfully express and secreted fungal-originated polyurethane degradation enzymes in E. aphidicola LJJL01. (FIG.7). [00156] The plasmid map of the LCC and MHETase is shown in FIG. 8, and the corresponding pBLT-2:Ptac-LCC_MHETase plasmid gene sequence is shown as SEQ ID NO. 7. [00157] The new synthetic LCC expression cassette sequence is shown as SEQ ID NO. 8. [00158] A plasmid map of the expression of fungal PU-degradation genes in E. aphidicola LJJL01design is shown in FIG. 9, and the corresponding ACE-540972 plasmid gene sequence is shown as SEQ ID NO.9. [00159] An additional plasmid map of the expression of fungal PU-degradation genes in E. aphidicola LJJL01 design is shown in FIG. 10, and the corresponding LAP_117292 plasmid gene sequence is shown as SEQ ID NO.10. [00160] FIG. 11 shows the developed in situ degradation of PET without chemical catalyst of external supplementation of chemical (diol/acids), and the integrated OHD and engineered microbes to recover the original plastic monomer (i.e., TPA). Example 11. Hybrid OHD-Microbe Process to Recycle or Upcycle PET. [00161] Given that high-crystallinity of PET, it is challenging to implement fully biological process to recover the monomer directly from commercial PET. Pyrolysis (high- temperature, costly process) and chemical deconstruction (expensive catalysts/ harsh solvents) process have been developed to recover the monomers [Bhaskar et al., 2004; Shojaei et al., 2020; Walker et al., 2020]. Also, researchers developed the microbes to utilize those monomers and enable the plastic upcycling [Tiso et al., 2020; Kenny et al., 2008]. [00162] We developed the OHD process to solubilize high-crystalline PET (> 45%) by mixing it with biomass. Thus, the novel process can apply to real-world waste (mix waste streams). [00163] We demonstrated the both P. putida and E. aphidicola LJJL01can grow on PET- Corn Stover OHD substrate. Also, if we use the engineered E. aphidicola LJJL01 or P. putida KT2440 harboring the PET degradation enzymes, they can convert BHET and MHET in the OHD to TPA selectively (FIGS. 12 and 13). (Enable OHD-microbes hybrid recirculation strategy of PET). The recovered TPA can be upcycled into various chemicals; indeed muconate and PDC (FIG.14). Several other upcycling routes can be developed in E. aphidicola LJJL01. [00164] FIG.15 shows the selectively designed microbe for depolymerization of PET. Example 12. Strain Construction. [00165] For the in vivo experiments, pBLT2-LCC-Mle046 plasmid (FIG. 20, SEQ ID NO: 11) was constructed using HiFi DNA assembly method. This plasmid was then transformed into E. coli DH5α-Iq strain using the heat shock method. Colonies were selected using LB agar plates supplemented with 50 μg/mL kanamycin. The colonies were screened by a colony PCR and visualized using gel electrophoresis. The plasmids were then extracted, and sequence verified. Q5 mutagenesis was performed to create the pBLT2-LCC-Mle046(mutant) (FIG.21, SEQ ID NO: 12 and SEQ ID NO: 13) with the glycine in position 131 swapped out for a serine using primers as shown in the table below. Transformants were selected using LB agar plates supplemented with 50 μg/mL kanamycin and sequence verified. Correct plasmids were transformed into E. aphidicola LJJL01 using electroporation and selection was done with LB-kanamycin plates. Table 3: Oligo (overlap-black, blue-PCR specific)-5'-3' Example 13. Shake Flask Experiments. [00166] Shake flask experiments for assaying the BHET degradation were performed using 2X M9 minimal medium at pH 8, supplemented with 10 g/L glucose, salts, 1 mM bis (2- hydroxyethyl) terephthalate (BHET), 50 μg/mL kanamycin and 1 μM IPTG. Strains used for the study were E. aphidicola-pBLT2, E. aphidicola-pBLT2-LCC, E. aphidicola-pBLT2-LCC- Mle046 and E. aphidicola-pBLT2-LCC-Mle046(mutant). Overnight cultures of strains in LB medium supplemented with the antibiotics were harvested and washed with M9 medium. A fresh culture of 25 mL in 250 mL flasks were prepared with an initial OD600 value of 1.0 and shake flasks were incubated at 30 °C shaking at 225 rpm. All the shake flask experiments were performed in 2 replicates. The conversions were monitored by means of a HPLC analysis using Shimadzu LC-2050C 3D coupled to a PDA detector. An isocratic elution program was used with 0.5% formaic acid in water and acetonitrile (80:20) as the mobile phases. The total run time was 10 minutes. Compounds were identified based on the retention times compared with that of the standards. Quantification was done based on the matrix matched calibration curves generated for each analyte using standard solutions prepared in 2XM9. Chromatograms were analyzed using LabSolutions DB software. HPLC analysis was done at time intervals 0, 24, 48 and 72 hours. Growth of the cultures were determined by TECAN Sunrise plate reader – Infinite M Flex. [00167] Shake flask results are expressed as average values of the duplicates data with their standard errors of the means (SEM). Multiple comparisons were done using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc honest significance difference test. (https://astatsa.com/OneWay_Anova_with_TukeyHSD/). [00168] The purified Mle046 mutant did not have a high concentration to register. Therefore no in vitro assays were completed, preventing us from getting biochemical properties of the Mle046 (mutant). Example 13. Results and discussion In-vivo investigation of the synergy of Mle046 and its mutant with LCC on BHET degradation. [00169] In FIG. 16, EA-LCC-Mle046 shows a synergistic effect against BHET compared to EA-LCC or other controls. Indeed, the TPA was detected only in the presence of Mle046, or its mutant. Notably, EA-LCC-Mle046 (mutant) yields 2X MHET (~ 0.5 mM vs 0.25 mM after 72 h) from BHET relative to the EA-LCC-Mle046, but did not improve the TPA yield. The results suggest that the newly developed mutant has PETase activity. Given that both LCC and Mle046 activities are optimum at pH 8, the strains incubated at pH 8 exhibit higher conversion of BHET relative to pH 7. 1 Of note, the host strain E. aphidicola LJJL01 can grow well in both pH 7 and pH 8, allowing this efficient conversion of BHET at pH 8.. We demonstrated the synergistic activity of the dual enzyme system (PETase, and MHETase from I. sakaiensis) on selective plastic degradation using an in-vitro study. 3 To the best of our knowledge, this is the first report demonstrating the synergistic activity of LCC and Mle046 on BHET degeneration by in-vivo study at ambient temperature. Of note, Mrigwani and coworkers demonstrate the synergy of LCC and thermostable Thermus thermophilus carboxylesterase at 60 0 C. 4 [00170] In FIG. 17, the samples at pH 7 versus the samples at pH 8 show that TPA conversion is increase by about 33% with the pH difference. Example 14. Investigation of the synergy of Mle046 and its mutant with LCC on MHET degradation. [00171] In FIG.18, the efficiency of the enzymes in MHET degradation is given. There is no statistically significant difference between the four variants (0.05 > p). In FIG.19, the efficiency of the enzymes in TPA production are demonstrated, and here it is clear that EA- LCC-Mle046 (wild-type) has a much better conversion yield than the other three variants and shows statistical significance (0.05 > p). Example 15. Discussion of Examples 12-14. [00172] We uncovered the Mle046 synergistic activity with LCC for selective degradation of PET and an engineering approach to modulate substrate specificity of Mle046 by mutating the catalytic domain (i.e., introducing PETase activity). Moving forward, more enzyme characterization through x-ray crystallography, in vitro assays to determine biochemical properties, and molecular dynamics studies will allow us to find better mutations to improve this enzyme and work towards a circular PET economy to serve our world better. Sequence Listing [00173] SEQ ID NO: 1 [00174] LENGTH: 2885 [00175] TYPE: RNA [00176] ORGANISM: artificial [00177] OTHER INFORMATION: gRNA plasmid sequence gaattctaaa gatctttgac agctagctca gtcctaggta taatactagt 50 tctggcgcag ttgatatgta gttttagagc tagaaatagc aagttaaaat 100 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt 150 tttgaagctt gggcccgaac aaaaactcat ctcagaagag gatctgaata 200 gcgccgtcga ccatcatcat catcatcatt gagtttaaac ggtctccagc 250 ttggctgttt tggcggatga gagaagattt tcagcctgat acagattaaa 300 tcagaacgca gaagcggtct gataaaacag aatttgcctg gcggcagtag 350 cgcggtggtc ccacctgacc ccatgccgaa ctcagaagtg aaacgccgta 400 gcgccgatgg tagtgtgggg tctccccatg cgagagtagg gaactgccag 450 gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta 500 tctgttgttt gtcggtgaac tggatcctta ctcgagtcta gactgcaggc 550 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg 600 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat 650 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg 700 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg 750 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga 800 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 850 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 900 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg 950 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc 1000 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa 1050 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga 1100 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 1150 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag 1200 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc 1250 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa 1300 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc 1350 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa 1400 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 1450 ctaaagtata tatgagtaaa cttggtctga cagttgacag cttatcatcg 1500 ataagcttta atgcggtagt ttatcacagt taaattgcta acgcagtcag 1550 gcaccgtgta tgaaatctaa caatgcgctc atcgtcatcc tcggcaccgt 1600 caccctggat gctgtaggca taggcttggt tatgccggta ctgccgggcc 1650 tcttgcggga tatcgtccat tccgacagca tcgccagtca ctatggcgtg 1700 ctgctagcgc tatatgcgtt gatgcaattt ctatgcgcac ccgttctcgg 1750 agcactgtcc gaccgctttg gccgccgccc agtcctgctc gcttcgctac 1800 ttggagccac tatcgactac gcgatcatgg cgaccacacc cgtcctgtgg 1850 atcctctacg ccggacgcat cgtggccggc atcaccggcg ccacaggtgc 1900 ggttgctggc gcctatatcg ccgacatcac cgatggggaa gatcgggctc 1950 gccacttcgg gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc 2000 cccgtggccg ggggactgtt gggcgccatc tccttgcatg caccattcct 2050 tgcggcggcg gtgctcaacg gcctcaacct actactgggc tgcttcctaa 2100 tgcaggagtc gcataaggga gagcgtcgac cgatgccctt gagagccttc 2150 aacccagtca gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc 2200 acttatgact gtcttcttta tcatgcaact cgtaggacag gtgccggcag 2250 cgctctgggt cattttcggc gaggaccgct ttcgctggag cgcgacgatg 2300 atcggcctgt cgcttgcggt attcggaatc ttgcacgccc tcgctcaagc 2350 cttcgtcact ggtcccgcca ccaaacgttt cggcgagaag caggccatta 2400 tcgccggcat ggcggccgac gcgctgggct acgtcttgct ggcgttcgcg 2450 acgcgaggct ggatggcctt ccccattatg attcttctcg cttccggcgg 2500 catcgggatg cccgcgttgc aggccatgct gtccaggcag gtagatgacg 2550 accatcaggg acagcttcaa ggatcgctcg cggctcttac cagcctaact 2600 tcgatcattg gaccgctgat cgtcacggcg atttatgccg cctcggcgag 2650 cacatggaac gggttggcat ggattgtagg cgccgcccta taccttgtct 2700 gcctccccgc gttgcgtcgc ggtgcatgga gccgggccac ctcgacctga 2750 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca 2800 tgacattaac ctataaaaat aggcgtatca cgaggcagaa tttcagataa 2850 aaaaaatcct tagctttcgc taaggatgat ttctg 2885 [00178] SEQ ID NO: 2 [00179] LENGTH: 2068 [00180] TYPE: DNA [00181] ORGANISM: artificial [00182] OTHER INFORMATION: repair DNA sequence acgaattcga gctcggtacc cggggatcct atatgtgccg gacgccgacc 50 gtgccgcccc agctgattga gcagatgctg caccggcccg tcaggggggc 100 agccgtaccc tgacggcacg acgctgcgga actgatagcg cccctgttca 150 tcggtaatga tggtacgacg caggttaaac gcactctgac ttttatcgaa 200 gaacgagtaa ttaccgaggg tattggcgtg ccagacctca acgctggcgc 250 cgggaagcgg ttgaccctct tcgttccata ccgtgccctg catcaacagc 300 acttcgccct ggttatcttc agaaccatcg tccagccgcg cccaggcctg 350 acagactggc gccccggcca catagagagg accttcgatg gtgcgcggtg 400 tcccaccgtg cagccccgcc ctcgcttcag cctcatccgc ccggatatcc 450 ataaaacgct ccagtccggt tcccgcagcc agcagcccca tctcctgagc 500 gataccggta tcggccagat attccagccc tttccagatc tcagtttgct 550 gcaggcccag atcctcaatc gctttaaaca gatcgctcag gagacgaacg 600 gtgacctgct gaatacgtgg gttcaccgtg cctttcgcgg ttttgacgat 650 aaagcagtta accaggttgt cgatctcttt gctgttcata ttgacgctcc 700 atttcaggta cagctcagga tgagaaagta tttaacgatc gtccacacgc 750 accgccgaag ggtggcggca cagcggcact atctgcatcg tcataaaggg 800 ataaagcggc agctcactca gcagctggtg caactcctca acgctatcga 850 cgtcaaatac gctgacgttg gcgtaatgcc cggcaatgcg ccagatgtgt 900 cgccacttgc cgcgacgctg aagcgcctgc gagtaggctt tttcccgtgc 950 cttaagttcg ctggcgacat tttccgccat cgtctgcggc agattcacat 1000 ccatagttac gtgaaatagc atggtgaaat ttcctcctac gctatagaat 1050 cccgccgatc ggggatagcc aaatcctgct gaaaatgatt gacccgctgt 1100 atgagaatgc gcagatcgcg cctgttcgcc ccagctcaca taacttgcga 1150 taaaaacgtc ttttagtgcc aatgacactt ttaggttatt ttttcgccgg 1200 tgaagtcagc accctgatga ctcaaactca ggaggagcga gatgatcgac 1250 aaaagtctgt ttttggccga tgaagccgtg gctgacattc acgatggtgc 1300 cacgctgatg attggcggat ttggcccggc aggacagccc tatgcgctgc 1350 tggacgcact gattcggcgg cggccacaga acctcacgct ggtcagcaac 1400 aacgctggca acgctgattc gggtctggcg ctactgctca aagcggggtg 1450 cgtgcgcaag atgatctgtt cctttccgcg ccagagtgac tcatgggtat 1500 ttgacgacct ctaccgccgc ggagagattg agctggagct ggtgccgcag 1550 ggcaatcttg tggcgcgtat tcaggcaggt ggcagcgggc tgggggccat 1600 ttttacccca acgggtttcg gtactgaact ggctgcgggc aaagagacgc 1650 gcgaaattga ggggcggcag tacgtgctgg aactggcgct gaaggcagac 1700 tttgccctga tcaaagcaca gtgcggtgat cgctggggta acctggtcta 1750 tagcaaagcg gcacgcaact tcaacccgat tatggcgatg gctgcccgct 1800 gcaccatcgc tgaagtctcc cgccacgtcg cgctgggcaa gctggacccg 1850 gaggcggtgg tgactcccgg tatttttgtg cagcgcgtgg tctgttcagc 1900 caatctcagc cccgcaacga tcgcctcagg agcgtgacac aatgcaactc 1950 atccgcctca cgcataacca gctggcgcag cgcatcgccc gcgatatccc 2000 ggacggagcc tacgtcaatc ttggcatcgg cctgccaacc tgcaggcatg 2050 caagcttggc actggccg 2068 [00183] SEQ ID NO: 3 [00184] LENGTH: 1110 [00185] TYPE: DNA [00186] ORGANISM: bacteria [00187] OTHER INFORMATION: E. aphidicola LJJL01muconate cycloisomerase gene sequence (Gene ID: 2752379084) ttaacggcgg cgcagcgccg cgatcctgtc gcgatcgagg gcaatgccca 50 gcccgggccc ggtcgggacc tgcagcataa aatcacgata aactagcggt 100 tcactgagaa tatcttcggt cagcagcagc gggccaaaca gctcagtgcc 150 gaaactcaaa tcgttgaagg tggcgcagag atgcgccgtg gcggccgtgc 200 ctacggcgcc ttccagcatg gtgcccccgt aaagcgcgat atctgccagc 250 tgggcaatat cggcaacccg gcgcgcctgc gtcagcccgc cggactgggt 300 aatttttatc gagaacacgt cggcggcagc gtggcgggcc agctcgaaag 350 catcgcgcgg ccctttcagc gcttcgtccg cgatcactgg caggctgaaa 400 cggcgggtca gtcgcgccag tccggcacga ttttctgccg caatcggctg 450 ctcaacggca tcaatgccgc cggcttccag tgctgccatt ccgcgctcgg 500 cctggcgctc gctccaggcc tggttgacgt cgacgcgcac gctgacctca 550 tcccccaacg cctgcctgat ggccagcgcg tgcgccacgt ccgcatcaac 600 atcgcgcagg ccgattttga gtttaaaaat acgatgacgg cgcagggcta 650 acatctgccg ggcttcggca atatctttat cggtgctgcc gctggccaac 700 gtccaggcca ccggtagcgc atcgcgcacc cgtcctccca gcagttcact 750 gagcgcaata ttcagacgcc gtgcctgagc gtccagcaag gcggtttcaa 800 ttgcgcattt agcaaagcgg ttgccctgca ctgatttatt caggcgcgcc 850 atcagctggg cgatgcggcg cgcatcctga ccgatcagta acggcgtcat 900 ccagcggtca atattgactt tgacgctctc ggggctttcg tcgccgtagc 950 tcaggccacc aatggtggtg gcttctcccc agccctcaaa cccgtcctca 1000 cacaccagat ggaccagcac cagcgtttgc acctgaagcg tggccattgc 1050 caggcgatgt ggacggatag caggaacatc aataagcagc gtttcaatag 1100 ttttgatcat 1110 [00188] SEQ ID NO: 4 [00189] LENGTH: 369 [00190] TYPE: protein [00191] ORGANISM: bacteria [00192] OTHER INFORMATION: E. aphidicola LJJL01Muconate cycloisomerase protein sequence (Gene ID: 2752379084) MIKTIETLLI DVPAIRPHRL AMATLQVQTL VLVHLVCEDG 40 FEGWGEATTI GGLSYGDESP ESVKVNIDRW MTPLLIGQDA 80 RRIAQLMARL NKSVQGNRFA KCAIETALLD AQARRLNIAL 120 SELLGGRVRD ALPVAWTLAS GSTDKDIAEA RQMLALRRHR 160 IFKLKIGLRD VDADVAHALA IRQALGDEVS VRVDVNQAWS 200 ERQAERGMAA LEAGGIDAVE QPIAAENRAG LARLTRRFSL 240 PVIADEALKG PRDAFELARH AAADVFSIKI TQSGGLTQAR 280 RVADIAQLAD IALYGGTMLE GAVGTAATAH LCATFNDLSF 320 GTELFGPLLL TEDILSEPLV YRDFMLQVPT GPGLGIALDR 360 DRIAALRRR 369 [00193] SEQ ID NO: 5 [00194] LENGTH: 320 [00195] TYPE: protein [00196] ORGANISM: bacteria [00197] OTHER INFORMATION: LCC protein sequence (signal peptide shown in bold) MNFPRASRLM QAAVLGGLMA VSAAATAMDG VLWRVRTAAL 40 MAALLALAAW ALVWASPSVE AQSNPYQRGP NPTRSALTAD 80 GPFSVATYTV SRLSVSGFGG GVIYYPTGTS LTFGGIAMSP 120 GYTADASSLA WLGRRLASHG FVVLVINTNS RFDGPDSRAS 160 QLSAALNYLR TSSPSAVRAR LDANRLAVAG HSMGGGGTLR 200 IAEQNPSLKA AVPLTPWHTD KTFNTSVPVL IVGAEADTVA 240 PVSQHAIPFY QNLPSTTPKV YVELCNASHI APNSNNAAIS 280 VYTISWMKLW VDNDTRYRQF LCNVNDPALC DFRTNNRHCQ 320 [00198] SEQ ID NO: 6 [00199] LENGTH: 613 [00200] TYPE: DNA [00201] ORGANISM: bacteria [00202] OTHER INFORMATION: MHET protein sequence (signal peptide shown in bold) MQTTVTTMLL ASVALAACAG GGSTPLPLPQ QQPPQQEPPP 40 PPVPLASRAA CEALKDGNGD MVWPNAATVV EVAAWRDAAP 80 ATASAAALPE HCEVSGAIAK RTGIDGYPYE IKFRLRMPAE 120 WNGRFFMEGG SGTNGSLSAA TGSIGGGQIA SALSRNFATI 160 ATDGGHDNAV NDNPDALGTV AFGLDPQARL DMGYNSYDQV 200 TQAGKAAVAR FYGRAADKSY FIGCSEGGRE GMMLSQRFPS 240 HYDGIVAGAP GYQLPKAGIS GAWTTQSLAP AAVGLDAQGV 280 PLINKSFSDA DLHLLSQAIL GTCDALDGLA DGIVDNYRAC 320 QAAFDPATAA NPANGQALQC VGAKTADCLS PVQVTAIKRA 360 MAGPVNSAGT PLYNRWAWDA GMSGLSGTTY NQGWRSWWLG 400 SFNSSANNAQ RVSGFSARSW LVDFATPPEP MPMTQVAARM 440 MKFDFDIDPL KIWATSGQFT QSSMDWHGAT STDLAAFRDR 480 GGKMILYHGM SDAAFSALDT ADYYERLGAA MPGAAGFARL 520 FLVPGMNHCS GGPGTDRFDM LTPLVAWVER GEAPDQISAW 560 SGTPGYFGVA ARTRPLCPYP QIARYKGSGD INTEANFACA 600 APPIIQDEPQ TPA 613 [00203] SEQ ID NO: 7 [00204] LENGTH: 5450 [00205] TYPE: DNA [00206] ORGANISM: artificial [00207] OTHER INFORMATION: pBLT-2:Ptac-LCC_MHETase plasmid gene sequence atcattcagg acgagcctca gactccagcg taactggact gaaaacaaac 50 taaagcgccc ttgtggcgct ttagttttgt tccgcggcca ccggctggct 100 cgcttcgctc ggcccgtgga caaccctgct ggacaagctg atggacaggc 150 tgcgcctgcc cacgagcttg accacaggga ttgcccaccg gctacccagc 200 cttcgaccac atacccaccg gctccaactg cgcggcctgc ggccttgccc 250 catcaatttt tttaattttc tctggggaaa agcctccggc ctgcggcctg 300 cgcgcttcgc ttgccggttg gacaccaagt ggaaggcggg tcaaggctcg 350 cgcagcgacc gcgcagcggc ttggccttga cgcgcctgga acgacccaag 400 cctatgcgag tgggggcagt cgaaggcgaa gcccgcccgc ctgccccccg 450 agcctcacgg cggcgagtgc gggggttcca agggggcagc gccaccttgg 500 gcaaggccga aggccgcgca gtcgatcaac aagccccgga ggggccactt 550 tttgccggag ggggagccgc gccgaaggcg tgggggaacc ccgcaggggt 600 gcccttcttt gggcaccaaa gaactagata tagggcgaaa tgcgaaagac 650 ttaaaaatca acaacttaaa aaaggggggt acgcaacagc tcattgcggc 700 accccccgca atagctcatt gcgtaggtta aagaaaatct gtaattgact 750 gccactttta cgcaacgcat aattgttgtc gcgctgccga aaagttgcag 800 ctgattgcgc atggtgccgc aaccgtgcgg caccctaccg catggagata 850 agcatggcca cgcagtccag agaaatcggc attcaagcca agaacaagcc 900 cggtcactgg gtgcaaacgg aacgcaaagc gcatgaggcg tgggccgggc 950 ttattgcgag gaaacccacg gcggcaatgc tgctgcatca cctcgtggcg 1000 cagatgggcc accagaacgc cgtggtggtc agccagaaga cactttccaa 1050 gctcatcgga cgttctttgc ggacggtcca atacgcagtc aaggacttgg 1100 tggccgagcg ctggatctcc gtcgtgaagc tcaacggccc cggcaccgtg 1150 tcggcctacg tggtcaatga ccgcgtggcg tggggccagc cccgcgacca 1200 gttgcgcctg tcggtgttca gtgccgccgt ggtggttgat cacgacgacc 1250 aggacgaatc gctgttgggg catggcgacc tgcgccgcat cccgaccctg 1300 tatccgggcg agcagcaact accgaccggc cccggcgagg agccgcccag 1350 ccagcccggc attccgggca tggaaccaga cctgccagcc ttgaccgaaa 1400 cggaggaatg ggaacggcgc gggcagcagc gcctgccgat gcccgatgag 1450 ccgtgttttc tggacgatgg cgagccgttg gagccgccga cacgggtcac 1500 gctgccgcgc cggtagtacg taagaggttc caactttcac cataatgaaa 1550 taagatcact accgggcgta ttttttgagt tatcgagatt ttcaggagct 1600 aaggaagcta aaatgagcca tattcaacgg gaaacgtctt gctcgaggcc 1650 gcgattaaat tccaacatgg atgctgattt atatgggtat aaatgggctc 1700 gcgataatgt cgggcaatca ggtgcgacaa tctatcgatt gtatgggaag 1750 cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa 1800 tgatgttaca gatgagatgg tcaggctaaa ctggctgacg gaatttatgc 1850 ctcttccgac catcaagcat tttatccgta ctcctgatga tgcatggtta 1900 ctcaccactg cgatcccagg gaaaacagca ttccaggtat tagaagaata 1950 tcctgattca ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc 2000 ggttgcattc gattcctgtt tgtaattgtc cttttaacgg cgatcgcgta 2050 tttcgtctcg ctcaggcgca atcacgaatg aataacggtt tggttggtgc 2100 gagtgatttt gatgacgagc gtaatggctg gcctgttgaa caagtctgga 2150 aagaaatgca taagcttttg ccattctcac cggattcagt cgtcactcat 2200 ggtgatttct cacttgataa ccttattttt gacgagggga aattaatagg 2250 ttgtattgat gttggacgag tcggaatcgc agaccgatac caggatcttg 2300 ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg 2350 ctttttcaaa aatatggtat tgataatcct gatatgaata aattgcagtt 2400 tcacttgatg ctcgatgagt ttttctgagg gcggatcccc ctcaagtcaa 2450 aagcctccgg tcggaggctt ttgactttct gctatggagg tcaggtatga 2500 ttacactCTA GAGAGCTGTT GACAATTAAT CATCGGCTCG 2540 TATAATGTGT GGAATTGTGA GCGGATAACA ATTTCACACC 2580 ATCAAGTCAA AACACTATAT AGGAACGAAA CCATGAACTT 2620 CCCTCGCGCG TCGCGCCTGA TGCAGGCGGC GGTCCTCGGT 2660 GGTCTGATGG CAGTCAGCGC CGCGGCCACC GCTATGGACG 2700 GTGTACTGTG GCGTGTGCGC ACCGCAGCCC TGATGGCCGC 2740 CCTGCTGGCA CTGGCTGCGT GGGCGCTGGT GTGGGCGTCG 2780 CCGAGCGTTG AGGCGCAGAG CAACCCTTAC CAGCGTGGCC 2820 CGAACCCGAC CCGCTCGGCC CTGACCGCCG ACGGTCCATT 2860 CTCTGTCGCC ACCTATACCG TCAGCCGCCT GTCGGTGTCG 2900 GGTTTCGGCG GCGGCGTGAT CTACTACCCG ACCGGCACCT 2940 CCTTGACCTT CGGCGGCATT GCCATGTCCC CTGGCTACAC 2980 CGCTGACGCC TCGAGCCTGG CCTGGCTGGG TCGCCGTCTG 3020 GCCAGCCACG GCTTCGTCGT ACTGGTCATC AACACCAACA 3060 GCCGTTTCGA CGGCCCTGAC TCCCGCGCCA GCCAGCTGTC 3100 GGCTGCTCTG AACTACCTGC GTACCTCGTC GCCGTCCGCA 3140 GTCCGTGCCC GCCTGGACGC CAACCGTCTG GCCGTAGCCG 3180 GCCACTCGAT GGGCGGCGGC GGCACCCTGC GCATCGCCGA 3220 ACAAAACCCA AGCCTGAAGG CCGCTGTCCC ACTGACCCCG 3260 TGGCACACCG ACAAGACCTT CAACACCTCG GTGCCAGTGC 3300 TGATCGTAGG TGCGGAGGCC GACACCGTGG CCCCAGTGTC 3340 CCAGCACGCC ATCCCGTTCT ACCAGAACCT GCCGTCCACC 3380 ACCCCAAAGG TTTATGTCGA GCTCTGCAAC GCCAGTCACA 3420 TCGCCCCGAA TTCGAACAAC GCTGCCATCT CCGTCTACAC 3460 CATCAGCTGG ATGAAGCTGT GGGTTGACAA CGACACCCGC 3500 TACCGCCAGT TCCTGTGTAA CGTGAACGAC CCGGCCCTGT 3540 GCGATTTCCG TACCAACAAT CGTCACTGCC AGTGAcaagg 3580 attacatata agggtatata aatgcagacc accgtcacca ctatgctgct ggcatcggtc 3640 gccctggccg cctgcgcagg cggcggcagc accccgctgc cgctgccgca gcaacagccg 3700 ccacagcagg agccgccgcc tcctccagtc ccgctggctt cccgtgctgc 3750 gtgtgaggcc ctgaaggacg gcaacgggga catggtttgg ccgaacgccg 3800 ccaccgtagt tgaagtggcc gcatggcgcg acgctgcccc ggctaccgcg 3850 tccgccgccg ctctgccgga acactgcgaa gttagcggcg ccatcgccaa 3900 gcgcactggt attgacggtt atccgtacga aatcaagttc cgcctgcgca 3950 tgccggcgga gtggaatggc cgtttcttca tggagggtgg ttccggcacc 4000 aacggctccc tgagcgcggc caccggcagc atcggtggcg gccagatcgc 4050 ctcggccctg tcccgcaact tcgccaccat cgcgaccgac ggtggccacg 4100 acaacgctgt caacgacaat ccagacgccc tgggtacggt agcgttcggc 4150 ctggacccac aggctcgcct ggacatgggt tacaattcgt acgaccaggt 4200 gacccaagct ggcaaagccg ccgttgcccg tttctacggc cgtgccgccg 4250 acaagtcgta cttcatcggc tgctcggaag gtggtcggga gggcatgatg 4300 ctcagccaac gcttcccatc ccactacgac ggtatcgtcg ccggtgcccc 4350 tggctaccag ctgcctaaag ccggtatctc gggcgcttgg accactcagt 4400 cgctggcccc ggcggcggtg ggcctggacg ctcagggcgt cccgctgatc 4450 aacaagagct tctccgatgc cgacctgcac ctgctgtcgc aggccatcct 4500 cggtacttgc gatgcgctgg acggcctggc tgacggcatc gttgacaact 4550 accgcgcgtg ccaggccgct ttcgacccgg ctaccgcggc taaccctgcc 4600 aacggtcaag ctctgcaatg tgtgggtgcc aaaaccgccg attgcctgag 4650 cccggtacag gttaccgcca tcaaacgtgc aatggccggc ccggtcaaca 4700 gcgccggcac cccgctgtac aaccgttggg cctgggacgc tggtatgagc 4750 ggcctgtccg gtaccaccta caatcagggc tggcgttcct ggtggctggg 4800 tagcttcaac tcctcggcga acaacgcgca gcgtgtttcg ggtttctccg 4850 cccgctcctg gctggtcgac ttcgccaccc caccagagcc tatgccgatg 4900 acccaggtgg ctgcacgcat gatgaaattc gacttcgaca tcgacccgct 4950 gaagatctgg gccaccagcg gccagttcac ccagtcgagc atggactggc 5000 acggggccac ctccaccgac ctggccgcct tccgcgatcg tggcggcaag 5050 atgatcctgt accacggtat gagcgacgca gccttctcgg ccctggacac 5100 cgctgactac tacgaacgcc tgggcgccgc tatgccgggc gccgcgggct 5150 tcgctcgtct gttcctcgtc ccaggcatga accactgttc gggcggtcca 5200 ggtaccgacc gtttcgacat gctgacccct ctggtggcgt gggttgagcg 5250 cggcgaagcc ccggaccaga tctcggcgtg gagcggcacc ccaggctact 5300 tcggcgtcgc tgcccgtacc cgcccgctgt gcccgtaccc gcaaatcgca 5350 cgctacaagg gttccggcga tatcaacacc gaagcaaact tcGCCTGCGC 5400 CGCGCCTCCG 5450 [00208] SEQ ID NO: 8 [00209] LENGTH: 1070 [00210] TYPE: DNA [00211] ORGANISM: artificial [00212] OTHER INFORMATION: new synthetic LCC expression cassette ctctagagag ctgttgacaa ttaatcatcg gctcgtataa tgtgtggaat 50 tgtgagcgga taacaatttc acaccatcaa gtcaaaacac tatataggaa 100 cgaaaccatg aacttccctc gcgcgtcgcg cctgatgcag gcggcggtcc 150 tcggtggtct gatggcagtc agcgccgcgg ccaccgctat ggacggtgta 200 ctgtggcgtg tgcgcaccgc agccctgatg gccgccctgc tggcactggc 250 tgcgtgggcg ctggtgtggg cgtcgccgag cgttgaggcg cagagcaacc 300 cttaccagcg tggcccgaac ccgacccgct cggccctgac cgccgacggt 350 ccattctctg tcgccaccta taccgtcagc cgcctgtcgg tgtcgggttt 400 cggcggcggc gtgatctact acccgaccgg cacctccttg accttcggcg 450 gcattgccat gtcccctggc tacaccgctg acgcctcgag cctggcctgg 500 ctgggtcgcc gtctggccag ccacggcttc gtcgtactgg tcatcaacac 550 caacagccgt ttcgacggcc ctgactcccg cgccagccag ctgtcggctg 600 ctctgaacta cctgcgtacc tcgtcgccgt ccgcagtccg tgcccgcctg 650 gacgccaacc gtctggccgt agccggccac tcgatgggcg gcggcggcac 700 cctgcgcatc gccgaacaaa acccaagcct gaaggccgct gtcccactga 750 ccccgtggca caccgacaag accttcaaca cctcggtgcc agtgctgatc 800 gtaggtgcgg aggccgacac cgtggcccca gtgtcccagc acgccatccc 850 gttctaccag aacctgccgt ccaccacccc aaaggtttat gtcgagctct 900 gcaacgccag tcacatcgcc ccgaattcga acaacgctgc catctccgtc 950 tacaccatca gctggatgaa gctgtgggtt gacaacgaca cccgctaccg 1000 ccagttcctg tgtaacgtga acgacccggc cctgtgcgat ttccgtacca 1050 acaatcgtca ctgccagtga 1070 [00213] SEQ ID NO: 9 [00214] LENGTH: 4965 [00215] TYPE: DNA [00216] ORGANISM: artificial [00217] OTHER INFORMATION: ACE-540972 plasmid gene sequence atggccacgc agtccagaga aatcggcatt caagccaaga acaagcccgg 50 tcactgggtg caaacggaac gcaaagcgca tgaggcgtgg gccgggctta 100 ttgcgaggaa acccacggcg gcaatgctgc tgcatcacct cgtggcgcag 150 atgggccacc agaacgccgt ggtggtcagc cagaagacac tttccaagct 200 catcggacgt tctttgcgga cggtccaata cgcagtcaag gacttggtgg 250 ccgagcgctg gatctccgtc gtgaagctca acggccccgg caccgtgtcg 300 gcctacgtgg tcaatgaccg cgtggcgtgg ggccagcccc gcgaccagtt 350 gcgcctgtcg gtgttcagtg ccgccgtggt ggttgatcac gacgaccagg 400 acgaatcgct gttggggcat ggcgacctgc gccgcatccc gaccctgtat 450 ccgggcgagc agcaactacc gaccggcccc ggcgaggagc cgcccagcca 500 gcccggcatt ccgggcatgg aaccagacct gccagccttg accgaaacgg 550 aggaatggga acggcgcggg cagcagcgcc tgccgatgcc cgatgagccg 600 tgttttctgg acgatggcga gccgttggag ccgccgacac gggtcacgct 650 gccgcgccgg tagtacgtaa gaggttccaa ctttcaccat aatgaaataa 700 gatcactacc gggcgtattt tttgagttat cgagattttc aggagctaag 750 gaagctaaaa tgagccatat tcaacgggaa acgtcttgct cgaggccgcg 800 attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg 850 gcaatcaggt gcgacaatct atcgattgta tgggaagccc gatgcgccag 900 agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat 950 gagatggtca ggctaaactg gctgacggaa tttatgcctc ttccgaccat 1000 caagcatttt atccgtactc ctgatgatgc atggttactc accactgcga 1050 tcccagggaa aacagcattc caggtattag aagaatatcc tgattcaggt 1100 gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat 1150 tcctgtttgt aattgtcctt ttaacggcga tcgcgtattt cgtctcgctc 1200 aggcgcaatc acgaatgaat aacggtttgg ttggtgcgag tgattttgat 1250 gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa 1300 gcttttgcca ttctcaccgg attcagtcgt cactcatggt gatttctcac 1350 ttgataacct tatttttgac gaggggaaat taataggttg tattgatgtt 1400 ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa 1450 ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat 1500 atggtattga taatcctgat atgaataaat tgcagtttca cttgatgctc 1550 gatgagtttt tctgagggcg gatccccctc aacggatccc cctcaagtca 1600 aaagcctccg gtcggaggct tttgactttc tgctatggag gtcaggtatg 1650 attacactCT AGAGAGCTGT TGACAATTAA TCATCGGCTC 1690 GTATAATGTG TGGAATTGTG AGCGGATAAC AATTTCACAC 1730 CATCAAGTCA AAACACTATA TAGGAACGAA ACCATGAACT 1770 TCCCTCGCGC GTCGCGCCTG ATGCAGGCGG CGGTCCTCGG 1810 TGGTCTGATG GCAGTCAGCG CCGCGGCCAC CGCTatgtgc 1850 ctgagcctgg cggcactgct gaacgcggtt ctggtgctgc gcgccgcacc 1900 ggccctgggt agcccggcaa agcgtagctg ctcccaggca gatctgaccg 1950 tggagctgga cagcggcatc gttcacggca ccgtggagag cggcaacccg 2000 gatgttcgtc agttcctggg tatcccgtac gcgaaaccgc cggtgggtga 2050 cctgcgcttc gcaccgagcg agccgattga gagcttcggc gagatcaccg 2100 cggataccct gccgccgagc tgtatgcagt acctgaccag cctggagagc 2150 atctggacca tcgacgtgct gcaattcaac gaagcgggtc tgaacaccac 2200 cggtccgcag agcgaggatt gcctgaccat cagcgtgtgg gcgccgcgtg 2250 gcgccaagaa agacctgccg gtcctgctgt ggatctacgg cggtagcttc 2300 aagaccggcg gtgaggatgt cccgtaccag atcccgaccc agtgggttca 2350 gcgtacccag gaccacatcg tggtcagctt caactaccgc gtcaacatct 2400 tcggtttccc gaacgcggcc ggtctggacg atgacaagca gaacctgggc 2450 ctgctggatc agcgtctggc ggtcgagtgg gttcgcgata acatcgccaa 2500 attcggcggt gacgttaacc gtatcggtct gtggggtcag agcgcgggcg 2550 gtatcagcgt cgcctactac agctacacct acccggagga cccgattgtg 2600 agcgcgctgc tgatgaacag cggtaacgag tacctggata tcaccagcag 2650 cgacaccacc cacagcaact tcaccttcat ggccagccag ttcggttgcg 2700 gtgatctggc cccggatgag gagctggcgt gtatgcgtac cgttgatgcg 2750 agcgccatcg aggagttcct gcacatctac atcgacaacg gcaccgagcc 2800 ggcggtgagc ttcgcaccga ttgtggacga gaagaccgtc ttcagcgatt 2850 tctacgaccg cgccgtcaac ggtagcgttg ccgcactgcc gaccatcctg 2900 ggcagcaacc tggatgacgg tgtgccgttc gtcacctaca gcccggatgg 2950 cgttaacgcg accgacgcct acgaggtgac caccgactac ttcttctgcc 3000 cggcgttcaa gagcgccaac aaccgtcacg cggccggtgc gccggtgttc 3050 cgctacgagt acagcggcaa cttcagcaac atcagcccga aaccgtggat 3100 gggtgcctgg cacaacagcg agctgccgct gctgttcggc acctacagca 3150 actaccgtgg tccgagcacc agcctggaga tcgagaccag catcgcgatg 3200 caggatgcct ggctgagcct ggtcgcgaac ggtgcggccg gtccgctggc 3250 cctgggttgg ccgctgtaca gcccggaggc gggcagcctg ctgcgcgatt 3300 tcggtaaaga cgtggcggcc cagaccacca acttcgagga ctgggagagc 3350 acctgctccg aggtgttcca gccgggaggt ggaggaggtt ctatgagcaa 3400 gggcgaggag ctctttaccg gcgtcgtccc cattctcgtt gagctggacg 3450 gcgacgtgaa cggacataag ttcagtgtct cgggcgaggg cgaaggagat 3500 gccacctatg ggaagctaac cctgaagttc atctgcacaa ccgggaagct 3550 gccggtcccc tggccgacgc tggttaccac cctgacctac ggcgtgcaat 3600 gcttctcgcg ctaccctgac cacatgaagc gccacgactt cttcaaatcc 3650 gctatgccgg agggctacgt ccaggaacgc accatattct tcaaggacga 3700 cggtaactac aagacgcgcg ccgaagtcaa gttcgagggg gataccctcg 3750 tgaaccgaat cgagttgaag gggatcgact tcaaagaaga tggcaacatc 3800 ctcggccaca aactggagta caactacaat tcgcataacg tgtacatcat 3850 ggccgacaag cagaagaatg gcatcaaggt gaacttcaag attcgccaca 3900 acatcgagga cgggtccgtt cagctggccg accactatca gcagaacaca 3950 ccaattggag acggccccgt cctgctcccc gataaccatt acctttcgac 4000 acagtcggcg ctgtcgaagg acccgaacga aaagcgggac cacatggtgc 4050 tcctggagtt cgtcacggcg gccgggatca cgcacggaat ggacgaactc 4100 tacaagtagG ATATCattca ggacgagcct cagactccag cgtaactgga 4150 ctgaaaacaa actaaagcgc ccttgtggcg ctttagtttt gttccgcggc 4200 caccggctgg ctcgcttcgc tcggcccgtg gacaaccctg ctggacaagc 4250 tgatggacag gctgcgcctg cccacgagct tgaccacagg gattgcccac 4300 cggctaccca gccttcgacc acatacccac cggctccaac tgcgcggcct 4350 gcggccttgc cccatcaatt tttttaattt tctctgggga aaagcctccg 4400 gcctgcggcc tgcgcgcttc gcttgccggt tggacaccaa gtggaaggcg 4450 ggtcaaggct cgcgcagcga ccgcgcagcg gcttggcctt gacgcgcctg 4500 gaacgaccca agcctatgcg agtgggggca gtcgaaggcg aagcccgccc 4550 gcctgccccc cgagcctcac ggcggcgagt gcgggggttc caagggggca 4600 gcgccacctt gggcaaggcc gaaggccgcg cagtcgatca acaagccccg 4650 gaggggccac tttttgccgg agggggagcc gcgccgaagg cgtgggggaa 4700 ccccgcaggg gtgcccttct ttgggcacca aagaactaga tatagggcga 4750 aatgcgaaag acttaaaaat caacaactta aaaaaggggg gtacgcaaca 4800 gctcattgcg gcaccccccg caatagctca ttgcgtaggt taaagaaaat 4850 ctgtaattga ctgccacttt tacgcaacgc ataattgttg tcgcgctgcc 4900 gaaaagttgc agctgattgc gcatggtgcc gcaaccgtgc ggcaccctac 4950 cgcatggaga taagc 4965 [00218] SEQ ID NO: 10 [00219] LENGTH: 5028 [00220] TYPE: DNA [00221] ORGANISM: artificial [00222] OTHER INFORMATION: LAP_117292 plasmid gene sequence atggccacgc agtccagaga aatcggcatt caagccaaga acaagcccgg 50 tcactgggtg caaacggaac gcaaagcgca tgaggcgtgg gccgggctta 100 ttgcgaggaa acccacggcg gcaatgctgc tgcatcacct cgtggcgcag 150 atgggccacc agaacgccgt ggtggtcagc cagaagacac tttccaagct 200 catcggacgt tctttgcgga cggtccaata cgcagtcaag gacttggtgg 250 ccgagcgctg gatctccgtc gtgaagctca acggccccgg caccgtgtcg 300 gcctacgtgg tcaatgaccg cgtggcgtgg ggccagcccc gcgaccagtt 350 gcgcctgtcg gtgttcagtg ccgccgtggt ggttgatcac gacgaccagg 400 acgaatcgct gttggggcat ggcgacctgc gccgcatccc gaccctgtat 450 ccgggcgagc agcaactacc gaccggcccc ggcgaggagc cgcccagcca 500 gcccggcatt ccgggcatgg aaccagacct gccagccttg accgaaacgg 550 aggaatggga acggcgcggg cagcagcgcc tgccgatgcc cgatgagccg 600 tgttttctgg acgatggcga gccgttggag ccgccgacac gggtcacgct 650 gccgcgccgg tagtacgtaa gaggttccaa ctttcaccat aatgaaataa 700 gatcactacc gggcgtattt tttgagttat cgagattttc aggagctaag 750 gaagctaaaa tgagccatat tcaacgggaa acgtcttgct cgaggccgcg 800 attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg 850 ataatgtcgg gcaatcaggt gcgacaatct atcgattgta tgggaagccc 900 gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga 950 tgttacagat gagatggtca ggctaaactg gctgacggaa tttatgcctc 1000 ttccgaccat caagcatttt atccgtactc ctgatgatgc atggttactc 1050 accactgcga tcccagggaa aacagcattc caggtattag aagaatatcc 1100 tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt 1150 tgcattcgat tcctgtttgt aattgtcctt ttaacggcga tcgcgtattt 1200 cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttggtgcgag 1250 tgattttgat gacgagcgta atggctggcc tgttgaacaa gtctggaaag 1300 aaatgcataa gcttttgcca ttctcaccgg attcagtcgt cactcatggt 1350 gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg 1400 tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca 1450 tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt 1500 tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca 1550 cttgatgctc gatgagtttt tctgagggcg gatccccctc aacggatccc 1600 cctcaagtca aaagcctccg gtcggaggct tttgactttc tgctatggag 1650 gtcaggtatg attacactCT AGAGAGCTGT TGACAATTAA 1690 TCATCGGCTC GTATAATGTG TGGAATTGTG AGCGGATAAC 1730 AATTTCACAC CATCAAGTCA AAACACTATA TAGGAACGAA 1770 ACCATGAACT TCCCTCGCGC GTCGCGCCTG ATGCAGGCGG 1810 CGGTCCTCGG TGGTCTGATG GCAGTCAGCG CCGCGGCCAC 1850 CGCTatgcgt gtcaccctgg gtctggcggc cctggccgtt ggtgccagcg 1900 ccatcccgac cgagctgcgt gagcgtcagg acggccacaa cagcaaaccg 1950 tgcccgaaga aaccgctggt gaccaaagac gccctggaga aggatatcac 2000 catcaagaaa ctgatgcgcg gtgcgcagca gctggaggat ttcgcctaca 2050 gctacccggc gcgtaaccgc atcatgggcg gtcaggcgca caacgacacc 2100 gtcaagtggc tgaagaaaga gctggagagc accagctact acgatgtgac 2150 cctgcaaccg ttcagcaact acgtcatgct gaacggcacc ctgaacgcct 2200 tcaccatcga cggtcgtgcg atcaacagca ccatcctgga gtacagcgcc 2250 agcaccccgg gtctggttac cgccccgatt gtgccggtga acaacctggg 2300 ttgcgaggcc agcgatttcc cggccgaggt gtccggtgcc atcgcgctga 2350 tcagccgtgg cacctgcgag ttcggtctga aaagcgccct ggcgggtagc 2400 gccggcgccg ttggtgccat catctacaac aacgtggccg gcaccatcag 2450 cgccaccctg ggcccgccgc cgcgtccgga gggtgactac gtggcgagcg 2500 tcaccctgag cctggaggaa ggccaggcca tcgttgccaa agtggccggc 2550 ggtgcgaccg tgaccggcac cctggacgtc ctgaccgatg tccaggttat 2600 caccaccaac aacgtcctgg cgaccagcaa atgcggcgac aagaacaacc 2650 agctgtggct gggtgcccac accgatagcg ttggtgccgg tccgggtatc 2700 aacgacgatg gtagcggcac cgttgccatc ctgaacgtgg ccaaaagcct 2750 ggcgaagtac aacgtgaaca acgcggtcag cttcggtttc tggagcggtg 2800 aagagagcgg tctgctgggt agcaccttct tcgtggagag cctgagcccg 2850 gaggccgccc tggacgttcg tgcctacctg aacttcgata tgatcgcgag 2900 cccgaactac gtgcaccaga tctacgatgg tgatggcagc gcctacggcc 2950 tgagcggtcc ggcgggcagc gatgatatcg aggccttctt cgcgggttac 3000 ttcaaagacc tgaacatccc gagcaacgag accgagttca acggtcgcag 3050 cgactacggc ccgttcctgg atgcaaacat cccggcgggc ggcaccacca 3100 ccggtgcaga tgaggtcaag accgttgaag agcaggccat ctggggcggt 3150 gtggccggcg agatcctgga ccagaactac caccaggcgg ccgataacgt 3200 caccaacctg aacgaggaag cgtggctgct gcacagccgc ggcatcgcgg 3250 cggcggtggc ccactacgcc accagctggg aaggcatccc ggagcgtgtt 3300 ccggcaaacg gcaccgccaa acgtagcgtg cgtgagccgg tggtccgtaa 3350 gggtgtcaaa ggcggtaaag ttaccctgaa gctgctggga ggtggaggag 3400 gttctatgag caagggcgag gagctcttta ccggcgtcgt ccccattctc 3450 gttgagctgg acggcgacgt gaacggacat aagttcagtg tctcgggcga 3500 gggcgaagga gatgccacct atgggaagct aaccctgaag ttcatctgca 3550 caaccgggaa gctgccggtc ccctggccga cgctggttac caccctgacc 3600 tacggcgtgc aatgcttctc gcgctaccct gaccacatga agcgccacga 3650 cttcttcaaa tccgctatgc cggagggcta cgtccaggaa cgcaccatat 3700 tcttcaagga cgacggtaac tacaagacgc gcgccgaagt caagttcgag 3800 ggggataccc tcgtgaaccg aatcgagttg aaggggatcg acttcaaaga 3850 agatggcaac atcctcggcc acaaactgga gtacaactac aattcgcata 3900 acgtgtacat catggccgac aagcagaaga atggcatcaa ggtgaacttc 3950 aagattcgcc acaacatcga ggacgggtcc gttcagctgg ccgaccacta 4000 tcagcagaac acaccaattg gagacggccc cgtcctgctc cccgataacc 4050 attacctttc gacacagtcg gcgctgtcga aggacccgaa cgaaaagcgg 4100 gaccacatgg tgctcctgga gttcgtcacg gcggccggga tcacgcacgg 4150 aatggacgaa ctctacaagt agGATATCat tcaggacgag cctcagactc 4200 cagcgtaact ggactgaaaa caaactaaag cgcccttgtg gcgctttagt 4250 tttgttccgc ggccaccggc tggctcgctt cgctcggccc gtggacaacc 4300 ctgctggaca agctgatgga caggctgcgc ctgcccacga gcttgaccac 4350 agggattgcc caccggctac ccagccttcg accacatacc caccggctcc 4400 aactgcgcgg cctgcggcct tgccccatca atttttttaa ttttctctgg 4450 ggaaaagcct ccggcctgcg gcctgcgcgc ttcgcttgcc ggttggacac 4500 caagtggaag gcgggtcaag gctcgcgcag cgaccgcgca gcggcttggc 4550 cttgacgcgc ctggaacgac ccaagcctat gcgagtgggg gcagtcgaag 4600 gcgaagcccg cccgcctgcc ccccgagcct cacggcggcg agtgcggggg 4650 ttccaagggg gcagcgccac cttgggcaag gccgaaggcc gcgcagtcga 4700 tcaacaagcc ccggaggggc cactttttgc cggaggggga gccgcgccga 4750 aggcgtgggg gaaccccgca ggggtgccct tctttgggca ccaaagaact 4800 agatataggg cgaaatgcga aagacttaaa aatcaacaac ttaaaaaagg 4850 ggggtacgca acagctcatt gcggcacccc ccgcaatagc tcattgcgta 4900 ggttaaagaa aatctgtaat tgactgccac ttttacgcaa cgcataattg 4950 ttgtcgcgct gccgaaaagt tgcagctgat tgcgcatggt gccgcaaccg 5000 tgcggcaccc taccgcatgg agataagc 5028 SEQ ID NO: 11 Length: Type: DNA Organism: Artificial Other information: pBLT-2-LCC-Mle046 plasmid gene sequence gtgtggccaataagctttgactggactgaaaacaaactaaagcgcccttgtggcgcttta gttttgttccgcggccaccggctggctcgc ttcgctcggcccgtggacaaccctgctggacaagctgatggacaggctgcgcctgcccac gagcttgaccacagggattgcccacc ggctacccagccttcgaccacatacccaccggctccaactgcgcggcctgcggccttgcc ccatcaatttttttaattttctctggggaaa agcctccggcctgcggcctgcgcgcttcgcttgccggttggacaccaagtggaaggcggg tcaaggctcgcgcagcgaccgcgca gcggcttggccttgacgcgcctggaacgacccaagcctatgcgagtgggggcagtcgaag gcgaagcccgcccgcctgcccccc gagcctcacggcggcgagtgcgggggttccaagggggcagcgccaccttgggcaaggccg aaggccgcgcagtcgatcaacaa gccccggaggggccactttttgccggagggggagccgcgccgaaggcgtgggggaacccc gcaggggtgcccttctttgggcac caaagaactagatatagggcgaaatgcgaaagacttaaaaatcaacaacttaaaaaaggg gggtacgcaacagctcattgcggcacc ccccgcaatagctcattgcgtaggttaaagaaaatctgtaattgactgccacttttacgc aacgcataattgttgtcgcgctgccgaaaag ttgcagctgattgcgcatggtgccgcaaccgtgcggcaccctaccgcatggagataagca tggccacgcagtccagagaaatcggc attcaagccaagaacaagcccggtcactgggtgcaaacggaacgcaaagcgcatgaggcg tgggccgggcttattgcgaggaaac ccacggcggcaatgctgctgcatcacctcgtggcgcagatgggccaccagaacgccgtgg tggtcagccagaagacactttccaag ctcatcggacgttctttgcggacggtccaatacgcagtcaaggacttggtggccgagcgc tggatctccgtcgtgaagctcaacggcc ccggcaccgtgtcggcctacgtggtcaatgaccgcgtggcgtggggccagccccgcgacc agttgcgcctgtcggtgttcagtgcc gccgtggtggttgatcacgacgaccaggacgaatcgctgttggggcatggcgacctgcgc cgcatcccgaccctgtatccgggcga gcagcaactaccgaccggccccggcgaggagccgcccagccagcccggcattccgggcat ggaaccagacctgccagccttgac cgaaacggaggaatgggaacggcgcgggcagcagcgcctgccgatgcccgatgagccgtg ttttctggacgatggcgagccgttg gagccgccgacacgggtcacgctgccgcgccggtagtacgtaagaggttccaactttcac cataatgaaataagatcactaccgggc gtattttttgagttatcgagattttcaggagctaaggaagctaaaatgagccatattcaa cgggaaacgtcttgctcgaggccgcgattaa attccaacatggatgctgatttatatgggtataaatgggctcgcgataatgtcgggcaat caggtgcgacaatctatcgattgtatgggaa gcccgatgcgccagagttgtttctgaaacatggcaaaggtagcgttgccaatgatgttac agatgagatggtcaggctaaactggctga cggaatttatgcctcttccgaccatcaagcattttatccgtactcctgatgatgcatggt tactcaccactgcgatcccagggaaaacagc attccaggtattagaagaatatcctgattcaggtgaaaatattgttgatgcgctggcagt gttcctgcgccggttgcattcgattcctgtttgt aattgtccttttaacggcgatcgcgtatttcgtctcgctcaggcgcaatcacgaatgaat aacggtttggttggtgcgagtgattttgatga cgagcgtaatggctggcctgttgaacaagtctggaaagaaatgcataagcttttgccatt ctcaccggattcagtcgtcactcatggtgat ttctcacttgataaccttatttttgacgaggggaaattaataggttgtattgatgttgga cgagtcggaatcgcagaccgataccaggatctt gccatcctatggaactgcctcggtgagttttctccttcattacagaaacggctttttcaa aaatatggtattgataatcctgatatgaataaatt gcagtttcacttgatgctcgatgagtttttctgagggcggatccccctcaagtcaaaagc ctccggtcggaggcttttgactttctgctatg gaggtcaggtatgattacactCTAGAGAGCTGTTGACAATTAATCATCGGCTCGTATAAT GTG TGGAATTGTGAGCGGATAACAATTTCACACCATCAAGTCAAAACACTATATAGG AACGAAACCATGAACTTCCCTCGCGCGTCGCGCCTGATGCAGGCGGCGGTCCTC GGTGGTCTGATGGCAGTCAGCGCCGCGGCCACCGCTATGGACGGTGTACTGTGG CGTGTGCGCACCGCAGCCCTGATGGCCGCCCTGCTGGCACTGGCTGCGTGGGCGC TGGTGTGGGCGTCGCCGAGCGTTGAGGCGCAGAGCAACCCTTACCAGCGTGGCC CGAACCCGACCCGCTCGGCCCTGACCGCCGACGGTCCATTCTCTGTCGCCACCTA TACCGTCAGCCGCCTGTCGGTGTCGGGTTTCGGCGGCGGCGTGATCTACTACCCG ACCGGCACCTCCTTGACCTTCGGCGGCATTGCCATGTCCCCTGGCTACACCGCTG ACGCCTCGAGCCTGGCCTGGCTGGGTCGCCGTCTGGCCAGCCACGGCTTCGTCGT ACTGGTCATCAACACCAACAGCCGTTTCGACGGCCCTGACTCCCGCGCCAGCCAG CTGTCGGCTGCTCTGAACTACCTGCGTACCTCGTCGCCGTCCGCAGTCCGTGCCC GCCTGGACGCCAACCGTCTGGCCGTAGCCGGCCACTCGATGGGCGGCGGCGGCA CCCTGCGCATCGCCGAACAAAACCCAAGCCTGAAGGCCGCTGTCCCACTGACCC CGTGGCACACCGACAAGACCTTCAACACCTCGGTGCCAGTGCTGATCGTAGGTG CGGAGGCCGACACCGTGGCCCCAGTGTCCCAGCACGCCATCCCGTTCTACCAGA ACCTGCCGTCCACCACCCCAAAGGTTTATGTCGAGCTCTGCAACGCCAGTCACAT CGCCCCGAATTCGAACAACGCTGCCATCTCCGTCTACACCATCAGCTGGATGAAG CTGTGGGTTGACAACGACACCCGCTACCGCCAGTTCCTGTGTAACGTGAACGACC CGGCCCTGTGCGATTTCCGTACCAACAATCGTCACTGCCAGTGAcaaggattacatataa gg gtatataaatgAACTTCCCtcgcgcgtcgcgcctgatgcaggcggcggtcctcggtggtc tgatggcagtcagcgccgcggc caccgctcatatggcagttgcaggtgacttcccgaatcagttcctgagctgcttcgatgc agccaatctgaccgaaattgaactgccgg cagatgttcagggcttccgtctgattgaaattgccgaacatgccggtgataaaggcatgc cggcccattgtgaaattgtgggcgccatta atgatcgcattagtccggtggatggccagcattatagcattaaattccgtctgcgtctgc cgcaggattggaatggtcgcttctatatgga aggcggtggcggtagcaatggtgttctgaaagatgcaatgggcccgaccggcctgaatca ggaagatagtgccctggaacgcggct tcgcagtggtgaccaccgatagcggccatgataatgataccaatagcgatagcaatgcca gcggccgcagcgccttcggtatggatc cgcaggcacgcctggacttcggttatatgagttatgatattgttacccgtgtgggtaaag caattgtggaaaaatattatggtgcagcccc ggaaaaaagttacttcattggttgtagtgaaggcggtcgtgaagcagccctgatgaccca gcgctatccggatctgtatgatggtgtggt tgccggtgccccgggtattcacttcagctatagtgcagcctatgccccgttcctgctgcg catcttcggtaatctggccgaaaccagaaa tcagagcggtccggatggtattccgctgctgaataaactgtatagtgataatgatgtgca gctgattgccgatgccgttgtgggcgcctg tgatgccctggatggcctggaagatcgtatgagcaataatattgaagcctgtaccaccgt taccgtgctgccgcgcctgcgcgctctga catgcagtggcgccaaagaagaaggttgcctgctggaagatcagattgatgccttcgtgg ccggtatggcaggtccggtgaccagtg atggtacccgtctgtatccgggtcatccgtgggatccgggtattggcggtcgtattggtg atagtgttaatgatggcttccgtagttggtg gttcggtagctatgatagcgatcagaataatgcccgcaaagtgaccctgagcaccccgca gcatgccatgctgtggcagaccccgcc ggttccgctgcgtcctgatgaatatgtgcgcttcgaaatgaacttcaatattgatgaaac acctgccctggcatacgctaccaccgatctg tatccggtgagcagtgcagaactgggcaatgccgatagtccggatctgagcgacttcgca agccgcggcggtaaactggtgatctat catggtgccgcagatgcagcattcagtgcactggataccattaaatattggaatgccgtt aatgaaaccgccgatggtcaggccgcag acttcgcacgtctgttcattattccgggtatgaatcattgccagggtggtccggccaccg atgatgttgatctgctgaccccgctgatggc atgggttgaagatgatctgccgattgaacgcctggaagccaccgtgagcaatccggatta cttcggtggtaaaaatctgagtcgcccg ctgtgtccgtatccgctgtatgccgaatatgatggtgaaggtgatccgagtagcgcagaa tcattcacct SEQ ID NO: 12 Length: Type: DNA Organism: Artificial Other information: Mutant Mle046 DNA sequence Ggatccccctcaagtcaaaagcctccggtcggaggcttttgactttctgctatggaggtc aggtatgattttgcattaggcaccccaggc ttgacaattaatcatcggctcgtataatgtgtggaattgtgagcggataacaatttcaca ctctagaggaggaatcttactctagaatgcat atggcagttgcaggtgacttcccgaatcagttcctgagctgcttcgatgcagccaatctg accgaaattgaactgccggcagatgttca gggcttccgtctgattgaaattgccgaacatgccggtgataaaggcatgccggcccattg tgaaattgtgggcgccattaatgatcgcat tagtccggtggatggccagcattatagcattaaattccgtctgcgtctgccgcaggattg gaatggtcgcttctatatggaaggcggtgg cggtagcaatggtgttctgaaagatgcaatgggcccgaccggCCTGAATCAGGAAGATAG TGCCCTGGAA CGCAGTTTCGCAGTGGtgaccaccgatagcggccatgataatgataccaatagcgatagc aatgccagcggccgcag cgccttcggtatggatccgcaggcacgcctggacttcggttatatgagttatgatattgt tacccgtgtgggtaaagcaattgtggaaaaa tattatggtgcagccccggaaaaaagttacttcattggttgtagtgaaggcggtcgtgaa gcagccctgatgacccagcgctatccgga tctgtatgatggtgtggttgccggtgccccgggtattcacttcagctatagtgcagccta tgccccgttcctgctgcgcatcttcggtaatc tggccgaaaccagaaatcagagcggtccggatggtattccgctgctgaataaactgtata gtgataatgatgtgcagctgattgccgat gccgttgtgggcgcctgtgatgccctggatggcctggaagatcgtatgagcaataatatt gaagcctgtaccaccgttaccgtgctgcc gcgcctgcgcgctctgacatgcagtggcgccaaagaagaaggttgcctgctggaagatca gattgatgccttcgtggccggtatggc aggtccggtgaccagtgatggtacccgtctgtatccgggtcatccgtgggatccgggtat tggcggtcgtattggtgatagtgttaatga tggcttccgtagttggtggttcggtagctatgatagcgatcagaataatgcccgcaaagt gaccctgagcaccccgcagcatgccatgc tgtggcagaccccgccggttccgctgcgtcctgatgaatatgtgcgcttcgaaatgaact tcaatattgatgaaacacctgccctggcat acgctaccaccgatctgtatccggtgagcagtgcagaactgggcaatgccgatagtccgg atctgagcgacttcgcaagccgcggc ggtaaactggtgatctatcatggtgccgcagatgcagcattcagtgcactggataccatt aaatattggaatgccgttaatgaaaccgcc gatggtcaggccgcagacttcgcacgtctgttcattattccgggtatgaatcattgccag ggtggtccggccaccgatgatgttgatctg ctgaccccgctgatggcatgggttgaagatgatctgccgattgaacgcctggaagccacc gtgagcaatccggattacttcggtggta aaaatctgagtcgcccgctgtgtccgtatccgctgtatgccgaatatgatggtgaaggtg atccgagtagcgcagaatcattcacctgtg tggccaataagctttgagatatcgatatcattcaggacgagcctcagactccagcgtaac tggactgaaaacaaactaaagcgcccttg tggcgctttagttttgttcc SEQ ID NO: 13 Length: Type: Protein Organism: bacteria Other information: Mle046 protein sequence MHMAVAGDFPNQFLSCFDAANLTEIELPADVQGFRLIEIAEHAGDKGMPAHCEIVGAIND RISPVDGQH YSIKFRLRLPQDWNGRFYMEGGGGSNGVLKDAMGPTGLNQEDSALERGFAVVTTDSGHDN DTNSDS NASGRSAFGMDPQARLDFGYMSYDIVTRVGKAIVEKYYGAAPEKSYFIGCSEGGREAALM TQRYPDL YDGVVAGAPGIHFSYSAAYAPFLLRIFGNLAETRNQSGPDGIPLLNKLYSDNDVQLIADA VVGACDAL DGLEDRMSNNIEACTTVTVLPRLRALTCSGAKEEGCLLEDQIDAFVAGMAGPVTSDGTRL YPGHPWDP GIGGRIGDSVNDGFRSWWFGSYDSDQNNARKVTLSTPQHAMLWQTPPVPLRPDEYVRFEM NFNIDETP ALAYATTDLYPVSSAELGNADSPDLSDFASRGGKLVIYHGAADAAFSALDTIKYWNAVNE TADGQAA DFARLFIIPGMNHCQGGPATDDVDLLTPLMAWVEDDLPIERLEATVSNPDYFGGKNLSRP LCPYPLYAE YDGEGDPSSAESFTCVANKL* SEQ ID NO: 14 Length: Type: Protein Organism: artificial Other information: mutant Mle046 (G117S) protein sequence MHMAVAGDFPNQFLSCFDAANLTEIELPADVQGFRLIEIAEHAGDKGMPAHCEIVGAIND RISPVDGQH YSIKFRLRLPQDWNGRFYMEGGGGSNGVLKDAMGPTGLNQEDSALERSFAVVTTDSGHDN DTNSDSN ASGRSAFGMDPQARLDFGYMSYDIVTRVGKAIVEKYYGAAPEKSYFIGCSEGGREAALMT QRYPDLY DGVVAGAPGIHFSYSAAYAPFLLRIFGNLAETRNQSGPDGIPLLNKLYSDNDVQLIADAV VGACDALD GLEDRMSNNIEACTTVTVLPRLRALTCSGAKEEGCLLEDQIDAFVAGMAGPVTSDGTRLY PGHPWDPG IGGRIGDSVNDGFRSWWFGSYDSDQNNARKVTLSTPQHAMLWQTPPVPLRPDEYVRFEMN FNIDETPA LAYATTDLYPVSSAELGNADSPDLSDFASRGGKLVIYHGAADAAFSALDTIKYWNAVNET ADGQAAD FARLFIIPGMNHCQGGPATDDVDLLTPLMAWVEDDLPIERLEATVSNPDYFGGKNLSRPL CPYPLYAEY DGEGDPSSAESFTCVANKL* References [00223] Bentley, G. J.; Narayanan, N.; Jha, R. K.; Salvachúa, D.; Elmore, J. R.; Peabody, G. L.; Black, B. A.; Ramirez, K.; De Capite, A.; Michener, W. E., Engineering glucose metabolism for enhanced muconic acid production in Pseudomonas putida KT2440. Metabolic engineering 2020, 59, 64-75. [00224] Bhaskar, T.; Kaneko, J.; Muto, A.; Sakata, Y.; Jakab, E.; Matsui, T.; Uddin, M. A., Pyrolysis studies of PP/PE/PS/PVC/HIPS-Br plastics mixed with PET and dehalogenation (Br, Cl) of the liquid products. Journal of analytical and applied pyrolysis 2004, 72 (1), 27-33. [00225] Jayakody, L. N.; Dissanayake, L., Engineering microbes to bio-upcycle polyethylene terephthalate. Frontiers in Bioengineering and Biotechnology 2021, 9, 356. [00226] R.K. Jha, J.M. Bingen, C.W. Johnson, T.L. Kern, P. Khanna, D.S. Trettel, C.E. Strauss, G.T. Beckham, T. Dale, A protocatechuate biosensor for Pseudomonas putida KT2440 via promoter and protein evolution, Metabolic engineering communications, 6 (2018) 33-38. [00227] Johnson, C. W.; Salvachúa, D.; Rorrer, N. A.; Black, B. A.; Vardon, D. R.; John, P. C. S.; Cleveland, N. S.; Dominick, G.; Elmore, J. R.; Grundl, N., Innovative chemicals and materials from bacterial aromatic catabolic pathways. Joule 2019, 3 (6), 1523- 1537. [00228] Kenny, S. T.; Runic, J. N.; Kaminsky, W.; Woods, T.; Babu, R. P.; Keely, C. M.; Blau, W.; O’Connor, K. E., Up-cycling of PET (polyethylene terephthalate) to the biodegradable plastic PHA (polyhydroxyalkanoate). Environmental science & technology 2008, 42 (20), 7696-7701. [00229] Leavitt, J. M.; Wagner, J. M.; Tu, C. C.; Tong, A.; Liu, Y.; Alper, H. S., Biosensor‐enabled directed evolution to improve muconic acid production in Saccharomyces cerevisiae. Biotechnology journal 2017, 12 (10), 1600687. [00230] Shojaei, B.; Abtahi, M.; Najafi, M., Chemical recycling of PET: A stepping‐ stone toward sustainability. Polymers for Advanced Technologies 2020, 31 (12), 2912-2938. [00231] Tiso, T.; Narancic, T.; Wei, R.; Pollet, E.; Beagan, N.; Schröder, K.; Honak, A.; Jiang, M.; Kenny, S.; Wierckx, N., Bio-upcycling of polyethylene terephthalate. BioRxiv 2020. [00232] Tournier, V.; Topham, C.; Gilles, A.; David, B.; Folgoas, C.; Moya-Leclair, E.; Kamionka, E.; Desrousseaux, M.-L.; Texier, H.; Gavalda, S., An engineered PET depolymerase to break down and recycle plastic bottles. Nature 2020, 580 (7802), 216-219. [00233] Walker, T. W.; Frelka, N.; Shen, Z.; Chew, A. K.; Banick, J.; Grey, S.; Kim, M. S.; Dumesic, J. A.; Van Lehn, R. C.; Huber, G. W., Recycling of multilayer plastic packaging materials by solvent-targeted recovery and precipitation. Science advances 2020, 6 (47), eaba7599. [00234] Sanders, M.M. 2017. The Application of Oxidative Hydrothermal Dissolution (OHD) to Organic-Rich Shales. Southern Illinois University at Carbondale. [00235] 1. Tournier, V.; Topham, C.; Gilles, A.; David, B.; Folgoas, C.; Moya- Leclair, E.; Kamionka, E.; Desrousseaux, M.-L.; Texier, H.; Gavalda, S., An engineered PET depolymerase to break down and recycle plastic bottles. Nature 2020, 580 (7802), 216-219. [00236] 2. Meyer-Cifuentes, I. E.; Öztürk, B., Mle046 is a marine mesophilic MHETase-like enzyme. Frontiers in Microbiology 2021, 12. [00237] 3. Knott, B. C.; Erickson, E.; Allen, M. D.; Gado, J. E.; Graham, R.; Kearns, F. L.; Pardo, I.; Topuzlu, E.; Anderson, J. J.; Austin, H. P., Characterization and engineering of a two-enzyme system for plastics depolymerization. Proceedings of the National Academy of Sciences 2020, 117 (41), 25476-25485. [00238] 4. Mrigwani, A.; Thakur, B.; Guptasarma, P., Enhancing high-temperature degradation of polyethylene terephthalate through a synergistic division of enzyme labour between a solid-degrading thermostable cutinase and a reaction intermediate-degrading thermostable carboxylesterase. bioRxiv 2022.